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Thyrotropin-Secreting Pituitary Adenomas

ABSTRACT

 

Thyrotropin-secreting pituitary tumors (TSH-omas) are a rare cause of hyperthyroidism and account for less than 1% of all pituitary adenomas. It is, however, noteworthy that the number of reported cases increased over the last few years as a consequence of the routine use of ultrasensitive immunometric assays for measuring TSH levels. Contrary to previous RIAs, ultrasensitive TSH assays allow a clear distinction between patients with suppressed and those with non-suppressed circulating TSH concentrations, i.e. between patients with primary hyperthyroidism (Graves’ disease or toxic nodular goiter) and those with central hyperthyroidism (TSH-oma or pituitary resistance to thyroid hormone action, PRTH). Failure to recognize the presence of a TSH-oma may result in dramatic consequences, such as improper thyroid ablation that may cause the pituitary tumor volume to further expand. The presence of neurological signs and symptoms (visual defects, headache) or clinical features of concomitant hypersecretion of other pituitary hormones (acromegaly, galactorrhea/amenorrhea) strongly supports the diagnosis of TSH-oma. Nevertheless, the differential diagnosis between TSH-oma and PRTH may be difficult when the pituitary adenoma is very small, or in the case of confusing lesions, such as an empty sella or pituitary incidentalomas. First-line treatment of TSH-omas is pituitary adenomectomy followed by irradiation in the case of surgical failure. However, medical treatment with somatostatin analogs, such as octreotide and lanreotide, are effective in reducing TSH secretion in more than 90% of cases with consequent normalization of FT4 and FT3 levels and restoration of the euthyroid state. For complete coverage of all related areas of Endocrinology, please visit our on-line FREE web-text, WWW.ENDOTEXT.ORG.

 

INTRODUCTION

 

Thyrotropin (TSH)-secreting pituitary adenomas (TSH-omas) are a rare cause of hyperthyroidism. In this situation, TSH secretion is autonomous and refractory to the negative feedback of thyroid hormones (inappropriate TSH secretion) and TSH itself is responsible for the hyperstimulation of the thyroid gland and the consequent hypersecretion of T4 and T3 (1, 2). Therefore, this entity can be appropriately classified as a form of "central hyperthyroidism". The first case of TSH-oma was documented in 1960 by measuring serum TSH levels with a bioassay (3). In 1970, Hamilton et al. (4) reported the first case of TSH-oma proved by measuring TSH by RIA.

 

Classically, TSH-omas were diagnosed at the stage of invasive macroadenoma and were considered difficult to cure. However, the advent of ultrasensitive immunometric assays, routinely performed as first line test of thyroid function, has greatly improved the diagnostic workup of hyperthyroid patients, allowing the recognition of the cases with unsuppressed TSH secretion. Therefore, TSH-omas are now more often diagnosed at an earlier stage, before they become a macroadenoma, and an increased number of patients with normal or elevated TSH levels in the presence of high free thyroid hormone concentrations have been recognized. Signs and symptoms of hyperthyroidism along with values of thyroid function tests similar to those found in TSH-oma may be recorded also among patients affected with resistance to thyroid hormones (5-7). This form of resistance to thyroid hormones is called pituitary resistance to thyroid hormones (PRTH), as the resistance to thyroid hormone action appears more severe at the pituitary than at the peripheral tissue level. The clinical importance of these rare entities is based on the diagnostic and therapeutic challenges they present.

 

Failure to recognize these different diseases may result in dramatic consequences, such as improper thyroid ablation in patients with central hyperthyroidism or unnecessary pituitary surgery in those with PRTH. Conversely, early diagnosis and the correct treatment of TSH-omas may prevent the occurrence of neurological and endocrinological complications, such as visual defects by compression of the optic chiasm, headache, and hypopituitarism, and should improve the rate of cure.

 

EPIDEMIOLOGY

 

TSH-producing adenoma is a rare disorder, accounting for about 0.5% to 2% of all pituitary adenomas, the prevalence in the general population being 1 to 2 cases per million (1, 2, 8-69). However, this figure is probably underestimated as the number of reported cases of TSH-omas has risen in the last decade. The increased incidence of TSH-omas has been further confirmed by data obtained from The Swedish Pituitary Registry (70), demonstrating an increased incidence of TSH-omas over time (0.05 per 1 million per year in 1990-1994 to 0.26 per 1 million per year in 2005-2009), the national prevalence in 2010 being 2.8 per 1 million inhabitants. The augmented number of reported cases principally results from the introduction of ultrasensitive TSH immunometric assays and from improved general practitioner awareness. Based on the finding of measurable serum TSH levels in the presence of elevated FT4 and FT3 concentrations, many patients, previously thought to be affected with primary hyperthyroidism (Graves' disease), can nowadays be correctly diagnosed as patients with TSH-oma or, alternatively, with PRTH (1, 2, 5-7).

 

The presence of a TSH-oma has been reported at ages ranging from 8 to 84 years (2, 58, 59). However, most patients are diagnosed around the fifth-sixth decade of life. TSH-omas occur with equal frequency in men and women, in contrast with the female predominance seen in other, more common thyroid disorders. Familial cases of TSH-oma have been reported as part of multiple endocrine neoplasia type 1 (MEN1) syndrome (10) and in familial isolated pituitary adenoma (FIPA) families with an AIPmutation (52).

 

PATHOLOGICAL ASPECTS

 

Immunostaining studies showed the presence of TSH beta subunit, either free or combined, in all adenomatous cells from every type of TSH-oma, with only few exceptions (1, 2, 8, 9, 71). The majority of TSH-secreting adenomas (72%) are secreting TSH alone, often accompanied by unbalanced hypersecretion of alpha-subunit of glycoprotein hormones (alpha-GSU) (Table 1). Interestingly, the existence of TSH-omas composed of two different cell types, one secreting alpha-GSU alone and another cosecreting alpha-GSU and the entire TSH molecules (mixed TSH/alpha-GSU adenomas), was documented by using double gold particle immunostaining (72). The presence of a mixed TSH/alpha-GSU adenoma is suggested by the finding of an extremely high alpha-GSU/TSH molar ratio and/or by the observation of dissociated TSH and alpha-GSU responses to TRH (1, 72). Classic mixed adenomas characterized by concomitant hypersecretion of other anterior pituitary hormones are found in about 30% of patients.

 

Table 1. Recorded Cases of TSH-Secreting Adenomas of Different Type
  Number  % of total
Total TSH-secreting adenomas (TSH-omas) 470 ---
Pure TSH-omas 332 70.6
TSH-omas with associated hypersecretion of other pituitary hormones (mixed TSH-omas) 138 29.4
Mixed TSH/GH-omas 83 17.7
Mixed TSH/PRL-omas 47 10.0
Mixed TSH/FSH/LH-omas 8  1.7

(updated end November 2018 and personal unpublished observations)

 

Hypersecretion of GH and/or PRL, resulting in acromegaly and/or an amenorrhea/galactorrhea syndrome, are the most frequent associations. This may be due to the fact that somatotroph and lactotroph cells share with thyrotropes common transcription factors, such as Prop-1 and Pit-1 (73). Rare is the occurrence of mixed TSH/gonadotropin adenoma, while no association with ACTH hypersecretion has been documented to date, probably due to the distant origin of corticotroph and thyrotroph lineages. Nonetheless, positive immunohistochemistry for one or more pituitary hormone does not necessarily correlate with its or their hypersecretion in vivo(9, 71, 74). Accordingly, clinically and biochemically silent thyrotropinomas have been reported (9, 75, 76). Moreover, true TSH-secreting tumors associated with Hashimoto’s thyroiditis and hypothyroidism have been documented (2, 39, 77, 78).

 

Microadenomas, with a diameter <1 cm, were recorded in less than 15% of the cases before 1996 (22), but their prevalence among all the TSH-omas is progressively increasing due to improved testing of thyroid function and awareness among endocrinologists and general practitioners. In particular, in the series recently published by our Institution (79), up to 30% of TSH-omas were microadenomas. Most TSH-omas had been diagnosed at the stage of macroadenomas and showed localized or diffuse invasiveness into the surrounding structures, especially into the dura mater and bone (1, 8, 9, 15, 22, 71). Extrasellar extension in the supra- and/or parasellar direction were present in the majority of cases. The occurrence of invasive macroadenomas is particularly high among patients with previous thyroid ablation by surgery or radioiodine (Figure 1) (2). This finding emphasizes the deleterious effects of incorrect diagnosis and treatment of these adenomas, and the relevant action on tumor growth exerted by the reduction of circulating thyroid hormone levels through an altered feedback mechanism. Such an aggressive transformation of the tumor resembles that occurring in Nelson's syndrome after adrenalectomy for Cushing's disease. Finally, some data suggest that somatic mutations of the thyroid hormone receptor beta may be responsible for the defect in negative regulation of TSH secretion in some

TSH-omas (56, 80-82). In addition, alteration in iodothyronine deiodinase enzyme expression and function may contribute to the resistance of tumor cells to the feedback mechanism of elevated thyroid hormone levels (83). However, these data were not confirmed by another study on this topic (84).

Figure 1. Clinical manifestations in patients with TSH-secreting adenomas. Patients have been divided into two categories according to previous thyroid surgery. The presence of goiter is the rule, even in patients with partial thyroidectomy. Hyperthyroid features may be overshadowed by those of associated hypersecretion/deficiency of other pituitary hormones. Invasive tumors are seen in about half of the patients with previous thyroidectomy and in 1/4 of untreated patients (P<0.01 by Fisher's exact test). Intrasellar tumors show an opposite distribution pattern.

The consistency of TSH-omas is usually very fibrous and sometimes so hard that they deserve the name of "pituitary stone" (85). Increased basic fibroblast growth factor (bFGF) levels were found in blood from two patients with invasive mixed PRL/TSH-secreting adenomas characterized by marked fibrosis (86). The tumoral origin of bFGF was confirmed by the finding of specific transcript in the tissues removed at surgery, suggesting a possible autocrine role for this growth factor in tumor development.

 

By light microscopy and appropriate staining, adenoma cells usually have a chromophobe appearance. Cells are often arranged in cords, and they frequently appear polymorphous and characterized by large nuclei and prominent nucleoli. Ultrastructurally, the well differentiated adenomatous thyrotrophs resemble the normal ones, while the poorly differentiated adenomas are composed of elongated angular cells with irregular nuclei, poorly developed RER, long cytoplasmic processes and sparse small secretory granules (50-200 nm) usually lining up along the cell membrane (9, 71). Generally, no exocytosis is detectable. Cells with abnormal morphology or mitoses are occasionally found which may be mistaken for a pituitary malignancy or metastases from distant carcinomas (87). Nevertheless, the transformation of a TSH-oma into a pituitary carcinoma with multiple metastases has seldom been reported (35, 60, 88). Future malignant behaviour might be predicted by the finding of very high circulating levels of free alpha-subunit, whereas a concomitant, spontaneous and marked decrease of both TSH and alpha-GSU serum concentrations might indicate that the tumor is becoming less differentiated and correlate with invasive and metastatic behaviour. Finally, in a mouse model of TSH-oma, the activation of phosphatidyl-inositol 3-kinase promoted aberrant pituitary growth that may induce transformation of the adenoma into a carcinoma (89).

MOLECULAR AND IN VITRO SECRETION STUDIES

 

The molecular mechanisms leading to the formation of TSH-omas are presently unknown, as is true for the large majority of pituitary adenomas. X-chromosomal inactivation analysis demonstrated that most pituitary adenomas, including the small number of TSH-omas investigated, derive from the clonal expansion of a single initially transformed cell (43). Accordingly, the general principles of tumorigenesis, that assume the presence of a transforming event providing gain of proliferative function followed by secondary mutations or alterations favoring tumor progression, presumably also apply to TSH-omas.

 

A large number of candidate genes, including common proto-oncogenes and tumor suppressor genes as well as pituitary specific genes, have been screened for mutations able to confer growth advantage to thyrotrope cells. In analogy with the other pituitary adenomas, no mutations in oncogenes commonly activated in human cancer, particularly RAS, have been reported in TSH-omas. In contrast, with GH-secreting adenomas in which the oncogene gsp(GNASmutation) is frequently present, none of the TSH-oma screened has been shown to express activating mutations of genes encoding for G protein subunits, such as alpha s, alpha q, alpha 11 or alpha i2 (90). Similarly, no mutations in the TRH receptor or dopamine D2 receptor genes (90-91) have been reported in 9 and 3 TSH-omas, respectively, while these tumors were not screened for alterations in protein kinase C, previously identified in some invasive tumors. In consideration of the crucial role that the transcription factor Pit-1 exerts on cell differentiation and PRL, GH and TSH gene expression, the Pit-1 gene has been screened for mutations in 14 TSH-omas and found to be wild-type (2). By contrast, as occurs in GH-omas, Pit-1 was demonstrated to also be overexpressed in TSH-omas, although the proliferative potential of these findings remains to be elucidated (2, 71, 73).

 

In addition to activating mutations or overexpression of protooncogenes, tumors may originate from the loss of genes with antiproliferative action. As far as the loss of tumour suppressor genes is concerned, no loss of p53 was found in one TSH-oma studied, while the loss of retinoblastoma gene (Rb), which is unaltered in other pituitary adenomas, was not investigated in TSH-omas. Another candidate gene is MEN1coding for menin. In fact, 3-30% of sporadic pituitary adenomas show loss of heterozygosity (LOH) on 11q13, where MEN1is located, and LOH on this chromosome seems to be associated with the transition from the non-invasive to the invasive phenotype. A screening study carried out on 13 TSH-omas using polymorphic markers on 11q13 showed LOH in 3, but none of them showed a MEN1mutation after sequence analysis (92). Interestingly, hyperthyroidism due to TSH-omas has been reported in five cases within a familial setting of multiple endocrine neoplasia type 1 syndrome (1, 2, 10). In addition, LOH and in particular polymorphisms at the somatostatin receptor type 5 gene locus, seem to be associated with an aggressive phenotype and resistance to somatostatin analog treatment (93). Moreover, germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) are known to be involved in sporadic pituitary tumorigenesis, but mutations were found in two patients with TSH-omas (52, 94). Finally, arecent study based on whole-exome sequencing identified several candidate somatic mutations and change in copy numbers in 12 sporadic TSH-omas, but with low number per tumor and without recurrence of mutations (95). Further studies in combination with epigenetic and transcriptomic approaches are necessary to reveal the underlying genetic lesions.

 

The extreme refractoriness of neoplastic thyrotrophs to the inhibitory action of thyroid hormones indicates mutant forms of thyroid hormone receptors (TR) as other potential candidate oncogenes. Absence of TR alpha1, TR alpha2 and TR beta1 expression was reported in two TSH-omas (82, 96), but aberrant alternative splicing of thyroid hormone receptor beta2 (THRB) mRNA encoding TR beta variant lacking T3 binding activity and other THRBmutations were shown as a mechanism for impaired T3-dependent negative regulation of both TSH and alpha-GSU in tumoral tissue (80, 81). Moreover, an aberrant expression of a novel thyroid hormone receptor βisoform (TRβ4) may partly contribute to the inappropriate secretion of TSH in TSH-omas (56). Several patients with THRBmutation and an PRTH phenotype have been described to bear pituitary lesions at imaging of the sella region, raising diagnostic and therapeutic dilemmas (30, 97-99). The results of dynamic testing of TSH secretion were consistent with PRTH, rather than TSH-omas, indicating that these lesions are likely to be pituitary incidentalomas, whose prevalence in not selected autopsy series reaches 20%.

 

Pharmacological manipulations in short-term cultures of TSH-omas indicate that these tumors express a large number of functioning receptors. Although in vivoTSH response to TRH is usually absent, several in vitrostudies showed either the presence or the absence of TSH response, indicating that the majority of tumors possess TRH receptors (2). Similarly, somatostatin binding experiments indicate that almost all TSH-omas express a variable number of somatostatin receptors, the highest somatostatin-binding site densities being found in mixed GH/TSH adenomas (57, 100, 101). Since somatostatin analogs are highly effective in reducing TSH secretion by neoplastic thyrotrophs (12, 13, 102, 103), the inhibitory pathway mediated by somatostatin receptors appears to be largely intact in such adenomas. Consistently, there is a good correlation between somatostatin binding capacity and maximal biological response, as quantified by inhibition of TSH secretion and in vivorestoration of euthyroid state (57, 102-104). The presence of dopamine receptors in TSH-omas was the rationale for therapeutic trials with dopaminergic agonists, such as bromocriptine (57, 105, 106). Several studies have shown a large heterogeneity of TSH responses to dopaminergic agents, either in primary cultures or in vivo(1, 41, 107, 108). The effects of these two inhibitory agents should be nowadays re-evaluated in light of the demonstration of the possible heterodimerization of somatostatin receptor subtype 5 (sst5) and dopamine D2 receptor (109).

 

CLINICAL FINDINGS

 

Patients with TSH-omas present with signs and symptoms of hyperthyroidism that are frequently associated with those related to the pressure effects of the pituitary adenomas, causing loss of vision, visual field defects, headache and/or loss of anterior pituitary function (Figure 1) (22, 49, 63, 110). TSH-omas may occur at any age and, in contrast with the common thyroid disorders, there is no preferential incidence in females (2, 8, 22, 59). Due to the long history of thyroid dysfunction, many patients had been mistakenly diagnosed as having primary hyperthyroidism (Graves' disease), and about one third had inappropriate thyroid ablation by thyroidectomy and/or radioiodine. True coexistence of Graves’ disease and TSH-oma has been reported in a few cases (54, 111). Clinical features of hyperthyroidism are usually present, sometimes milder than expected given the level of thyroid hormones, probably due to their longstanding duration (112). Consistently, several untreated patients with TSH-oma were described as clinically euthyroid (59, 113). Moreover, hyperthyroid features can be overshadowed by those of acromegaly in patients with mixed TSH/GH adenomas (79, 114-118), thus emphasizing the importance of the systematic measurement of TSH and FT4 in patients with pituitary tumors. Acromegaly itself can often be associated with multinodular goitre, presenting a further possible differential diagnostic scenario.

 

Cardiotoxicosis with atrial fibrillation, cardiac failure, massive pleural and pericardial have been reported in sporadic cases (119-123) and typical episodes of periodic paralysis have also been described in two patients (20, 124). A highprevalenceof radiological vertebral fracture has been recently documented in a series of patients with TSH-omas (125).

 

The presence of a goiter is the rule, even in the patients with previous partial thyroidectomy, since thyroid residue may regrow as a consequence of TSH hyperstimulation. The occurrence of uni- or multinodular goiter is frequent (about 72% of reported cases), but progression towards functional autonomy seems to be rare (126, 127). The monitoring of the thyroid nodule(s) and the performance of fine needle aspiration biopsy are indicated in TSH-omas since differentiated thyroid carcinomas were documented in several patients (1, 11, 55, 128-131). A recent publication evaluating sixty-two patients who underwent surgery for TSH-oma showed an estimated incidence of thyroid carcinoma of 4.8%, thus suggesting a possible role of TSH hypersecretion in the development of thyroid tumors (130). The prevalence of circulating antithyroid autoantibodies (anti-thyroglobulin: Tg-Ab, and anti-thyroid peroxidase: TPO-Ab) is similar to that found in the general population, but some patients developed Graves' disease after pituitary surgery and a few others presented bilateral exophthalmos due to autoimmune thyroiditis (2, 55, 132), while unilateral exophthalmos due to orbital invasion by the pituitary tumor has also been reported (3, 133).

 

Dysfunction of the gonadal axis is not rare, with menstrual disorders present in one third of the reported cases, mainly in the mixed TSH/PRL adenomas. Central hypogonadism, delayed puberty and decreased libido were also found in a number of males with TSH-omas and/or mixed TSH/FSH adenomas (1, 107, 134, 135).

 

As a consequence of suprasellar extension or invasiveness, signs and symptoms of an expanding tumor mass are predominant in many patients. Partial or total hypopituitarism was seen in about 25% of cases, headache reported in 20-25% of patients and visual field defects are present in about 50% of patients (Figure 1).

 

BIOCHEMICAL FINDINGS

 

High concentrations of circulating free thyroid hormones in the presence of detectable TSH levels characterize the hyperthyroidism secondary to TSH-secreting pituitary adenomas. Normal levels of total T4 were recorded in several patients with TSH-omas despite the presence of clinical signs and symptoms of hyperthyroidism. This observation indicates that the measurement of circulating free thyroid hormones (FT4 and FT3) is mandatory. In fact, these measurements show the highest sensitivity for the correct diagnosis of central hyperthyroidism and prevent misclassification in the case of excess of circulating levels of thyroxine-binding globulin (26). Many different physiological or clinical conditions, such as pregnancy or PRTH, may present with hyperthyroxinemia and detectable serum TSH levels, and should be distinguished from TSH-omas. Most of these conditions may be recognized on the basis of either a patient's clinical history or by measuring the concentrations of FT4 and FT3 with direct "two-step" methods, i.e. the methods able to avoid possible interference due to the contact between serum factors and tracer at the time of the assay (e.g. equilibrium dialysis+RIA, adsorption chromatography+RIA, and back-titration) (136, 137). In fact, some factors may interfere with the measurement of either thyroid hormones or TSH. The presence of anti-iodothyronine autoantibodies (anti-T4 and/or anti-T3) or abnormal albumin/transthyretin forms, such as those circulating in familial dysalbuminemic hyperthyroxinemia, may cause FT4 and/or FT3 to be overestimated, particularly when "one-step" analog methods are employed (137, 138). The more common factors interfering in TSH measurement and giving spuriously high levels of TSH are the circulating heterophilic antibodies, i.e. antibodies directed against mouse gamma-globulins (36, 139).

 

About 30% of TSH-oma patients with an intact thyroid gland showed TSH levels within the normal range (2). In this respect, it is worth noting that TSH with “reflex FT4 strategy” (i.e. measurement of FT4 test only in the presence of an abnormal TSH result) fails to recognize both central hypo and hyperthyroidism thus leading to TSH deficiency or TSH-oma misdiagnosis (140, 141). The diurnal rhythm is preserved in TSH-omas and TSH secretion shares many characteristics (cross-approximate entropy, cross-correlation and cosinor regression) of other pituitary hormone-secreting adenomas (142). Interestingly, a case of TSH-oma with cyclic fluctuations in serum TSH levels has recently been reported (143).

 

Furthermore, despite the TSH-dependent origin of hyperthyroidism, there is no direct correlation between free thyroid hormone and immunoreactive TSH levels (Figure 2). An increased biological activity of secreted TSH molecules likely accounts for the finding of normal TSH in the presence of high levels of FT4 and FT3 (114). TSH molecules secreted by pituitary tumors are heterogeneous and may have either a normal, reduced, or increased ratio between their biological and immunological activities, probably due to modification of glycosylation processes secondary to alterations of the post-translational processing of the hormone within the tumor cell (144, 145). Interestingly, TSH levels in patients previously treated with thyroid ablation were 6-fold higher than in untreated patients, though free thyroid hormone levels were still in the hyperthyroid range (115). Moreover, tumoral thyrotrophs may undergo more active cellular proliferation in response to even small reductions in circulating thyroid hormone levels, as documented by the higher number of invasive macroadenomas found in previously treated patients (Figure 1), beyond the increase of TSH secretion.

Figure 2. Absent correlation between immunoreactive concentrations of circulating TSH and FT3 in 14 patients with TSH-secreting adenomas. Dotted lines indicate the upper limits of normal ranges for both parameters.

Independently of previous thyroid ablation, circulating free alpha-GSU levels and alpha-GSU/TSH molar ratio were clearly elevated in the majority of patients with TSH-oma, either due to unbalanced secretion of the subunit or to the presence of a mixed TSH/alpha-GSU adenoma (72). The calculation of the alpha-GSU/TSH molar ratio increases the diagnostic sensitivity of hormone measurement, and an alpha-GSU/TSH molar ratio above 1.0 was associated with the presence of a TSH-secreting pituitary adenoma (2, 8). However, data from our group show that the individual values must be compared with those of control groups matched for TSH and gonadotropin levels before drawing any diagnostic conclusions. Controls with normal levels of TSH and gonadotropins may have alpha-GSU/TSH molar ratios as high as 5.7, and values as high as 29.1 can be found in euthyroid postmenopausal women (146). Indeed, hypersecretion of alpha-GSU is not unique to TSH-omas, being present in the majority of true gonadotropinomas, in a subset of non-functioning pituitary adenomas, and in a number of GH- or PRL-secreting tumors. Moreover, high alpha-GSU levels may be observed in conditions other than pituitary adenomas, such as in patients with inflammatory bowel disease (e.g., ulcerative colitis, Crohn disease) or with other neuroendocrine tumors (e.g., carcinoids) (146).

 

PARAMETERS OF THYROID HORMONE ACTION

 

Patients with central hyperthyroidism may present with mild signs and symptoms of thyroid hormone overproduction. Therefore, the measurements of several parameters of peripheral thyroid hormone action have been proposed to quantify the degree of tissue hyperthyroidism (2, 5-8, 98). Some of them are measured in vivo(basal metabolic rate, cardiac systolic time intervals, "Achilles" reflex time) and others in vitro(sex hormone-binding globulin: SHBG, cholesterol, angiotensin converting enzyme, soluble interleukin-2 receptor, osteocalcin, carboxyterminal cross-linked telopeptide of type I collagen (ICTP), etc.). In particular, liver (SHBG) and bone parameters (ICTP) have been successfully used to differentiate hyperthyroid patients with TSH-omas from those with pituitary RTH (Figure 3). In fact, as seen in the common forms of hyperthyroidism, patients with TSH-omas have high SHBG and ICTP levels, while they are in the normal range in patients with hyperthyroidism due to PRTH (147, 148).

Figure 3. Values of sex hormone-binding globulin (SHBG) and carboxyterminal cross-linked telopeptide of type 1 collagen (ICTP) in patients with PRTH or TSH-omas. Shaded areas represent the normal ranges either in premenopausal women or in postmenopausal women and men. The combined measurement of parameters from different tissues may be useful for the differential diagnosis and by-pass possible interference by different factors (age, liver or bone diseases, combined alteration of pituitary functions, treatments, etc.). Tx TSH-omas, treated TSH-omas

DYNAMIC TESTING

 

Although both stimulatory and inhibitory tests had been proposed for the diagnosis of TSH-omas, none of them is of clear-cut diagnostic value. Classically, T3 suppression test has been used to assess the presence of a TSH-oma. A complete inhibition of TSH secretion after T3 suppression test (80-100 ug/day for 8-10 days) has never been recorded in patients with TSH-oma. T3 suppression test can be combined with TRH testing, where normal subjects and TSH-oma patients do not show elevation of TSH in response to TRH, while PRTH patients show a brisk elevation (1). In patients with previous thyroid ablation, T3 suppression seems to be the most sensitive and specific test in assessing the presence of a TSH-oma (2, 8, 98). However, this test is strictly contraindicated in elderly patients or in those with coronary heart disease.

 

TRH testing has been widely used to investigate the presence of a TSH-oma. In the vast majority of patients, TSH and alpha-GSU levels do not increase after TRH injection. In patients with hyperthyroidism, discrepancies between TSH and alpha-GSU responses to TRH (i.e. higher al [ha-GSU than TSH response) are pathognomonic of TSH-omas cosecreting other pituitary hormones. Such a discrepancy is also found in an opposite clinical condition, i.e. the congenital hypothyroidism due to a TSH beta gene mutation (149).

 

The majority of TSH-omas maintain sensitivity to native somatostatin and its analogs. Indeed, administration of native neuropeptide or its analogues (octreotide or lanreotide) induces a reduction of TSH levels in the majority of cases, and these tests may be predictive of the efficacy of long-term treatment (30, 89, 102, 104).

 

We recommend the use of both T3 suppression and TRH tests whenever possible, because the combination of their results increases the specificity and sensitivity of the diagnostic work-up.

 

IMAGING STUDIES AND LOCALIZATION OF THE TUMOR

 

As for other tumors of the region of the sella turcica, nuclear magnetic resonance imaging (MRI) is nowadays the preferred tool for the visualization of a TSH-oma. High-resolution computed tomography (CT) is the alternative investigation in the case of contraindications, such as patients with pacemakers. Most TSH-omas have been diagnosed in the past at the stage of macroadenomas, and various degrees of suprasellar extension or sphenoidal sinus invasion are seen in two thirds of cases.

 

Microadenomas are now reported with increasing frequency, accounting for about 15-30% of all recorded cases in both clinical and surgical series. Pituitary scintigraphy with radiolabeled octreotide (Octreoscan) has been shown to successfully localize TSH-omas that express somatostatin receptors (8, 150, 151). However, the specificity of the Octreoscanis low, since positive scans can be seen in the case of a pituitary mass of different types, either secreting or non-secreting.

 

Finally, ectopic localization of a TSH-oma has been reported by four groups who found a nasopharyngeal mass in few patients with clinical and biochemical features of central hyperthyroidism (21, 32, 152-157). Histological and immunohistochemical studies of both specimens collected during the operation showed unequivocally that the tumor was a TSH-oma, and the resection of the mass restored TSH and alpha-GSU levels to normal.

DIFFERENTIAL DIAGNOSIS

 

If FT4 and FT3 concentrations are elevated in the presence of measurable TSH levels, it is important to exclude methodological interference due to the presence of circulating autoantibodies (e.g. against T3 and T4) or heterophilic antibodies (e.g. for TSH). In a patient with signs and symptoms of hyperthyroidism, the confirmed presence of elevated FT4/FT3 and detectable TSH levels rules out Graves' disease or other forms of primary hyperthyroidism. In patients on levothyroxine replacement therapy, the finding of measurable TSH in the presence of high FT4/FT3 levels may be due to poor compliance or to an incorrect high L-T4 dosage, probably administered before blood sampling.

 

When the existence of central hyperthyroidism is confirmed, several diagnostic steps have to be carried out to differentiate a TSH-oma from PRTH (Table 2) (1, 5-8, 30, 97, 98, 158). The presence of neurological signs and symptoms (visual defects, headache) of an expanding intra-cranial mass or clinical feature of concomitant hypersecretion of other pituitary hormones (acromegaly, galactorrhea/amenorrhea) points to the presence of a TSH-oma. The presence of alterations of the pituitary content on MRI or CT scanning strongly supports the diagnosis of a TSH-oma. Nevertheless, the differential diagnosis may be difficult when the pituitary adenoma is very small, or in the case of confusing lesions, such as an empty sella. Moreover, the possibility of pituitary incidentalomas should always be considered, due to their high prevalence. In our series, about 20% of PRTH patients have a pituitary lesion on MRI.

 

No significant differences in age, sex, previous thyroid ablation, TSH levels or free thyroid hormone concentrations were seen between patients with TSH-oma and those with PRTH. However, in contrast to PRTH patients, familial cases of TSH-oma have never been documented. Serum TSH levels within the normal range are more frequently found in PRTH, while elevated alpha-GSU concentrations and/or a high alpha-GSU/TSH molar ratio are typically present in patients with TSH-omas. Moreover, TSH unresponsiveness to TRH stimulation and/or to T3 suppression tests favours the presence of a TSH-oma. We have shown that chronic administration of long-acting somatostatin analogues in patients with central hyperthyroidism caused a marked decrease of FT3 and FT4 levels in all patients but one with TSH-oma, while patients with PRTH did not respond at all (30). Thus, administration of long-acting somatostatin analogs for at least 2 months can be useful in the differential diagnosis in problematic cases of central hyperthyroidism.

 

Indexes of thyroid hormone action at the tissue level (such as SHBG or ICTP levels) are in the hyperthyroid range in most patients with TSH-oma, while they are generally normal/low in PRTH (Figure 3). Exceptions are the findings of normal SHBG levels in patients with mixed GH/TSH adenoma, due to the inhibitory action of GH on SHBG synthesis and secretion, and of high SHBG in PRTH patients treated with estrogens or showing profound hypogonadism. Genetic analysis of the TR beta gene may be useful in the differential diagnosis, as TR beta mutations in leukocyte DNA have been found only in patients with PRTH.

 

Another parameter that can be useful for the differential diagnosis is the evaluation of the sensitivity to long-acting somatostatin analogs (30). More than 90% of TSH-oma are sensitive and 2 or more administrations of analog are usually sufficient to induce significant decreases or normalization of circulating free thyroid hormone. These modifications have never been observed in PRTH patients (Table 2).

 

Table 2. Differential Diagnosis Between TSH-Secreting Adenomas (TSH-omas) and Resistance to Thyroid Hormones (RTH).
Parameter TSH-omas RTH P
Female/Male ratio 1.3 1.4 NS
Familial cases 0 % 85 % <0.0001
TSH mU/L 3.0 ±0.4 2.3 ±0.3 NS
FT4 pmol/L 38.8 ±3.9 29.9 ±2.3 NS
FT3 pmol/L 14.0 ±1.2 11.3 ±0.8 NS
Lesions at CT or MRI 99 % 23 % <0.0001
Germline THRB mutation 0% 84% <0.0001
High biological activity of circulating serum TSH 38% 90% NS
High alpha-GSU levels 69 % 3 % <0.0001
High alpha-GSU/TSH molar ratio 81 % 2 % <0.0001
Elevated SHBG and/or ICTP 90% 8% <0.0001
Blunted TSH response to TRH test (cutoff <3 mU/L in males and <5 mU/L in females) 87 % 2 % <0.0001
Abnormal TSH response to T3 suppressiona 100 % 100 % NS
FT4/FT3 reduction/normalization during long-acting somatostatin analogc 92% 0% <0.0001

aWerner's test (80-100 µg T3 for 8-10 days). Quantitatively normal responses to T3, i.e. complete inhibition of both basal and TRH-stimulated TSH levels, have never been recorded in either group of patients.

bAlthough abnormal in quantitative terms, TSH response to T3 suppression test was qualitatively normal in 45/47 PRTH patients.

c Two or more injections of somatostatin analogs (e.g., Octreotide-LAR 20-30 mg every month or Lanreotide Autogel 120 mg every 6-8 weeks)

Only patients with intact thyroid were taken into account. Data are obtained from patients followed at our Department and are expressed as mean ± SE.

 

TREATMENT AND OUTCOME

 

As stated in the published guideline by European Thyroid Association (158), surgical resection is the recommended therapy for TSH-secreting pituitary tumors, with the aim of removing neoplastic tissue and restoring normal pituitary/thyroid function. However, radical removal of large tumors, that still represent the majority of TSH-omas, is particularly difficult because of the marked fibrosis of these tumors and the local invasion involving the cavernous sinus, internal carotid artery or optic chiasm. Considering this high invasiveness, surgical removal or debulking of the tumor by transsphenoidal or subfrontal adenomectomy, depending on the tumor volume and its suprasellar extension, should be undertaken as soon as possible. Particular attention has to be paid to presurgical preparation of the patient, particularly in the preanesthetic period (159): antithyroid drugs or octreotide along with propranolol should be used, aiming at restoration of euthyroidism. In patients with very severe hyperthyroidism, the administration of iopanoic acid may be successfully employed (25). The preoperative treatment with somatostatin analogs may be useful to reduce hyperthyroid signs and symptoms, as well as the adenoma size (160, 161). After surgery, partial or complete hypopituitarism may result (162, 163). However, a case of thyroid storm after pituitary surgery was documented (164). Evaluation of pituitary function, particularly ACTH secretion, should be carefully undertaken soon after surgery and hormone replacement therapy initiated, if needed. In case of failure of pituitary surgery and in the presence of life-threatening hyperthyroidism, total thyroidectomy or thyroid ablation with radioiodine is indicated (165). According to the largest published series, pituitary surgery is effective in restoring euthyroidism in 75% to 83% of patients with TSH-omas (61, 79).

 

If pituitary surgery is contraindicated or declined, as well as in the case of surgical failure, pituitary radiotherapy and/or medical treatment with somatostatin analogs are two valid alternatives (158). In the case of radiotherapy, the recommended dose is no less than 45 Gy fractionated at 2 Gy per day or 10-25 Gy in a single dose if a stereotactic gamma knife is available (158, 166). Radiotherapy is effective in normalizing thyroid function in 37% of patients within 2 years (79). The successful experience of an invasive TSH-oma associated with an unruptured aneurysm treated by two-stage operation and gamma knife has been reported (167). Although earlier diagnosis has improved the surgical cure rate of TSH-omas, several patients have required medical therapy in order to control the hyperthyroidism. Dopamine agonists, and particularly cabergoline, have been employed in some TSH-omas with variable results, positive effects being mainly observed in some patients with mixed PRL/TSH adenoma (121, 168, 169). Today, the medical treatment of TSH-omas rests on long-acting somatostatin analogs, such as octreotide or lanreotide (12, 13, 85, 158, 170-172). Indeed, many papers suggest the use of somatostatin analogs as first-line therapy for patients with TSH-omas, particularly for invasive macroadenomas (173-176). Treatment with these analogs leads to a reduction of TSH and alpha-GSU secretion in almost all cases, with restoration of the euthyroid state in the majority of them and it is safe even during pregnancy (18, 24, 177, 178). In some cases, inhibition of tumoral TSH secretion may be so profound that hypothyroidism may even be seen. During somatostatin analog therapy tumor shrinkage occurs in about 50% of patients and vision improvement is seen in 75% (61, 79, 179). Very rapid shrinkage of the tumor has been described (34). Resistance to octreotide treatment has been documented in a few cases. Patients on somatostatin analogs have to be carefully monitored, as untoward side effects, such as cholelithiasis and carbohydrate intolerance, may become manifest. The dose administered should be tailored for each patient, depending on therapeutic response. Tolerance is usually very good, as gastrointestinal side effects are transient with long-acting analogs (12, 13, 57, 179, 180).

 

CRITERIA OF CURE AND FOLLOW-UP

 

Due to the rarity of the disease and the great heterogeneity of the methods used, the criteria of cure of patients operated or irradiated for TSH-omas has not been clearly established. Previous thyroid ablation makes some of these criteria inapplicable (Table 3).

 

Table 3. Criteria for the Evaluation of the Outcome of Treatment.
Criteria  Comments

Remission from hyperthyroid

manifestations (clinical and biochemical)

Clinical improvement may be transient

No predictive value

Disappearance of neurological

manifestations (adenoma imaging,

visual field defects, headache)

May be transient

Poor predictive value

Normalization of free thyroid hormone levels

Biochemical remission may be transient

Poor predictive value

Normalization of circulating TSH levels

Not applicable to patients with normal TSH

Poor predictive value

Undetectable TSH one week after

neurosurgery

Applicable to hyperthyroid patients that stopped treatments at least 10 days before surgery

Good prognostic value

Normalization of alpha-GSU levels and

alpha-GSU/TSH molar ratio

Not applicable to patients with normal values before neurosurgery

Lack of sensitivity

Positive T3-suppression test with

undetectable TSH and no response to TRH (or central hypothyroidism)

Not applicable to elderly patients or in

those with cardiac diseases

Optimal sensitivity/specificity and predictive

 value

 

 

In untreated hyperthyroid patients, it is reasonable to assume that cured patients have clinical and biochemical reversal of thyroid hyperfunction. However, the findings of normal free thyroid hormone concentrations or indices of peripheral thyroid hormone action (SHBG, ICTP, etc.) are not synonymous with complete removal or destruction of tumoral cells, since transient clinical remission accompanied by normalization of thyroid function is possible (62, 63, 67, 98, 115). Disappearance of neurological signs and symptoms is a good prognostic event, but lacks both sensitivity and specificity, as even an incomplete debulking of the tumor may cause visual field defects and headache to vanish. The resolution of specific neuroradiological abnormalities is confusing, since the pituitary imaging performed soon after surgery is often difficult to interpret. The criteria of normalization of circulating TSH is not applicable to previously thyroidectomized patients and to the 26% of patients with normal basal values of TSH. In our experience, undetectable TSH levels one week after surgery are likely to indicate complete adenomectomy, provided that the patient was hyperthyroid and presurgical treatments were stopped before surgery (115). Normalization of alpha-GSU and/or the alpha-GSU/TSH molar ratio is in general a good index for the evaluation of therapy efficacy (8, 115). However, both parameters are characterized by less than optimal sensitivity, as they are normal in about 25% of patients with TSH-oma. The most sensitive and specific test to document the complete removal of the adenoma remains, in the absence of contraindication, the T3 suppression test (115). In fact, only patients in whom T3 administration completely inhibits basal and TRH-stimulated TSH secretion, appear to be truly cured.

 

Few data on the recurrence rates of TSH-oma in patients judged cured after surgery or radiotherapy have been reported. However, the recurrence of the adenoma does not appear to be frequent, at least in the first years after successful surgery (62, 115). In general, the patient should be evaluated clinically and biochemically 2 or 3 times the first year postoperatively, and then every year. Pituitary imaging should be performed every two or three years, but should be promptly done whenever an increase in TSH and thyroid hormone levels, or clinical symptoms occur. In the case of a persistent macroadenoma, close visual field follow-up is required, as visual function could be threatened. Emergency surgical decompression is not always able to reverse even a recent visual deficit.

 

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The Effect of Inflammation and Infection on Lipids and Lipoproteins

ABSTRACT

 

Chronic inflammatory diseases, such as rheumatoid arthritis, systemic lupus erythematosus, and psoriasis and infections, such as periodontal disease and HIV, are associated with an increased risk of cardiovascular disease. Patients with these disorders also have an increase in coronary artery calcium measured by CT and carotid intima media thickness measured by ultrasound. Inflammation and infections induce a variety of alterations in lipid metabolism that may initially dampen inflammation or fight infection, but if chronic could contribute to the increased risk of atherosclerosis. The most common changes are decreases in serum HDL and increases in triglycerides. The increase in serum triglycerides is due to both an increase in hepatic VLDL production and secretion and a decrease in the clearance of triglyceride rich lipoproteins. The mechanisms by which inflammation and infection decrease HDL levels are uncertain. There is also a consistent increase in lipoprotein (a) levels due to increased apolipoprotein (a) synthesis. LDL levels are frequently decreased but the prevalence of small dense LDL is increased due to cholesterol ester transfer protein (CETP) mediated exchange of triglycerides from triglyceride rich lipoproteins to LDL followed by triglyceride hydrolysis. In addition to affecting serum lipid levels, inflammation also adversely effects lipoprotein function. LDL is more easily oxidized as the ability of HDL to prevent the oxidation of LDL is diminished. Moreover, there are a number of steps in the reverse cholesterol transport pathway that are adversely affected during inflammation.  The greater the severity of the underlying inflammatory disease, the more consistently these abnormalities in lipids and lipoproteins are observed. Treatment of the underlying disease leading to a reduction in inflammation results in the return of the lipid profile towards normal. The changes in lipids and lipoproteins that occur during inflammation and infection are part of the innate immune response and therefore are likely to play an important role in protecting the host. The guidelines for the management of lipid disorders and the standard risk calculators for predicting cardiovascular disease (ACC/AHA, Framingham, etc.) underestimate the risk in patients with inflammation. It has been recommended to increase the calculated risk by approximately 50% in patients with severe inflammatory disorders. The treatment of lipid disorders in patients with inflammatory disorders is similar to patients without inflammatory disorders. Of note statins, fibrates, and fish oil have anti-inflammatory properties and have been reported to have beneficial effects on many of these inflammatory disorders.

 

INTRODUCTION

 

A number of chronic inflammatory diseases, including rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, Sjögren's syndrome, polymyalgia rheumatica, inflammatory bowel disease, and psoriasis are associated with an increased risk of cardiovascular disease (1-9). For example, in a meta-analysis of twenty-four studies comprising 111,758 patients with 22,927 cardiovascular events it was observed that there was a 50% increased risk of CVD death in patients with rheumatoid arthritis (10). Patients with rheumatoid arthritis have a similar risk for a cardiovascular event as patients with diabetes (11). Similarly, women with systemic lupus erythematosus in the 35- to 44-year age group were over 50 times more likely to have a myocardial infarction than were women of similar age in the Framingham Offspring Study (12). As a final example, a meta-analysis of 14 studies reported that in individuals with severe psoriasis the risk for cardiovascular mortality was 1.37, the risk for myocardial infarction was 3.04, and the risk for stroke was 1.59 times higher than the general population (13). It should be noted that the pathology in psoriasis is localized to the skin but nevertheless even this disorder, by inducing systemic inflammation, is associated with an increased risk of cardiovascular disease.

 

Further, supporting the link of rheumatoid arthritis, systemic lupus erythematosus, and psoriasis with atherosclerosis are studies showing that patients with these disorders have an increase in coronary artery calcium measured by CT and carotid intima media thickness measured by ultrasound (14-20). Finally, even children and adolescents with systemic lupus erythematosus have an increase in carotid intimal-medial thickness (21). Thus, it is clear that patients with a number of different chronic inflammatory diseases have an increased risk of atherosclerotic cardiovascular complications.

 

In addition, chronic infections are also associated with an increased risk of atherosclerosis (22-24). Since the development of effective anti-viral agents, it has been widely recognized that a major cause of morbidity and mortality in HIV infected patients is due to cardiovascular disease (25). Moreover, numerous studies have demonstrated an association of periodontal infections with an increased risk of atherosclerotic vascular disease (26). Additionally, carotid intima-media thickness is increased in patients with periodontal disease (27-30). The link between various chronic infections, such as HIV, dental infections, Helicobacter pylori, chronic bronchitis, and urinary tract infections with cardiovascular disease is presumably due to the chronic inflammation that accompanies these infections (31). For certain infections such as chlamydia pneumonia and cytomegalovirus it is possible that the association with cardiovascular disease is due to a direct role in the vessel wall.

 

To definitively link inflammation with cardiovascular disease studies determining the effect of anti-inflammatory drugs on cardiovascular events have been carried out. The Cantos study has provided data supporting a link between inflammation and cardiovascular disease (32). In this trial 10,061 patients with a previous myocardial infarction and a hsCRP level of 2 mg/L or more were randomized to canakinumab, a monoclonal antibody targeting interleukin-1β, or placebo. At 48 months canakinumab did not reduce lipid levels from baseline but did reduce hsCRP levels by approximately 30-40% indicating a decrease in inflammation. Most importantly, canakinumab administration led to a significantly lower rate of recurrent cardiovascular events than placebo. These results support the hypothesis that inflammation increases the risk of cardiovascular events. In contrast, a recent trial using methotrexate to inhibit inflammation failed to reduce cardiovascular event (33). However, in this trial methotrexate did not reduce levels of interleukin-1β, interleukin-6, or C-reactive protein raising the possibility that methotrexate did not effectively inhibit inflammation and therefore did not reduce cardiovascular events. Clearly further studies determining the effect of drugs that reduce inflammation on cardiovascular events are required.

 

The mechanisms by which chronic inflammation and infection increase the risk of atherosclerotic cardiovascular disease are likely multifactorial. As will be discussed below inflammation and infection induce a variety of alterations in lipid and lipoprotein metabolism that could contribute to the increased risk of atherosclerosis.

LIPID AND LIPOPROTEIN ABNORMALITIES IN PATIENTS WITH INFLAMMATORY DISORDERS AND INFECTIONS  

Rheumatoid Arthritis

 

The most consistent abnormality in patients with rheumatoid arthritis is a decrease in HDL cholesterol and apolipoprotein A-I levels (9,34-37). In particular, small HDL particles are decreased in patients with rheumatoid arthritis (38). Patients with more severe rheumatoid arthritis have the greatest reductions in HDL cholesterol levels (34-37,39). There is an inverse correlation of CRP levels with HDL cholesterol levels (i.e. higher CRP levels are associated with lower HDL cholesterol levels). With regards to total cholesterol and LDL cholesterol, there is more variability with many studies showing a decrease, other studies showing no change, and some studies showing an increase in patients with rheumatoid arthritis (34-37,39). The more severe the rheumatoid arthritis the greater the likelihood that the LDL cholesterol levels will be decreased. Serum triglyceride levels and small dense LDL levels tend to be increased in patients with rheumatoid arthritis (34-37,39,40). Levels of lipoprotein (a) are characteristically elevated in patients with rheumatoid arthritis and correlate with CRP levels (41-43).

 

Systemic Lupus Erythematosus

 

The changes in serum lipids and lipoproteins seen in patients with systemic lupus erythematosus are very similar to those observed in patients with rheumatoid arthritis (44-46). Specifically, there is a decrease in HDL cholesterol levels and an increase in serum triglyceride levels. LDL cholesterol levels are variable and maybe increased, normal, or low but small dense LDL levels tend to be increased. Lipoprotein (a) levels are also increased (47). Similar to rheumatoid arthritis the more severe the disease state the greater the alterations in serum lipid levels.

 

Psoriasis

 

A large number of studies have compared serum lipid levels in controls and patients with psoriasis (48). However, many of these studies included only a small number of subjects and the results have therefore been extremely variable with some studies showing alterations in serum lipid levels in patients with psoriasis and other studies showing no changes. In general, there is a tendency for an increase in serum triglycerides and a decrease in HDL cholesterol levels in patients with psoriasis (49-53). Additionally, a number of studies showed an increase in LDL cholesterol and lipoprotein (a) levels in patients with psoriasis (49,50,52). Small dense LDL levels and oxidized Lp(a) are also increased in psoriasis (40) (54). This variability between studies is most likely due to differences in the severity of the psoriasis with more severe disease demonstrating more robust alterations in lipid levels. The prevalence of other abnormalities that affect lipid metabolism such as obesity and abnormalities in glucose metabolism could also account for the variability in results.

 

Other Inflammatory Disease

 

Decreased HDL cholesterol levels have also been observed in patients with inflammatory bowel disease, Sjögren's syndrome, and ankylosing spondylitis (55-58). LDL cholesterol and triglyceride levels varied but LDL cholesterol levels tended to be decreased and triglyceride levels increased.

 

Periodontal Disease

 

Differences exist between studies but in general patients with periodontitis tend to have increased LDL cholesterol and triglyceride levels and decreased HDL cholesterol levels (59-63). Additionally, the prevalence of small dense LDL is increased in patients with periodontitis (62,64). The severity of the periodontitis correlated with the changes in the in the lipid profile with patients with increased periodontal disease having higher triglyceride levels, lower HDL cholesterol levels, and smaller LDL particle size (65). Moreover, treatment of periodontitis improved the dyslipidemia, with the HDL cholesterol levels increasing and the LDL cholesterol levels decreasing (62,66,67).

 

Acute Infections

 

Patients with a variety of different infections (gram positive bacterial, gram negative bacterial, viral, tuberculosis) have similar alterations in plasma lipid levels. Specifically, total cholesterol, LDL cholesterol, and HDL cholesterol levels are decreased while plasma triglyceride levels are elevated or inappropriately normal for the poor nutritional status (31,68-74). As expected apolipoprotein A-I, A-II, and B levels are reduced (68,73,74). While LDL cholesterol levels were decreased, the concentration of small dense LDL has been found to be increased during infections (75-77).That plasma cholesterol levels decrease during infection has been known for many years as it was described by Denis in 1919 in the Journal of Biological Chemistry (JBC 29: 93, 1919). The alterations in lipids correlate with the severity of the underlying infection i.e. the more severe the infection the more severe the alterations in lipid and lipoprotein levels (78,79). The decreases in plasma cholesterol levels can be quite profound and a recent case report described HDL cholesterol levels < 10mg/dl and LDL cholesterol levels < 3mg/dl in sepsis (80).

 

Of note studies have demonstrated that the degree of reduction in total cholesterol, HDL cholesterol, and apolipoprotein A-I are predictive of mortality in patients with severe sepsis (81-85). Moreover, epidemiologic studies have suggested that low cholesterol, LDL cholesterol, and HDL levels increase the chance of developing an infection (86-89). During recovery from the infection plasma lipid and lipoprotein abnormalities return towards normal. The changes in lipid and lipoproteins that occur during infection can be experimentally reproduced in humans and animals by the administration of endotoxin and lipoteichoic acid (31,90).

 

Summary  

 

Thus, in these different inflammatory disorders and infectious diseases, the alterations in plasma lipid and lipoprotein levels are very similar with decreases in plasma HDL being consistently observed. Also of note is the consistent increase in lipoprotein (a) levels and small dense LDL (31,91). There is also a tendency for plasma triglyceride levels to be elevated and LDL cholesterol levels decreased. The greater the severity of the underlying disease the more consistently these abnormalities in lipids are observed. Additionally, treatment of the underlying disease leading to a reduction in inflammation results in a return of the lipid profile towards normal. This is best illustrated in periodontal disease where intensive dental hygiene can reverse the abnormalities in the lipid profile (66,67).

 

Table 1. Effect of Inflammation and Infection on Lipid and Lipoprotein Levels

Triglycerides- Tend to be increased
HDL cholesterol- Decreased
LDL cholesterol- Variable but with more severe inflammation or infection they are decreased
Small dense LDL- Increased
Lp(a)- Increased

 

EFFECT OF ANTI-INFLAMMATORY DRUGS ON LIPID LEVELS

 

As noted above, treatments that reduce inflammation will return the lipid profile towards normal resulting in an increase in plasm HDL levels and a decrease in triglyceride levels. If LDL levels were reduced at baseline, treatment that reduces inflammation will also result in an increase in LDL levels (i.e. a return towards “normal” levels) (92-94). Many of the drugs used for the treatment of rheumatoid arthritis, systemic lupus erythematosus, and psoriasis decrease inflammation and have been shown to increase both HDL and LDL levels (9,92,93,95). The increase in HDL tends to be more robust. In a few instances, drugs used to treat inflammatory disorders have effects on lipid metabolism that are independent of the reduction in inflammation. For example, high dose glucocorticoid treatment results in an increase in serum triglyceride and LDL levels due to the increased production and secretion of VLDL by the liver (96-98) and hydroxychloroquine has been reported to lower total cholesterol, LDL, and triglycerides in patients with rheumatoid arthritis and systemic lupus erythematosus (99-101).

 

PATHOPHYSIOLOGY OF THE DYSLIPIDEMIA OF INFLAMMATION AND INFECTION

 

Inflammation and infections increase the production of a variety of cytokines, including TNF, IL-1, and IL-6, which have been shown to alter lipid metabolism (31). Many of the changes in plasma lipids and lipoproteins that are seen during chronic inflammation and infections are also observed following the acute administration of cytokines (31).

 

Increased Triglyceride Levels

 

Multiple cytokines increase serum triglyceride and VLDL levels (TNF, IL-1, IL-2, IL-6, etc.) (31). Following a single administration of a cytokine or LPS (a model of gram-negative infections), which stimulates cytokine production, an increase in serum triglyceride and VLDL levels can be seen within 2 hours and this effect is sustained for at least 24 hours. The increase in serum triglycerides is due to both an increase in hepatic VLDL production and secretion and a decrease in the clearance of triglyceride rich lipoproteins (figure 1) (31). The increase in VLDL production and secretion is a result of increased hepatic fatty acid synthesis, an increase in adipose tissue lipolysis with the increased transport of fatty acids to the liver, and a decrease in fatty acid oxidation in the liver. Together these changes provide an increased supply of fatty acids in the liver that stimulate an increase in hepatic triglyceride synthesis (31). The increased availability of triglycerides leads to the increased formation and secretion of VLDL. The decrease in the clearance of triglyceride rich lipoproteins is due to a decrease in lipoprotein lipase, the key enzyme that metabolizes triglycerides in the circulation (31). A variety of cytokines have been shown to decrease the synthesis of lipoprotein lipase in adipose and muscle tissue (31). Studies have also shown that inflammation also increases angiopoietin like protein 4, an inhibitor of lipoprotein lipase activity, which would further block the metabolism of triglyceride rich lipoproteins (102). In systemic lupus erythematosus, antibodies to lipoprotein lipase have been reported and are associated with increased triglyceride levels (103,104).

 

Figure 1: Pathogenesis of Hypertriglyceridemia

 

Production of Small Dense LDL and HDL

 

The elevation in triglyceride rich lipoproteins in turn has effects on other lipoproteins (31). Specifically, cholesterol ester transfer protein (CETP) mediates the exchange of triglycerides from triglyceride rich VLDL and chylomicrons to LDL and HDL. The increase in triglyceride rich lipoproteins per se leads to an increase in CETP mediated exchange, increasing the triglyceride content of both LDL and HDL. The triglyceride on LDL and HDL is then hydrolyzed by hepatic lipase and lipoprotein lipase leading to the increased production of small dense LDL and HDL. The affinity of Apo A-I for small HDL particles is reduced leading to the disassociation of Apo A-I and the consequent accelerated clearance and breakdown of Apo A-I by the kidneys. These changes, along with other factors described below, result in a reduction in the levels of Apo A-I and HDL in patients with inflammation.

 

Decreased HDL Levels

 

In addition to a decrease in HDL, inflammation can also lead to structural changes in this lipoprotein (31). During inflammation HDL particles tend to be larger with a decrease in cholesterol ester and an increase in free cholesterol, triglycerides, and free fatty acids. Furthermore, there are marked changes in HDL associated proteins and the enzymes and transfer proteins involved in HDL metabolism and function (figure 2 and 3).

Figure 2: Changes in HDL Protein Composition During Inflammation

 

Figure 3: Changes in Enzymes and Transfer Proteins During Inflammation

 

The precise mechanism by which inflammation and infection decrease HDL levels is uncertain and is likely to involve multiple mechanisms (31). Decreases in apolipoprotein A-I synthesis in the liver occur during inflammation and would result in the decreased formation of HDL. However, in acute infection and inflammation HDL decreases faster than would be predicted from decreased synthesis of apolipoprotein A-I. Increased serum amyloid A (SAA) production by the liver and other tissues occurs during inflammation and infection and the SAA binds to HDL displacing apolipoprotein A-I, which can accelerate the clearance of HDL. However, the overexpression in SAA in the absence of the acute phase response does not result in a decrease in HDL levels (105). Inflammation results in a decrease in LCAT leading to decreased cholesterol ester formation, which would prevent the formation of normal HDL, leading to decreased cholesterol carried in HDL. Elevations in triglyceride rich lipoproteins that accompany inflammation and infection can lead to the enrichment of HDL with triglycerides that can accelerate the clearance of HDL. Finally, cytokine induced increases in enzymes such as secretory phospholipase A2 (sPLA2) and endothelial cell lipase, which metabolize key constituents of HDL, could alter the stability and metabolism of HDL. Given the complexity of HDL metabolism it is not surprising that multiple pathways could be affected by inflammation, which together may account for the decrease in HDL levels.

 

Increased Lipoprotein (a)

 

The mechanism accounting for the increase in lipoprotein (a) (Lp(a)) during inflammation is likely due to increased apolipoprotein (a) synthesis, as apolipoprotein (a) is a positive acute phase protein whose expression is up-regulated during inflammation (31,106). The apolipoprotein (a) gene contains several IL-6 responsive elements that enhance transcription (107). Tocilizumab an antibody against IL-6, that is used to treat rheumatoid arthritis, has been shown to decrease Lp(a) levels (108) .

 

FUNCTIONAL CHANGES IN LIPOPROTEINS THAT INCREASE THE RISK OF ATHEROSCLEROSIS

 

LDL

 

While the levels of LDL do not consistently increase and may even decrease with inflammation and infection, many studies have indicated that inflammation and infection are associated with small dense LDL (31). These small dense LDL particles are believed to be more pro-atherogenic for a number of reasons (109). Small dense LDL particles have a decreased affinity for the LDL receptor resulting in a prolonged period of time in the circulation. Additionally, they more easily enter the arterial wall and bind more avidly to intra-arterial proteoglycans, which traps them in the arterial wall. Finally, small dense LDL particles are more susceptible to oxidation, which could result in an enhanced uptake by macrophages (110).

 

Several markers of lipid peroxidation, including conjugated dienes, thiobarbituric acid-reactive substances, malondialdehyde, and lipid hydroperoxides are increased in serum and/or circulating LDL during inflammation and infection (31,65,111-113). Moreover, LDL isolated from LPS-treated animals is more susceptible to oxidation in vitro (31). Oxidized LDL is taken up very efficiently by macrophages and is thought to play a major role in foam cell formation in the arterial wall (114). Additionally, antibodies to oxidized LDL are present in patients with systemic lupus erythematosus and could facilitate the uptake of an antibody LDL complex via the Fc-receptor in macrophages (111). Finally, studies have shown that LDL isolated from patients with periodontal disease leads to enhanced uptake of cholesterol esters by macrophages (65)

 

HDL

 

In addition to a decrease in serum HDL, inflammation and infection affects the anti-atherogenic properties of HDL (31,115,116). Reverse cholesterol transport plays a key role in preventing cholesterol accumulation in macrophages thereby reducing atherosclerosis. Most steps in the reverse cholesterol transport pathway are adversely affected during inflammation and infection (figure 4 and 5)  (39,117). First, cytokines induced by inflammation and infection decrease the production of Apo A-I, the main protein constituent of HDL. Second, pro-inflammatory cytokines decrease the expression of ABCA1, ABCG1, SR-B1, and apolipoprotein E in macrophages, which will lead to a decrease in the efflux of phospholipids and cholesterol from the macrophage to HDL. Third, the structurally altered HDL formed during inflammation is a poor acceptor of cellular cholesterol and in fact may actually deliver cholesterol to the macrophage (39,55,117-124). HDL isolated from patients with rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, psoriasis, ankylosing spondylitis, periodontal disease, and acute sepsis are poor facilitators of cholesterol efflux (55,118-123,125). Similarly, the experimental administration of endotoxin to humans also results in the formation of HDL that is a poor facilitator of the efflux of cholesterol from macrophages (126). Of note treatments that reduce inflammation in patients with rheumatoid arthritis, psoriasis, or periodontitis can restore towards normal the ability of HDL to remove cholesterol from cells (123,127-129). Fourth, pro-inflammatory cytokines decrease the production and activity of LCAT, which will limit the conversion of cholesterol to cholesterol esters in HDL. This step is required for the formation of a normal spherical HDL particle and facilitates the ability of HDL to transport cholesterol. Fifth, pro-inflammatory cytokines decrease CETP levels, which will decrease the movement of cholesterol from HDL to Apo B containing lipoproteins, an important step in the delivery of cholesterol to the liver. Sixth, pro-inflammatory cytokines decrease the expression of SR-B1 in the liver. SR-B1 plays a key role in the uptake of cholesterol from HDL particles into hepatocytes. Finally, inflammation and infection decrease both the conversion of cholesterol to bile acids and the secretion of cholesterol into the bile, the two mechanisms by which cholesterol is disposed of by the liver.

 

 

Figure 4: Effect of Inflammation on Reverse Cholesterol Transport (from reference 61)

Figure 5. Effect of Inflammation on the Factors Involved in Reverse Cholesterol Transport (from reference 61)

 

Another important function of HDL is to prevent the oxidation of LDL. Oxidized LDL is more easily taken up by macrophages and is pro-atherogenic (114). Paraoxonase is an enzyme that is associated with HDL and plays a key role in preventing the oxidation of LDL. Inflammation and infection decrease the expression of paraoxonase 1 in the liver resulting in a decrease in circulating paraoxonase activity (31). Plasma paraoxonase levels are decreased in patients with patients  rheumatoid arthritis, systemic lupus erythematosus, psoriasis, and infections (130-138) Studies have shown that HDL isolated from patients with rheumatoid arthritis and systemic lupus erythematous have a diminished ability to protect LDL from oxidation and in fact may facilitate LDL oxidation (115). Moreover, in patients with rheumatoid arthritis, reducing inflammation and disease activity with methotrexate treatment restored HDL function towards normal (139). Additionally, treatment with atorvastatin 80mg improved the function of HDL in patients with rheumatoid arthritis (140).

 

Thus, it should be recognized that in patients with inflammatory disorders and infections the absolute levels of lipids and lipoproteins may not be the only factor increasing the risk of atherosclerosis (31,48,112,115-117). Rather functional changes in LDL and HDL maybe pro-atherogenic and thereby contribute to the increased risk of atherosclerosis in inflammatory disorders and infections. Additionally, the increase in lipoprotein (a) may also play a role.

 

Table 2. Pro-Atherogenic Changes During Inflammation

Increased triglycerides
Decreased HDL
Increased small dense LDL
Increased Lp(a)
Oxidized LDL
Dysfunctional HDL

 

 

BENEFICIAL EFFECTS OF LIPIDS DURING INFECTIONS AND INFLAMMATION

 

The changes in lipids and lipoproteins that occur during inflammation and infection are part of the innate immune response and therefore are likely to play an important role in protecting from the detrimental effects of infection and inflammatory stimuli (31,141-143). Some of the potential beneficial effects are listed in Table 3. Thus, the changes in lipid and lipoprotein metabolism that occur during inflammation may initially be protective but if chronic can increase the risk of atherosclerosis.

 

Table 3. Beneficial Effects of Lipoproteins

Redistribution of nutrients to immune cells that are important in host defense
Lipoproteins bind endotoxin, lipoteichoic acid, and other biological agents and prevent their toxic effects
Lipoproteins bind urate crystals
Lipoproteins bind and target parasites for destruction
Apolipoproteins neutralize viruses
Apolipoproteins lyse parasites

 

LIPID MANAGEMENT IN A PATIENT WITH AN INFLAMMATORY DISEASE

 

Deciding When to Treat

 

As noted earlier, patients with inflammatory disorders are at an increased risk for atherosclerosis and this is not totally accounted for by standard lipid profile measurements and other risk factors (1-3,9). Some authors have advocated considering inflammatory disorders as a cardiovascular risk equivalent similar to diabetes; however many of the guidelines and risk calculators commonly used for deciding on lipid lowering therapy do not take into account this increased risk in patients with inflammatory disorders (3,144). For example, both the 2013 American College of Cardiology/American Heart Association and National Lipid Association recommendations do not specifically address this patient population (145,146). In the 2018 American College of Cardiology/American Heart Association recommendations, the presence of inflammatory disease is included as a risk factor, which can influence decisions on whether to initiate treatment (147). Additionally, the standard risk calculators for predicting cardiovascular disease (ACC/AHA and Framingham) underestimate the risk in this population (148-152). Even the Reynolds Risk Calculator (http://www.reynoldsriskscore.org/Default.aspx), which uses measurements of hsCRP levels, a marker of inflammation, underestimates the risk of cardiovascular events in patients with inflammatory disorders (148-152). Thus, if one follows the standard protocols one will undertreat patients with inflammatory disorders. It should be noted that the QRISK calculator (http://qrisk.org/) does factor in the presence of rheumatoid arthritis when calculating risk (153).

 

A reasonable approach is to use the standard approach and calculators but increase the calculated risk by approximately 50% in patients with severe inflammatory disorders. For example, if a patient with rheumatoid arthritis has a 5% ten-year risk and 40% lifetime risk one might increase the ten-year risk to 7.5% and lifetime risk to 60%. This approach has been recommended by an expert committee who advocated introducing a 1.5 multiplication factor (i.e. 50% increase) in patients with rheumatoid arthritis (9). Alternatively, one could carry out imaging studies such as obtaining a coronary artery calcium score to better define risk. Whatever the approach taken, it is crucial to recognize that patients with inflammatory diseases have an increased risk of cardiovascular disease and therefore one needs to be more aggressive.

 

Recent guidelines from the American College of Cardiology and American Heart Association are summarized in table 4 (147).

 

Table 4. ACC/AHA Guidelines

In patients with clinical ASCVD initiate high intensity statin therapy or maximally tolerated statin therapy. High intensity statin therapy is atorvastatin 40-80mg per day or rosuvastatin 20-40mg per day.
In very high-risk ASCVD, use an LDL cholesterol > 70 mg/dL (1.8 mmol/L) to consider addition of non-statins (ezetimibe or PCSK9 inhibitors). Very high-risk includes a history of multiple major ASCVD events or 1 major ASCVD event and multiple high-risk conditions.
In patients with LDL cholesterol ≥190 mg/dL [≥4.9 mmol/L]) begin high-intensity statin therapy. If the LDL-C level remains ≥100 mg/dL (≥2.6 mmol/L), adding ezetimibe is reasonable.
In adults 40 to 75 years of age without diabetes mellitus and with LDL-C levels ≥70 mg/dL (≥1.8 mmol/L) start a moderate-intensity statin if the 10-year ASCVD risk is ≥7.5%. Moderate intensity therapy is atorvastatin 10-20mg, rosuvastatin 5-10mg, simvastatin 20-40mg, pravastatin 40mg.

 

It should be noted that the ACC/AHA guidelines do not emphasize LDL cholesterol or non-HDL cholesterol goals while other organizations such as the National Lipid Association and American Association of Clinical Endocrinologists do provide LDL cholesterol and non-HDL cholesterol goals in their recommendations (Figures 6 and 7) (145,154). Our approach is to combine these approaches. In patients who have pre-existing cardiovascular disease we initiate intensive statin therapy. In patients 40-75 years of age without pre-existing cardiovascular disease we calculate the 10-year risk of developing cardiovascular disease and initiate moderate statin therapy if the 10-year risk is > 7.5% (note we adjust the results by multiplying by 1.5 to account for the effects of inflammation).  In patients < 40 years of age we calculate both the 10-year risk and lifetime risk and discuss with the patients the various options. In the presence of a high 10-year risk or lifetime risk we tend to recommend therapy. In patients >75 years of age we examine the 10-year risk and the patient’s functional status. If there is uncertainty, we will often obtain a cardiac calcium scan to help decide the best course of action. A zero calcium indicates a very low 10-year risk.

Figure 6. American Association of Clinical Endocrinologists LDL-C, Non-HDL-C, and Apo B Treatment Goals (154)

 

Figure 7. National Lipid Association LDL-C and Non-HDL-C Treatment Goals (145)

 

Eight to twelve weeks after initiating statin therapy we obtain a lipid panel to determine if the LDL and non-HDL cholesterol levels are at goal. In patients with pre-existing cardiovascular disease our goal is an LDL cholesterol < 70mg/dl and a non-HDL cholesterol < 100mg/dl. In patients with cardiovascular disease that is progressive or who have multiple risk factors a goal LDL cholesterol of < 55mg/dl and a non-HDL c < 80mg/dl should be strongly considered as suggested by the American Association of Clinical Endocrinologists (154). In patients without pre-existing cardiovascular disease our goal is an LDL cholesterol < 100mg/dl and a non-HDL cholesterol < 130mg/dl. On occasion in patients without pre-existing cardiovascular disease but either a high ten-year risk or multiple risk factors we will aim for an LDL cholesterol < 70mg/dl and a non-HDL cholesterol < 100mg/dl. If the LDL cholesterol or non-HDL cholesterol levels are not at goal, we either adjust the statin dose or consider adding additional medications.

 

Treatment Approach

 

As in all patients with lipid abnormalities the initial approach is lifestyle changes. Dietary recommendations are not unique in patients with inflammatory disorders. Exercise is recommended but depending upon the clinical situation the ability of patients with certain inflammatory disorders to participate in an exercise regimen may be limited. Exercise programs will need to be tailored for each patient’s capabilities. Treatment of the underlying disease to decrease inflammation is likely to be beneficial (9). Studies have shown that increased disease activity is associated with a greater risk of cardiovascular disease while lower disease activity is associated with a lower risk (9,155-161). Moreover, treatments that reduce disease activity can decrease cardiovascular risk (9).

 

Drug Therapy

 

This section on drug therapy will focus solely on the studies that are unique to patients with inflammatory diseases. Detailed information on the use of these drugs can be found in the chapters on cholesterol lowering drugs and triglyceride lowering drugs (162,163).

 

STATIN THERAPY

 

As expected, studies have demonstrated that statins lower LDL cholesterol levels in patients with inflammatory disorders to a similar degree as patients without inflammatory disorders. For example, in a randomized trial in 116 patients with rheumatoid arthritis with a mean LDL cholesterol level of 125mg/dl, the effect of atorvastatin 40mg was compared to placebo (164). Atorvastatin reduced LDL cholesterol by 54mgdl vs. 3mg/dl in the placebo group (164). Similarly in the IDEAL trial there was a small subgroup of patients with rheumatoid arthritis (165). The IDEAL trial compared the ability of atorvastatin 80mg vs. simvastatin 20-40mg to reduce cardiovascular events. The lowering of LDL cholesterol with either simvastatin or atorvastatin was similar in the patients with and without rheumatoid arthritis (165). Finally, a combined analysis of the IDEAL, Treat to New Target (TNT), and CARDS trials reported that the decrease in LDL cholesterol levels with statin therapy was similar in patients with or without psoriasis (166). Studies have shown similar reductions in LDL cholesterol levels with statin therapy in patients with systemic lupus erythematosus (see below). The effects of statin treatment on other lipid parameters were also similar in patients with and without inflammatory diseases. Thus, as expected statins improve the lipid profile in patients with inflammatory disorders. In some studies, the incidence of statin associated side effects have been increased in the patients with inflammatory disorders. Specifically, in the IDEAL trial rheumatoid arthritis patients reported myalgia more frequently than patients without rheumatoid arthritis (10.4% and 7.7% in rheumatoid arthritis patients vs 1.1% and 2.2% in non-rheumatoid arthritis patients receiving simvastatin and atorvastatin respectively) (165). Note that this does not necessarily indicate that statins induce myalgias more frequently in patients with rheumatoid arthritis as there was not a placebo group in the IDEAL trial. Rather it is likely that patients with rheumatoid arthritis have an increased prevalence of myalgias.

 

A key question is whether statin therapy will reduce cardiovascular events in patients with inflammatory diseases. A number of studies have looked at surrogate markers for events such as changes in carotid intima-media thickness or changes in cardiac calcium scores in patients treated with statins. The results have varied with some studies showing benefits and other studies showing no effects. Rollefstad et al measured changes in carotid plaque size in 86 patients with inflammatory joint disease treated with rosuvastatin for 18 months (167). The LDL cholesterol levels decreased from 155mg/dl to 66mg/dl and plaque height was significantly reduced (167). Similarly, Mok et al treated 72 patients with systemic lupus erythematosus with rosuvastatin 10mg or placebo for 12 months and reported that carotid intima-media thickness appeared to decrease (168). Moreover, Plazak et al treated 60 patients with systemic lupus erythematosus with atorvastatin 40mg or placebo for 1 year and measured changes in coronary calcium score (169). They observed an increase in coronary calcium in the placebo group while there was no change in the patients treated with statin therapy (169).  In contrast, Petri et al treated 200 patients with systemic lupus erythematosus with atorvastatin 40mg or placebo for 2 years and measured both carotid intima-media thickness and coronary calcium score (170). In this study no beneficial effects of statin therapy were observed (170). Similarly, Schanberg et al treated 221 children with systemic lupus erythematosus with atorvastatin 10-20mg or placebo for 36 months and did not observe a beneficial effect of statin treatment on carotid intima-media thickness (171). Thus, the effect of statin therapy in patients with inflammatory disorders on these surrogate markers of atherosclerosis is uncertain.

 

There are no large randomized controlled trials evaluating the impact of statin therapy on cardiovascular disease outcomes in patients with inflammatory disease. A subgroup analysis of a small number of patients with systemic lupus erythematosus in the ALERT study has been reported (172). The ALERT study was a randomized placebo-controlled trial examining the effect of fluvastatin 40-80mg on cardiovascular events after kidney transplantation. In this trial fluvastatin therapy reduced the risk of cardiovascular events by 74% in the patients with systemic lupus erythematosus (172). Additionally, a post hoc analysis of patients with inflammatory arthritis in the IDEAL and TNT trial has been reported (173). The IDEAL trial compared atorvastatin 80mg vs simvastatin 20-40mg and the TNT compared atorvastatin 80mg vs. atorvastatin 10mg. In these trials, statin therapy resulted in a decrease in lipid levels in the patients with inflammatory arthritis to a similar degree as patients without inflammatory arthritis (173). Moreover, there was an approximate 20% reduction in the risk of cardiovascular events in patients treated with atorvastatin 80mg compared to moderate dose statin therapy in patients with and without inflammatory arthritis (173). Similarly, a post hoc analysis of the IDEAL and TNT trials reported a similar reduction in cardiovascular events with high dose statin therapy compared to low dose statin therapy in patients with psoriasis (166). A trial that focused solely on patients with rheumatoid arthritis was initiated (TRACE RA) but was terminated early after a median of 2.5 years (Kitas GD, et al. Trial of atorvastatin for the primary prevention of cardiovascular events in patients with rheumatoid arthritis [abstract]. Arthritis Rheumatol 2015;67 Suppl 10; available from: http://acrabstracts.org/abstract/trial-of-atorvastatin-for-the-primary-prevention-of-cardiovascular-events-in-patients-with-rheumatoid-arthritis/). In this trial 2,986 patients with RA were randomized to atorvastatin 40mg/day vs. placebo.  As expected, the reduction in LDL cholesterol levels was significantly greater in the atorvastatin group compared to placebo (-42mg/dL vs. -5mg/dL, p<0.001). There was a 34% (unadjusted) risk reduction for major cardiovascular events in the atorvastatin group compared to placebo that was not statistically significant. Of note, the decrease in events was in the range expected based on the Cholesterol Treatment Trialists’ Collaboration meta-analysis of the effect of statins in other populations.  The number and type of adverse events were similar in the atorvastatin and placebo groups. Taken together these results suggest that patients with inflammatory diseases will have a reduction in cardiovascular events with statin therapy but clearly further studies are indicated.

 

It is well recognized that statins have anti-inflammatory properties and studies have consistently demonstrated a decrease in CRP levels in patients treated with statins. Two meta-analyses have explored the effect of statin therapy on disease activity in patients with rheumatoid arthritis. A meta-analysis by Ly et al included 15 studies with 992 patients and reported that statin therapy decreased erythrocyte sedimentation rate, CRP, tender joint count, swollen joint count, and morning stiffness (174). Similarly, a meta-analysis by Xing et al included 13 studies with 737 patients (175). They reported that statin therapy decreased erythrocyte sedimentation rate, CRP, tender joint count, and swollen joint count (175). Additionally, the disease activity score 28 (DAS28), which focuses on joint pathology, decreased significantly in the patients treated with statin therapy and the patients with the most active disease benefited the most (175,176).

 

In contrast to the beneficial effects seen in patients with rheumatoid arthritis, in randomized placebo controlled trials in patients with systemic lupus erythematosus studies by Plazak et al and Petri et al failed to show a decrease in disease activity with statin therapy (169,170). In psoriasis treatment with statins has produced mixed results with some studies showing a decrease in skin abnormalities and others showing no significant effect or even an increase in disease activity (177). Finally, treatment with statins has been shown to improve periodontal disease and reduce inflammation (178-180). Thus, statins can decrease the clinical manifestations of rheumatoid arthritis, periodontitis, and perhaps psoriasis but have no effect on the clinical manifestations of systemic lupus erythematosus. These differences could be due to the relative severity of the inflammatory response and/or the specific pathways that induce inflammation in these different disorders.

 

The effect of statins on outcomes in patients with sepsis has been extensively studied. Numerous observational studies have shown that patients treated with statins have a marked reduction in morbidity and mortality (181,182). For example, in a meta-analysis by Wan et al of 27 observational studies with 337,648 patients, statins were associated with a relative mortality risk of 0.65 (CI 0.57-0.75) (182). However, in randomized placebo controlled clinical trials statin administration has not been shown to reduce mortality or improve outcomes (181-183). For example in a meta-analysis by Wan et al of 5 randomized controlled trials with 867 patients the relative risk was 0.98 (182). Similarly, a meta-analysis by Pertzov et al of fourteen randomized trials evaluating 2628 patients also did not observe any benefits of statin therapy in patients with sepsis (184). Additionally, a recent study examining the effect of rosuvastatin on sepsis associated acute respiratory distress also failed to demonstrate a benefit of statin therapy (185). Thus, while observational data suggested that statins may be beneficial the more rigorous randomized placebo-controlled trials have not provided evidence of benefit.

 

FIBRATE THERAPY

 

Fibrates, gemfibrozil and fenofibrate, are used to lower triglycerides and raise HDL cholesterol levels. However, fibrates, by activating PPAR alpha, are well known to have anti-inflammatory effects. Several studies have shown that fibrate therapy improves the clinical manifestations in patients with rheumatoid arthritis. For example Shirinsky et al treated 27 patients with rheumatoid arthritis with fenofibrate and reported a significant reduction in disease activity score (DAS28) (186). A recent review described 4 randomized trials and 2 observation trials of fibrates in patients with rheumatoid arthritis and in general these studies showed that fibrate therapy decreased disease activity in patients with rheumatoid arthritis (187). There have been no clinical trials of fibrate therapy in patients with sepsis, psoriasis, systemic lupus erythematosus, and periodontal disease. Thus, there is a suggestion that the anti-inflammatory properties of fibrates may beneficially impact disease activity, but clearly further studies are required.

 

BILE ACID SEQUESTRANT THERAPY

 

Bile acid binders are used to lower LDL cholesterol levels. While there are no studies of the effect of bile acid binders in patients with either rheumatoid arthritis, systemic lupus erythematosus, or periodontal disease, there are two studies in patients with psoriasis. Both Roe and Skinner et al reported that the treatment of patients with psoriasis with bile acid binders improved the skin condition (188,189). The mechanism for this beneficial effect is unknown.

 

EZETIMIBE THERAPY

 

Ezetimibe is used to lower LDL cholesterol levels. There is a single six week trial in 20 patients with rheumatoid arthritis that demonstrated that ezetimibe treatment decreased total cholesterol, LDL cholesterol, and CRP levels (190). Moreover, ezetimibe treatment reduced disease activity (190). The mechanism for this beneficial effect is unclear.

 

FISH OIL THERAPY

 

Fish oil is widely used to reduce serum triglyceride levels and is recognized to have anti-inflammatory properties. There are numerous studies examining the effect of fish oil therapy on inflammatory diseases. A meta-analysis of 17 randomized controlled trials by Goldberg and Katz of the effect of omega-3-polyunsaturated fatty acids in patients with rheumatoid arthritis reported that treatment with omega-3-fatty acids reduced joint pain intensity, morning stiffness, number of painful and/or tender joints, and the use of non-steroidal anti-inflammatory medications (191). Similarly, meta-analyses by Lee et al and Gioxari et al also demonstrated that fish oil had beneficial effects in patients with rheumatoid arthritis (192,193). In psoriasis, a recent review of 15 trials reported that overall there was a moderate benefit of fish oil supplements with 12 trials showing clinical benefit and 3 trials showing no benefit (194). In contrast, Gamret et al evaluated fish oil treatment in patients with psoriasis in 20 studies (12 randomized controlled trials, 1 open-label nonrandomized controlled trial, and 7 uncontrolled studies) (195). They reported that most of the randomized controlled trials showed no significant improvement in psoriasis, whereas most of the uncontrolled studies showed benefit when fish oil was used daily. In systemic lupus erythematosus four randomized trials have demonstrated clinical benefit with fish oil therapy, while three trials failed to show disease improvement (196-202). Finally, there are data suggesting that treatment with fish oil reduces periodontal disease (203-205). A major limitation of the studies in patients with periodontal disease is that in these trials the experimental group treated with fish oil also was simultaneously treated with aspirin making it difficult to be sure that the beneficial effects were solely due to fish oil supplementation (203,204). Taken together these studies indicate that in addition to lowering serum triglyceride levels, fish oil therapy may have beneficial effects on the underlying inflammatory disorder in some instances.

 

NIACIN THERAPY

 

Niacin is used to lower LDL cholesterol levels and triglycerides and raise HDL cholesterol levels.  There are no reported clinical trials of niacin in patients with rheumatoid arthritis, systemic lupus erythematosus, psoriasis, or periodontal disease.

 

PCSK9 INHIBITORS

 

PCSK9 inhibitors are used to lower LDL cholesterol level. In addition, PCSK9 inhibitors also lower Lp(a) levels. There are no reported clinical trials of PCSK9 inhibitors in patients with rheumatoid arthritis, systemic lupus erythematosus, psoriasis, or periodontal disease.

 

Treatment Strategy

 

The first priority in treating lipid disorders is to lower the LDL cholesterol levels to goal, unless triglycerides are markedly elevated (> 500-1000mg/dl), which increases the risk of pancreatitis. LDL cholesterol is the usual first priority because the database linking lowering LDL cholesterol with reducing cardiovascular disease is extremely strong and we now have the ability to markedly decrease LDL cholesterol levels in the vast majority of patients. Dietary therapy is the initial step but, in many patients, will not be sufficient to achieve the LDL cholesterol goals. If patients are willing and able to make major changes in their diet it is possible to achieve remarkable reductions in LDL cholesterol levels but this seldom occurs in clinical practice (for details see the chapter on the effect of lifestyle changes on lipids and lipoproteins) (206).

 

Statins are the first-choice drugs to lower LDL cholesterol levels and many patients with inflammatory disorders will require statin therapy. Currently four statins are available as generic drugs, lovastatin, pravastatin, atorvastatin, and simvastatin, and these statins are relatively inexpensive. The choice of statin will depend on the magnitude of LDL cholesterol lowering required and whether other drugs that the patient is taking might alter statin metabolism thereby increasing the risk of statin toxicity. For example, cyclosporine affects the metabolism of many of the statins and in patients taking cyclosporine fluvastatin appears to be the safest statin (207).

 

If a patient is unable to tolerate statins or statins as monotherapy are not sufficient to lower LDL cholesterol to goal the second-choice drug is either ezetimibe or a PCSK9 inhibitor. Ezetimibe can be added to any statin. PCSK9 inhibitors can also be added to any statin and are the drugs of choice if a large decrease in LDL cholesterol is required to reach goal (PCSK9 inhibitors will lower LDL cholesterol levels by 50-60% when added to a statin, whereas ezetimibe will only lower LDL cholesterol by approximately 20%).  Bile acid sequestrants are an alternative particularly if a reduction in A1c level is also needed. Ezetimibe, PCSK9 inhibitors, and bile acid sequestrants additively lower LDL cholesterol levels when used in combination with a statin, because these drugs increase hepatic LDL receptor levels by different mechanisms, thereby resulting in a reduction in serum LDL cholesterol levels. Niacin and the fibrates also lower LDL cholesterol levels but are not usually employed to lower LDL cholesterol levels

 

The second priority should be non-HDL cholesterol (non-HDL cholesterol = total cholesterol – HDL cholesterol), which is particularly important in patients with elevated triglyceride levels (>150mg/dl). Non-HDL cholesterol is a measure of all the pro-atherogenic apolipoprotein B containing particles. Numerous studies have shown that non-HDL cholesterol is a strong risk factor for the development of cardiovascular disease. The non-HDL cholesterol goals are 30mg/dl greater than the LDL cholesterol goals. For example, if the LDL goal is <100mg/dl then the non-HDL cholesterol goal would be <130mg/dl. Drugs that reduce either LDL cholesterol or triglyceride levels will reduce non-HDL cholesterol levels.

 

The third priority in treating lipid disorders is to decrease triglyceride levels. Initial therapy should focus on lifestyle changes including a decrease in simple sugars and ethanol intake. Fibrates, niacin, statins, and omega-3-fatty acids all reduce serum triglyceride levels. Typically, one will target triglyceride levels when one is trying to lower non-HDL cholesterol levels to goal. Patients with very high triglyceride levels (> 500-1000 mg/dl) are at risk of pancreatitis and therefore lifestyle and triglyceride lowering drug therapy should be initiated early. Note that there is limited evidence demonstrating that lowering triglyceride levels reduces cardiovascular events. With a recent study demonstrating that adding omega-3-fatty acids (EPA) to statins in patients with elevated triglyceride levels reduces cardiovascular events one can anticipate an increased use of omega-3-fatty acids in patients with elevated triglyceride levels and a non-HDL cholesterol level above goal (208). In addition, the potential beneficial effects of fish oil on disease activity in many patients with inflammatory diseases make the use of omega-3-fatty acids an attractive choice in patients with inflammatory diseases and elevated triglyceride levels.

 

The fourth priority in treating lipid disorders is to increase HDL cholesterol levels. There is strong epidemiologic data linking low HDL cholesterol levels with cardiovascular disease, but whether increasing HDL levels with drugs reduces cardiovascular disease is unknown and studies have not been encouraging (209). Life style changes are the initial step and include increased exercise, weight loss, and stopping cigarette smoking. The role of recommending ethanol, which increases HDL levels, is controversial but in patients who already drink moderately there is no reason to recommend that they stop. The most effective drug for increasing HDL levels is niacin, but studies have not demonstrated a reduction in cardiovascular events when niacin is added to statin therapy (210,211). Fibrates and statins also raise HDL cholesterol levels but the increases are modest (usually less than 15%). Additionally, the ACCORD-LIPID trial failed to demonstrate that adding fenofibrate to statin therapy reduces cardiovascular disease (212). Unfortunately, given the currently available drugs, it is very difficult to significantly increase HDL cholesterol levels and in many of our patients we are unable to achieve HDL cholesterol levels in the recommended range. Furthermore, whether this will result in a reduction in cardiovascular events is unknown.

 

Note that there is very limited evidence that adding fibrates or niacin to lower triglyceride levels and/or increase HDL cholesterol levels will reduce cardiovascular events.  However, the recent studies of fibrates or niacin in combination with statins did not specifically target patients with high triglycerides, high non-HDL cholesterol, and low HDL cholesterol levels. The only drugs in combination with statin therapy that has been shown to further reduce cardiovascular events beyond statins alone is ezetimibe, PCSK9 inhibitors, and EPA, an omega-3-fatty acid.

 

Many patients with inflammatory diseases have multiple lipid abnormalities. As discussed above life style changes are the initial therapy. If life style changes are not sufficient in patients with both elevations in LDL cholesterol levels and triglycerides (and elevations in non-HDL cholesterol) one approach is to base drug therapy on the triglyceride levels (Figure 8). If the serum triglycerides are very high (greater than 500- 1000mg/dl), where there is an increased risk for pancreatitis and hyperviscosity syndromes, initial pharmacological therapy is directed at the elevated triglycerides and the initial drug choice is either a fibrate or high dose fish oil (3 grams EPA/DHA per day). After lowering triglyceride levels to < 500mg/dl statin therapy should be initiated if the LDL cholesterol and/or non-HDL cholesterol is not at goal. If the serum triglycerides are less than 500mg/dl, statin therapy to lower the LDL cholesterol levels to goal is the initial therapy (see Figure 8). Studies have clearly demonstrated that statins are effective drugs in lowering triglyceride levels in patients with elevated triglycerides. In patients with low triglyceride levels statins do not greatly affect serum triglyceride levels. If the non-HDL cholesterol levels remain above goal after one reaches the LDL cholesterol goal one should then consider combination therapy to lower triglyceride levels which will lower non-HDL cholesterol levels.

 

Figure 8. Combined Hyperlipidemia. Increased LDL and TG

 

Often monotherapy is not sufficient to completely normalize the lipid profile. For example, with statin therapy one may often lower the LDL cholesterol to goal but the non-HDL cholesterol and triglycerides remain in the abnormal range. Currently, there are no randomized controlled trials demonstrating that combination therapy with fibrates or niacin reduces cardiovascular disease to a greater extent than statin monotherapy. In fact three recent outcome studies adding either niacin or fenofibrate to statin therapy failed to demonstrate additional benefit (210-212). In contrast, adding EPA to statin therapy did reduce cardiovascular events (208). Despite the limited data based on randomized outcome trials many experts believe that further improvements in the lipid profile will be beneficial and that the studies completed so far should not be considered definitive as they had flaws such as not treating patients with the appropriate lipid profile.

 

When using combination therapy, one must be aware that the addition of either fibrates or niacin to statin therapy may increase the risk of myositis. The increased risk of myositis is greatest when gemfibrozil is used in combination with statins. Fenofibrate has a much more modest risk and the FDA approved the use of fenofibrate in combination with moderate doses of statins. Additionally, in the ACCORD LIPID trial the combination of simvastatin and fenofibrate was well tolerated (212). The increased risk with niacin appears to be modest. In the AIM-HIGH trial the risk of myositis was not increased in patients on the combination of Niaspan and statin, whereas in the HPS2-Thrive trial myopathy was increased in the group treated with the combination of niacin and statin (210,211). The absolute risks of combination therapy are relatively modest if patients are carefully selected; in many patients at high risk for cardiovascular disease combination therapy may be appropriate. Table 5 indicates the type of patient that one should consider the use of combination therapy. As with many decisions in medicine one needs to balance the benefits of therapy with the risks of therapy and determine for the individual patient the best approach. In deciding to use combination therapy a key focus is the non-HDL cholesterol level. When the LDL cholesterol is at goal but the non-HDL cholesterol is still markedly above goal it may be appropriate to resort to combination therapy in patients at high risk.

 

Table 5. When to Use Combination Therapy

·       Clinical Evidence of Arteriosclerosis

·       High Risk Patient

o   Hypertension

o   Family History of CAD

o   Cigarettes

o   Proteinuria

o   Microalbuminuria

o   Central Obesity

o   Inactivity

o   Elevated CRP

·       No Contraindications

o   Renal or Liver Disease

o   Non-compliant patient

o   Use of other drugs that effect statin metabolism

o   Other medical disorders that increase risk of toxicity

 

In summary, modern therapy of patients with inflammatory diseases demands that we aggressively treat lipids to reduce the high risk of cardiovascular disease in this susceptible population and in those with very high triglycerides to reduce the risk of pancreatitis. Furthermore, treatment with lipid lowering drugs in some instances may improve the underlying inflammatory disorder.

 

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Adrenal Androgens

ABSTRACT

 

Adrenal androgens (AA) are 19 carbon (C19) steroids that are secreted by the adrenal cortex through complicated biosynthetic pathways, which are regulated by complex mechanisms not completely understoodas of yet. Adrenal steroidogenesis differs between the fetal and adult adrenal not only in regard tothe site of production, but also in their significance for the human organism. The production of the AA is coordinated bya large number of adrenal and non-adrenal regulators. These steroids exerta number of effects in normal physiology and their excess may cause a number of different kinds of disorders.

 

INTRODUCTION

 

Adrenal androgens (AAs), normally secreted by the fetal adrenal zone and the zona reticularis of the adrenal cortex, are steroid hormones with weak androgenic activity. They includedehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), androstenedione (A4), androstenediol (Α5) and 11β-hydroxyandrostenedione (11βOHA4) (1). DHEA and DHEAS are secreted in greater quantities than the other adrenal androgens. Although these steroids have little androgenic activity, theyprovide a pool of circulating precursors for peripheral conversion to more potent androgens (e.g. testosterone, T) and estrogens, (e.g. estradiol) (2-6). The production of T by the adrenal glands is minimal (7). Although adrenal androgens do not appear to play a major role in the fully androgenized adult man, they seem to play a role in the adult woman and in both sexes before puberty.  Girls, women, and prepubertal boys may be negatively affected by AA hypersecretion in contrast to adult men. This chapter reviews AA biosynthesis, regulation, physiology and biological action. New data suggest that the principal androgen made by the human adrenal is 11-ketotestosterone (11-KT), a rarely studied steroid.

 

ADRENAL GLAND ANATOMY

 

Fetal Adrenal Gland(Figure 1)

Figure 1: Ontogenesis of steroidogenic enzymes in the human fetal adrenal gland. This schematic representation is divided into portions showing the fetal adrenal gland (right) at the first, second and third trimesters of pregnancy, and the adult adrenal gland (left). During the first trimester, the fetal gland is composed of a definitive zone (DZ, light grey) and a fetal zone (FZ, darker grey). Fetal zone (FZ) - expressing the P450C17 cytochrome, is responsible for massive secretion of DHEA and DHEA/S, used by the placenta as estrogen precursors. Second trimester - chromaffin cells (CC, darkest grey) originating from the neural crests migrate through the fetal cortex to progressively colonize the center of the gland to form the future medulla (Med). Third trimester - the newly constituted transitional zone (TZ, medium grey) acquires the enzyme 3ß-HSD while the expression of P450C17 remains, thus allowing the production of fetal cortisol. Near birth, cells of the definitive zone which express only 3ß-HSD, acquire the P450aldo and begin to secrete mineralocorticoids such as aldosterone. Neonatal - the fetal adrenal regresses strongly (mainly due to the regression of the fetal zone) and recovers progressively during the first years of extra-uterine life. Adult - adult adrenal gland is composed of the zona glomerulosa (ZGlo, light grey), zona fasciculata (ZFasc, medium grey) and zona reticularis (ZRet, darker grey) responsible for the production of mineralocorticoids (aldosterone), glucocorticoids (cortisol) and androgens (DHEA-DHEA/S), respectively. P450scc - cytochrome P450 side chain cleavage; Pregn. – pregnenolone; P450C17 - cytochrome P450 17a-hydroxylase, 17-20 lyase; 17OHP5 - 17-hydroxy-pregnenolone; DHEA/S - dehydroepiandrosterone-sulfate; S-Tfase - DHEA sulfotransferase; 3ß-HSD - 3ß-hydroxysteroid dehydrogenase; Prog. – progesterone; 17OHP4 - 17-hydroxyprogesterone; P450C21 - cytochrome P450 21-hydroxylase; P450C11 - cytochrome P450 11ß-hydroxylase; P450aldo - cytochrome P450 aldosterone synthase.

Fetal adrenal cortex arises from mesodermal cells migrating from the celomic epithelium very early in the embryonic period. Thus, adrenocortical tissue can be found in the ovaries, spermatic cord and testes. By the second month of gestation, the developing human fetal adrenal acquires two rudimentary, but distinct, zones: the inner fetal zonewhich consists of large eosinophilic cells, and the outer definitive zone, which is comprised of small, densely packed basophilic cells (8-9). At about the ninth week of gestation, the developing human fetal adrenal is completely encapsulated. Ultrastructural studies also have revealed a third zone between the inner fetal zone and the definitive zone, the transitional zone(10). Cells in this zone show intermediate characteristics (11) and they demonstrate the capacity to synthesize cortisol, being histologically similar to cells of the zona fasciculataof the adult adrenal cortex. By the 30th week of gestation, the human fetal adrenal cortex manifests a rudimentary form of the adult adrenal cortex; the definitive zoneand the transitional zonebegin to resemble the zona glomerulosaand the zona fasciculata, respectively (12). Although the fetal zone is functionally similar to the adult zona reticularis(where DHEA-S is produced), it produces, unlike the adult zona reticularis, largeamounts of other sulfated D5 steroids, including pregnenolone sulfate and 17a-hydroxypregnenolone sulfate.

 

Soon after birth, human fetal adrenal undergoes rapid involution due to the rapid regression of the inner fetal zone followed by a decrease in androgen secretion (12-17). Thus, the total weight of the glands decreases by approximately 50%. (11,18). Dramatic remodeling of the postnatal adrenal gland involves a complex combination of inner fetal zone regression and development of the zone glomerulosaand fasciculate(12,19). Because morphological studies have identified rudimentary zone glomerulosaand fasciculataduring late gestation, the development of these zones may occur from their primordial structures, although there has been a general belief that the adult cortical zones develop from the persistent definitive zone (13).

 

Various genetic disorders of steroidogenesis, which constitute human “gene knockout experiments of nature”, indicate that fetal adrenal steroidogenesis, and the fetal adrenal zone itself, are not essential for fetal development, survival, or parturition (20). Aging results in tissue-rearrangements within the adrenal cortex while there is a relative increase of the outer cortical zones (21). As far it regards to the zona reticularis, after a continuous growth until young adulthood (20 to 25 years), it remains at a plateau for 5 to 10 years, and it regresses gradually after the reproductive period of life (22-23).

 

Adult Adrenal Gland

 

The adult adrenal glands, consisting of cortex and medulla, have a roughly pyramidal shape, lie above the upper poles of the kidneys in the retroperitoneum and weigh approximately 4g each. They are well supplied with arterial blood from branches of the phrenic arteries, the aorta, and the renal arteries, which give rise to the superior, middle, and inferior adrenal arteries, respectively. Arterial blood enters from the outer cortex, flows through fenestrated capillaries between the cords of cells, and drains into venules in the medulla. On the right side, the adrenal vein directly enters the inferior vena cava; on the left side, it usually drains into the left renal vein. The adrenal cortex is divided into three histologic and functional zones: the outer, aldosterone-secreting, called zona glomerulosa; the intermediate, predominantly cortisol and corticosterone secreting, called zona fasciculata; and the inner, predominantly androgens secreting called zona reticularis (Figure 2). Each one is characterized by the expression of specific steroidogenic enzymes, which result in the production of different steroid hormones. Zona glomerulosa constitutes about 15% of cortical volume. Zona fasciculata is the thickest part of the adrenal cortex, constructing about 75% of the cortex, produces cortisol as well as small amounts of androgens and estrogens. Zona reticularis surrounds the medulla and produces the adrenal androgens and small amounts of cortisol and estrogens (24). Zona glomerulosa is deficient in 17a – hydroxylase activity and thus cannot produce cortisol and androgens. Whereas zona glomerulosais primarily regulated by angiotensin II and corticotropin (ACTH), both zona fasciculataand zona reticularisare regulated by ACTH (25).Both of these zones become hypofunctional and atrophic when ACTH is deficient while they become hypertrophic and hyperplastic when ACTH is secreted in excess.

Figure 2: Schematic presentation of the adrenal zones and the main products of each zone. Downloaded from: georgiahealth.edu

The anatomical alterations of the adrenal cortex that occur during lifespan are followed by a marked decline in circulating adrenal C19 steroids and their resulting androgen metabolites. This decline takes place mainly between the age groups of 20-30 and 50-60 yr, with smaller changes observed after the age of 60 yr (26).

 

ADRENAL STEROIDS AND BIOSYNTHESIS OF ADRENAL ANDROGENS

 

Main Biosynthetic Pathway of Adrenal Steroids

 

All human steroid hormones derive from cholesterol. Plasma lipoproteins are the major source of adrenal cholesterol. Low–density lipoprotein (LDL) accounts for about 80% of cholesterol delivered to the adrenal gland. There are specific cell surface LDL receptors on the adrenal tissue. Synthesis within the gland from acetyl-coenzyme A also occurs. A small pool of free cholesterol within the adrenal is available for acute response when stimulation occurs. Acute stimulation leads to hydrolysis of stored cholesteryl esters to free cholesterol, increased uptake from plasma lipoproteins and increased cholesterol synthesis within the gland (27). In addition, there is evidence that the adrenal can utilize high density lipoprotein HDL cholesterol through HDL receptor, SR-B1 (28).

 

Cholesterol enters the steroidogenic pathwayby the action of the enzyme cholesterol esterase and transferred from the outer mitochondrial membrane of steroidogenic cells to the inner mitochondrial membrane by the steroid acute regulatory (STAR) protein. This transport is followed by the conversion of cholesterol to pregnenolone which is the first step of steroid synthesis and the major action of ACTH on adrenals (Figure 3). The conversion of cholesterol to pregnenolone requires the action of the cholesterol side-chain cleavage enzyme, commonly referred to as P450scc, encoded by the CYP11A gene located on chromosome 15 in mitochondria. This cleavage gives birth to 21-carbon (C21) molecules resulting from the C27 cholesterol molecule. These reactions require molecular oxygen and a pair of electrons. The electrons are donated by nicotinamide adenine dinucleotide phosphate (NADPH) to adrenodoxin reductase (flavoprotein) and then to adrenodoxin (an iron-sulfur protein) and finally to P450scc. Electron transport to microsomal cytochrome P450 involves the enzyme P450 reductase (Figure 4).The steroid hormones produced by the adrenal cortex are members of a large family of compounds derived from the cyclopentanoperhydrophenanthrene ring structure that comprises three cyclohexane rings and one cyclopentane ring. P450scc is needed for the production of all steroids in the human body, including those produced by the adrenal. It is expressed in all adrenal cortex zones, and although its expression is obligatory for the synthesis of C19 steroids (DHEA, DHEA-S, and A4) it is the presence of downstream enzymes that determines whether these cells produce C21 corticosteroids or C19 steroids (Figure 5).

Figure 3: The role of the mitochondrion in the adrenal steroidogenesis.

Figure 4: Reaction mechanism of hydroxylations catalyzed by cytochrome P-450s of adrenal cortex mitochondria. Abbreviations: XH = substrate; XOH = product; Fp = flavoprotein; ISp = iron-sulfur protein.

Figure 5: Adrenal androgen biosynthetic pathway.

Biosynthesis of Adrenal Androgens.

 

The second step of adrenal steroidogenesis is mediated by cytochrome P450 17A1 enzyme which acts both as a hydroxylase hydroxylating pregnenolone, and as a lyase splitting the C17–C20 bond of 17-hydroxypregnenolone (17OHP5),resulting in the production of DHEA (29-31). Specifically, CYP17A1 gene encodes a protein that catalyzes two metabolic pathways, the 17a-hydroxylation (principal for the androgen and glucocorticoid pathway) and the 17,20 lyase reaction (specific for androgen pathway). Even though the affinity of the human Cytochrome P450 17A1 is similar either forΔ5 steroid substrate (pregnenolone) or Δ4 steroid substrate (progesterone), the predominant pathway for the 17,20 lyase reaction is viathe 17 OH pregnenolone (Δ5 substrate) (32). This 17,20-lyase activity is predominant in zona reticularis. There, the presence of cytochrome b5, form Aenzyme (encoded by the CYB5A gene), a cofactor/regulator of cytochrome P450 17A1 function, promotes 17,20-lyase activity (33).

 

DHEA is then converted to DHEAS sulfate by an adrenal sulfokinase (encoded by the SULT2A1 gene). This enzyme, present mostly in the cytoplasm of adrenocortical cells in zona reticularis, mediates the sulfo conjunctionof the Δ5 steroids (pregnenolone, 17α-hydroxypregnenolone, DHEA, and A5. Although all of these adrenal steroids, in fetal life, act as substrates for the corresponding sulfated products; in adult life the main substrate for the production of DHEAS is DHEA (34). During embryonic development DHEAS is supplied in maternal circulation from the fetal adrenals and acts as a substrate for estrogen synthesis from the placenta in such a way that the concentration of maternal estriol (produced in the placenta) reflects fetoplacental steroidogenesis (5). After birth however the sulfation of DHEA to DHEAS has a preventive role for androgen production by preventing excessive amounts of DHEA, a substrate for HSD3B2, to produce increased A4 and finally T (35). Of note, the expression of SULT2A1 in zona reticularisincreases during adrenarche (36).

 

DHEA is also converted to A4 by the enzyme 3-β-hydroxysteroid dehydrogenase (3βHSD) encoded by the HSD3B2 gene. This pathway represents the predominant pathway for the production of DHEA in humans.3-β-Hydroxysteroid dehydrogenasehas a major role in the synthesis of androgens but also of mineralocorticoids and glucocorticoids as it catalyzes the conversion of Δ5 (pregnenolone, 17α-hydroxypregnenolone, DHEA and A5) to Δ4 steroids (progesterone, 17α-hydroxyprogesterone, Α4 and T). In fetal adrenal the expression of 3βHSDpeaks at the 8thto 9thgestational week, resulting in the production of cortisol at 8thto 10thgestational week, decreasing thereafter and being undetectable at 14thgestational week.The decrease of HSD3B2 expression is followed by a decrease of cortisol synthesis. The transient cortisol synthesis by the 10thgestational week may exert a negative feedback on ACTH secretion suppressing adrenal synthesis of androgenic C19 steroids during the time of genital differentiation. Thus, in humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development (9,37). Given that fetal adrenal cell has a low expression of HSD3B2 gene until the end of 2ndtrimester (38-39) it becomes evident that the fetal adrenal gland is more likely to produce Δ5 steroids, especially DHEA, than Δ4 steroids with mineralocorticoid and glucocorticoid activities. This low HSD3B2 expression is apparent also in adrenarche where the characteristic expansion of zona reticularis, demonstrating lower concentration of HSD3B2 compared to the adjacent zona fasciculata, facilitates the increased amount of DHEAS which marks the prepubertal to adult life transition (40-42,36).

 

Finally, Δ4 can be converted to T, although adrenal secretion of this hormone is minimal (Figure 5) (43-45). Human type 5 17β-hydroxysteroid dehydrogenase (encoded by the 17β-HSD) catalyzes the conversion of A4 to T (46-47). The fetal as well as the postnatal adrenal also expresses AKR1C3 gene in zona reticularis, which appears to be responsible for the small amount of T produced directly by the adrenal glands (48) and is likely responsible for the larger amounts of androgens produced in congenital adrenal hyperplasia.

 

 CIRCULATIONAND METABOLISM

 

Adrenal androgens are secreted from the adrenal cortex in an unbound state. Bound steroids are biologically inactive. Androstenedione, DHEA and DHEAS bind mainly to albumin. About 90% of adrenal androgens are bound to albumin and 3% approximately are bound to sex hormone-binding globulin (SHBG). The binding globulins have high affinity and low capacity, whereas, albumin has low affinity and high capacity for steroids. Adrenal androgens can follow two different pathways after entering the circulation. Their metabolism turns either towards degradation and inactivation or towards peripheral conversion to their more potent derivatives T and dihydrotestosterone (DHT).Adrenal androgens and their metabolites are inactivated or degradedin various tissues, including liver and kidney (49). Major biochemical routes for inactivation and excretion are conjugation of androgens to glucuronate or sulfate residues to produce hydrophilic glucuronides or sulfates, respectively, excreted in the urine (Figure 6A).

Figure 6: Metabolism of adrenal androgens in the pilosebaceous unit (A) and adipocyte tissue (B). Abbreviations: A, Δ4-androstenedione; T, testosterone; DHT, dihydrotestosterone; R, receptor; 3β-HSD, 3β-hydroxysteroid dehydrogenase; 17β-HSD, 17β-hydroxysteroid dehydrogenase; 5α-r, 5α-reductase; 3α-, 3β -diol, 3α-androstenediol, 3β-androstenediol; 3α-, 3β- diol-G, 3α-diol-glucuronide, 3β-diol-glucuronide; androsterone-G, androsterone glucuronide; ar, aromatase; E1, estrone; E2, 17β-estradiol

DHEA, DHEAS, and A4 are converted to the potent androgens T and DHT in peripheral tissues. Major conversions are those of A4 to T and T to DHT by the enzymes 17β-hydroxysteroid dehydrogenase (17β-HSD) and 5α-reductase, respectively. Major peripheral sites of androgen conversion are the hair follicles, the sebaceous glands (Figure 6A), the prostate, the external genitalia and the adipose tissue (50-51). DHEASis the sulfated version of DHEA. This conversion is catalyzed by sulfotransferase (SULT2A1) primarily in the adrenals, the liver, the kidney and small intestine. The concentrations of DHEAS in the circulation are about 300 times greater than those of free DHEA. The former show no diurnal variation, whereas the latter reach their peak in the early morning hours. DHEA secreted by the adrenal gland can be also converted to A4. Both DHEA and DHEAS are also metabolized to 7αand 16α– hydroxylated derivatives and by 17βreduction to Α5 and its sulfate. Androstenedione is converted either to T or by reduction of its 4,5-double bond to etiocholanolone or androsterone. Testosterone is converted to DHT in androgen-sensitive tissues by 5βreduction. The product is mainly metabolized by 3αreduction to 3α androstanediol. The metabolites of these androgens are conjugated either as glucuronides or sulfates and excreted in the urine.Active uptake of androgens and in situestrogen synthesis occur in peripheral adipose tissue (Figure 6B) through the enzymes 17β-HSD and aromatase, respectively (52-55). Peripheral conversion contributes significantly to circulating T concentration in women, but not in men, in whom T is largely produced by the testis. Three main enzyme complexes are involved in the synthesis of estrogens in peripheral tissues (56-58):

  • Aromatase for the aromatization of androstenedione to estrone.
  • Estrone sulfatase (E1-STS), which catalyses the formation of estrone from estrone sulfate.
  • Estradiol-17-β-hydroxysteroid dexydrogenase (17β-HSD) Type 1 which is responsible for the reduction of estrone to the biologically active estrogen, estradiol.

 

Finally, according to recent studies, 11-KT has been found to be the principal androgen made by the human adrenal. Both A4 and T may undergo 11-hydroxylation catalyzed by P450c11b (CYP11B1 gene) to yield 11OHA4 and 11OH-testosterone (11OH-T), respectively. These 11-hydroxysteroids may be oxidized by 11β-hydroxysteroid dehydrogenase type 2 (HSD-11B2), which is more known for its role in the oxidation of cortisol to cortisone, to 11-ketoandrostenedione (11-KA4) and 11-KT, respectively (Figure 7). These 11-keto steroids may then be 5α-reduced by 5α-reductase type 2 (SRD5A2 gene) in peripheral tissues, and possibly also by 5α-reductase type 1 (SRD5A1 gene) in the adrenal itself, to 5α-androstanedione and 5α-dihydrotestosterone (5αDHT), respectively. Both 11-KT and 11-ketodihydrotestosterone (11-KDHT) are bona fideandrogens that bind to and transactivate the androgen receptor. Whereas most studies have addressed the synthesis of these steroids in castration resistant prostate cancer, other studies showed that they may have an important role also in other disease states (e.g. congenital adrenal hyperplasia).

Figure 7: Novel Adrenal Androgens. 3bHSD2, 3b-hydroxysteroid dehydrogenase type 2; 11KA4, 11-ketoandrostenedione; 11KT, 11-ketotestosterone; 11OHA4, 11b-hydroxyandrostenedione; 11OHT, 11b-hydroxytestosterone; 17OH-PREG, 17a-hydroxypregnenolone; 17OH-PROG, 17a-hydroxyprogesterone; A4, androstenedione; AKR1C3, aldo-keto reductase 1C3; CYB5A, cytochrome b5; CYP11A1, cytochrome P450 cholesterol side-chain cleavage; CYP11B1, cytochrome P450 11b-hydroxylase; CYP17A1, cytochrome P450 17a-hydroxylase/17,-20-lyase; DHEA, dehydroepiandrosterone; DHEAS, DHEA sulfate; PREG, pregnenolone; StAR, steroidogenic acute regulatory protein; T, testosterone, SULT2A1, Sulfotransferase Family 2A Member 1; PROG, progesterone; CYP17A2 cytochrome P450 family 17 polypeptide 2; 11βHSD2, 11-β-hydroxysteroid dehydrogenase type 2.

ANDROGEN RECEPTOR

 

The inactive androgen precursors secreted by the adrenal glands are converted to T and DHT and exert their effects in most peripheral tissues by interacting with high-affinity receptor proteins. The androgen receptor (AR), member of the steroid receptor superfamily, also known as NR3C4 (nuclear receptor subfamily 3, group C, member 4) is a type of nuclear receptor that is activated by binding to T or DHT in the cytoplasm and then trans-locates into the nucleus. The AR is most closely related to the progesterone receptor, while progestins in higher dosages can block AR. Androgen receptors are encoded by the AR gene located on the X-chromosome at Xq11-12 (59). This gene contains a polymorphic CAG microsatellite repeat within exon 1, encoding for a variable length of polyglutamine chain at the amino terminal, the transactivation domain of the AR protein. Triplet-repeat DNA sequences can be sites of genetic instability, and their expansion in a variety of genes has been associated with human genetic diseases, such as fragile X-syndrome (60-61) and myotonic dystrophy (62). In the case of the AR gene, an inverse correlation of the number of CAG repeats with the risk for prostate cancer was described (63-66) and its expansion was documented in Kennedy's disease (spinal and bulbar muscular atrophy), a disorder associated with primary hypogonadism due to androgen insensitivity(67). In vitrostudies showed that progressive expansion of the repeat length in the AR was associated with a linear decrease in its transactivation function (68). These observations support the idea that there is an optimal number of repeats, which varies in the population from 11 to 31 (average size: 21±2) (63). Methylation of deoxycytosine residues is another process involved in the modulation of gene expression. Belmont et al. (69) showed that the methylation of HpaII and HhaI sites near the polymorphic CAG repeats in the first exon of the human AR (HUMARA) locus correlated with X-inactivation.

 

Patients with idiopathic hirsutism were shown to have a normal number of CAG repeats but with a preponderance of the shortest and most active alleles (70). These patients had also a preferential methylation of the longer AR allele compared to normal subjects, leading to inactivation of the functionally weaker gene. This skewing could allow the shorter, more active AR allele (64,68) to be preferentially expressed explaining the peripheral hypersensitivity to androgens in hirsute patients.

 

Multiple "coactivators" were identified enhancing transcription of the AR gene (71) including AP-1 (72), Smad3 (73-74), nuclear factor kB (NF-kB) (75-76) sex-determining region Y (SRY) (77) and the Ets family of transcription factors (78). The relative importance of these molecules for any particular cell type remains unclear, since the ability of a putative coregulator to alter the transcriptional activity is typically examined in transient transfection experiments. Although AR is normally thought to function as a homodimer, it was also shown to heterodimerize with other nuclear receptors including the estrogen receptor (ER) (79) glucocorticoid receptor (GR) (80) and testicular orphan receptor 4 (TR4) (81). One of the major mechanisms through which coregulators might function is by forming a bridge between the DNA-bound nuclear receptor and the basal transcriptional machinery (type I regulators) (82). Coactivators may also facilitate ligand binding, promote receptor nuclear translocation, or mediate signal transduction (type II coregulators). The role of "corepressors" in AR function is poorly defined. Three corepressors of androgen-bound AR have been identified to date, cyclin D1, calreticulin, and HBO1. However, relatively little is known about the mechanism of their repressive effect.

 

ADRENAL ANDROGEN PHYSIOLOGY AND REGULATION

 

Regulation

 

Adrenal androgens are secreted by the adrenal glands in response to ACTH, a 39-amino acid peptide synthesized and secreted by the anterior pituitary (Figure 8).It is derived from proopiomelanocortin (POMC), a large precursor molecule from which β-lipotropin hormone and β-endorphin are also derived (83-84). ACTH is the predominant form of corticotropin in plasma and has a half-life of approximately 10 minutes (85). Its synthesis and secretion are primarily regulated by corticotropin-releasing hormone (CRH) and arginine-vasopressin (AVP), both of which are produced by parvocellular neurons of the paraventricular nucleus of the hypothalamus and act in synergy with each other (86-87). Under ACTH regulation, adrenal androgens are secreted synchronously with cortisol. There are three mechanisms of neuroendocrine control: [1] episodic secretion and the circadian rhythm of ACTH, [2] stress responsiveness of the hypothalamic-pituitary-adrenal axis (HPA), [3] feedback inhibition of ACTH secretion by cortisol. [1]

Figure 8: Schematic presentation of the adrenal androgen regulation. Downloaded from: wikis.lib.ncsu.edu

The circadian rhythm is the result of the central nervous system regulation of CRH and ACTH nyctohemeral secretory episodes. The major secretory episodes begin in the sixth to eighth hour of sleep and then begin to decline as wakefulness occurs. Cortisol secretion then gradually declines during the day with fewer secretory episodes (88). The circadian rhythm of adrenal androgens is typical in different physiologic and pathologic conditions. Patients with nonclassical 21-hydroxylase deficiency, for example, have a distinct pattern of adrenal steroid secretion characterized by a high-frequency 17-hydroxyprogesterone release accompanied by a relative nocturnal cortisol deficiency (89-90). [2] Plasma ACTH and cortisol secretion are secreted within minutes following the onset of physical stress. This response abolishes circadian periodicity if the stress is prolonged. Stress responses originate in the CNS and result to CRH and ACTH secretion. [3] Corticotropin-stimulated cortisol exerts major feedback inhibitory influences at the concentration of both the hypothalamus and the anterior pituitary by suppressing CRH and ACTH synthesis and secretion.

 

Plasma DHEA, A4, and T concentrations parallel closely the circadian rhythm of plasma cortisol. Plasma DHEA-S concentrations do not exhibit a circadian rhythm because of the much longer circulating half-life of this sulfated steroid (91-92). Numerous other endocrine signals (93) were proposed as coregulators of adrenal androgen secretion. Among these are prolactin (PRL) (94), estrogen (95-99), epidermal growth factor (100), prostaglandins (101), angiotensin (102), GH (103), gonadotropins (104-105), β-lipotropin, and β-endorphin.  Glasow et al. reported the presence of PRL receptors in the human adrenal gland and suggested a direct effect of PRL on adrenal steroidogenesis that may be of particular relevance in clinical disorders characterized by hyperprolactinemia (106). Interestingly, adults with hyperprolactinemia have increased secretion of AAs by the zona reticularis, which is corrected by reduction of PRL secretion with bromocriptine (107). In women with PRL-secreting tumors there is a correlation between PRL concentration and DHEA-S (108). Pabon et al. (109) have detected the presence of LH- HCG receptors in zona reticularis and fasciculata. The receptor bearing cells were positive for steroidogenic enzymes, indicating that the receptors could be coupled to DHEAS secretion (110-112).

 

Cytokines interfere with steroidogenesis at the level of the adrenals, testes and ovaries. Within the adrenal adrenocortical and chromaffin cells cytokines such as interleukin (IL) 1, IL-6, tumor necrosis factor (TNF), leukemia inhibitory factor (LIF) and IL-18 are produced. They have a key role in the immune-adreno-cortical communication. Thus, in autoimmune and inflammatory diseases an adequate adrenal stress response is observed. In addition, cytokines such as IL-8 and monocyte chemotactic protein-1 (MCP-1) are involved in steroidogenesis (113). ΙL-6 also is known to activate the HPA axis by stimulating both the CRH and the AVP -secreting neurons of the paraventricular nucleus of the hypothalamus, and their terminals at the median eminence, the corticotrophs of the anterior pituitary, and the cortisol-secreting adrenal cells in rats. In the latter it acts through specific receptors expressed mainly in the zona fasciculata and reticularis, but also with lower density in the zona glomerulosa (114-115). The ability of IL-6 to stimulate glucocorticoids, mineralocorticoids, and androgens suggests that this cytokine might have a role in coordinating the response of all adrenocortical zones. Its secretion is regulated by different substances, such as CRH, ACTH, angiotensin II, or immune products such as IL-1 α/βindicating that IL-6 may play a major role in the interaction of the adrenal function with the immune/inflammatory reaction (116).

 

Interleukin 1 and TNF regulate the activity of HPA axis at several levels. Studies investigated their action on adrenal steroidogenesis and indicated that IL-1αand IL-1βincrease cortisol, A4, DHEA, DHEAS production and the accumulation of mRNAs for STAR, 17α-hydroxylase/17,20-lyase (CYP17A1) and HSD3B2 in these cells. TNF induced cortisol production (117).

 

Both ACTH and PRL stimulate AAs secretion by the fetal adrenal zone. In addition, placental CRH appears to play a major role in sustaining this zone and stimulating androgen secretion together with corticotropin and/or PRL (118).

 

Physiology

 

Adrenal androgens are secreted in small amounts during infancy and early childhood. DHEAS is maintained at minimum concentrations for 5 years in both male and females, after which a gradual increase is observed (115). Their secretion gradually increases with age, paralleling the growth of zona fasciculata and zona reticularis.Disturbances in both enzymatic activity in zona fasciculata and zona reticularis and its regulators (ACTH or peptides of hypothalamic – pituitary origin, such as PRL) may result in syndromes of hirsutism and virilization in females. Adrenal cortex normally secretes androgens in increasing amounts beginning at about 6-7 years of age in girls and 7-8 years of age in boys. This rise continues until late puberty. Adrenarche (secretion of adrenal androgens) occurs years before gonadarche (secretion of gonadal sex steroids). The appearance of pubic hair (pubarche) results from a rise in adrenal androgen concentration (adrenarche) (116-117). The mechanism(s) by which zona fasciculata and zona reticularis develop with age, as well as the regulation of adrenarche onset are not understood. The biochemical hallmark of adrenarche is accelerated DHEAS production from the adrenal gland. The axillary and pubic hair regions are the most sensitive androgen-dependent regions and they represent the clinical manifestation of adrenarche. Children with premature pubarche demonstrate hormonal responses to CRH stimulation test similar in magnitude to those of prepubertal children of comparable age, ruling out a prominent role of CRH in premature pubarche (118). Gell et al. suggested that as children mature, a decrease of HSD3B2 activity in the adrenal zona reticularis occurs, leading to an increased production of DHEA and DHEA-S, as seen during adrenarche, by shifting pregnenolone through the 17α-hydroxylase/17, 20 lyase pathway (Figure 5) (39).

 

Activation of the type 1 insulin-like growth factor (IGF1) receptor was shown to enhance steroidogenic responsiveness of the fetal zone cells to ACTH by modulating the ACTH signal transduction pathway at some point downstream from ACTH receptor binding (119). Also, locally produced IGF2 modulates fetal adrenocortical cells function by increasing responsiveness to ACTH viaactivation of the IGF1 receptor and increases the capacity of those cells for androgen synthesis by directly augmenting the expression of P450c17 (119). Thus, IGF2 may play a pivotal role in AA production, both physiologically in uteroand at adrenarche, as well as in conditions of hyperandrogenemia (119). All together, these data indicate that the IGF system is important in the regulation of the differential function of adult human adrenocortical cells (120). The rise in plasma concentrations of the AAs at adrenarche occurs in the presence of constant cortisol concentrations, suggesting that factors other than corticotropin are involved. The influences of sex and age are minor in the modulation of adrenal steroidogenesis supporting the conceptthat extra-adrenal factors prevail in the differential modulation of AAs and cortisol (121). These may include POMC-derived or other still uncovered peptides. An increased serine phosphorylation of human P450c17 might have a role in the development of both the excessive adrenarche and hyperandrogenism of patients with the polycystic ovary syndrome (PCOS) resulting in a substantial increase in 17-20-lyase activity (122-124) (Figure 5). P450c17 is the key enzyme that regulates androgen synthesis. (125). It is the only enzyme known to be able to convert C21-precursors to the androgen pro-hormones, the 17-ketosteroids. It is a single enzyme with two activities, 17 a-hydroxylase and 17,20-lyase (Figure 5) and serine phosphorylation appears to modulate the activity of P450c17. In particular, it promotes the 17,20-lyase activity, and at the same time it inhibits the activity of the insulin receptor (123-124,126-128). It was postulated that a single abnormal serine kinase might hyperphosphorylate both P450c17 and the insulin receptor, accounting for the hyperandrogenism and hyperinsulinism responsible for both premature pubarche and PCOSlater in life(129). In vitrostudies, however, failed to find evidence for increased autophosphorylation of the insulin receptor-βsubunit and P450c17 in PCOS (130). The reason for this might be related to the many different factors needed for P450c17 optimal activity and not normally expressed in the cell line used for that study (48).

 

BIOLOGIC EFFECTS

 

In adult men, the conversion of adrenal A4 to T accounts for less than 5% of the production rate of the latter, making its participation in the physiologic androgenization of the male negligible. Excessive AA secretion appears to have no major clinical consequences in the adult man, although this may be debated. Adrenal androgens hypersecretion in prepubertal boys, on the other hand has clearly been associated with isosexual precocious puberty.

 

In adult women, adrenal A4 and A4 generated from peripheral conversion of DHEA contribute substantially to total androgen production and effects. In the follicular phase of the menstrual cycle, adrenal precursors account for two thirds of Tproduction and half ofDHTproduction. At midcycle, the ovarian contribution increases, and the adrenal precursors account for 40% of T production. In women, increased AA production may be manifested as cystic acne, hirsutism, male type baldness, menstrual irregularities, oligoovulation or anovulation, infertility, and/or frank virilization. Excessive adrenal androgen secretion in prepubertal or pubertal girls can cause heterosexual precocious puberty.

 

Abnormalities in the timing and intensity of adrenarche are associated with PCOS, congenital adrenal hyperplasia (CAH) and insulin resistance conditions. Recently, we have shown that in postmenopausal PCOS women, androgen concentration at baseline are greater in PCOS than control women and remain increased after ACTH stimulation, while the results of the dexamethasone suppression test in postmenopausal PCOS women suggest that DHEAS and total T are partially of adrenal origin (131). Although the ovarian contribution was not fully assessed, increased A4 production suggests that the ovary also contributes to hyperandrogenism in postmenopausal PCOS women. In conclusion, this study indicates that postmenopausal PCOS women are exposed to higher adrenal and ovarian androgen concentrations than non-PCOS women (131).

 

Studies conducted over the past few years have investigated the use of DHEA to treat female infertility (132-133). Women with poor ovarian reserve, after DHEA supplementation 4 to 12 weeks prior an in vitrofertilization (IVF) cycle, had a 50-80% reduction in miscarriages (134). However, its efficacy in treating infertility remains controversial (135-137).

 

Reports demonstrate DHEA as a replacement therapy in the elderly (138-139). At 70-80 years of age, peak DHEA concentrations are about 10-20% of those in young adults. These reports suggest DHEA as replacement treatment in menopausal women; it has been reported to restore both the androgenic and estrogenic environment and reduce most of the symptoms of menopause (140-142). Other reports have suggested that oral DHEA in doses of 25-50 mg/d may restore plasma T concentrations to normal in some women with hypopituitarism who have diminished libido despite adequate estrogen therapy (143-145). In addition, DHEA replacement therapy has been investigated for the conditions of adrenopause and adrenal insufficiency (146-149). In spite of these few reports so far DHEA does not appear to be effective for perimenopausal symptoms (135) nor has it been shown to be effective as an “anti-aging” agent, as its effects in trials on cognitive function, body composition, insulin resistance, and well-being have been inconsistent (150-157,146). Based upon available data, the Endocrine Society guidelines, suggested against the routine use of DHEA for sexual function (or other indications) in postmenopausal women because of its limited efficacy and lack of long-term safety data (158). Clinical trial data on the efficacy of DHEA therapy in women with primary adrenal insufficiency are mixed.

 

In several studies of women with premature ovarian insufficiency (POI), serum ovarian androgen concentrations (A4and/or T) were lower than those of age-matched women without ovarian insufficiency, but similar to those seen in older postmenopausal women (159-161). In contrast, DHEAS concentrations were normal (although they would be expected to be low in those women with coexisting primary adrenal insufficiency).Potential side effects of androgen replacement include hirsutism and acne, and with oral preparations (e.g. DHEA), dyslipidemia. However, in women with autoimmune ovarian failure and coexisting adrenal insufficiency, adrenal androgen therapy with DHEA may be beneficial.

 

Other studies investigate the role of DHEA and DHEAS in the immune response and suggest that adrenal androgens have opposite biological effects to those of corticosteroids (162). DHEA is a cortisol antagonist (163). Research studies indicate DHEA supplementation has an anti-depressant effect (164-166). Exogenous DHEA has been proposed to have a number of potential benefits (on sexual function, depression, cognition, and inflammation), but available clinical trial data do not support these claims (115,167-168,137). It is widely available in some countries as a dietary supplement; however, quality control of these products has been shown to be quite poor (169,170).

 

Both gonadal and AAs contribute to the positive impact of androgenic steroids on bone cell metabolism in vitro(171). Interestingly, a study found that the potential anabolic effect of androgens on bone might not be mediated at the level of the mature osteoblast but at the level of fetal, less differentiated, osteoblastic cell lines (172).

 

Finally, although A4 seems to increase serum T and estrone concentrations when administered acutely to women, (173) the impact of regular use on sexual function or its potential androgenic side effects in women are unknown

 

CONCLUSIONS

 

The physiology of adrenal androgens follows the different periods of life starting from the fetal period. During this period, the secretion of these hormones from the fetal adrenal is important. It is not clarified as yet its role in the fetal development or survival, while it is of major importance for parturition. DHEA is the most prevalent steroid hormone in the body. After birth DHEA(S) concentrations fall rapidly with the involution of the fetal adrenal and rise slowly during childhood accelerating at adrenarche before the onset of puberty. The physiology of adrenarche is well described although its trigger has not been identified yet. DHEA concentrations drop dramatically with aging. There are pronounced differences in the average DHEA concentrations between men and women, with women on average having lower DHEA concentrations. The spectrum of women and men that would benefit from DHEA therapy is not clearly defined. Further studies are needed to investigate the side effects of the DHEA replacement therapy and to define the range of dosage that is more effective without complications. During menopause transition mean circulating DHEAS concentrations exhibit a positive inflection starting in the early perimenopause, continuing through the early post menopause and returning to early perimenopausal concentrations by late post menopause. This rise in mean DHEAS is accompanied by concomitant rises in T, DHEA, A4, and an equal rise in A5. Studies have shown that the mean A4 and T concentrations changed the least while mean DHEAS and A5 changed the most. The role of these changes in altering the estrogen/androgen balance in menopause is not known.

 

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  143. Kasperk CH, Wakley GK, Hierl T et al. Gonadal and adrenal androgens are potent regulators of human bone cell metabolism in vitro. J. Bone Miner’s. 1997;12:464-471
  144. Hofbauer LC, Hicok KC, Khosla S. Effects of gonadal and adrenal androgens in a novel androgen-responsive human osteoblastic cell line. J. Cell Biochem. 1998; 71:96-108.
  145. Leder BZ, Leblanc KM, Longcope C et al. Effects of oral androstenedione administration on serum testosterone and estradiol levels in postmenopausal women J Clin Endocrinol Metab. 2002;87(12):5449.

Inpatient Diabetes Management

CLINICAL RECOGNITION

Background

 

Appropriate inpatient glycemic management limits the risks of severe hypo- and hyperglycemia.  Preventing and treating hyperglycemia reduces infections and minimizes fluid and electrolyte abnormalities. Specific glucose goals remain fluid.  Hyperglycemia and hypoglycemia are associated with poor outcomes but the few prospective randomized studies have failed to demonstrate consistent improvements.  For example, intensive glycemic control in the ICU increased mortality in one large trial.  At this point, glucose goals should be thoughtful and tailored to the institution and its resources.  To successfully manage inpatient diabetes, institutional infrastructure must be in place with institution specific guidelines and protocols for which all nursing staff, pharmacy staff, physicians and others must be educated. The general guidelines below are appropriate at most institutions.

Physiologic Insulin Regimen

 

All patients have “basal, nutritional, and correctional” requirements which they must meet with endogenous or exogenous insulin.

  • Basal: insulin needed even when patient is not eating (to control gluconeogenesis). Use glargine (usually once daily in AM or at bedtime), NPH (at bedtime or AM and bedtime), detemir (once daily or q 12 hours), or a continuous insulin infusion.
  • Nutritional: insulin to cover carbohydrate intake from food, dextrose in IV fluid, tube feeds, TPN. Use rapid-acting insulin (aspart, lispro, or glulisine) or short-acting insulin (regular).
  • Correctional: insulin given to bring a high blood glucose level down to target range (with target usually below 150 mg/dL pre-meal, and below 200mg/dL at bedtime or 2am). Use rapid-acting insulin (aspart, lispro, or glulisine) or short-acting insulin (regular).

General Rules

 

  • a patient with type 1 DM will always need exogenous basal insulin, even if NPO. Failure to give such a patient insulin will lead to DKA.
  • Arbitrary sliding scale insulin should be avoided as it is not only ineffective but also potentially dangerous.
  • Ad hoc insulin orders should not be used. Comprehensive electronic medical record (EMR) order sets, or pre-printed order forms should only be used to order subcutaneous insulin and insulin infusions.  This standardization will decrease the risks of insulin dosing and administration errors.
  • Check blood glucose (BG) before meals and at bedtime. Check BG q 4 or q 6 hours in a patient who is NPO or is receiving continuous tube feeds or TPN.
  • Involve the diabetes educator or nurse specialist if available.
  • On admission, begin planning discharge, especially if the discharge plan will require new outpatient insulin use. Identify whether the patient will need a new glucose meter. Prescribe insulin, insulin pens with pen needles, syringes/needles, lancets, glucose strips, glucose tablets, and glucagon kit in the discharge prescription if needed.

Oral Hypoglycemic Agents

 

In general, oral diabetes medications and injectables other than insulin (e.g. GLP agonists) are inappropriate for initial management of the hyperglycemia patient.  Hospitalized patients often have the potential for renal impairment, tissue hypoxia, or need IV contrast, and these are all contraindications for using metformin.  Sulfonylureas should be held on admission because of current or potential NPO status resulting in a high risk of hypoglycemia.  As a patient’s status improves, however, it may be appropriate to restart oral medications.  DPP4 inhibitors may be useful for patients who have minimally elevated glucoses as there is a minimal risk for hypoglycemia.

 

Miscellaneous Guidelines

 

  • Nutritional coverage: Regular insulin is given 30 min before each meal.  Lispro, aspart, or glulisine are given with each meal or immediately after eating (can base on amount eaten).
  • Infection and glucocorticoids increase insulin needs; renal insufficiency decreases insulin needs.
  • Total daily dose of insulin needed: Type 1 patients require approximately 0.4 units/kg/day; type 2 patients vary in their insulin resistance and may require from 0.5 to 2 units/kg/day.

THERAPY

 

Insulin Regimens

 

The guidelines below assist with initial determination and subsequent adjustment of insulin doses. Insulin doses must be reevaluated on a daily basis and orders should be rewritten in order to achieve goals and to adapt to the patients’ changing clinical situation.

 

INSULIN REGIMEN FOR A PATIENT CONTROLLED WITH DIET AT HOME BUT NEEDING INSULIN IN HOSPITAL

 

Day 1:  Order a correctional sliding scale for before meals and bedtime (with lispro, aspart, glulisine or regular) based on BMI – see Table 1.

Day 2:  If BG pre-meals are >150 mg/dL, add nutritional insulin (with lispro, aspart, glulisine or regular) based on appetite).  Also, if AM fasting BG is >150 mg/dL, add bedtime basal insulin (with glargine, detemir, or NPH) dosed 0.1-0.2 unit/kg.

Day 3:  Adjust insulin doses based on BG pattern:  Increase or decrease basal insulin based on AM fasting BG, and adjust nutritional insulin based on pre-meal BG levels (see below for details).

 

Table 1: Correctional Insulin (lispro, aspart, glulisine or regular)

BG

(mg/dL)

Pre-meal:

Sensitive (BMI <25 or <50 units/d)

Pre-meal:

Average (BMI 25-30 or 50-90 units/d)

Pre-meal:

Resistant (BMI >30 or >90 units/d)

Bedtime

and 2 a.m.

131-150  0 units 1 unit 2 units 0 units
151-200 1 unit 2 units 3 units 0 units
201-250 2 units 4 units 6 units 1 unit
251-300 3 units 6 units 9 units 2 units
301-350 4 units 8 units 12 units 3 units
351-400 5 units 10 units 15 units 3 units
>400 6 units 12 units 18 units 3 units

 

INSULIN REGIMEN FOR A PATIENT ON ORAL AGENT(S) BUT REQUIRING INSULIN IN HOSPITAL BECAUSE OF HYPERGLYCEMIA OR CONTRAINDICATIONS TO THE ORAL AGENT(S)

 

Day 1:  Start nutritional insulin (lispro, aspart, glulisine or regular) based on appetite – generally about 0.1-0.2 units per kg, divided between the three meals for the day.  Also, order a correctional sliding scale (lispro, aspart, glulisine or regular) based on BMI – see Table 1.

Day 2:  If AM fasting BG is >150 mg/dL, add bedtime basal (glargine, detemir or NPH) dose of 0.1-0.2 units/kg.

Day 3:  Adjust insulin doses based on BG pattern:  Increase or decrease basal insulin based on AM fasting BG, and adjust nutritional insulin based on pre-meal BG levels (see below for details).

 

INSULIN REGIMEN FOR A PATIENT ON INSULIN AT HOME

 

  • If possible, consider home BG control, appetite, renal function, and risk for hypoglycemia.
  • All three components of insulin replacement must be addressed: basal, nutritional and correctional.
  • Basal requirements: Continue home regimen if patient has been well-controlled at home, but consider decreasing the total dose by 20-30% to reduce the risk of in-hospital hypoglycemia. Alternatively, start bedtime glargine, detemir or NPH at a dose of 0.2 units/kg
  • Nutritional requirements: Order nutritional insulin (lispro, aspart, glulisine or regular) based on appetite, or consider pre-meal dosing of 0.2 units/kg divided by 3 for the dose at each meal.
  • Correctional need: Order a correctional sliding scale based on total insulin dose or BMI – see Table 1.

 

INSULIN REGIMEN WHEN A PATIENT IS MADE NPO FOR A PROCEDURE

 

A patient will always require his or her basal insulin, even while NPO, and should not become hypoglycemic if that basal insulin is dosed appropriately.  For safety purposes, however:

  • The night before, give the usual dose of bedtime NPH, if applicable, or decrease the usual dose of bedtime glargine by 25%.
  • The morning of, if applicable, decrease the usual dose of morning NPH by 50%, or decrease the usual dose of morning glargine by 25%.
  • Do not give nutritional insulin (as patient is not eating), but continue the usual correctional insulin.
  • (an online resource to determine patient specific instructions when preparing for an NPO episode is at http://ucsf.logicnets.com)

 

INSULIN REGIMEN FOR AN ICU OR SURGICAL PATIENT WHO IS NPO

 

Consider insulin infusion therapy.

 

INSULIN REGIMEN FOR A PATIENT STARTING CONTNUOUS TUBE FEEDING

 

  • Consider insulin infusion therapy.
  • If moving from IV to SQ see below.
  • Basal need: The daily basal dose (glargine, detemir or total bid NPH dose) is the estimated total daily dose divided by 2.
  • Nutritional need: Divide the estimated total daily dose by 10 for the total nutritional (lispro, aspart, glulisine or regular) dose, to be given q 4 hours while tube feeding is active.
  • Correctional need: Order a correctional scale (lispro, aspart, glulisine or regular) based on total insulin dose or BMI (Table 1)
  • If not using IV insulin to start:
  • Estimate the tube feed formula’s 24-hour carbohydrate load.
  • Estimate the total daily dose (TDD) of insulin, starting with 1unit insulin for every 10 grams carbohydrate.

 

INSULIN REGIMEN FOR A PATIENT RECEIVING TPN

 

  • Standard TPN often contains 25% glucose, which, if 100 ml/hour, yields 25 g glucose/hour.
  • Basal and nutritional needs: Adding insulin to the TPN is safest, as the unexpected discontinuation of TPN will also mean the discontinuation of the insulin.  Start with 0.1 unit per gram glucose.  If patient previously needed high doses of basal insulin, divide that total daily dose by the number of TPN bottles to be administered daily, and add that to the prior calculation.
  • Correctional: Order a correctional sliding scale (lispro, aspart, glulisine or regular) based on BMI (Table 1).

 

INSULIN REGIMEN TO TRANSITION FROM AN INSULIN INFUSION TO SUBCUTANEOUS INSULIN

 

  • Calculate the patient’s total daily dose (TDD) of insulin, based on the most recent insulin infusion rate. For safety purposes, take 80% of that dose.
  • Basal need: Divide the 80% of the TDD by 2 and give half for the daily glargine, detemir or total NPH dose.
  • Nutritional need: If the patient is eating, divide the 80% of the TDD by 6 for the pre-meal lispro, aspart. Glulisine or regular dose.  If the patient is receiving tube feeds, divide the 80% of the TDD by 10 for the nutritional (lispro, aspart. Glulisine or regular) dose, to be given q 4 hours.  If the patient is not receiving nutrition, do not order nutritional insulin.
  • Correctional need: Order a q4h correctional scale (lispro, aspart, glulisine or regular) based on total insulin dose or BMI (Table 1).
  • Give the first basal insulin SQ injection 1-2 hours before the infusion is discontinued. If the transition is being made in the morning, consider using a one-time AM NPH injection or ½ of daily glargine or detemir dose to bridge until bedtime glargine, detemir or NPH begins.

 

INSULIN REGIMEN FOR A PATIENT RECEIVING GLUCOCORTICOIDS

 

  • Glucocorticoids may dramatically increase postprandial BG levels but have little effect on gluconeogenesis (fasting glucose levels). Often, BG levels are very high during the day, then lower overnight.
  • Anticipate post-prandial hyperglycemia by increasing the nutritional insulin doses.
  • The insulin dose will typically increase by 50% from before glucocorticoid use and the total amount may be 0.5 to significantly >1 Unit/kg

 

DAILY INSULIN ADJUSTMENTS

 

There are no validated formulas for making these adjustments, but the following rules generally work well.

  • Basal Insulin: Generally, the basal insulin dose is adjusted based on fasting glucose levels.  For example, if FBS <140, no change.  If FBS 141-160, increase basal dose by 2-3 units.  If FBS 160-180, increase basal dose by 4-5 units. If FBS 180-200, increase basal dose by 6-7 units.  If FBS >200, increase basal dose by 8 units.  With this approach, the basal insulin can be titrated up to the patient’s actual requirement relatively quickly.
  • Nutritional Insulin: The adequacy of the nutritional insulin dose is based on the glucose level prior to the next meal. For example, the glucose level just before lunch will indicate whether the insulin given at breakfast was appropriate.  The glucose level at bedtime will indicate whether the insulin given at dinner was appropriate.  A simple approach is as follows: If there was no significant change in the glucose level from before breakfast to before lunch, then the total dose of insulin the patient received at breakfast (nutritional plus correctional) should be used as the nutritional dose for breakfast the next day.  If there was a significant increase in the glucose level from before breakfast to before lunch, then the total dose of insulin the patient received at breakfast (nutritional plus correctional) should be increased and should become the nutritional dose for breakfast the next day.  If the glucose level before breakfast was high, and the glucose level at lunch was at goal, then no change in the nutritional dose will be required for the next day.  Finally, no matter what the glucose level was at breakfast, if the glucose level after breakfast or before lunch was low, then the breakfast nutritional dose should be decreased for the next day.

 

Hypoglycemic Protocols

  • BG <70 mg/dL: If patient taking po, give 20 grams of oral fast-acting carbohydrate either as glucose tablets or 6 oz. fruit juice.  If patient cannot take po, give 25 mL D50 IV push.
  • Check BG every 15 minutes and repeat above treatment until BG is ≥100 mg/dL.

 

Insulin Infusions

 

  • Use your hospital’s pre-printed order form or protocol in EMR and hospital-specific protocol for insulin infusions. Using an insulin infusion without a standardized protocol and trained providers can be unsafe.
  • Continuous glucose intake (in IV fluid or continuous TPN or tube feeds) is required during the infusion. Remember to manually adjust the infusion rate and/or the algorithm if there are changes in nutrition (e.g., if tube feeding or TPN is held) or other rapid changes in medical status.
  • When converting to SQ insulin, give the basal SQ dose 1-2 hours before discontinuing the insulin infusion.

 

GUIDELINE

 

Umpierrez GE, Hellman R, Korytkowski MT, Kosiborod M, Maynard GA, Montori VM, Seley JJ, Van den Berghe G; Endocrine Society. Management of hyperglycemia in hospitalized patients in non-critical care setting: an endocrine society clinical practice guideline. J Clin Endocrinol Metab. 2012 Jan;97(1):16-38.

 

REFERENCES

 

Society for Hospital Medicine Diabetes Resource Room:   http://www.hospitalmedicine.org/ResourceRoomRedesign/GlycemicControl.cfm

American Diabetes Association Diabetes Care in the Hospital: Standards of Medical Care in Diabetes—2019   Diabetes Care 2019 Jan; 42(Supplement 1): S173-S181.

 

Corsino L, Dhatariya K, Umpierrez G. Management of Diabetes and Hyperglycemia in Hospitalized Patients. Management of Diabetes and Hyperglycemia in Hospitalized Patients. In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-2017 Oct 1.

 

The Role of Lipids and Lipoproteins in Atherosclerosis

ABSTRACT

 

Atherosclerosis is the underlying cause of heart attack and stroke. Early observations that cholesterol is a key component of arterial plaques gave rise to the cholesterol hypothesis for the pathogenesis of atherosclerosis. Population studies have demonstrated that elevated levels of LDL cholesterol and apolipoprotein B (apoB) 100, the main structural protein of LDL, are directly associated with risk for atherosclerotic cardiovascular events (ASCVE). Indeed, infiltration and retention of apoB containing lipoproteins in the artery wall is a critical initiating event that sparks an inflammatory response and promotes the development of atherosclerosis. Arterial injury causes endothelial dysfunction promoting modification of apoB containing lipoproteins and infiltration of monocytes into the subendothelial space. Internalization of the apoB containing lipoproteins by macrophages promotes foam cell formation, which is the hallmark of the fatty streak phase of atherosclerosis. Macrophage inflammation results in enhanced oxidative stress and cytokine/chemokine secretion, causing more LDL/remnant oxidation, endothelial cell activation, monocyte recruitment, and foam cell formation. HDL, apoA-I, and endogenous apoE prevent inflammation and oxidative stress and promote cholesterol efflux to reduce lesion formation. Macrophage inflammatory chemoattractants stimulate infiltration and proliferation of smooth muscle cells. Smooth muscle cells produce the extracellular matrix providing a stable fibrous barrier between plaque prothrombotic factors and platelets. Unresolved inflammation results in formation of vulnerable plaques characterized by enhanced macrophage apoptosis and defective efferocytosis of apoptotic cells resulting in necrotic cell death leading to increased smooth muscle cell death, decreased extracellular matrix production, and collagen degradation by macrophage proteases. Rupture of the thinning fibrous cap promotes thrombus formation resulting in clinical ischemic ASCVE. Surprisingly, native LDL is not taken up by macrophages in vitro but has to be modified to promote foam cell formation. Oxidative modification converts LDL into atherogenic particles that initiate inflammatory responses. Uptake and accumulation of oxidatively modified LDL (oxLDL) by macrophages initiates a wide range of bioactivities that may drive development of atherosclerotic lesions. Lowering LDL-cholesterol with statins reduces risk for cardiovascular events, providing ultimate proof of the cholesterol hypothesis. All of the apoB containing lipoproteins are atherogenic, and both triglyceride rich remnant lipoproteins and Lp(a) promote atherothrombosis. Non-HDL cholesterol levels capture all of the apoB containing lipoproteins in one number and are useful in assessing risk in the setting of hypertriglyceridemia. Measures of apoB and LDL-P are superior at predicting risk for ASCVE, when levels of LDL-C and LDL-P are discordant.  Here, we also describe the current landscape of HDL metabolism. Epidemiological studies have consistently shown that HDL-C levels are inversely related to ASCVE. We highlight recent clinical trials aimed at raising HDL-C that failed to reduce CVE and the shifting clinical targets of HDL-C, HDL particle numbers, and HDL function (e.g. cholesterol efflux capacity). Furthermore, we describe many beneficial properties of HDL that antagonize atherosclerosis and how HDL dysfunction may promote cardiometabolic disease.

 

PATHOPHYSIOLOGY OF ATHEROSCLEROSIS

 

Atherosclerosis in Cardiovascular Disease

 

As the underlying cause of heart attack, stroke, and peripheral vascular disease, atherosclerosis is the major cause of death and morbidity in the United States and the industrial world (1). The discovery by Virchow more than 100 years ago that atheroma contained a yellow fatty substance, later identified as cholesterol by Windaus, suggested a role for lipids in the pathogenesis of atherosclerosis (2). Indeed, the goal of this chapter is to focus on the role of lipids and lipoproteins in the pathogenesis of atherosclerosis as well as their critical roles in risk assessment and as targets of therapy. The recognition that atherosclerosis is an inflammatory disease has led to tremendous progress in our understanding of the pathogenesis of atherosclerosis (3). First, we provide brief description of the cellular and molecular events in the key stages of atherosclerosis.

 

Initiation and Fatty Streak Phase of Atherosclerotic Lesions

 

The endothelial lining of arteries responds to mechanical and molecular stimuli to regulate tone, (4)hemostasis, (5)and inflammation (6)throughout the circulation. Endothelial cell dysfunction is an initial step in atherosclerotic lesion formation and is more likely to occur at arterial curves and branches that are subjected to low shear stress and disturbed blood flow (atherosclerosis prone areas) (7,8). These mechanical stimuli activate signaling pathways leading to a dysfunctional endothelium lining that is barrier compromised, prothrombotic, and proinflammatory (9). In atherosclerosis susceptible regions, the endothelial cells have cuboidal morphology, a thin glycocalyx layer, and a disordered alignment (8,10,11). In addition, these regions have increased endothelial cell senescence and apoptosis as evidenced by ER stress markers (12-14).In contrast, less atherosclerosis prone endothelium is exposed to laminar shear stress causing activation of signaling pathways that maintain endothelial cell coaxial alignment, proliferation, (13,14)glycocalyx layer, (15)and survival (12,16). In atherosclerosis resistant regions, the transcription factors, Kruppel-like factors (KLF) 2 and 4, are activated via MEK5/ERK5/MEF2 signalingwhich enhances expression of endothelial nitric oxide synthase (eNOS) (17-19). The increased nitric oxide (NO) production promotes endothelial cell migration and survival thereby maintaining an effective barrier (20). In addition, the expression of superoxide dismutase (SOD) is increased to reduce cellular oxidative stress (18). In atherosclerosis susceptible regions, reduced expression of eNOS and SOD leads to compromised endothelial barrier integrity (Figure 1), leading to increased accumulation and retention of subendothelial atherogenic apolipoprotein B (apoB)-containing lipoproteins (low-density lipoproteins (LDL)) and remnants of very low-density lipoproteins (VLDL) and chylomicrons) (21,22). KLF2, KLF4, and NO production inhibit activation of the nuclear factor kappa B (NF‐κB) pathway.Increased NF‐κBactivation in atherosclerosis susceptible areas leads to endothelial cell activation (Figure 1), as evidenced by increased expression of monocyte adherence proteins (VCAM-1, ICAM-1,and P-selectin) and proinflammatory receptors (toll-like receptor 2, TLR2) and cytokines (MCP-1 and IL-8) (19,23,24). In addition, endothelial cell activationleads to increased production of reactive oxygen species (25)that can cause oxidative modification of apoB-containing lipoproteins (26). Besides mechanical stimuli, endothelial cell activation is increased by various molecular stimuli, including oxidized LDL, cytokines,advanced glycosylation end products, and pathogen-associated molecules (27-30). In contrast, an atheroprotective function of HDL is to prevent endothelial activation and enhance NO production to maintain barrier integrity (see details below) (31).

Figure 1. Initiation of the atherosclerotic lesion. The fatty streak phase of atherosclerosis begins with dysfunctional endothelial cells and the retention of apoB-containing lipoproteins (LDL, VLDL, and apoE remnants) in the subendothelial space. Retained lipoproteins are modified (oxidation, glycation, enzymatic), which, along with other atherogenic factors, promotes activation of endothelial cells. Activated endothelial cells have increased expression of monocyte interaction/adhesion molecules (selectins, VCAM-1) and chemoattractants (MCP-1) leading to attachment and transmigration of monocytes into the intimal space. Activated endothelial cells also promote the recruitment of other immune cells including dendritic cells, mast cells, regulatory T (T-reg) cells, and T helper 1 (Th-1) cells. The monocytes differentiate into macrophages and express receptors that mediate the internalization of VLDL, apoE remnants, and modified LDL to become foam cells. In addition, inflammatory signaling pathways are activated in macrophage foam cells leading to more cell recruitment and LDL modification.

Immune Cell Recruitment and Foam Cell Formation

 

Activation of endothelial cells causes a monocyte recruitment cascade involving rolling, adhesion, activation and transendothelial migration (Figure 1). Selectins, especially P-selectin, mediate the initial rolling interaction of monocytes with the endothelium (32). Monocyte adherence is then promoted by endothelial cell immunoglobulin-G proteins including VCAM-1 and ICAM-1(32). Potent chemoattractant factors such as MCP-1 and IL-8 then induce migration of monocytes into the subendothelial space (33-35). Ly6himonocytes, versus Ly6lo, preferentially migrate into the subendothelial space to convert to proinflammatory macrophages in mice (36-38). The enhanced migration of Ly6hiversus Ly6lomonocytes likely results from increased expression of functional P-selectin glycoprotein ligand-1 (39). In addition, the number of blood monocytes originating from the bone marrow and spleen, especially Ly6hicells, increases in response to hypercholesterolemia (36). Furthermore, hypercholesterolemia and atherosclerosis increase monocytosis in humans (40,41). Importantly, increased numbers of inflammatory CD14++CD16+monocytes independently predicted cardiovascular death, myocardial infarction, and stroke in patients undergoing elective coronary angiography (42). Intimal macrophages also result from proliferation of monocyte/macrophages, especially in more advanced lesions (43). During the initial fatty streak phase of atherosclerosis (Figure 1), the monocyte-derived macrophages internalize the retained apoB-containing lipoproteins, which are degraded in lysosomes, where excess free cholesterol is trafficked to the endoplasmic reticulum (ER) to be esterified by acyl CoA:cholesterol acyltransferase (ACAT), and the resulting cholesteryl ester (CE) is packaged into cytoplasmic lipid droplets, which are characteristic of foam cells (42)(Figure 2) (44,45). Modification of apoB lipoproteins via oxidation and glycation enhances their uptake through a number of receptors not down-regulated by cholesterol including CD36, scavenger receptor A, and lectin-like receptor family (see details below) (Figure 2) (46,47). Enzyme-mediated aggregation of apoB lipoproteins enhances uptake via phagocytosis (Figure 2) (48,49). In addition, native remnant lipoproteins can induce foam cell formation via a number of apoE receptors (LRP1 and VLDLR) (Figure 2) (50,51). Uptake of native LDL by fluid phase pinocytosis may also contribute to foam cell formation (Figure 2) (52,53).

 

Figure 2. Macrophage Cholesterol Metabolism. Native LDL is recognized by the LDL receptor (LDLR). The LDL is endocytosed and trafficked to lysosomes, where the cholesteryl ester (CE) is hydrolyzed to free cholesterol (FC) by the acid lipase. The FC is transported to the endoplasmic reticulum (ER) to be esterified by acyl CoA:cholesterol acyltransferase (ACAT). Increased FC in an ER regulatory pool initiates a signaling cascade resulting in down-regulation of the LDL receptor. Cholesterol regulation of the LDLR prevents foam cell formation via this receptor in the setting of hypercholesterolemia. ApoB containing lipoproteins that also contain apoE (apoE remnants, VLDL) can cause cholesterol accumulation via interaction of apoE with apoE receptors including the LRP1 and the VLDL receptor, which are not regulated by cellular cholesterol. Uptake of native LDL by fluid phase pinocytosis may also contribute to foam cell formation. Modifications of apoB containing lipoproteins induce significant cholesterol accumulation via a number of mechanisms. Enzyme-mediated aggregation of apoB lipoproteins enhances uptake via phagocytosis. Oxidation and/or glycation enhances internalization via a number of receptors that are not regulated by cholesterol, including CD36, scavenger receptor A (SRA), lectin-like receptors (LOX), and toll-like receptors (TLR4). The CE generated by ACAT is stored in cytoplasmic lipid droplets, where there is a continual cycle of hydrolysis to FC by neutral cholesterol esterase and re-esterification by ACAT. Cytoplasmic CE is cleared by two main pathways. In one pathway, removal of FC from the plasma membrane stimulates transport of FC that has been generated by neutral cholesterol esterase away from ACAT to the plasma membrane. Alternatively, cytoplasmic CE is packaged into autophagosomes, which are transported to fuse with lysosomes, where the CE is hydrolyzed by acid lipase and the resulting FC is then transported to the plasma membrane. The efflux of FC to lipid-poor apolipoproteins or HDL occurs by a number of mechanisms to reduce foam cell formation. Exogenous lipid-free apoA-I or endogenous apoE that is produced by the macrophages interacts with ABCA1 to stimulate the efflux of phospholipid and FC to form nascent HDL particles (e.g. apoA-I or apoE containing phospholipid discs). ApoE produces the most buoyant, FC-enriched particles. ABCA1 plays a major role in the clearance of cytoplasmic CE via autophagy. The apoA-I/apoE discs as well as mature HDL containing apoA-I and/or ApoE stimulate FC efflux via three major mechanisms including ABCG1, SR-BI, and aqueous diffusion. ABCG1 may also play a role in the intracellular trafficking of cholesterol.

The triggering of macrophage inflammatory pathways is also a critical event in lesion development. Inflammatory M1 phenotype macrophages exhibit increased oxidative stress, impaired cholesterol efflux and enhanced cytokine/chemokine secretion, leading to more LDL/remnant oxidation, endothelial cell activation, monocyte recruitment, and foam cell formation (54-59). Oxidative stress, modified lipoproteins, and other lesion factors (bioactive lipids, pattern recognition molecules, cytokines) are capable of inducing inflammation via receptors (54,55,60). In addition, plasma membrane cholesterol in macrophage foam cells enhances signaling via inflammatory receptors (61,62). Recently, inflammasome activation of IL-1β and IL-18 has been implicated in atherogenesis (63,64). Indeed, a recent clinical trial showed that subjects treated with the IL-1β monoclonal antibody, canakinumab, had a significantly lower rate of recurrent cardiovascular events which were independent of cholesterol lowering (65). Macrophage foam cell formation and cholesterol dependent inflammatory receptor signaling can be reduced by the removal of cholesterol by atheroprotective HDL and apoA-I via a number of mechanisms including ABCA1, ABCG1, SR-BI, and aqueous diffusion (Figure 2) (61,66-68)(see details below). Lipid-poor apoA-I stimulates efflux via ABCA1, whereas lipidated apoA-I or mature HDL are the main drivers of efflux via ABCA1, ABCG1, SR-BI, and aqueous diffusion (Figure 2) (61,69-71). Cytoplasmic CE is cleared by two major pathways. One route involves the hydrolysis of cytoplasmic CE by neutral cholesterol esterase and the resulting free cholesterol is mobilized away from the ACAT pool (72,73)and made available for efflux via ABCA1, ABCG1, SR-BI, and aqueous diffusion (Figure 2). Alternatively, cytoplasmic CE is packaged into autophagosomes, which are trafficked to lysosomes, where the CE is hydrolyzed by acid lipase(73,74), generating free cholesterol that is made available for efflux mainly via ABCA1(Figure 2) (73,74). Furthermore, HDL and apoA-I protect against atherosclerosis by reducing inflammation via mechanisms independent of cholesterol efflux (31,75)(see details below). In addition, small non-coding RNAs have been found to impact atherosclerosis development by regulating inflammation and/or cholesterol homeostasis in different cell types in lesions (76,77). MiR-33a and MiR-33b promote atherosclerosis by impairing cholesterol efflux and promoting inflammatory M1 macrophage conversion (78-80).Other microRNAs including MiR-223 and MiR-93 exhibit atheroprotective effects by increasing cholesterol efflux and conversion to the anti-inflammatory M2 macrophage phenotype (76,81-83). HDL carry small non-coding RNAs (77), which can also reduce or promote atherosclerosis development depending upon composition of individual non-coding RNAs (see details below).

 

Although macrophages are the main infiltrating cells, other cells contribute to the development of lesions including dendritic cells (84,85), mast cells, T cells, and B cells (Figure 1) (86,87). Dendritic cells promote the priming of reactive T cell clones and secrete cytokines, functioning in a largely pro-inflammatory capacity(88). They also take up lipid, which leads to inflammasome activation and increased pro-inflammatory cytokine secretion (89). Mast cells produce interferon-g(IFNg) and IL-6 and appear to promote lesion development(90). Atherosclerotic plaques also contain a significant number of adaptive immune cells, including T and B lymphocytes. The role of T cells is subset-dependent and atherosclerotic plaques have been shown to contain CD4+and CD8+effector T cells as well as T helper 1 (Th-1), Th-2, Th-17, and regulatory T (T-reg) cells. Antigen-specific Th-1 cells produce IFNgthat converts macrophages to a proinflammatory M1 phenotype. Th-17 cells have also been identified in atherosclerotic plaques and have been shown to produce IFNg. However, their specific role in atherosclerosis has not yet been elucidated (91). Classical T-reg cells produce anti-inflammatory cytokines (TGF-β and IL-10) and inhibit activation of Th-1 cells, leading to more anti-inflammatory M2 macrophages. As atherosclerosis progresses, T effector cell numbers increase or remain constant, while T-reg numbers decline. This reduction in T-regs is due in part to their heightened susceptibility to cell death as well as their impaired trafficking into lesions (91). Further, T-regs may appear fewer in number because they undergo phenotypic switching into other T-reg subtypes. Several subclasses of these ‘former’ T-regs have been identified in the atherosclerotic lesions of mice, including Th1-Tregs (CD4+CCR5+IFN-g+FoxP3+T-bet+) and T follicular helper cells (CXCR5+PD1+Bcl6+CD62LloCD44hiCD4+Foxp3-), and these have been shown to have both impaired regulatory and enhanced inflammatory function, therefore contributing to atheroprogression (91,92). B cells preferentially reside in the adventitial layer of arteries neighboring sites of plaque, in regions known as tertiary lymphoid organs (TLOs). The function of B lymphocytes is also subset dependent, with B-1 cells being atheroprotective and B-2 cells being atherogenic. B-1 cells undergo limited or no affinity maturation and produce natural antibodies (NAbs) that have broad specificity and low binding affinity. Among these are NAbs, found within atherosclerotic plaques, that can bind to oxidation motifs in LDL and block the uptake of oxLDL by macrophages (93). Mice engineered to overexpress a single-chain variable fragment of E06, an IgM NAb directed against oxidized phospholipids (oxPL), were found to have reduced atherosclerosis and features consistent with greater overall plaque stability, confirming the atheroprotective nature of these B-1 cell-derived antibodies (94). B-2 cells produce high-affinity IgA, IgE and IgG antibodies. While the role of IgA in atherosclerosis remains controversial, IgG and IgE are atherogenic. IgG forms immune complexes with oxLDL and promotes an inflammatory macrophage phenotype while IgE also stimulates macrophages and mast cells to produce proatherogenic cytokines (95).

 

ApoE in Atherosclerosis

 

In addition to apoA-I and HDL, the endogenous production of apoE by macrophages is critical in preventing atherosclerotic lesion formation. The majority of apoE in plasma is produced by the liver, but macrophages are responsible for producing 5 -10% of apoE in plasma (96). ApoE serves as the ligand for clearance of all of the apoB containing lipoproteins from the blood by the liver except for LDL. Gene knockout of apoE in mice results in hypercholesterolemia and spontaneous atherosclerotic lesion development (97,98). Hence, ApoE deficient mice have been used widely to study mechanisms of atherosclerotic lesion development. Bone marrow transplantation studies were used to examine the role of macrophage apoE in lipoprotein metabolism[Linton, 1998 #5676]. Transplantation of Apoe-/-mice with wildtype bone marrow, resulted in normalization of plasma cholesterol levels and protection from atherosclerosis (99), demonstrating the ability of macrophage apoE to exchange between lipoproteins and to serve as a vehicle for cellular gene therapy of atherosclerosis. Furthermore, reconstitution of wildtype (100)or LDL receptor deficient mice(Ldlr-/-)(101)with Apoe-/-bone marrow accelerates atherosclerotic lesion development without affecting plasma cholesterol levels, demonstrating an atheroprotective role for macrophage apoE. Interestingly, ApoE protects against atherosclerosis via several mechanisms. Expression of apoE by hematopoietic stem cells reduces monocyte proliferation and infiltration into the intima (102). In addition, apoE on apoB lipoproteins reduces the lysosomal accumulation of cholesterol by enhancing the expression of acid lipase (103). Importantly, secretion of apoE by macrophages stimulates efflux in the absence and presence of exogenous acceptors, including HDL and lipid-free apoA-I (Figure 2) (104-107). Recent studies demonstrated that macrophage apoE facilitates reverse cholesterol transport in vivo(108). Macrophage apoE stimulates phospholipid and cholesterol efflux via ABCA1, and the apoE particles formed then promotecholesterol efflux through ABCG1, SR-BI, and aqueous diffusion (104,109-111). Endogenous apoE is required for efficient formation of the most buoyant, cholesterol-enriched particles by macrophages (Figure 2) (104,112-116). In addition to cholesterol efflux, macrophage apoE prevents inflammation (117-120)and oxidative stress (121-124). The local production of apoE is likely a critical atheroprotective mechanism considering that areas of atherosclerotic lesions have limitedaccessibilityto plasma apoA-1 and HDL (100,101,125). Humans express three common apoE polymorphisms that predict CAD rates independently from plasma cholesterol levels (126). ApoE3 (C112, R158) is the most common isoform and is functionally similar to mouse apoE. Compared to apoE3 and apoE2 (C112, C158), apoE4 (R112, R158) are impaired in stimulating cholesterol efflux (127-130)and in preventing inflammation and oxidation (117,124,131). Consistent with the compromised function of apoE4, human carriers exhibit increased risk of CAD compared to humans expressing apoE3 or apoE2 (heterozygous) (126,132,133).

 

Progression to Advanced Atherosclerotic Lesions

 

Fatty streaks do not result in clinical complications and can even undergo regression. However, once smooth muscle cells infiltrate, and the lesions become more advanced, regression is less likely to occur (134,135). Small populations of vascular smooth muscle cells (VSMCs) already present in the intima proliferate in response to growth factors produced by inflammatory macrophages (136). In addition, macrophage-derived chemoattractants cause tunica media smooth muscle cells to migrate into the intima and proliferate (Figure 3). Critical smooth muscle cell chemoattractants and growth factors include PDGF isoforms, (137)matrix metalloproteinases, (138)fibroblast growth factors, (139)and heparin-binding epidermal growth factor (Figure 3) (140). HDL prevents smooth muscle cell chemokine production and proliferation. The accumulating VSMCs produce a complex extracellular matrix composed of collagen, proteoglycans, and elastin to form a fibrous cap over a core comprised of foam cells (Figure 4) (141). A vital component of the fibrous cap is collagen, and macrophage-derived TGF-bstimulates its production (Figure 4) (142). In addition, HDL maintains plaque stability by inhibiting degradation of the fibrous cap extracellular matrix through its anti-elastase activity (143).A subset of VSMCs accumulates CE and resides in the lesion core (Figures 3 and 4). This smooth muscle cell phenotype produces less a-actin and expresses macrophage markers, including CD68, F4/80 and Mac2 (144-146). While studies have shown that VSMCs express the VLDL receptor and various scavenger receptors, (145,147,148)data showing that these cells robustly load with CE, (147)similar to macrophages via these mechanisms is lacking. As lesions advance, substantial extracellular lipid accumulates in the core, in part due to large CE-rich particles arising from dead macrophage foam cells (149,150). Earlier in vitrostudies showed that these CE-rich particles effectively cholesterol load VSMCs (151,152). Regardless of the mechanisms of cholesterol enrichment, VSMCs compared to macrophages are inefficient at lysosomal processing and trafficking of cholesterol (152,153)and express much less ABCA1(154), which all contribute to impaired cholesterol efflux (155). However, macrophages in more advanced plaques also have reduced lysosome function and trapping of free and esterified cholesterol within their lysosomes contributes to the overall sterol accumulation in the lesion (156-158). The reduced lysosome function appears multifactorial but includes direct and indirect inhibition of lysosomal acid lipase, the enzyme responsible for hydrolysis of cholesteryl esters in lysosomes, and a reduced capacity for transferring cholesterol from lysosomes (159-162). In cell culture models of human macrophage foam cells, the inability to clear cholesterol from macrophages with compromised lysosome function continues even in the presence of compounds that stimulate efflux (161,163). Proteomic analysis of foam cells shows that changes in a number of lysosome proteases are related to macrophage sterol accumulation (164). Thus, at least in the advanced stages of atherosclerosis, lysosome dysfunction contributes to the overall lesion severity. As the intimal volume enlarges due to accumulating cells, there is vascular remodeling to lessen protrusion of the lesion into the lumen (Figure 4), thereby decreasing occlusion and the appearance of clinical symptoms for much of the life of the lesion (165-167).

Figure 3. Progression of the atherosclerotic plaque. Macrophage foam cell and endothelial cell inflammatory signaling continues to promote the recruitment of more monocytes and immune cells into the subendothelial space. Transition from a fatty streak to a fibrous fatty lesion occurs with the infiltration and proliferation of tunica media smooth muscle cells. Macrophage foam cells and other inflammatory cells produce a number of chemoattractant and proliferation factors, including transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF) isoforms, matrix metalloproteinases, fibroblast growth factors (FGF), and heparin-binding epidermal growth factor (HB-EGF). Smooth muscle cells are recruited to the luminal side of the lesion to proliferate and generate an extracellular matrix network to form a barrier between lesional prothrombotic factors and blood platelets and procoagulant factors. A subset of smooth muscle cells express macrophage receptors and internalize lipoproteins to become foam cells. Fibrous fatty lesions are less likely to regress than fatty streaks.

Figure 4. Features of the stable fibrous plaque. As the cell volume of the intima increases, there is vascular remodeling so that the lumen is only partially occluded, substantially lessening clinical events resulting from occlusion. The stable plaque contains a generous fibrous cap composed of layers of smooth muscle cells ensconced in a substantial extracellular matrix network of collagen, proteoglycans, and elastin. The thick fibrous cap of the stable plaque provides an effective barrier preventing plaque rupture and exposure of lesion prothrombotic factors to blood, thereby limiting thrombus formation and clinical events. Maintenance of a thick fibrous cap is enabled by regulation of the inflammatory status of the foam cell core of the lesion. Regulatory T (T-reg) cells produce transforming growth factor-β (TGF-β) and IL-10. In addition, T-reg cells inhibit antigen-specific activation of T helper 1 (Th-1) cell to produce interferon gamma (IFNg). Increased TGF-β and IL-10 and decreased IFNg reduce the proinflammatory macrophage phenotype leading to reduced cell death, effective efferocytosis (phagocytosis of dead cells), and anti-inflammatory cytokine production (i.e. TGF-β, IL-10). Thus, stable plaques have small necrotic cores containing macrophage debris and extracellular lipid resulting from secondary necrosis of noninternalized apoptotic macrophage foam cells. The production of TGF-β by T-reg cells and macrophages maintains fibrous cap quality by being a potent stimulator of collagen production in smooth muscle cells.

Vulnerable Plaque Formation and Rupture

 

The advanced atherosclerotic lesion is essentially a nonresolving inflammatory condition leading to formation of the vulnerable plaque, increasing the risk of plaque rupture. The vulnerable plaque is characterized by two fundamental morphological changes: 1) Formation of a necrotic core and 2) Thinning of the fibrous cap. Sections of the atheroma with a deteriorated fibrous cap are subject to rupture (Figures 4 and 5) (168,169). A recent lipidomics study showed that stable versus unstable plaques have different lipid subspecies profiles (170). Compared to plasma and control arteries, stable plaques have increased CE containing polyunsaturated fatty acids (170), which have increased susceptibility to oxidation. The CE containing polyunsaturated fatty acids are decreased in unstable plaques compared to stable plaques of the same subjects (170). In addition, 18:0 containing lysophosphatidylcholine is increased in unstable plaques indicating enhanced oxidation (170). Plaque rupture leads to acute exposure of procoagulant and prothrombotic factors from the necrotic core of the lesion to platelets and procoagulant factors in the lumen, thereby causing thrombus formation (Figure 5) (168,169). Thrombus formation at sites of plaque rupture accounts for the majority of clinical events with acute occlusive luminal thrombosis causing myocardial infarction, unstable angina, sudden cardiac death, and stroke (168,169).

Figure 5. Formation of the vulnerable plaque. The vulnerable plaque results from a heightened, unresolved inflammatory status of the lesion foam cell core. Antigen-specific activation of T helper 1 (Th-1) cells produces interferon gamma (IFNg) resulting in a proinflammatory macrophage phenotype. The proinflammatory macrophage foam cells exhibit enhanced inflammatory cytokine secretion and apoptosis susceptibility. There is less secretion of the anti-inflammatory cytokines, TGF-β and IL-10. In addition, proinflammatory macrophages have impaired atheroprotective functions including cholesterol efflux and efferocytosis. The defective efferocytosis of inflammatory apoptotic macrophages results in secondary necrosis leading to an enlarged necrotic core composed of leaked oxidative and inflammatory components. This unresolved inflammation causes thinning of the fibrous cap resulting from increased smooth muscle cell death, enhanced extracellular matrix degradation and decreased extracellular matrix production. Areas of thin fibrous cap are prone to rupture exposing prothrombotic components to platelets and procoagulation factors leading to thrombus formation and clinical events.

Macrophage Cell Death and Efferocytosis Influence Plaque Stability

 

The necrotic core results from a combination of accelerated macrophage death and impaired efferocytosis (receptor-mediated phagocytosis of apoptotic cells) (Figure 5) (171,172). As apoptotic cells accumulate and fail to be internalized by phagocytes, they undergo secondary necrotic death leading to the leakage of intracellular oxidative and inflammatory components, which then propagate more inflammation, oxidative stress, and death in neighboring cells (Figure 5) (173). Multiple triggers likely occur in lesions to accelerate macrophage death, including oxidative stress, death receptor activation, and nutrient deprivation (174). Prolonged ER stress and activation of the unfolded protein response (UPR) contribute to macrophage apoptosis as substantiated by studies showing that apoptosis and the UPR effector, CHOP, increase with each stage of atherosclerosis in humans, but the largest increase is observed in the vulnerable plaque (175). In diabetes and obesity, accelerated formation of an enlarged necrotic core is likely instigated by defective macrophage insulin signaling (176)and saturated fatty acids (177,178), which are potent inducers of ER stress. In addition, other triggers act in tandem with ER stress to accelerate apoptosis. In particular, activation of toll-like receptors (TLR) (TLR2 and TLR4) and scavenger receptors (CD36 and SR-A) by oxidized phospholipids induces apoptotic signaling (178-181). Death is also accelerated by simultaneous suppression of survival pathways such as pAkt and NF‐κB via these same receptors. Accelerated apoptotic macrophage death is not sufficient to promote necrosis. Apoptotic cells undergo secondary necrotic death if they are not internalized by phagocyte efferocytosis receptors. Necrotic death leads to the leakage of intracellular oxidative and inflammatory components, which then propagate more inflammation, oxidative stress, and death in neighboring cells (Figure 5) (173). The presence of necrotic tissue together with apoptosis is consistent with defective efferocytosis in human plaques. Studies have shown that the majority of apoptotic cells are free in advanced human lesions, whereas in tonsils apoptotic cells are macrophage-associated (182). Efferocytosis also becomes defective in advanced atherosclerosis through several different mechanisms. First, accumulating evidence has shown that the expression and function of key efferocytosis receptors,MerTK (183),  LRP1 (184), and SR-BI (185)are impaired in advanced atherosclerosis. These receptors recognize apoptotic cell ligands such as phosphatidylserine (185,186). and efferocytosis efficiency is enhanced by bridging molecules such as apoE and MFG-E8 that interact with efferocytosis receptors to enhance their efficiency and also have reduced expression in advanced lesions (186-190). Compared to apoE3, apoE4 is defective at facilitating efferocytosis of apoptotic cells (191). Efferocytosis via LRP1 (187)and SR-BI (185)also stimulates signaling pathways leading to pAkt production to promote phagocyte survival. In addition, anti-inflammatory signaling (185,192)is activated so that phagocytes secrete TGF-β and IL-10 (Figures 4 and 5). In addition, efferocytosis may be limited by competition for apoptotic cell binding. For example, oxPLs bind efferocytosis receptors and effectively compete for apoptotic cell recognition. In addition, lesional autoantibodies to oxPL and oxLDL are able to bind to ligands on the apoptotic cell themselves in order to prevent their binding and ingestion. Finally, apoptotic cells in advanced lesions appear to become poor substrates for efferocytosis. CD47, which typically acts as a “don’t eat me” signal expressed by live cells, is upregulated by apoptotic cells within human and murine atherosclerotic plaques, allowing them to evade uptake by phagocytes. When given to atheroprone Apoe-/-mice, a CD47-blocking antibody enhanced lesional efferocytosis and resulted in smaller necrotic cores(193). Similarly, mice that express low levels of the “eat me” signal, calreticulin, have increased necrotic cores compared to control mice and apoptotic cells from these mice demonstrate resistance to uptake by phagocytes (194).

 

Components of the necrotic core promote thinning of the fibrous cap. Loss of extracellular matrix is in part due to death of fibrous cap smooth muscle cells, resulting from macrophage-derived Fas receptor ligand (195), inflammatory cytokines (196), and oxidation products (Figure 5) (197,198). Smooth muscle cells are inefficient at efferocytosis (199)relying on macrophages to internalize apoptotic smooth muscle cells. As such, the impaired efferocytosis by lesional macrophages likely leads to uncontrolled VSMC death (Figure 5). In addition, impaired production of TGF-β by phagocytes (185,200)reduces collagen production by healthy smooth muscle cells (Figures 4 and 5). The extracellular matrix components are degraded by macrophage-derived matrix metalloproteinases, (201-203)elastases, and cathepsins (Figure 5) (204). HDL can reduce VSMC apoptosis and elastin degradation induced by elastases (143,205).

 

Importantly, HDL can prevent efferocyte apoptosis via ER stress by its cholesterol efflux and anti-oxidant functions (179,206,207). Furthermore, HDL drives conversion to the anti-inflammatory M2 macrophages which have enhanced efferocytosis ability compared to inflammatory M1 macrophages (56,208)leading to increased plaque stability. Once plaque rupture occurs, critical HDL functions may also include prevention of platelet activation and thrombus formation. In addition to the role of HDL in stabilizing plaques, recent studies have focused on the lesional loss of specialized proresolving mediators (SPM) versus proinflammatory factors (i.e. leukotriene B4)in promoting uncontrolled inflammation and formation of vulnerable plaques (209). Studies on human atherosclerotic lesions have shown that unstable versus stable plaques have decreased lipid-derived SPM including resolvin D1 and lipoxin A4 (210). In addition, resolving D1 treatment ofLdlr-/-mice with established atherosclerosis increased lesional efferocytosis and collagen content and reduced the necrotic area and reactive oxygen species content (210). Similar results were observed in Apoe-/-micetreated with the phospholipase D derived proresolving lipid, palmitoylethanolamide(211).Other lipid derived resolving mediators which impact atherosclerotic plaques include maresin 1 and resolvin D2 (212). Protein SPM have also been identified including annexin 1 and IL-10 (209). Administration of lesion targeting nanoparticles containing the bioactive annexin 1 peptide, Ac2-26, to Ldlr-/-mice with atherosclerosis reduced both lesional oxidative stress and necrosis while increasing collagen content and fibrous cap thickness (213). Enhancing the lesional IL-10 content also improved atherosclerotic lesion stability (214). In addition, Treg cells likely control atherosclerotic lesion inflammation resolution as recent studies demonstrated that Treg cells regulate efferocytosis in atherosclerotic lesions by secreting IL-13 to stimulate macrophage production of IL-10 to induce Vav-1 activation of Rac1 and increased efferocytosis (215).

 

Summary

 

Atherosclerotic lesions initiate with endothelial cell dysfunction causing modification of apoB containing lipoproteins (LDL, VLDL, remnants) and infiltration of immune cells, particularly monocytes, into the subendothelial space (Figure 1). The macrophages internalize the retained apoB containing lipoproteins to become foam cells forming the fatty streak (Figure 1). Macrophage inflammatory pathways are also activated leading to increased oxidative stress and enhanced cytokine/chemokine secretion, causing more LDL/remnant oxidation, endothelial cell activation, monocyte recruitment, and foam cell formation (Figure 1). HDL, apoA-I, and endogenous apoE reduce lesion formation by preventing endothelial cell activation, inflammation, and oxidative stress and also by promoting cholesterol efflux from foam cells. As the lesion progresses to fibrotic plaques as a result of continued inflammation, macrophage chemoattractants stimulate infiltration and proliferation of smooth muscle cells (Figure 3). Smooth muscle cells produce the extracellular matrix providing a stable fibrous barrier between plaque prothrombotic factors and platelets (Figure 4). Unresolved inflammation results in formation of vulnerable plaques, which have large necrotic cores and a thinning fibrous cap (Figure 5). Enhanced macrophage apoptosis and defective efferocytosis of apoptotic cells results in necrotic cell death causing heightened inflammation leading to increased smooth cell death, decreased extracellular matrix production, and collagen degradation by macrophage proteases. An imbalance between inflammatory factors and SPMs is prominent in facilitating formation of the vulnerable plaque.  Rupture of the thinning fibrous cap promotes thrombus formation resulting in clinical ischemic cardiovascular events (Figure 5).

 

 

 

THE ROLE OF CHOLESTEROL AND LIPOPROTEINS IN ATHEROGENESIS

 

Metabolism of ApoB Containing Lipoproteins

 

Apolipoprotein B (apoB) occurs in two isoforms, apoB100 and apoB48. ApoB100 is the main structural apolipoprotein of low-density lipoproteins (LDL), and there is only one molecule of apoB100 per LDL particle (216). ApoB100 is produced mainly by the liver, where it is required for the synthesis and secretion of triglyceride-rich very low-density lipoprotein (VLDL) particles (Figure 6). In the circulation, VLDL is metabolized to the cholesteryl ester-enriched intermediate low-density lipoprotein (IDL) and LDL particles through the progressive hydrolysis of triglycerides by lipoprotein lipase (LPL) and hepatic lipase (Figure 6). In humans, apoB48 is produced exclusively in the intestine through an unique RNA editing mechanism by the apobec-1 enzyme complex (217). ApoB100 is the full-length protein, which contains 4536 amino acids, whereas apoB48 contains the first 48% of the amino terminal amino acids. ApoB48 is required for the synthesis and secretion of triglyceride-rich chylomicrons, which play a critical role in the intestinal absorption of dietary fats and fat-soluble vitamins. Similar to the metabolism of VLDL, chylomicrons are metabolized in the circulation through the hydrolysis of triglycerides by LPL and hepatic lipase to form cholesteryl ester-enriched chylomicron remnants, which release free fatty acids that can be used for energy by the tissues.

Figure 6. Metabolism of ApoB100 containing lipoproteins. ApoB100 is critical for the production and secretion of very low-density lipoprotein (VLDL) by the liver. Plasma VLDL is metabolized to cholesteryl ester-enriched intermediate low-density lipoprotein (IDL) and LDL particles via hydrolysis of triglycerides by lipoprotein lipase (LPL) and hepatic lipase (HL). In addition, cholesteryl ester transfer protein (CETP) transfers CE from HDL to VLDL in exchange for triglyceride (TG) to HDL. ApoCII and apoCIII are transferred from HDL to VLDL and act as an activator or inhibitor of LPL activity, respectively. ApoB100 is the ligand for hepatic LDL receptor-mediated clearance of LDL. VLDL acquires apoE from HDL, and apoE mediates the clearance of triglyceride-enriched remnants and IDL. In addition, HDL can directly transfer cholesterol to liver via interaction with SR-BI. VLDL and IDL remnants can induce foam cell formation by internalization via apoE receptors on macrophages. LDL, IDL, and VLDL can be modified (oxidation, glycation) and internalized by a number of macrophage receptors including scavenger receptors and lectin-like receptors. HDL and lipid-poor apoA-I reduce foam cell formation by stimulating cholesterol efflux.

Static measurements of cholesterol in the LDL pool (LDL-C) represent the steady state of production of VLDL, its metabolism to LDL, and the receptor-mediated clearance of LDL by the LDL receptor (LDLR). Mutations in the Ldlrgene are the most common cause of familial hypercholesterolemia (FH), an autosomal dominant disorder associated with elevated levels of LDL-C and increased risk for premature cardiovascular disease (218). ApoB100 serves as the ligand for receptor-mediated clearance of LDL by the liver (Figure 6). In contrast, apoE mediates the clearance of triglyceride-rich remnants (IDL and chylomicron remnants) either through the LDLR or the remnant receptor pathway (Figure 6). The existence of the remnant receptor pathway was suggested by the fact that patients with homozygous FH, who completely lack LDLR function, have severely elevated levels of LDL-C but normal blood levels of triglycerides. The clearance of these remnant lipoproteins involves binding to heparin sulfate proteoglycans and the LDLR like protein -1 (LRP1) in the hepatic space of Disse, in a process called secretion capture that requires local enrichment by hepatic expression of apoE

(96,219).

 

The Cholesterol Hypothesis

 

Studies by Anitschkow showing that feeding cholesterol in oil to rabbits caused the formation of atheroma, similar to those seen in humans, demonstrated a causal role of cholesterol in the pathogenesis of atherosclerosis in 1913 (220). In 1939, Muller described families with inherited high cholesterol and increased risk for cardiovascular disease (221). Yet it would take several decades before compelling evidence from epidemiological studies, such as Framingham (221)and MRFIT (222), demonstrated that elevated blood cholesterol levels were associated with increased risk of cardiovascular events (CVE). Subsequently, LDL-C levels were found to be directly associated with CVE (223); whereas HDL-C levels were shown to be inversely related to risk of CVE (224). The Seven Countries Studies by Ancel Keys showed that coronary heart disease (CHD) mortality rates were higher in countries with higher blood levels of cholesterol (e.g. Finland, Norway, and the USA) than in countries of southern Europe and Japan with lower blood levels of cholesterol (225). The high levels of cholesterol were proposed to be associated with the amount of saturated fat in the diet. As such, the cholesterol hypothesis was born, proposing that lowering LDL-C would reduce CVE(226).

 

Response to Retention Hypothesis for the Initiation of Atherosclerosis

 

The response to retention hypothesis holds that retention of atherogenic lipoproteins in the artery wall is a critical initiating event that sparks an inflammatory response and promotes the development of atherosclerosis (Figure 1). First articulated in 1995 by Williams and Tabas (227), the hypothesis was based on more than two decades of work demonstrating that apoB-containing lipoproteins are retained in the artery wall by interaction with proteoglycans (228,229). Proteoglycans consist of a protein core bound covalently to one or more glycosaminoglycans (GAGs). The most common proteoglycans in the artery wall are decorin, biglycan, perlecan, versican, and syndecan-4 (230). There is ionic binding between the positively charged GAGs and negatively charged amino acids of apoB100 (229). Boren et al.identified the principal proteoglycan-binding site in LDL and showed that a single point mutation in apoB100 impaired binding to proteoglycans (231). The major proteoglycan binding site consists of residues 3359-3369 in apoB100 (site B), which is in the C-terminal half of apoB100. Furthermore, mutation of “site B” in mice resulted in reduced retention of apoB100 in the artery wall and reduced atherosclerosis, providing in vivosupport for the response to retention hypothesis (232). Subsequently, proteoglycan binding sites were identified for apoB48 (233)and a second site (site A) on apoB100, which is exposed when LDL is modified by secreted phospholipase A2 (sPLA2), forming a small dense LDL particle (234).

 

Surprisingly, native LDL, despite the strong evidence for its critical role in promoting atherosclerosis, does not induce macrophage foam cell formation or much in the way of inflammation in vitro. These observations led to the hypothesis that LDL has to be modified to promote foam cell formation and induce inflammation. Binding of proteoglycans induces structural changes in LDL impacting both the configuration of apoB100 and the lipid composition (234). Hence, the binding of LDL to proteoglycans makes the LDL more susceptible to oxidation and aggregation, which promotes foam cell formation and a proinflammatory response, and the process is self-perpetuating. Oxidized LDL (oxLDL) can induce further production of proteoglycans by vascular smooth muscle cells, retaining more LDL in the arterial wall. Furthermore, macrophages express LPL, which can serve as bridging molecules, binding both lipoproteins and proteoglycans (235,236). Consistent with an important role for LPL in atherogenesis, the loss of macrophage LPL expression protects mice from atherosclerosis (237,238). In addition, macrophages secrete sphingomyelinase, which has been reported to act synergistically with LPL to promote binding of LDL and lipoprotein (a) (Lp(a)) to vascular smooth muscle cells (VSMC) and the extracellular matrix promoting their retention in the artery (239,240). Furthermore, sphingomyelinase induces aggregation and fusion of LDL particles, promoting increased binding to proteoglycans and induces foam cell formation (241). Thus, interfering with the retention of apoB-containing lipoproteins in the artery wall is a potential strategy for preventing atherosclerosis.

 

OXIDATION OF PHOSPHOLIPIDS AND PROTEINS IN LIPOPROTEINS AND THEIR ROLE IN ATHEROSCLEROSIS

 

Overview

 

The response to retention hypothesis for the initiation of atherosclerosis posits that retention of LDL in the artery wall leads to its modification into highly atherogenic particles that initiate inflammatory responses. A key overall point is that retention of LDL leads to oxidative modification of LDL, allowing this oxidized LDL (oxLDL) to be recognized by scavenger receptors on macrophages and other cells. Uptake of oxLDL by macrophages leads to marked accumulation of cholesterol, converting them to foam cells and initiating development of atherosclerotic lesions. In addition to serving as a substrate for cholesterol accumulation, oxLDL exerts a wide range of bioactivities that are consistent with it being critical for driving atherogenesis (Table 1). In mouse models, loss of enzymes that modulate LDL oxidation increases atherosclerosis, and dietary antioxidants that reduce levels of oxLDL also inhibit atherosclerosis. Although human trials with dietary antioxidants have failed to reduce disease outcomes, it is important to recognize that these interventions are less efficacious in reducing oxLDL levels in humans than in rodent models. Additional studies are needed to determine optimal interventions for lowering oxLDL levels and whether such interventions will be effective for preventing or treating atherosclerosis.

 

Table 1-Potential Atherogenic Activities of Oxidized LDL (oxLDL)
Macrophages Smooth muscle cells
Serves as ligand for recognition by scavenger receptors 256, 257, 258 Induces proliferation, migration, and transition to inflammatory phenotype 276, 277, 278, 279
Serves as substrate for unregulated cholesterol uptake 262
Induces expression and secretion of inflammatory cytokines 280, 281, 282, 283 Lymphocytes
Induces polarization to M1 (minimally oxidized LDL) or M2-phenotype (highly oxidized LDL) 284 Serves as a neo-antigen 274
Inhibits egress from atherosclerotic lesions 289 Induces chemotaxis 275
Induces macrophage apoptosis and rupture of atherosclerotic plaques 290, 291, 292 Increases antibody production 275
Other cell types
Endothelial cells Induces chemotaxis of monocytes, PMN, and eosinophils 285, 286, 287, 288
Induces surface expression of adhesion molecules 266, 268, 269, 270 Increases platelet aggregation 293, 294, 295, 296
Induces inflammatory genes including cytokine release 271, 272 Activates dendritic cells and induces their release of T cells stimulating cytokines 284

 

Peroxidation of Polyunsaturated Fatty Acids Generates Oxidatively Modified Lipoproteins

 

The outer shell of lipoproteins is composed of phospholipids with polyunsaturated fatty acid (PUFA) side chains. These PUFAs (and to a lesser extent the PUFAs of cholesterol esters and triglycerides in the lipoprotein core) are highly vulnerable to oxidation by free radical species, particularly hydroxyl radicals (OH). This vulnerability results from the relatively low energy required for free radicals to abstract hydrogen atoms located between two adjacent double bonds (bis-allelic hydrogens). Hydrogen abstraction by free radicals creates a lipid radical that reacts nearly instantaneously with any molecular oxygen present in the environment.

 

The resulting lipid peroxide radical (LOO) can then propagate the radical reaction by abstracting hydrogens from neighboring phospholipids or can react with itself to create a large number of secondary peroxidation products (Figure 7). Secondary products that may be relevant to atherogenesis can be thought of in two broad classes: oxidized lipids (primarily oxidized phospholipids but also oxidized cholesterol esters) and reactive lipid aldehydes that exert their effects by modifying proteins and other macromolecules. Oxidized phospholipids (oxPL) include chain shortened oxPL such as 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC)(242), 1-O-alkyl-2-azelaoyl-sn-glycero-3-phophorylcholine (azPAF) (243), and 1-(Palmitoyl)-2-(5-keto-6-octene-dioyl) phosphatidylcholine (KOdiA-PC)  and cyclized oxPL such as  1-palmitoyl-2-(5,6)-epoxyisoprostane E2-sn-glycero-3-phosphocholine (PEIPC) (244)and 1-palmitoyl-2-F2-isoprostane-sn-glycero-3-phosphocholine (F2IsoP-PC) (245). Reactive lipid species include malondialdehyde  (246), 4-hydroxynonenal (246), and isolevuglandins (247)that modify proteins associated with lipoprotein particles including ApoB100 (Figure 7).

Figure 7. Oxidation of Phospholipid Polyunsaturated Fatty Acids. Oxidation of phospholipids containing polyunsaturated fatty acids present in plasma lipoproteins results in formation of a variety of reactive lipid aldehydes and oxidized phospholipids that convert these lipoproteins to atherogenic particles. Reactive lipid species include malondialdehyde (MDA), isolevuglandins (IsoLG), methyglyoxal (MGO), 4-oxononenal (ONE), and 4-hydroxynonenal (HNE). Oxidized phospholipids include 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC), 1-O-alkyl-2-azelaoyl-sn-glycero-3-phophorylcholine (azPAF), 1-(Palmitoyl)-2-(5-keto-6-octene-dioyl) phosphatidylcholine (KOdiA-PC), 1-palmitoyl-2-F2-isoprostane-sn-glycero-3-phosphocholine (F2IsoP-PC), and 1-palmitoyl-2-(5,6)-epoxyisoprostane E2-sn-glycero-3-phosphocholine (PEIPC).

It is critical to keep in mind that oxidatively modified LDLs (oxLDLs) are in fact highly heterogeneous and complex particles, even though oxLDL is usually referred to as a discrete entity. Oxidation of LDL in vitro has been used extensively to study the biological activities of oxLDL, but, even here, the actual species present varies significantly based on the oxidation method (exposure to air, to copper, or to oxidases) and length of oxidation. Many of the methods commonly used to measure the concentration of oxLDL in vivo only measure general characteristics of oxLDL. For instance, because the reaction of reactive lipid species with lysine residues of ApoB100 converts LDL from a positively charged particle to a negatively charged particle, oxLDL is often detected by increased mobility during agarose gel electrophoresis. Alternatively, oxLDL in plasma and other tissues can be quantified by the immunoreactivity of the natural IgM autoantibody E06. However, while E06 recognizes a variety of oxidized phosphatidylcholines, it does not necessarily recognize all oxPL equally. Therefore, equivalent E06 immunoreactivity does not necessarily mean exposure to identical oxLDL particles. While modification of LDL with malondialdehyde (MDA-LDL) (248)is often used as a model of oxLDL for bioactivity assays, modification of LDL by other reactive lipid species can exert unique effects from MDA-LDL, and MDA-LDL does not include any of the various oxPL species. Therefore, it is important to keep in mind that in vivo oxLDL is a mixture of many different compounds and that the atherogenic activities of oxLDL represent the net cellular responses to the full range of compounds present.

 

While oxLDL has been studied in greatest detail, all lipoproteins are vulnerable to oxidation at least in vitro, and this oxidative modification alters their biological activities in ways that may be atherogenic. The species of plasma lipoprotein that has the highest content of oxidized phospholipids (oxPL) depends on the species of oxPL under consideration. This suggests that not all oxPL are formed in situ on the lipoprotein where they are found and might instead be transferred from other lipoproteins or tissues. Lp(a) is the major carrier in plasma of oxPLs that are detected by E06 immunoreactivity (249)and these oxPLs associate with Lp(a) in preference to native LDL particles in human plasma (250). E06 immunoreactive oxPL generated in chemically oxidized LDL can rapidly transfer to Lp(a) (249), so the high content of these lipids in Lp(a) isolated from human plasma may be due either to direct oxidation of Lp(a) or by transfer of the oxPL from oxLDL to Lp(a). In LDL and Lp(a) isolated from human plasma, levels of MDA-modified lysine (based on E014 immunoreactivity) are higher in LDL than Lp(a), while E06 immunoreactivity is much greater for Lp(a) than for LDL (249). Because MDA-modified proteins do not readily transfer between particles, these findings suggest that oxidation initially occurs in LDL with subsequent transfer of oxPL to Lp(a). Thus, a physiological role has been proposed for Lp(a) in binding and transporting oxPL in the plasma (251). Unlike oxPL detected by E06 immunoreactivity that are highest in Lp(a), the F2-IsoP-phospholipids forms of oxPL are highest in HDL (252). As with Lp(a), the high levels of these oxPLs in HDL may well be the result of transfer from other oxidized lipoproteins and tissues. Because oxidation is unlikely to occur in the circulation, the rate that oxPL are transferred from tissue to various plasma lipoproteins could potentially be an important determinant of the risk for atherosclerosis.

 

Significant correlations have been found between levels of oxLDL and extent of atherosclerosis in human patients. Measurement of oxLDL using E06 antibody showed that: 1) significant elevation of oxLDL in acute coronary syndromes (250), 2) treatment with a statin markedly reduced these levels (253), 3) oxLDL levels are higher in children with familial hypercholesterolemia compared to their siblings (254), and 4) oxLDL levels predict the presence and progression of atherosclerosis and symptomatic cardiovascular disease (255). Measurement of oxLDL using antibodies against MDA-LDL found that oxLDL were elevated in patients with coronary artery disease (CAD) (256), that elevated levels of oxLDL predicted future cardiac events in diabetic patients with CAD, that oxLDL were particularly elevated in patients with rheumatoid arthritis and CAD compared to either alone (257), and that treatment with fibrates decreased levels of oxLDL (258). Thus, there is a clear correlation between the presence of oxLDL and cardiovascular disease.

 

Mechanisms of Lipoprotein Oxidation In Vitro And In Vivo

 

The precise mechanisms that generate oxidized lipoproteins in vivo are still only partially understood. LDL circulating in the plasma appears to be protected from oxidation, both by dietary antioxidants such as vitamin E and C (259)and by protective enzymes including glutathione peroxidases (260,261), peroxiredoxins, PAF-acetylhydrolase (also known as lipoprotein-PLA2) (262,263), and paraoxonases (PON) (264,265). Penetration of LDL into the artery wall occurs at branch points in the aorta and other places with turbulent flow and shear stress. Retention of LDL in the intima, due to interactions with extracellular matrix such as chondroitin sulfate-rich proteoglycans, sequesters LDL away from the antioxidant environment of the plasma and exposes LDL to oxidation. A variety of oxidases and peroxidases generate strong oxidants that can readily oxidize LDL. These include myeloperoxidase (MPO) (266), xanthine oxidase (XO) (267), NADPH oxidases (NOXs) (268), and inducible nitric oxide synthase (iNOS) (269). Oxygenases such as lipoxygenases (LOX) have also been shown to oxidize LDL in vitro (270,271). The extent that each of these enzymes contributes to lipoprotein oxidation in vivo and thus to atherosclerosis remains to be fully elucidated, and there is much we do not understand about these individual processes. This is illustrated by studies on MPO and the 12/15-Lipoxygenase, the two enzymes most closely linked to lipoprotein oxidation.

 

MYELOPEROXIDASE (MPO)

 

MPO released from activated neutrophils (and to a lesser extent from monocytes/macrophages) can accumulate in the subintimal space of the artery wall(272), so neutrophil activation indirectly increases the chance for lipoprotein oxidation. Increased plasma levels of MPO correlate with increased levels of oxLDL in hypercholesterolemic children (273). Increased MPO blood levels also associate with increased risk for atherosclerosis (274-277)and polymorphisms in the MPO gene that lower MPO activity reduce the risk for atherosclerosis (278,279).

 

Incubation of lipoproteins with MPO generates oxidized phospholipids that serve as ligands for CD36(280). A putative binding site for MPO with the apoB of LDL has been identified (281), although further verification is needed. Of interest, MPO also associates with HDL via binding to ApoAI and PON1 in a ternary complex(282), so that the binding of MPO to HDL and subsequent generation of reactive oxygen species may account for the high levels of oxidized lipids carried by HDL. Association of MPO with HDL leads to modification of tyrosine 71 of paraoxonase, reducing PON1 activity (282). It also generates reactive lipid dicarbonyls such as isolevuglandins that modify ApoAI (283)and phosphatidylethanolamine (284). In the presence of small molecules that scavenge lipid dicarbonyls, the ability of MPO to crosslink ApoAI is markedly reduced (283).  Modification of HDL by lipid dicarbonyls such as isolevuglandins and MDA reduce its ability to drive cholesterol efflux from macrophages and protect against inflammatory stimuli such as LPS (283,285).

 

Mouse models have been used to directly examine the contribution of MPO to atherosclerosis, although these studies carry the caveat that mouse MPO levels are only 10-20% that of humans (286,287). Transplantation of bone marrow from genetically altered mice into atherosclerosis susceptible strains (e.g. Ldlr-/- and Apoe-/- mice) after lethal irradiation to ablate host hematopoietic cells is commonly used to study the effect of specific genes expressed by macrophages and other hematopoietic cells on atherogenesis (99). Reconstitution of Ldlr-/- mice with macrophages overexpressing MPO markedly increased their susceptibility to atherosclerosis (288). However, transplantation of MPO-/- macrophages into Ldlr-/- mice, also markedly increased atherosclerosis, and this was confirmed in MPO-/-/Ldlr-/- double knockout mice compared to MPO+/+/Ldlr-/- controls (289). The reasons for these paradoxical findings with both MPO overexpression and deletion remain unclear. Perhaps the complete lack of MPO activity is harmful because it allows overgrowth of specific microbes that incite atherosclerosis via alternative mechanisms. In contrast to effects of complete ablation, a recently developed selective MPO inhibitor (e.g. INV315) that only partially reduces MPO activity markedly reduced atherosclerosis in Apoe-/- mice (290). Thus, clinical studies with selective MPO inhibitors are needed to determine if this will be a meaningful therapeutic approach to the treatment of atherosclerosis in humans.

 

12/15-LIPOXYGENASES

 

Although the primary substrates for lipoxygenases are non-esterified fatty acids, exposure of LDL to 15-LOX also leads to oxidation of phospholipids and cholesterol esters (270,271). In mice, the gene analogous to the human 15-LOX encodes a lipoxygenase that converts arachidonyl chains to both 12-HPETE and 15-HPETE and is thus a 12/15-LOX. 12/15-LOX-/-  mice on Apoe-/-  background have reduced atherosclerosis compared to Apoe-/- mice (291). Importantly, they also have lower levels of autoantibodies against oxLDL and MDA-LDL (291). These results support the notion that 12/15-LOX can directly contribute to atherosclerosis via LDL oxidation. Nevertheless, the role of 15-LOX in human atherogenesis is less clear-cut. While homozygotes of an Alox15 variant that almost completely ablates 15-LOX activity tended to have a reduced risk for coronary artery disease, heterozygotes paradoxically have increased risk of disease (292). Other polymorphisms in the Alox15 gene encoding 15-LOX increase risk for coronary artery calcification (293), yet others have no effect (294). Direct correlations between Alox15 polymorphisms and biochemical measurements of oxidized lipoproteins or oxPL and oxidized cholesterol esters have not been reported to date in humans, but are clearly needed.

 

Biological Activities of OxLDL And Receptors That Mediate These Activities

 

Perhaps the most important atherogenic effect of LDL oxidation is that this modification of LDL shifts recognition and internalization of the lipoprotein from the LDL receptor (LDLR) to scavenger receptors (295-297). While internalization of LDL by the LDLR in hepatocytes downregulates cholesterol synthesis to maintain cholesterol homeostasis, internalization of oxLDL by scavenger receptors fails to trigger this inhibition (298,299,300). Thus, cholesterol synthesis continues unabated despite the fact that peripheral cells are accumulating large amounts of cholesterol. In particular, macrophages express scavenger receptors and gluttonously take up large quantities oxLDL to form foam cells in the initial atherosclerotic lesion (301).

 

OxLDL also activates a number of cellular responses in macrophages, dendritic cells, endothelial cells, T cells, and smooth muscle cells that in aggregate promote inflammation, lesion formation, atherogenesis, and unstable atherosclerotic plaques (302-304). OxLDL induces surface expression of adhesion molecules and the release of chemokines from endothelial cells (305-311),  all of which are important steps in recruitment of leukocytes to sites of lesions. Exposure to oxLDL activates dendritic cells so that they induce T-cell proliferation and production of IL-17 (312). OxLDL itself also serves as a neo-antigen (313). OxLDL also induces increased antibody generation by lymphocytes (314). OxLDL also promotes smooth muscle cell proliferation, migration, and transition to a proinflammatory phenotype (315-318). OxLDL induces secretion by macrophages of inflammatory cytokines (e.g. TNF, IL-1, MCP-1, and IL-8) that activate other inflammatory cell types (319-322). OxLDL polarizes macrophages towards the M1-like phenotype or M2-like phenotype depending on its extent of oxidation (323). OxLDL promotes the chemotaxis of monocytes, neutrophils,  eosinophils, and T cells (314,324-327), bringing them into the arterial wall. In contrast, oxLDL inhibits macrophage emigration out of atherosclerotic lesions, because it induces netrin-1 (328). OxLDL induces apoptosis of macrophages and development of unstable plaques prone to rupture (329-331). Thrombotic arterial occlusion in the aftermath of plaque rupture is a critical cause of mortality, therefore the fact that oxLDL increases platelet aggregation (332-335)suggests an additional mechanism whereby elevated circulating oxLDL may increase risk of mortality during acute coronary events (336). As discussed in detail below, identification of cognate receptors for various components of oxLDL and other oxidized lipoproteins has provided important insight into the mechanisms by which these oxidized lipoproteins exert their pathophysiological effects.

 

MACROPHAGE SCAVENGER RECEPTOR (SR-AI)

 

In 1979, Brown and Goldstein demonstrated that macrophages had specific binding sites for acetylated LDL (AcLDL) that allowed uptake of this modified LDL even in the presence of high cellular cholesterol levels (298). This was in contrast to LDL uptake by the LDLR, which is markedly downregulated when cellular cholesterol levels rise (Figure 2). Cholesterol synthesis is also downregulated by LDL uptake by LDLR (337). The lack of feedback inhibition during uptake of modified LDL by this unidentified receptor suggested a plausible mechanism for the massive accumulation of cholesterol in macrophages that generates foam cells. The putative receptor mediating this binding was named the macrophage scavenger receptor (MSR). Later, oxLDL (338)and MDA-LDL (248)were shown to compete with AcLDL for binding and uptake by macrophages, suggesting they were native ligands for MSR. In 1990, Kodama et al. purified and sequenced this scavenger receptor, allowing identification of the MSRgene (339). Through alternative gene splicing, this gene gives rise to Scavenger Receptor A–I (SR-AI), SRA-II, and SRA-III.  Deletion of the MSR gene in C57BL6 mice fed butterfat diet substantially reduced atherosclerotic lesions and deletion of MSR in Ldlr-/-mice also reduced lesion formation (340).

 

CD36 AND OTHER SCAVENGER RECEPTORS

 

Subsequent work has shown that in addition to SR-AI, macrophages express a wide range of scavenger receptors that recognize oxidized lipoproteins including MARCO, scavenger receptor-B1, -B2, -B3 (CD36), and Lectin-like oxLDL Receptor-1 (LOX-1) (341). These scavenger receptors belong to a larger family of pattern recognition receptors, all of which are individually capable of binding to a wide spectrum of ligands. Quantitatively, SR-A1 and CD36 account for the vast majority of all oxLDL uptake by macrophages (342). The specific ligands of the two receptors on oxLDL appear to diverge (342).  SR-AI appears to preferentially recognize more rigorously oxidized LDL and seems to primarily recognize modified lysine residues like MDA-lysines. In contrast, the primary ligands of CD36 on oxLDL appear to be oxidized phospholipids, in particular fragmented phosphatidylcholine including azPAF (243), POVPC (343)and KOdiA-PC (280). Apoe-/- mice lacking CD36 are more vulnerable to some bacterial infections (344)but also have less atherosclerosis when fed a high cholesterol diet (345).

 

Recent findings suggest that SR-AI and other scavenger receptors have both pro- and anti-atherosclerotic effects, depending on the context. For instance, deletion of the MSR gene actually increased lesion size in male Apoe-/-mice (346); however, deletion of both MSR and CD36 greatly reduces lesion complexity and vulnerable plaques, the most critical aspect of lesion development (347). The complex results of scavenger receptor deletion should not be surprising given that scavenger receptors have multiple ligands and that an important role of scavenger receptors expressed by macrophages is to allow these macrophages to remove bacteria and damaged cells from surrounding tissues. Under normal physiological conditions, uptake of oxLDL by macrophages is probably generally protective, because subsequent efflux of the cholesterol from the macrophages to HDL via reverse cholesterol transport as well as emigration of these macrophages from the arterial wall to lymph nodes serves to minimize the accumulation of cholesterol-laden macrophages in the arterial wall. However, under conditions where reverse cholesterol transport capacity is reduced or where emigration of macrophages is inhibited, uptake of oxLDL by macrophages leads to its accumulation and initiation of pathophysiological processes.

 

TOLL-LIKE RECEPTORS AND OTHER TARGETS OF OXLDL

 

In addition to scavenger receptors, other pattern recognition receptors also recognize components of oxLDL. Perhaps most important among these are the Toll-like Receptors (TLR) including TLR-2 (348,349), TLR-4 (350), TLR-6 (351), TLR-7 (352), and TLR-9 (352).  TLRs can interact with scavenger receptors, for instance, CD36 forms complexes with TLR4 and TLR6 that recognize oxLDL and activate NFkappaB (351).While bacterial components such as bacterial lipopolysaccharide (LPS) are full agonists for TLRs, oxLDL components like POVPC often appear to act functionally as partial agonists of TLRs, so that activation of macrophages and dendritic cells by full agonists like LPS is reduced in the presence of oxLDL (353,354).

 

In addition to TLRs, another important pattern recognition receptor for oxLDL is the receptor for advanced glycation end-products (RAGE) (355). Other factors of the innate immune response that bind oxidized phospholipids including C-reactive protein(CRP) (356,357)and natural IgM antibodies like E06 (358,359). While scavenger and pattern recognition receptors tend to recognize broad classes of compounds, a number of G-protein coupled receptors (GPCRs) recognize specific oxidized phospholipids. These include the receptor for platelet-activating factor (PAFR) (360-362), prostaglandin receptor EP2 (363,364), and sphingosine-1-phosphate receptor 1 (S1P1) (365). Intracellular receptors for oxidized phospholipids include nuclear hormone receptors PPAR alpha (366)and PPAR gamma (243). Non-receptor, intracellular targets for oxLDL include c-SRC (367)and NRF-2 (368,369).

 

Mechanisms Protecting Against LDL Oxidation In Vivo

 

Given the susceptibility of LDL to oxidation, it is perhaps not surprising that a number of mechanisms appear to exist in order to protect LDL from oxidation. These include small molecule antioxidants circulating in plasma and enzymes that catabolize oxidized lipids. How essential each of these mechanisms are to the control of oxLDL levels and preventing the development of atherosclerosis remains an area of active investigation. Obviously, a better understanding of the relationship between changes in protective mechanism and atherogenesis might allow identification of particularly vulnerable individuals and the development of novel therapeutic approaches.

 

SMALL MOLECUE ANTIOXIDANTS

 

Circulating small molecule antioxidants such as ascorbate (vitamin C), alpha-tocopherol (vitamin E), urate, and bilirubin serve as sacrificial targets reacting with free radicals and reactive oxygen species to prevent lipid and protein oxidation. Thus, even when strong oxidants are added to plasma ex vivo, there is relatively little generation of oxLDL until the oxidants have depleted these small molecule antioxidants, most specifically ascorbate (370). Depletion of vitamin C and vitamin E increase atherosclerosis in Apoe-/-mice(371). Importantly, plasma ascorbate levels inversely correlate with prevalence of cardiovascular disease in humans (372). Supplementation with vitamin C appears to play a role in preventing endothelial dysfunction in humans (373). However, it is not clear that supplementing dietary antioxidants beyond those typically obtained in a well-balanced diet endows any additional atheroprotective effects. Supplementation with dietary antioxidants inhibits development of atherosclerosis in susceptible mice (374-378). While a few human trials with dietary antioxidants have demonstrated reduced atherosclerosis and cardiovascular disease (379-382), most large-scale trials have failed to demonstrate any disease reduction (383-387). The reasons underlying these failures continue to be investigated and debated (388,389). Because it had not been fully appreciated that relatively high doses of these antioxidants were needed to markedly alter lipid peroxidation rates in humans (390), one possibility is that the doses used in most large scale prevention trials were simply insufficient (390,391) . However, the ability to use very high doses of small molecule antioxidants like vitamin E for extended periods of times may be limited by the toxicity of these high doses (392).

 

ANTIOXIDANT ENZYMES

 

Antioxidant enzymes appear to play a more critical role than dietary antioxidants in limiting lipoprotein oxidation. Two families of nonheme peroxidases, the glutathione peroxidases and the peroxiredoxins, appear to be the most critical. Glutathione peroxidases (Gpx) 1-4 are selenoproteins that convert glutathione to glutathione disulfide while reducing peroxides (including lipid peroxides) to water (393,394). Polymorphisms in glutathione peroxidase 1 (Gpx1) are associated with increased risk for atherosclerosis in various human populations (395-397). Furthermore, genetic deletion of Gpx1 markedly exacerbates atherosclerosis in Apoe-/- mice(398,399), while overexpression of Gpx4 in Apoe-/-mice inhibits atherogenesis (400). Peroxiredoxins (Prdx) are cysteine containing proteins where the cysteine is oxidized to sulfenic acid during reduction of peroxides (401). Deletion of either Prdx1 or Prdx2 increases atherosclerosis in Apoe-/- mice(402,403). Overexpression of Prdx4 inhibits atherosclerosis in Apoe-/- mice (404). In contrast, overexpression of Prdx6 failed to inhibit atherosclerosis in C57BL6 mice fed an atherogenic diet (405).

 

In general, studies looking for associations between risk for atherosclerosis and polymorphisms or deficiencies in other major antioxidant genes including catalase, SOD-1, -2, and -3, and glutathione S-transferase have been negative (406,407). In fact, SOD-1 overexpression may even increase fatty streak lesions in mice (408). However, SOD-1 does inhibit proliferation and migration of smooth muscle cells induced by oxLDL in vitro   (315), and overexpression of both SOD-1 and catalase reduce atherosclerosis in Apoe-/-mice (409). Sod2+/-mice crossed with Apoe-/-mice have increased atherosclerosis compared to control Apoe-/-mice (410), but there is little effect on atherosclerosis of crossing Sod3-/-mice with Apoe-/-mice (411).  Several studies have demonstrated an association between SOD2and hypertriglyceridemia (412,413).

 

ENZYMES THAT CATABOLIZE LIPIDS

 

In addition to anti-oxidant enzymes, several enzymes specifically catabolize oxidized phospholipids including secreted Platelet-Activating Factor Hydrolases (sPAF-AH) and Paraoxonases (PON). sPAF-AH, also known as lipoprotein associated PLA2 (LP-PLA2) is a calcium independent PLA2secreted by macrophages that primarily circulates on LDL and to a lesser extent on HDL (414,415). sPAF-AH does not hydrolyze phospholipids with the typical long-chain fatty acids, but efficiently cleaves phospholipids with oxidatively fragmented (e.g. azPAF and POVPC) (362,416,417)or oxidatively cyclized (e.g. F2-isoprostane-PC) sn-2 chains (418). Whether this effect results in a net gain of pro- or anti-inflammatory lipids is controversial, because only some of these oxPL are highly potent inflammatory mediators, while others are partial agonists that might therefore antagonize inflammatory responses to other mediators like LPS. Furthermore, this hydrolysis generates lysoPC and lysoPAF, which are proinflammatory at high concentrations. This ambivalent effect is also seen in vivo. While a large number of clinical studies have found that increased sPAF-AH predicts increased risk for atherosclerosis (419,420), whether increased sPAF-AH actually contributes to atherogenesis or simply reflects a compensatory increase in response to elevated oxLDL is unclear (421,422). Some gene polymorphisms in sPAF-AH that reduce its activity (i.e. Val279Phe) appear to increase the risk of myocardial infarction (423), yet another polymorphism (i.e. Ala379Val) appears to have little effect (424). The interpretation that increased sPAF-AH activity caused an increased risk of atherosclerotic cardiovascular disease (ASCVD) led to the development of selective sPAF-AH inhibitors and their clinical trials (425). However, two recently completed phase III trials with one such inhibitor, darapladib, found that while this drug significantly reduced circulating PAF-AH activity, it had no effect on ASCVD events (426,427).

 

Paraoxonases (PONs) were originally named for their ability to hydrolyze the neurotoxin paraoxon and this activity is still routinely used to assay paraoxonase activity in plasma. However, in terms of atherosclerosis, the most important physiological function of PONs appears to be their ability to protect against LDL oxidation (428). PON-1 and PON-3 circulate bound to HDL. HDL treated with specific inhibitors of PON fails to protect LDL from oxidation (429). Treatment of oxLDL with purified PON1 markedly decreases its ability to induce endothelial cell activation and monocyte binding (264). Genetic deletion of PON1 markedly increases atherosclerosis in C57BL6 mice(430), and this is further exacerbated in Apoe-/-mice (431). Conversely, overexpression of PON-1 reduces atherosclerotic lesions in both wild-type mice fed high cholesterol diets and Apoe-/- mice (432). Adenovirus expression of PON-2 and PON-3 also inhibits atherosclerosis in Apoe-/-mice (433,434), indicating that all three PON enzymes have protective effects. However, transgenic Apoe-/- mice overexpressing the entire gene cluster of PON genes (PON-1, -2, -3) were not further protected compared to Apoe-/-mice with transgenic expression of PON-1 or PON-3 alone (435), suggesting these effects are redundant rather than additive. These mouse studies appear relevant to human disease, as a large number of studies have shown that polymorphisms in PON1 are associated with increased risk for atherosclerosis (265). It should be noted that PON activity varies greatly even in persons with the same polymorphism, suggesting that environmental factors leading to PON inactivation may also be important in determining disease risk.

 

Summary for Oxidized Lipoproteins

 

In summary, substantial evidence has accumulated over the past several decades for a causative role for oxidized lipoproteins in the initiation and progression of atherosclerosis and the need to reduce lipoprotein oxidation in order to reduce disease burden. Nevertheless, significant questions remain including which mechanisms are most important for driving lipoprotein oxidation, what treatment strategies can effectively reduce lipoprotein oxidation, and what are the key components of oxidized lipoproteins that drive atherogenesis?

 

ELEVATED LDL-C AND RISK FOR ASCVD

 

Genetic Causes of Elevated LDL-C

 

As described above, FH is an autosomal dominant inherited disorder associated with elevated levels of LDL-C and premature ASCVD, and provides some of the most compelling evidence for a causal role for LDL-C in atherosclerosis. Brown and Goldstein discovered the LDLR pathway and found that mutations in the Ldlrgene cause FH (300). Heterozygotes for loss-of-function mutations have cholesterol levels that are about twice normal, and these subjects are at increased risk of premature CVE. In contrast, individuals with homozygous FH have extremely high levels of LDL-C (> 500 mg/dL) and often develop severe coronary atherosclerosis and supravalvular aortic stenosis in early childhood. The prevalence of heterozygous FH is around 1/200-250 in the USA, whereas homozygous FH is extremely rare affecting only about 1/160,000 to 1/250,000 individuals (436). Nonetheless, about 20% of people having myocardial infarctions (MI) before 40 years of age have heterozygous FH. Thus, FH offers an important opportunity to target therapies to prevent atherosclerosis (437), but FH remains under recognized with recent evidence suggesting that only 1-10% of subjects with FH have been identified (438). Most individuals with significant hypercholesterolemia do not have classic monogenic autosomal dominant inherited dyslipidemias, but polygenic factors contributing to susceptibility to environmental factors underlie the observed increase in LDL-C levels.  A recent study suggests that among individuals with LDL cholesterol ≥190 mg/dl, gene sequencing identified a monogenic FH mutation in only <2%of subjects (439). However, for any observed LDL cholesterol, FH mutation carriers are at substantially increased risk for CAD (439). Pathogenic variants in three genes (LDLR, APOB, and PCSK9) account for the majority of monogenic FH cases. Recent genome-wide association studies (GWAS) have identified more than 50 discrete genetic loci that are associated with an increased risk of CVE(440,441). Many of these genetic loci are associated with genes previously known to impact LDL-C levels and cardiovascular risk (e.g. Ldlr, APOB, PCSK9), but novel loci that impact both LDL-C levels and risk for MI have also been identified, e.g. sortilin-1 (SORT1)(442,443). Most importantly, inherited low levels of LDL-C due to loss-of-function mutations in the PCSK9gene have been shown to be associated with dramatic reductions in risk for ASCVD events in the Atherosclerosis Risk in Communities study (444). Hence, genetic disorders of lipoprotein metabolism provide strong evidence that the impact of LDL-C on the development of atherosclerosis is dose- and time-dependent (445), supporting a causal role for LDL-C in atherosclerosis.

 

Lowering LDL-C Reduces ASCVD

 

Large randomized outcomes trials of cholesterol lowering drugs have provided critical proof of the cholesterol hypothesis (446). The Coronary Drug Project, conducted between 1966 and 1975, found niacin treatment showed modest benefit in decreasing definite nonfatal recurrent myocardial infarction by 26% (10.2% for niacin group vs 13.8% for placebo group) (447). However, there was no benefit in primary endpoint, total mortality. Impressively, with a mean follow-up of 15 years, nearly 9 years after termination of the trial, all-cause mortality was 11% lower in niacin group than in the placebo group (448). The Lipid Clinics Research trial was another early major outcomes trial to show that lowering cholesterol reduced cardiovascular events. Treatment with cholestyramine, a bile acid binding inhibitor, resulted in a 12% reduction in LDL-C levels and a 19% reduction in CHD events (449). The early lipid lowering cardiovascular outcomes trials were limited by a lack of highly effective approaches for lowering LDL-C levels, and several trials raised concerns that cholesterol lowering did not reduce total mortality and might increase the risk of cancer, accidental death and suicide (446). The advent of the statin drug class (HMG-CoA reductase inhibitors) provided a much more effective approach to lowering LDL-C and laid to rest the concerns raised by the earlier trials. The 4S trial was a landmark clinical trial of cholesterol lowering with simvastatin in patients with coronary artery disease (CAD) and severely elevated levels of LDL-C that was designed to look at total mortality as the primary endpoint (450). The 4S showed for the first time that lowering LDL-C levels by 35% with simvastatin resulted in a 30% reduction in total mortality with a 42% reduction in CHD deaths and a 34% reduction in the risk of Major Coronary Events (450). A large number of subsequent trials extended these results to populations with CHD with low levels of LDL-C and to subjects without known CAD (primary prevention) with high or low levels of LDL-C (451). It is important to note that the relationship between on-treatment LDL-C lowering and reduction in cardiovascular events in secondary prevention trials was similar for both statin and non-statin approaches to lowering LDL-C levels. A large meta-analysis of 26 statin trials involving over 170,000 subjects demonstrated that statin treatment for 5–years reduced the combined incidence of major coronary events, coronary revascularization, and stroke by 20% per every 1 mmol/l (38.7 mg/dL) reduction in LDL-C (452). These results have been extended by a recent large meta-analysis of 49 trials involving 9 different interventions to lower LDL that included more than 300,000 patients and approximately 40,000 major vascular events, each 1mmol/l (38.7mg/dl) reduction in LDL-C was associated with 23% relative reduction in the risk of major vascular events (453). This raised the question of whether further lipid lowering would be of additional benefit. With the recent development of proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, dramatic additional LDL lowering up to 50 -70% is now possible. In the FOURIER trial, patients with prior stable CAD who received the PCSK9 inhibitor, evolocumab, in combination with statin therapy achieved median LDL-C levels of 30 mg/dl. This was associated with a 15% reduction in the composite endpoint of cardiovascular death, MI, stroke, hospitalization for unstable angina or coronary revascularization (454). Similarly, ODYSSEY demonstrated that administration of alirocumab to acute coronary syndrome patients already on maximally tolerated statin therapy led to LDL-C values <50 mg/dl and was associated with a 15% reduction in the composite endpoint of death from coronary heart disease, nonfatal MI, ischemic stroke or unstable angina requiring hospitalization, and this benefit approached 24% in the subgroup of patients with initial LDL-C values >100 mg/dL (455). Together, the results of these PCSK9 trials reinforce the “lower is better” hypothesis.

 

Although statins are very effective in preventing CVE, many patients on statins do still have CVE, a phenomenon referred to as residual risk (456). This residual risk is likely attributable at least in part to inflammation. Indeed, definitive support for this hypothesis recently came from the Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS), where administration of an anti-IL1bantibody to patients with prior MI and elevated serum hsCRP successfully reduced recurrent CVE independent of lipid lowering (>90% of patients were receiving concurrent statin therapy) (457). A secondary analysis of the FOURIER trial also demonstrated that, while relative risk for the primary cardiovascular endpoint was consistent across groups, the absolute risk reduction with evolocumab was greatest in patients with elevated hsCRP (458). These results suggest that targeting both LDL and inflammation will provide the most robust strategy for lowering ASCVD risk.

 

Levels of LDL-C, ApoB-100, Non-HDL Cholesterol and LDL-P as Markers for ASCVD Risk.

 

Based on the strength of the direct association of LDL-C levels and risk for ASCVD, the guidelines for treatment of hypercholesterolemia have focused on LDL-C levels for risk assessment, stratification and treatment recommendations. Indeed, the terms LDL-C and LDL are often, though incorrectly, used interchangeably in practice. It is important to understand that LDL is a collection of particles defined by density (d = 1.019 – 1.063 g/ml) that are heterogeneous, consisting of a large variety of lipids and proteins (459). In addition, LDL particles vary in size and cholesterol content. The relationship between LDL-C levels and risk for ASCVD is “J-shaped”, and the predictive value of LDL-C levels is better at higher levels of LDL-C. Surprisingly, the majority of subjects presenting to the hospital with acute coronary artery syndrome do not have elevated levels of LDL-C, but tend to have low levels of HDL-C and elevated triglycerides (460). There has been tremendous interest in whether other measures of LDL, including subpopulations, apoB100, or particle number, might serve as a better predictors of CVE than quantifying LDL cholesterol content.

 

Groundbreaking studies by Krauss and co-workers (461)described two major patterns for LDL subpopulations based on size and density of the LDL particles. Pattern A is characterized by large buoyant LDL (lbLDL) particles, whereas Pattern B is associated with small dense LDL (sdLDL). Importantly, sdLDL is associated with increased triglyceride levels and low HDL-C, which is referred to as the lipid triad, a phenotype common in insulin resistance. Hence, Pattern B is commonly seen in subjects with obesity, metabolic syndrome and type 2 diabetes mellitus. A number of studies have reported that Pattern B is associated with an increased risk of CVE (462). Several different approaches have been used to characterize LDL phenotypes, including gradient gel electrophoresis, ultracentrifugation (sequential and vertical), ion mobility and nuclear magnetic resonance (NMR) (462,463). A number of mechanisms have been proposed to underlie the proatherogenic properties of sdLDL, including increased susceptibility of oxidation (464)and glycation (465), promoting arterial retention and increased macrophage foam cell formation. Cholesteryl ester transfer protein (CETP), which transfers CE from HDL to VLDL/LDL and triglycerides in the opposite direction, and hepatic lipase, which hydrolyses triglycerides, impacts the lipid composition and size of sdLDL. As such, increased levels of sdLDL have the potential to provide additional information regarding risk of CVE in individuals with normal LDL-C levels but elevated triglycerides and low HDL. Alternatively, it has been proposed that the real impact of sdLDL is due to increased LDL particle number.

 

Each LDL particle contains one molecule of apoB100, and the majority of apoB100 in plasma is on LDL particles (466). Hence, levels of apoB100 correlate directly with LDL particle (LDL-P) number. A large number of studies have shown that levels of apoB100 are superior markers of ASCVD risk compared to LDL-C (467). Because the mass of cholesterol in LDL particles varies, LDL-C levels will result in overestimation apoB levels and the number of LDL particles, when LDL particles are cholesterol-enriched (Figure 8) and underestimate apoB and LDL particle number when the particles are cholesterol depleted (Figure 8) (468).

Figure 8. Schematic of the Relationship Between Measurements of LDL-C Versus ApoB100 Particle Number in Concordant and Discordant Human Populations. Subjects with hypertriglyceridemia have enhanced numbers of small dense LDL where each particle is enriched in triglyceride (TG) and relatively poor in cholesteryl ester (CE) content compared to normal subjects, and measurement of LDL-C underestimates particle numbers and apoB100 levels. Other subjects have enlarged CE-enriched LDL particles and measurement of LDL-C overestimates the number of LDL particles and apoB100 molecules. Thus, LDL particle number or apoB100 levels are a more accurate predictor of cardiovascular risk in the setting of discordance between the percentiles for measures of cholesterol carried by LDL (LDL-C or Non-HDL-C) and particle number (apoB100 or LDL-P). Adapted from Sniderman, A.D. et al. Curr Opin Lipidol 2014, 25:461–467.

Furthermore, all of the major atherogenic lipoproteins contain apoB (LDL, triglyceride rich remnants of VLDL, IDL, chylomicron remnants, and Lp(a)). LDL-C is routinely calculated using the Friedewald formula (LDL-C = TC – HDL-C – TG/5), but this formula is not accurate when serum TG levels are > 400 mg/dl. It has long been recognized that LDL-C underestimates risk of ASCVD in the setting of hypertriglyceridemia (467). Non-HDL cholesterol is the mass of cholesterol in all of the apoB-containing particles: Non-HDL-C = TC – HDL-C. The ATPIII guidelines recommended using Non-HDL-C to estimate risk of ASCVD, when TG > 200 mg/dL (469,470). A meta-analysis by Sniderman et al.found that Non-HDL-C was a slightly better marker of ASCVD risk than LDL-C, but apoB was far superior to Non-HDL-C (471). NMR spectroscopy is another way to measure LDL-P concentrations. Table 2 includes selected percentiles for mean levels for the various LDL-related markers from the Framingham Offspring Study (472). In an analysis of the Framingham Offspring Study, LDL-P determined by NMR was more strongly related to incident CVD events than LDL-C levels, and the ability of Non-HDL-C to predict risk was less than LDL-P, but better than LDL-C (473). In addition, they found that low LDL-P numbers were a better index of low CVD risk than low LDL-C (473). In contrast, an earlier meta-analysis from the Emerging Risk Factors Collaboration found LDL-C, Non-HDL and apoB to be equivalent markers of CVE (474). The lack of difference may relate to the population studied. When the LDL particles have normal cholesterol content, then LDL-C, Non-HDL and apoB are equivalent markers (Figure 8) of risk (471,473). Interestingly, data from the Multi-Ethnic Study of Atherosclerosis (MESA) demonstrated that when LDL-C and LDL-P are discordant (Figure 8), then LDL-P proves to be a better predictor of risk for incident CVD events than LDL-C (475).

 

Table 2. Equivalent Percentiles in the Framingham Offspring Study
Percentile % LDL-C mg/dL Non-HDL-C mg/dL ApoB mg/dL LDL-P nmol/L
2 70 83 54 720
20 100 119 78 1100
50 130 153 97 1440
80 160 187 118 1820
95 191 224 140 2210
Adapted from Contois JH, et al. Clinical Chemistry 2009; 55:407-419

 

For several decades the guidelines for treatment of hypercholesterolemia have focused on LDL-C levels both for risk stratification and as the principal target of therapy to prevent ASCVD. Indeed, therapeutic goals for LDL-C of < 100 mg/dL and < 70 mg/dL for subjects at high-risk and very high risk of CVE, respectively, were recommended by the 2004 update of the NCEP ATPIII, guidelines (469,470). The 2013 ACC/AHA guidelines for treatment of hypercholesterolemia abandoned these targets in favor of recommending the use of high-intensity statins in high risk individuals (476), there are numerous sets of guidelines that have maintained the recommendation for LDL-C targets, including those of the National Lipid Association (NLA) (477), the American Association for Clinical Endocrinologists (478), and the European Guidelines (479). The Canadian guidelines include targets for levels of apoB(480), and the NLA guidelines include targets for both LDL-C and Non-HDL-C(477). Table 2 includes the percentiles for mean levels for these various LDL markers from the Framingham Offspring Study. The levels of LDL-C shown in Table 2 closely coincide with levels that have been widely used in guidelines for lipid management for decision-making regarding levels at which to initiate therapy and goals of therapy. The recent NLA guidelines recommend using both LDL-C and Non-HDL-C as targets of therapy with only two sets of targets: LDL-C < 70 mg/dL and Non-HDL-C < 100 mg/dL for very high-risk subjects and LDL-C < 100 mg/dL and Non-HDL-C < 130 mg/dL for high, moderate or low risk subjects (who qualify for drug therapy). Most recently, AHA/ACC published a new version of guidelines for cholesterol management (481). These guidelines included evidence from recent 2 large randomized clinical end-point trials of PCSK9 inhibitors (454,455)and a long-awaited ezetimibe trial in patients with recent acute coronary syndromes (482). The new guidelines re-introduced LDL-C treatment goals in some high-risk patient groups, such as those with high risk of ASCVD and those with very high baseline LDL-C. The new AHA/ACC guidelines also stated that elevated apoB particle number, elevated Lp(a) and hypertriglyceridemia are all additional risk factors for ASCVD.

 

Lp(a)

 

Lp(a) has been shown to be an independent risk factor for atherothrombotic events, including heart attack, stroke and peripheral vascular disease, in multiple prospective studies (451,483). The new AHA/ACC guidelines list elevated Lp(a) a one of the risk-enhancing factors for developing ASCVD (481). Lp(a) consists of an LDL particle in which apoB100 is covalently linked via a disulfide bridge to apo(a), a glycoprotein with repeating Kringle units that share homology with plasminogen. Although apo(a) is synthesized by the liver, the Lp(a) particles are not formed in the liver but in the plasma. Despite being a modified LDL particle, Lp(a) levels are independent of LDL-C levels. The catabolism of Lp(a) is poorly understood, but Lp(a) is not cleared by the LDLR (484).  The number of repeating Kringle units is highly variable but largely genetically determined, and this contributes to tremendous heterogeneity in size of Lp(a). The plasma levels of Lp(a) vary tremendously in humans, and plasma Lp(a) levels are generally inversely related to the size of the apo(a) isoform (485). Thus, smaller Lp(a) particles with fewer Kringle repeats are present at higher levels in the plasma. In American Caucasians, the increased levels of smaller Lp(a) particles is largely explained by the size of the LPAgene, based on the size of the repeated KIV2domain (486), which is believed to be due to difficulty of hepatic secretion of larger apo(a) isoforms. Nevertheless, this relationship varies in different ethnic populations. Early studies suggested that even though Lp(a) levels are higher in African Americans that Lp(a) levels did not appear to be an independent risk factor for cardiovascular events in this group(487). However, by determining allele specific Lp(a) concentrations, a larger more recent analysis demonstrated that elevated Lp(a) levels associated with small apo(a) isoform sizes serve as an independent risk factor for CHD in both African Americans and Caucasians (488). Similarly, a 20 year follow up study of the ARIC cohort found that elevated levels of Lp(a) are associated with a similar degree of risk in in both African Americans and Caucasians (489). A recent meta-analysis by the Emerging Risk Factors Collaboration evaluated 36 prospective studies with 126,634 subjects found that Lp(a) is an independent risk factor for CHD (490). In contrast to previous studies that suggested Lp(a) was only relevant as a risk factor when levels were extremely elevated, the meta-analysis demonstrated that risk and that Lp(a) levels are continuously associated with CHD risk (490). The Ile4399Met polymorphism (rs3798220) in the protease-like domain of apo(a) is particularly associated with increased risk for severe CAD (491). Subsequently, Clarke et al.found that the rs3798220 and rs10455872 variants were associated with small apo(a) isoform size, increased Lp(a) levels and substantially increased risk of CAD (492). Furthermore, a Mendelian randomization study by Kamstrup et al.demonstrated that a genetically determined doubling of Lp(a) plasma levels leads to a 22% increase in the risk of MI, strongly supporting a causal role for elevated levels of Lp(a) and risk for MI (493).

 

The proatherogenic mechanisms for Lp(a) remain incompletely understood, but recent studies suggest an important role for oxidative modification of Lp(a) by oxidized phospholipids (OxPL) (251). Mounting evidence supports an important role for OxPLs in the development of atherosclerosis (251). Interestingly, OxPLs associate with Lp(a) in preference to native LDL particles in human plasma (250). Hence a physiological role has been proposed for Lp(a) for binding and transporting OxPL in the plasma (251). Although, Lp(a) is found only in humans and Old-World monkeys, mice expressing human Lp(a) have been developed to examine the role of Lp(a) in atherogenesis and lipoprotein metabolism. The first transgenic mice expressing high levels of human apoB100 were created using a 79.5-kb human genomic DNA fragment containing the entire human APOBgene that was isolated from a P1 bacteriophage library, and crossing these mice with apo(a) transgenic mice produced high levels of human Lp(a) in plasma (494). In a study of transgenic mice expressing high and low concentrations of Lp(a), high levels of OxPLs were found in transgenic mice with very high levels of Lp(a), but not in LDL of apoB transgenic control mice (495). These studies support the concept of preferential transfer of OxPL to Lp(a). In the Dallas Heart Study, levels of OxPL on apoB were strongly correlated with Lp(a) levels, and inversely related to the size of the apo(a) isoforms (496). In the European Prospective Investigation of Cancer (EPIC)–Norfolk prospective study the impact of OxPL and Lp(a) levels on CHD risk was additive (497). Further studies are needed to define the extent to which the preferential binding of OxPL by Lp(a) is responsible for mediating the increased risk of atherothrombotic events attributable to Lp(a).

 

Lp(a) is considered an emerging risk factor, but the approach to managing patients with elevated levels of Lp(a) has not been well established. Elevated levels of Lp(a) do not respond well to changes in diet or statin therapy. Analysis of data from the Familial Atherosclerosis Treatment Study (FATS) showed that substantial lowering of LDL-C (with lovastatin plus colestipol or niacin plus colestipol) in subjects with CAD and high apoB100 eliminated the increased risk attributable to having very high Lp(a) (498). The JUPITER trial showed that treatment of subjects with low levels of LDL-C, but increased hsCRP, with rosuvastatin (20 mg) reduced CVE. In JUPITER, elevated Lp(a) was a significant determinant of residual risk, but the reduction in relative risk with rosuvastatin was similar among participants with high or low Lp(a) (499,500). Treatment with niacin reduces Lp(a) by 20-30%, and the European guidelines recommend treating patients with elevated Lp(a) who are at intermediate to high risk of CVD with extended release niacin to obtain levels of Lp(a) < 50 mg/dL (501). Nonetheless, the recent failure of the AIM-HIGH and HPS-2 THRIVE studies have cast doubt on the use of extended release niacin in subjects fitting the profile of those studies (CAD with LDL well treated on a statin). LDL apheresis is approved and effective for lowering Lp(a) in individuals with recurring CVE in the setting of very high levels of Lp(a). There are a number of new therapies that may prove useful in treating patients with elevated levels of Lp(a). The recently approved monoclonal antibodies to PCSK9 significantly lower Lp(a) by around 30% in addition to lowering LDL-C by 30-50%. Furthermore, a Phase 1 clinical trial of a second-generation antisense to apo(a) has recently reported potent, dose-dependent, selective reductions of plasma Lp(a) (502). This approach has the appeal of specifically targeting apo(a) to reduce Lp(a) levels. Hopefully, these new approaches will ultimately yield an effective approach to lower levels of Lp(a) that translates into reduced cardiovascular events.

 

INTESTINAL LIPID METABOLISM AND CHYLOMICRON ASSEMBLY

 

Intestinal Lipid Absorption

 

Through absorption of dietary lipids, the intestine is a key regulator of stored and circulating lipids. Primarily it is enterocytes in the small intestine that actively regulate the release of dietary lipids into circulation (503-505). The predominant lipids derived from diet are triglycerides, phospholipids and cholesteryl esters. In the intestinal lumen, ingested lipids are emulsified by bile salts to enhance their hydrolysis by lipases (Figure 9) (506-509). Triglycerides make up the largest percentage of the intestinal lipids. Lipolysis of triglycerides releases free fatty acids (non-esterified fatty acids) and monoacylglycerides (Figure 9). These are absorbed on the luminal surface of the enterocytes both by free diffusion and actively by protein-mediated transport into the enterocyte cytosol (Figure 9) (508-510). The principal transporters identified to date are CD36 (now known as SR-B2 (511)) and several fatty acid binding and transport proteins (512-514).

Figure 9. Intestinal Triglyceride and Cholesterol Metabolism. In the intestinal lumen, dietary triglyceride (TG) and cholesterol are emulsified by bile salts which enhance their uptake. Lipases in the intestinal lumen digest triglycerides to free fatty acids (FFA) and monoacylglycerides (MAG). These are absorbed into the enterocyte where they are used in the synthesis of TG, phospholipid and cholesteryl ester (CE). Much of the synthesized TG in enterocytes is packaged, along with phospholipids, cholesterol and proteins into chylomicrons, which are secreted at the basolateral surface of the enterocyte and enter the lymphatic system. The assembly of chylomicrons begins in the endoplasmic reticulum. During the synthesis of apolipoprotein B48 (apoB48), the protein acquires phospholipid from the endoplasmic reticulum membrane and also cholesterol and TG to form a primordial chylomicron. Continued acquisition of TG and CE and smaller, exchangeable proteins (e.g. apolipoprotein A-IV and apolipoprotein C-III) in the endoplasmic reticulum enlarges the particle to form a prechylomicron. Prechylomcirons are transported to the Golgi apparatus in specialized COPII vesicles. In the Golgi apparatus, the prechylomicron matures into a chylomicron. The maturation process includes the glycosylation of apoB48, the acquisition of additional proteins (e.g. apolipoprotein A-I) and lipid. Secretory vesicles formed from the Golgi carry the mature chylomicrons to the basolateral surface of the enterocyte. Fusion of the secretory vesicle membrane with the plasma membrane releases the chylomicron into the extracellular space where it is taken up into lacteals near the enterocyte and, thus, enters the lymphatic circulation. Dietary cholesterol in the intestinal lumen is taken into the enterocyte by a process involving Niemann-Pick C1-like protein 1 (NPC1L1). Enterocyte cholesterol and CE can be incorporated into chylomicrons and secreted with TG. In addition, enterocyte cholesterol can be directly excreted into the intestinal lumen using the heterodimer ATP-binding cassette transporter G5 and G8 (ABCG5/G8). Enterocyte cholesterol can also be transported to and incorporated into the basolateral membrane for efflux into the circulation.

Chylomicron Assembly and Secretion

 

In the enterocyte, the free fatty acids and monoacylglycerides are used to synthesize triglycerides, phospholipids, and cholesteryl esters (Figure 9) (508,509,513,515-517). The majority of the triglycerides formed in the enterocytes are repackaged into large, buoyant lipoproteins, called chylomicrons, and secreted from the basolateral surface of the cell (Figure 9). These particles play a central role in the transport of triglycerides and fat-soluble vitamins to the rest of the body (518).

 

The assembly of the chylomicron particle from precursors is a complex process. Each particle contains a single copy of apolipoprotein B48 and assembly begins with the synthesis of this protein in the rough endoplasmic reticulum. Apolipoprotein B48 is a truncated form of apolipoprotein B100 that is formed by posttranscriptional editing (519,520). As apolipoprotein B48 is synthesized and translocated across the endoplasmic reticulum membrane, it becomes lipidated to form a phospholipid-rich, dense primordial chylomicron in the lumen of the endoplasmic reticulum (Figure 9). The primordial chylomicron contains apolipoprotein B48, phospholipid, cholesterol and minor amounts of cholesteryl ester and triglyceride (513,521,522). The assembly process requires microsomal triglyceride transfer protein(523). In the absence of sufficient lipid, or if microsomal triglyceride transfer protein function is impaired, apolipoprotein B48 is ubiquitinated and targeted for proteasome degradation (524). The importance of this initiating assembly step is seen in patients with a defect in the MTP gene leading to the rare recessive disorder abetalipoproteinemia. Individuals with abetalipoproteinemia have almost undetectable levels of apoB or and very low total cholesterol levels in their plasma because of the inability to assemble apoB-containing lipoproteins in their enterocytes or hepatocytes. Among the sequelae experienced by these patients are accumulation of triglycerides in their intestines and livers and a deficiency of lipid-soluble vitamins in their plasma (525,526). If untreated, these patients develop severe neurological problems; mostly related to vitamin E and A deficiency.

 

After formation, the initial primordial particle expands by the acquisition of additional triglyceride and cholesteryl ester (Figure 9). The additional lipid is acquired by fusion with non-apolipoprotein B48 containing particles that are rich in triglyceride and cholesteryl ester. The exact origin of these lipid particles and their precise composition is currently actively debated (504,505,513,527,528), but the fusion of the primordial chylomicron with the apolipoprotein B48-free particles occurs in the endoplasmic reticulum (513). The resulting particle is a prechylomicron (Figure 9).  In addition to apolipoprotein B48, the prechylomicron surface can contain multiple copies of other small, exchangeable apoproteins including apolipoprotein A-IV and apolipoprotein C-III. Exchangeable apoproteins are soluble proteins that are not as tightly adherent to the particle surface and so can be exchanged between lipid particles.

 

Prechylomicrons are transported out of the endoplasmic reticulum and delivered to the Golgi apparatus for further processing (Figure 9). Transport occurs in specialized vesicles that can accommodate their large size. The unique vesicles contain a number of specific proteins necessary for the transport and docking process. Vesicle-associated membrane protein-7, coatomer protein II and Sar1b, a small GTPase component of the coatomer protein II vesicle assembly machinery (Figure 9)  are among the specialized proteins on the lipid transport vesicles (505,529-531). The maturation of the particle in the Golgi apparatus includes further glycosylation of apolipoprotein B48 and the addition of apolipoprotein A-I to the surface (505,532,533). After processing, the mature chylomicron is packaged into Golgi-derived secretory vesicles and transported to the basolateral surface and exocytosed into the lymph (Figure 9) (527,534,535).

 

The assembly of chylomicrons in enterocytes is a complex process requiring a number of coordinated steps and specific factors to work in unison. A failure in any of these can lead to lipid-related disease states. For instance, mutations in the SAR1B gene lead to retention of prechylomicrons within membrane-bound structures in the enterocytes (529). The condition is marked in childhood by decreased blood cholesterol levels, lipid accumulation in the enterocytes, chronic fat malabsorption with steatorrhea, and deficiencies in fat-soluble vitamin and essential fatty acids.

 

Chylomicron Cholesterol

 

Although chylomicrons are triglyceride-rich, they also carry substantial amounts of cholesterol (536,537). The cholesterol in chylomicrons comes from the general pool of enterocyte cholesterol. Enterocytes acquire cholesterol by uptake at the luminal surface, acquisition from lipoproteins at the basal lateral surface, and by de novo synthesis within the enterocyte. Niemann-Pick C1-Like 1 protein is a key component of the luminal acquisition machinery (Figure 9) (538), while the low density lipoprotein receptor appears to be a major mediator of cholesterol acquisition at the basolateral surface (539,540). The incorporation of cholesterol into chylomicrons contributes to the circulating levels of cholesterol, and increases in intestinal synthesis of chylomicrons due to increased dietary lipids contributes to cardiovascular risk and atherosclerosis, albeit by complex mechanisms (516,541,542).

 

Non-Chylomicron Intestinal Lipid Metabolism

 

Enterocytes can also regulate circulating lipids by means other than chylomicron secretion.  In the presence of excess fatty acids or cholesterol, the enterocyte can store excess lipid in their esterified forms (triglycerides and cholesteryl esters, respectively) within cytoplasmic lipid droplets (543-545). The neutral lipids in the droplets can subsequently be mobilized by hydrolysis as needed by the cell. The free fatty acids liberated from storage droplets can be incorporated into the chylomicron production pathway to become part of secreted chylomicrons.

 

Finally, the intestine also regulates circulating cholesterol levels by taking up excess circulating cholesterol and excreting it into the intestinal lumen for clearance in the feces. This process is known as trans-intestinal cholesterol excretion. It acts as an adjunct to liver biliary secretion and can account for as much as 30% of neutral sterol excretion (546). Trans-intestinal cholesterol excretion occurs at the luminal surface of the enterocytes by a process that primarily utilizes the ATP-binding cassette transporter pair ABCG5/G8 (Figure 9) but can use other pathways as well (547).

 

Summary

 

It is clear that intestinal lipid processing is a key contributor to the circulating levels of both triglyceride and cholesterol. Dietary, genetic and metabolic factors that disrupt the process of enterocyte lipid metabolism potentially can alter lipid homeostasis and produce disease states.

 

 

 

TRIGLYCERIDES, CARDIOVASCULAR DISEASE AND ATHEROSCLEROSIS

 

Causes of Hypertriglyceridemia

 

The prevalence of high circulating triglyceride levels is increasing worldwide, particularly in developed countries. In the United States there has been a greater than 7 fold increase in average plasma triglyceride concentration over the last 30 years (548). This increase coincides, in part, with increased instances of obesity and type 2 diabetes (T2DM) although the relationship of these conditions to hypertriglyceridemia is complex (549-553). Most classifications of hypertriglyceridemia are based, at least in part, on the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) (469). These guidelines classified circulating triglyceride levels <150 mg/dL as normal. Values between 150 mg/dL and 199 mg/dL are considered borderline high and anything above 200 mg/dL are classified as high, with those above 500 mg/dL deemed to be very high (469,470). Hypertriglyceridemia is generally the result of increases in one or more of the triglyceride-rich lipoproteins; chylomicrons, VLDL, or their remnants. The increase occurs because of increased synthesis, decreased catabolism or both, with the underlying cause generally being the result of alterations in metabolic factors such as apolipoprotein C-II, apolipoprotein C-III, CETP and lipoprotein lipase. However, hypertriglyceridemia can also be secondary to other disease states (e.g. diabetes mellitus, hypothyroidism, renal disease, and nephrotic syndrome) (548,554). Not surprisingly, environmental conditions, particularly a diet with high fat or high glycemic index content and in which energy intake is out of balance with energy utilization, are associated with hypertriglyceridemia as is excess alcohol consumption (548,554). In fact, dietary choices and lack of exercise are widely held to be a major contributor to the recent rise in circulating triglyceride levels in developed countries.

 

Hypertriglyceridemia as an Independent Risk Factor for Cardiovascular Disease

 

Individuals with elevated triglyceride levels are at increased risk for cardiovascular complications, particularly atherosclerosis (555,556). The Framingham Study was one of the first large studies to associate hypertriglyceridemia with cardiovascular disease, particularly in women (557). However, many other studies before and since have also shown a univariate association of high triglycerides and increased risk of cardiovascular disease. In many of these studies, however, the affect went away after accounting for other major risk factors (558-561),calling into question whether triglycerides represent an independent risk factor. For instance, a meta-analysis of the Emerging Risk Factors Collaboration data revealed triglycerides as a strong risk factor for cardiovascular disease and stroke, but, after adjusting for standard risk factors (primarily lipoprotein-associated cholesterol), the researchers concluded that triglyceride levels provided no additional predictive value (474). The authors did note, as have other studies (562-565), that patients with triglyceride levels above 500 mg/dL are at increased risk of pancreatitis; providing impetus for measuring triglyceride levels in patients and treating those with high levels irrespective of cardiovascular risk. The lack of strong association of triglyceride concentration with cardiovascular disease (after accounting for other risk factors such as elevated LDL-C and low HDL-C) has led some to question whether measuring triglyceride levels has any utility for cardiovascular patient management. In contrast, we argue below that there are a number of important reasons for evaluating triglyceride levels in patients, particularly those with cardiovascular disease, metabolic syndrome or diabetes.

 

First, we would point out that the difficulty in substantiating an independent association of triglycerides with cardiovascular disease may simply reflect the fact that a number of interrelated risk factors make it difficult to determine to what extent triglycerides independently contribute to cardiovascular events. One key issue is that, even in studies suggesting independence, the effect size has been small compared to traditional risk factors like LDL-C (548). Therefore, independence is very hard to detect in small studies. There are also issues regarding the way triglyceride levels are determined, the high variability of triglyceride concentrations in a single individual, and the association of triglyceride levels with other atherogenic conditions such as low HDL-C, obesity and T2DM (548,566-571). These confounding issues are not always considered by authors when drawing conclusions. Moreover, endpoints have differed widely among studies. Despite the confounding issues, an increasing number of case control studies do indicate triglycerides as an independent risk factor for cardiovascular disease even when adjusting for total cholesterol, LDL-C and HDL-C (572-578). The PROCAM study, for instance, found increases in risk for cardiovascular events as triglyceride levels increased and residual risk remained after accounting for other major risk factors (579), and the PROVE IT-TIMI 22 study revealed that triglyceride levels had a substantial impact on cardiovascular outcomes in patients with acute coronary syndrome that was independent of LDL-C (580). Moreover, Mendelian randomization studies strongly suggest a causal relationship between factors involved in regulating triglyceride rich lipoprotein levels and cardiovascular disease (581-583). For instance, analysis of data from the Copenhagen City Heart Study showed that genetic variants of lipoprotein lipase that resulted in reduced circulating triglyceride levels also reduced all-cause mortality (583).

 

Meta-analyses of randomized, prospective trials probably provide the strongest evidence for triglyceride levels as an independent risk factor. One such analysis assessing the effects of lowering circulating cholesterol levels with statins, indicated that in patients with preexisting coronary heart disease, there was a reduction in residual risk not associated with lowering LDL-C that could be related to other lipoproteins, such as triglyceride-rich lipoproteins (584). Most convincingly, a recent meta-analysis of 29 prospective studies showed that considering triglyceride concentrations yielded an adjusted odds ratio of 1.72 (95% Confidence interval=1.56-1.90) for those in the top tertile of triglyceride levels even after adjusting for other common risk factors (556). A similar odds ratio was reported in a meta-analysis that included data from 26 prospective studies in Asian and Pacific populations (585).

 

Given the increasing evidence that hypertriglyceridemia is indicative of increased cardiovascular disease risk, a key question is whether reducing triglyceride levels are protective. The results of several studies do suggest that reducing TG levels can reduce risk of cardiovascular events. An analysis of two secondary prevention trials of pravastatin suggests that high HDL-C and low triglycerides were significant predictors of reduced risk for CHD events (586). A recent meta-analysis of 18 trials evaluating the effects of fibrates on cardiovascular outcomes reported a 10% relative risk reduction for major cardiovascular events in individuals with hypertriglyceridemia alone or in combination with low HDL-C (587). Other meta-analyses have generally shown small but significant associations of low triglycerides and protection from cardiovascular events independent of other major risk factors (588).

 

Thus, the evidence is mounting for an independent role of circulating triglyceride levels in mediating cardiovascular risk and certainly has established the utility of determining triglyceride levels in at-risk patients. However, the studies also suggest that the association between high triglycerides and cardiovascular disease is complicated, multidimensional, and possibly indirect.

 

Is There a Direct Role for Triglycerides in Promoting Cardiovascular Disease?

 

If hypertriglyceridemia does directly affect cardiovascular disease, the mechanism(s) remain to be fully elucidated. Nonetheless, several hypotheses have been put forward. As the most prevalent form of cardiovascular disease, atherosclerosis has been the target for most explorations of a direct role for triglycerides in cardiovascular disease, and there is growing evidence, albeit circumstantial, that triglycerides can directly influence specific aspects of atherosclerotic lesion development. Many of the hypotheses are based on the fact that triglyceride rich lipoproteins (VLDL, chylomicron) also contain significant amounts of cholesterol (536)and could promote foam cell formation by contributing cholesterol to the lesion. Remnants of VLDL and chylomicrons are created by partial hydrolysis of their triglycerides through the action of lipoprotein lipase. These particles have an increased percentage of cholesterol(537,589)and can acquire additional cholesterol by transfer from HDL through the action of cholesterol ester transfer protein(CETP) (590). In hypertriglyceridemia, there is increased VLDL synthesis, delayed clearance and often increases in remnant particles (591,592). In fact, it has been argued that nonfasting triglyceride levels primarily reflect remnant lipoproteins, particularly in hypertriglyceridemia, and these particles may be the atherogenic moiety (593). Although chylomicrons and, to some extent, very low density lipoproteins are generally too large to cross the endothelial layer and invade the arterial intima, conversion to remnants allows these particles to accumulate within atherosclerotic lesions and to deposit their cholesterol (594-596). This would imply that levels of lipoprotein lipase, by increasing remnants, could influence atherosclerotic lesion development and there are animal studies showing just such a correlation (237,238,597). Evidence for the importance of remnants in atherogenesis also comes from individuals with type III hyperlipoproteinemia. Patients with type III hyperlipoproteinemia have decreased clearance of remnant lipoproteins and develop premature atherosclerosis (598).  ApoE is crucial for the normal clearance of chylomicrons and VLDL remnants, but the ApoE-2 isoform has reduced ability to bind to lipoprotein receptors and mediate clearance (599). Type III hyperlipoproteinemia occurs most often in subjects who are homozygous for APOE2, but the majority of E2/E2 individuals do not have the Type III phenotype, suggesting that a second hit is required to express the phenotype (600). Interestingly, rare genetic variants of APOE have been described that cause an autosomal dominant form of Type III hyperlipoproteinemia (601,602)and  ApoE deficiency in humans is extremely rare but is associated with the Type III phenotype  (600,603).

 

One mitigating factor in evaluating how much delivery of cholesterol in triglyceride-rich particles contributes to atherosclerosis is the fact that, although triglyceride-rich particles and their remnants contain large amounts of cholesterol, they also contain significant amounts of triglyceride. At least with respect to cellular cholesterol accumulation in macrophage foam cells (a hallmark of atherosclerosis), the presence of triglyceride in cells actually promotes the hydrolysis of cholesteryl esters to cholesterol (604,605). Cholesterol stored in foam cells is primarily in the form of cholesteryl esters. In order to be removed from the cell and eventually from the plaque, esterified cholesterol must first be converted to unesterified cholesterol (606). The presence of triglyceride intermixed with cholesteryl esters in foam cells facilitates the hydrolysis and removal of cholesterol (604,605,607). The differing effects of circulating triglyceride levels on cardiovascular disease risk and their cellular effects on cholesterol metabolism have yet to be reconciled.

 

There are mechanisms other than cholesterol delivery by which triglycerides could influence atherosclerosis. Lipolysis of triglyceride rich particles not only concentrates cholesterol in the particles it also produces free fatty acids and monoglycerides. Cell culture studies have demonstrated that long-chain fatty acids, particularly saturated fatty acids like palmitate and stearate, are cytotoxic (608-610). Thus, the presence of triglyceride lipolysis within atherosclerotic lesions could raise toxic free fatty acid levels in cells of the arterial wall, which would promote cell death and resulting inflammation. Both increased cell death and increased inflammatory signaling are key attributes of atherogenesis (611-614). In support of triglyceride lipolysis as an atherogenic driver, macrophages make and secrete lipoprotein lipase (lipoprotein lipase) and it is estimated that macrophages are the primary source of lipoprotein lipase in atherosclerotic plaques (615). Localized lipolysis of triglyceride-rich lipoproteins and their remnants can also liberate other oxidized fatty acids, which can promote cytotoxicity and inflammation (616-619); key players in atherosclerotic lesion development. Increases in macrophage lipoprotein lipase do stimulate macrophage cytotoxicity (620), while diminution of macrophage lipoprotein lipase in mice reduces atherosclerotic plaque size (237,621,622). Thus, localized hydrolysis of triglyceride-rich particles by macrophages have the potential to produce cytotoxic and inflammatory effects.

 

It is also becoming clear that the dietary fatty acid composition of lipoproteins, including triglyceride-rich lipoproteins, affects their metabolism in complex and not completely understood ways. The fatty acid composition of lipoproteins (as well as phospholipids and cholesteryl esters) is strongly influenced by dietary intake of fatty acids. Although dietary intake of saturated fatty acids is popularly believed to be bad, whether consuming saturated fat, per se, increases cardiovascular risk is somewhat controversial based on available evidence (623,624). However, in subjects with FH, increased saturated fat in the diet clearly increases LDL-C levels. What also appears clear is that replacing saturated fatty acids in the diet with polyunsaturated fatty acids (PUFA) reduces cardiovascular events (623-627). Omega-6 PUFA are the primary PUFA found in western diets. There is evidence these lower triglyceride levels, in part, by increasing lipolysis of triglyceride-rich lipoproteins (628). Omega-3 PUFA are the other major source of dietary PUFA. Fish are a rich source of long-chain omega-3 PUFA, and there is compelling evidence that omega-3 PUFA (at least from marine sources) reduce both triglyceride levels and cardiovascular risk (629-631). A recent large scale randomized controlled trial (REDUCE-IT) using an EPA only fish oil product reduced major cardiovascular event by 25% in patients who have hypertriglyceridemia (632). Replacing saturated fat with monounsaturated fatty acids may provide some reduction in cardiovascular events, but PUFA appear to have a stronger correlation with improved cardiovascular risk compared to monounsaturated fatty acids (633-635). In contrast to cis fatty acids, trans unsaturated fatty acids, which are common in processed foods, have been convincingly associated with increased cardiovascular risk (623,636,637). Given this and other evidence, a recent report from the National Lipid Association’s Expert Panel recommends, for patients with low or moderate risk for cardiovascular disease, that intake of saturated fatty acids be reduced to <7% of total energy and trans fatty acids should be avoided (638). The reduction in saturated and trans fats should be replaced with PUFA, protein and carbohydrate (638). The guidelines also suggest eating fish twice weekly. For individuals with high triglyceride levels, the Expert Panel also recommends supplementation with omega-3 polyunsaturated fatty acids from marine sources (638). The 2018 AHA/ACC listed persistent hypertriglyceridemia as a risk enhancer for developing ASCVD and recommend using omega-3 fish oil for individuals with high triglyceride levels to prevent pancreatitis. However, the AHA/ACC guidelines did not include the evidence of the REDUCE-IT trial. Therefore, in these individuals with hypertriglyceridemia and other risk factors for ASCVD, one should consider initiating omega-3 fish oil or intensifying statin therapy(481).

 

Lipoprotein lipase-mediated hydrolysis of triglyceride is not the only mechanism in the artery wall for the metabolism of triglyceride rich particles to produce potentially atherogenic compounds. The foam cell macrophages are also capable of the endocytic uptake of VLDL and remnant particles, which can then be catabolized in the lysosome (Figure 2) (639-642). Interestingly, there is evidence that under atherogenic influences, including macrophage sterol engorgement, the route of triglyceride metabolism in macrophages can shift to favor endocytic delivery of triglyceride-rich lipoproteins rather than surface hydrolysis (641,643). Whereas surface hydrolysis of triglycerides by surface lipases primarily delivers only free fatty acids to cells, endocytic uptake of particles would include the delivery of the particle’s full content, including its sterol, which would exacerbate foam cell sterol accumulation.

 

Another potential way that triglyceride-rich lipoproteins could influence atherosclerosis focuses on the apolipoprotein CIII content of VLDL and remnants. ApoCIII inhibits lipoprotein lipase, inhibits remnant uptake by the liver, and its levels are associated with hypertriglyceridemia (644-648). Thus, high apolipoprotein CIII concentrations could promote arterial retention of VLDL and remnants making them more atherogenic, suggesting apolipoprotein CIII as a therapeutic target. In fact, individuals with certain mutations in APOC3 have low triglycerides and LDL-C (649,650). Two recent studies show that loss-of-function mutations in apoCIII lowered serum triglycerides by >39%, significantly reduced LDL-C and raised HDL-C, and lowered the incidence of cardiovascular events by >36% (651,652). An antisense oligonucleotide selective inhibitor of apoCIII has been developed that lowers serum apoCIII and triglycerides in mice, non-human primates, and humans and is currently in a phase 2 clinical trial (653).  These studies indicate that reduction of apoCIII by antisense oligonucleotide inhibition significantly reduces circulating triglyceride levels (654,655). Besides their effects on circulating lipids, Apo CIII-containing lipoproteins also stimulate a range of processes including activation of monocytes, inflammation, endothelial cell NO production resulting in vascular dysfunction and increased lipid oxidation and binding of lipoproteins to PG which can stimulate macrophage foam cell formation (227,239,656-658).

 

A final way in which triglyceride levels could influence atherogenesis is related to the finding that patients with hypertriglyceridemia also tend to have increased circulating levels of thrombotic factors such as fibrinogen and plasminogen activator inhibitor and inflammatory mediators (TNF-alpha, IL-6, VCAM-1 and MCP-1) (659-661). Thrombosis and inflammation are key factors in atherosclerosis and its progression to heart attack and stroke.

 

Reducing Circulating Triglyceride Levels

 

It is clear, therefore, that there are a variety of ways in which the triglyceride-containing particles in hypertriglyceridemic plasma could contribute either directly or indirectly to multiple aspects of atherosclerotic lesion development. Regardless of whether triglycerides are directly causative of cardiovascular disease, the evidence is mounting that assessment of triglyceride levels has an important role in evaluating and managing cardiovascular risk, and treating elevated triglyceride levels may reduce risk for cardiovascular events (548,662). This is particularly true for patients with coronary heart disease or diabetes (548,662-664). Several agents have shown efficacy in reducing triglyceride levels and also in reducing cardiovascular disease risk. The reduced risk is thought to occur to a large extent by reducing atherosclerosis. Currently, therapeutic agents recommended for treating hypertriglyceridemia are fibrates, statins, niacin and omega-3 PUFA but others are being developed. Unfortunately, clinical trials of the impact of triglyceride lowering medications on cardiovascular events in subjects with severe hypertriglyceridemia have not been undertaken.

 

Fibrates are the most effective approach for directly lowering triglyceride levels. Fibrates have been shown to lower triglyceride levels by 30%-50% depending on the baseline levels (548). More importantly, fibrate therapy with gemfibrozil has been shown to reduce cardiovascular risk in patients with elevated triglycerides (665-667). Unfortunately, the trials of combination therapy of statins with fenofibrate have failed to meet their primary endpoints in terms of reducing cardiovascular events (668,669). However, posthoc analysis of all of the fibrate trials show significant benefits in terms of reducing CVD events, when looking at the subgroup of patients with elevated triglycerides and low HDL-C and features of the metabolic syndrome or diabetes (670,671).

 

Statins inhibit HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis, and produce dramatic reductions in LDL-C levels; typically from 20%-60% depending upon the particular statin used and dosage (672). However, they also reduce circulating levels of triglycerides (673)and Non-HDL-C (477). Levels of LDL-C are often low in the setting of hypertriglyceridemia, so Non-HDL-C levels are a more useful measure of the burden of atherogenic apoB-containing lipoproteins than LDL-C in patients with hypertriglyceridemia. Indeed, the National Lipid Association recommends using goals for Non-HDL-C levels of < 130 mg/dl or < 100 mg/dl, in subjects at high- and very-high risk of cardiovascular events, respectively (477).  Niacin is a B vitamin that can lower overall circulating lipid levels when given in high doses. The mechanism of action is not entirely clear but niacin reduces VLDL production by the liver. Unfortunately, clinical trial data regarding the use of niacin on cardiovascular outcomes in severe hypertriglyceridemia is lacking. Although in a subgroup analysis of the AIM-HIGH trial, niacin showed a trend toward benefit in the tertile of subjects with the highest triglycerides (>198mg/dl) and lowest HDL-C (<33mg/dl), which is consistent with the post hoc analysis of fibrate trials (674). Finally, evidence indicates that daily intake of omega-3 PUFA from marine sources (which primarily containing eicosapentaenoic and docosahexaenoic acids) can significantly reduce circulating triglyceride levels (675-678). Treatment with 3 – 4 grams a day of omega-3 PUFA (EPA+DHA) is effective in lowering triglycerides. A large meta-analysis of omega-3 FA in 20 studies including 63,000 participants did not see an impact on a combined cardiovascular endpoint or coronary events, but there was a reduction in vascular death (679). However, most omega-3 outcome trials used less than one gram of omega-3 PUFA, which was probably too low of a dose to have meaningful triglyceride lowering effects that could yield clinical efficacy. The JELIS trial, compared the effect of 1.8 g EPA vs. placebo on top of statin in a hypercholesteremic but relatively normal triglycerides patient population (mean LDL-C 182 mg/dl and triglycerides 151 mg/dl) (95). JELIS found a 19% relative risk reduction in CV events but a more pronounced 53% reduction in the subgroup with mixed dyslipidemia, specifically the subgroup with triglycerides >150mg/dl and HDL<40 mg/dl(680). Most recently, a large randomized controlled trial (REDUCE-IT) of an EPA only fish oil product reduced major cardiovascular event by 25% in patients who have hypertriglyceridemia (632). Whether the clinical benefit was confined to EPA only or it can be generalized to all omega-3 PUFA is still yet to be determined. In contrast to marine-derived omega-3 PUFA, plant-derived omega-3 PUFA have generally not shown efficacy for lowering triglycerides(681,682). A number of new approaches for treating hypertriglyceridemia are in development including antisense oligonucleotides to ApoC3(654,655).

 

Summary

 

The association of elevated triglyceride levels and cardiovascular disease has been well established (555,556,579,588). What remains a subject of ongoing debate is the extent to which triglycerides directly promote atherogenesis or, alternatively, simply represent a biomarker for other processes that influence cardiovascular risk. (542,548,554,561,570,592,683,684). Nonetheless, the evidence supports measuring triglycerides and including triglyceride and Non-HDL-C reduction in treatment regimens is strengthening especially in patients with metabolic syndrome, diabetes, or cardiovascular disease.

 

HDL METABOLISM AND ATHEROSCLEROSIS

 

HDL and Reverse Cholesterol Transport

 

Apolipoprotein A-I (apoA-I) is the major protein on HDL and provides both structure and function. Lipid-poor apoA-I and mature HDL both contribute to removing cholesterol from macrophages and prevent foam cell formation (Figure 2). Although cholesterol flux from macrophages to HDL (or apoA-I) alleviates cholesterol-accumulation in lesions, the net flux of cholesterol from the lesion has little to no effect on systemic cholesterol levels. Nevertheless, macrophage cholesterol efflux to HDL reduces inflammation and the atherosclerotic burden, and is the first step in reverse cholesterol transport (RCT) (Figure 10) (685-687). This pathway was first described in 1966 (688). The rate at which cholesterol flows through the RCT pathway is of greater importance than steady state levels of HDL-cholesterol (HDL-C). Interestingly, cholesterol movement from macrophages to HDL occurs through at least 4 routes (70). First, lipid-poor apoA-I stimulates the efflux of phospholipid and free cholesterol through interaction with ATP-binding cassette transporter A1 (ABCA1) (Figure 2), which generates pre-beta HDL and nascent discoidal particles (689). The more lipidated the apoA-1 becomes, the discoidal HDL particles transition into a spherical structure and lose their ability to interact with ABCA1 and stimulate cholesterol efflux through ABCA1. Both discoidal HDL particles and mature spherical HDL particles can also promote free cholesterol efflux from another transporter, ATP-binding cassette transport G1 (ABCG1), which is thought to reside on sub-cellular organelles as opposed to the plasma membrane (Figure 2) (690,691). This transporter is a critical regulator of intracellular cholesterol trafficking cellular cholesterol availability, and cholesterol export (690,692). HDL’s primary receptor for cholesteryl ester (CE) uptake, scavenger receptor BI (SR-BI), is also a bidirectional free cholesterol transporter in that it facilitates the efflux and influx of free cholesterol between cells and mature HDL (693-695)(Figure 2). The net direction of cholesterol flux is determined by the cholesterol concentration gradient (plasma membrane and HDL ratio of free cholesterol to phospholipid) (696)as well as by the phospholipid subspecies (697,698). Finally, cholesterol can simply move from the plasma membrane to HDL through passive aqueous diffusion, which is a major route of cholesterol efflux from macrophages (Figure 2) (70,687,695). On HDL free cholesterol is solubilized in the phospholipid surface layer and is rapidly esterified by lecithin:cholesterol acyltransferase (LCAT) (Figure 6), and the hydrophobic CE is then mobilized to HDL’s core (699,700).

Figure 10. Beneficial Functions of HDL. HDL mediates a number of atheroprotective processes. HDL is critical in reverse cholesterol transport where it mediates the first step of removing cholesterol from the periphery and macrophage foam cells for clearance by the liver. HDL can directly mediate the last step in reverse cholesterol transport by delivering cholesterol to the liver via interaction with SR-BI. HDL reduces LDL oxidation and cell oxidative status by removing lipid hydroperoxides from LDL and cells. HDL also prevents LDL oxidation via its anti-oxidant enzymes (PON1, LCAT, and Lp-PLA2) and by the reduction of lipid hydroperoxides by apoA-I. HDL maintains the endothelial cell barrier by stimulating vasorelaxation resulting from enhanced nitric oxide production from HDL induced signaling via a number of endothelial cell receptors (SR-BI, S1P, ABCG1). HDL prevents thrombus formation by inhibiting coagulation factors and by stimulating efflux of cholesterol from platelets via SR-BI to reduce platelet aggregation. HDL prevents endothelial cell and macrophage apoptosis by signaling pathways which modulate expression of the pro-apoptotic protein, Bid, and the anti-apoptotic factor, Bcl-xl. HDL also reduces apoptosis susceptibility by alleviating endoplasmic reticulum stress by removing excess free cholesterol and lipid hydroperoxides from cells. HDL limits atherosclerotic lesion inflammation by inhibiting endothelial cell activation resulting in less monocyte recruitment. HDL also reduces lesion inflammation by promoting the macrophage anti-inflammatory M2 phenotype via ABCA1/ JAK2 signaling to enhance anti-inflammatory cytokine production (IL-10, TGF-β). HDL inhibits conversion to the macrophage inflammatory M1 phenotype by preventing antigen-specific activation of T helper 1 (Th-1) cell to produce interferon gamma. HDL contains an array of proteins and bioactive lipids that regulate HDL function. In addition, HDL controls a number of atheroprotective processes by modulating gene expression by transferring microRNAs to recipient cells.

Spherical mature HDL then transports CE to peripheral cells and tissues, and back to the liver as part of the RCT pathway (Figure 10). HDL delivers CE to the liver through 2 primary routes. HDL delivers CE to the liver through binding to SR-BI (Figure 6), which drives selective uptake of core lipids (694). Another major route of cholesterol delivery to the liver is mediated through LDL and the LDL receptor (LDLR) (Figure 6) (701). In the circulation, HDL exchanges CE for TG from VLDL and LDL through cholesteryl ester transfer protein (CETP) activity (Figure 6), and this action is responsible for directing CE through the LDL receptor pathway (702). Besides these major routes holoparticle uptake of HDL may also contribute to delivery of HDL-CE to the liver. Hepatocytes, and many other cell types in other tissues, likely participate in HDL retro-endocytosis where apoA-I or HDL particles are taken up by endocytosis and resecreted without degradation in late endosomes and lysosomes (703,704). SR-BI and CD36 may participate in this process as well as other potential HDL receptors (705-707). For example, the F0F1ATPase and P2Y13receptor have been reported to facilitate the uptake of the entire HDL particle(703,704,708,709). The liver then excretes both cholesterol and bile acids-derived from cholesterol into the bile which are removed from the body in feces, thus completing RCT from peripheral macrophages to bile through HDL and the liver (710). Recent evidence suggests there is also likely an HDL-independent pathway for systemic cholesterol removal through transintestinal cholesterol excretion (TICE) (711). Historically, HDL’s anti-atherogenic properties were largely attributed to HDL’s role in RCT and removing excess cholesterol from macrophages and peripheral tissues; however, continually emerging alternative HDL functions likely significantly contribute to HDL’s protection against CVD.

 

HDL Levels and Risk of CVD

 

Historically, HDL-C was synonymous with the term HDL; however, the amount of cholesterol in the HDL pool (HDL-C) and the number and quality of HDL particles (HDL-P) are independent concepts that are important to consider in the context of HDL function. Several decades of high-quality epidemiological studies have clearly shown that HDL-C levels are inversely correlated to CVD risk and events, independent of race, gender, and ethnicity (712). In well-controlled studies assessing CVD risk using multivariate approaches to adjust for covariates, both apoA-I and HDL-C are strong independent predictors of CVD risk (474). Nonetheless, HDL-C levels are also inversely correlated to insulin resistance, obesity, and triglycerides. As such, HDL-C’s causality in protection from CVD is difficult to define and is somewhat controversial, mainly due to epidemiological discrepancies between the dose-response of HDL-C levels to CVD outcomes. It is possible that HDL-C levels may simply be a biomarker for CVD and not play a causal role in atherosclerosis; however, an increasing number of functional studies clearly support HDL’s functional relevance in biochemical mechanisms of atherosclerosis. In any case, epidemiological studies over the past 50 years have provided many insights into HDL-C and CVD risk. The first evidence came from the Framingham Heart Study in 1966 demonstrating a link between HDL-C and ASCVD (713). In 1975, HDL-C levels were found to be inversely associated with CVD in a Norwegian trial (Tromso Heart Study) (714). In subsequent years, the Honolulu Heart Study (1976) (715)and Framingham Heart Study (1977) (559)both reported that many CVD patients had low HDL-C levels. Over the years, low HDL-C levels have consistently been reported to be associated with increased risk of ASCVD and events (716-718). By the late 1980s and early 1990s, the relationship between HDL-C and CVD was generally accepted, as studies during this period established that low HDL-C levels were associated with CVD risk independent of other risk factors even in patients with normal total cholesterol levels (719-721).

 

Clinical Outcomes Trials

 

Prior to the statin-era, results from randomized controlled clinical trials suggested that increasing HDL-C levels 1 mg/dL or 1% reduces mortality from CVD by 3-4% (722,723). In the Air Force/Texas Coronary Atherosclerosis Prevention Study (AFCAPS/TexCAPS), treatment of men and women withaverage TC and LDL-C levels and below-average HDL-C levels with lovastatin (20-40 mg) reduced LDL-C by 25% and raised HDL-C 6%, resulting in a 37% reduction in the risk for the first major acute coronary event (724). These results showed that statin therapy was effective in reducing risk for CVE in subjects with low-HDL-C. The extent to which the benefit came from HDL-C raising is unclear. Studies completed in subjects on statins have yielded inconsistent results with regard to the importance of raising HDL-C partly due to evidence suggesting that statin (fluvastatin) use in low HDL-C subjects decreased coronary artery disease (CAD) with little to no increase in HDL-C levels (725-727). In the fluvastatin regression study, low HDL-C subjects on placebo showed increased disease (angiographic) progression compared to subjects with high HDL-C levels (727). Collectively, evidence from these and a large number of epidemiological studies overwhelmingly support a clear inverse association between HDL-C levels and CVD risk. This is demonstrated clinically as raising HDL levels through injections of reconstituted HDL (rHDL) resulted in atherosclerotic plaque regression, as determined by intravascular ultrasound (728). A number of animal studies clearly support the HDL-C hypothesis. For example, raising HDL in mice and rabbits consistently blocks atherogenesis( 729-731). However, raising HDL-C levels by mono- or combined therapy to reduce risk and events has proven challenging. Two major clinical outcomes trials of raising HDL with niacin failed to show a benefit. In subjects with CAD with LDL-C levels well controlled with a statin, the addition of extended release niacin in AIM-HIGH (732)and extended release niacin plus laropiprant (prostaglandin D2 receptor blocker to inhibit flushing) in HPS-2THRIVE (733)failed to reduce cardiovascular outcomes. However, structural limitations of the two Niacin trials design complicated their interpretation (734). In addition, major cardiovascular outcomes trials of 3 CETP inhibitors torcetrapib (735), dalcetrapib (736), evacetrapib have now failed to show a benefit in reducing cardiovascular events. More recently, the CETP inhibitor (anacetrapib) was tested in the REVEAL trial, which was a positive outcomes trial (737). However, the benefit of anacetrapib in reducing CVE seems to be largely explained by lowering of non-HDL, rather than increases in HDL-C (738). More recently, two recombinant apoA-I products MDCO-216 and CER001 showed no benefit in imaging studies (739,740). Collectively, the failure of these clinical studies has raised doubts about the HDL hypothesis.  Indeed, raising HDL-C is presently not a primary target for therapeutic intervention. Nevertheless, HDL infusion in humans has been reported to improve endothelial function, which should contribute to inhibiting atherogenesis (741). At this time, HDL particle infusion therapies have not been proven to be an effective approach to reduce cardiovascular events (742); however, clinical trials with reconstituted HDL are still ongoing. Furthermore, recent studies indicate that HDL particle number and cholesterol efflux capacity are better indicators of CHD risk than HDL-C levels (743,744). The therapeutic targeting of HDL non-cholesterol cargo, quality, and function are emerging and gaining support, as HDL have many other biological properties that likely contribute to prevention of atherosclerosis and CVD (745). In addition, quantifying HDL function, including cholesterol efflux capacity, will provide a better risk index than steady-state HDL-C levels (746).

 

Particle Number and Cholesterol Efflux

 

A major blow to HDL causality in atherosclerosis comes from genetic studies. Mendelian disorders resulting in very low HDL-C levels have yielded conflicting data, as mutations to critical lipoprotein genes (e.g. apoA-I) were found to be associated with protection from atherosclerosis in one study (747)and increased risk in another study (748). The ApoA-I Milano mutation is associated with low levels of HDL-C and reduced risk of CVD (747). Infusion of recombinant apoA-I Milano was reported to induce regression of atherosclerosis (749), but there has not been clear progress in developing it as an approach to therapy since the initial regression study was published. The evidence that some genetic causes of low HDL-C are associated with increased risk for premature atherosclerosis, whereas others are not, supports the notion that HDL function may be more important than HDL-C levels. Nonetheless, Mendelian disorders of low HDL-C levels are rare, and thus the sample sizes in these studies are limited and it is difficult to draw accurate conclusions. To address this issue, genome-wide association studies were completed to attempt to resolve if HDL-C is a risk index or causal factor. These studies are limited in that many variants that raise or lower HDL-C levels also affect other lipoproteins, namely LDL-C levels. For example, variants in CETPraise HDL-C levels and reduce LDL-C levels, which complicates risk prediction based on HDL-C levels (750). Nevertheless, studies have found that variance solely associated with HDL-C levels is not linked to cardiovascular events. For example, single nucleotide polymorphisms (SNPs) in endothelial lipase (LIPG), which raises HDL-C levels, are not associated with decreased CVD(751).

 

As failed clinical trials aimed at raising HDL-C levels and genetic studies do not uniformly support causality for HDL-C in CVD, HDL functional tests in future prospective studies will likely provide more resolution to HDL’s causal role in CVD. Cholesterol efflux capacity, a marker of HDL function, has been reported to be inversely associated with CVD risk independent of HDL-C levels (744,746). This was first demonstrated in a cross-sectional study using radio-tracing of cholesterol efflux (746). A subsequent study also found an inverse association between HDL efflux capacity and atherosclerosis, but reported a positive link to cardiovascular events (752). In a third study assessing HDL cholesterol efflux in a US cohort using a fluorescence method, efflux was again linked to decreased risk of CVD (743). Recently, HDL cholesterol efflux capacity was found to be inversely associated with CVD risk and events in a large nested case-control prospective study (n=3,494 subjects) from the EPIC-Norfolk Study (744,753). These associations were independent of many other co-founding factors, including HDL-C, T2DM, obesity, LDL-C, and age amongst others (744).

 

In addition to HDL cholesterol efflux and functional indices as risk predictors, HDL particle number (HDL-P) has also been reported to provide biomarker potential. HDL-P numbers can be quantified using nuclear magnetic resonance (754)or calibrated ion mobility assays (755). HDL-P was found to be inversely associated with carotid intima medial thickness (cIMT) and coronary heart disease (CHD) independent of LDL particle numbers and HDL-C levels in the large multi-ethnic study of atherosclerosis (MESA) (756). Importantly, HDL-P remains inversely associated to CHD after adjusting for triglycerides and apolipoprotein B (apoB), thus suggesting that HDL-P is far superior to HDL-C levels as a biomarker of ASCVD and events (757,758). Furthermore, neither HDL-C levels nor HDL-P levels correlate to cholesterol efflux from macrophages; therefore, the rate of cholesterol efflux is still critical to understanding RCT and HDL function. Likewise, HDL quality is more important than apoA-I levels, which also do not correlate with HDL function, e.g. RCT (759). Serum samples with identical apoA-I and HDL-C levels were found to have differing cholesterol acceptance capacities, mostly due to pre-beta HDL levels, which contributed to altered ABCA1-mediated cholesterol efflux (759). These studies strongly suggest that HDL function (cholesterol efflux capacity), as opposed to HDL-C, HDL-P, and apoA-I levels, provide a more important risk assessment and better predictor of future events as well as a more reasoned therapeutic target for reducing CVD risk and events. However, clinical assays for apoA-I and HDL-P are widely available and well-established, whereas assays for cholesterol efflux capacity have not been standardized and remain a research tool at present.

 

HDL Composition and Analysis

 

Historically, HDL have been isolated by density-gradient ultracentrifugation (DGUC) based on isopycnic equilibrium, and HDL have been defined by their density 1.063-1.21 g/mL since the 1950s (760,761). Based on mass, HDL can also be separated from other lipoproteins by size-exclusion chromatography (fast protein liquid chromatography, FPLC), and HDL’s molecular weight ranges from 175,000 - 360,000 Da (762). In addition to DGUC and FPLC, affinity chromatography can also be used to purify HDL from plasma using antibodies against apoA-I (763)or apoA-II, as HDL heterogeneity includes particles containing apoA-I:apoA-II (75%) or apoA-I only (25%) (763,764). Furthermore, asymmetric flow field-flow fractionation is now being used to isolate and characterize HDL (765). HDL can also be separated by non-denaturing gradient gel electrophoresis, e.g. polyacrylamide gel electrophoresis. Large HDL (HDL2, 8.8-12.9 nm in diameter) and small HDL (HDL3, 7.2-8.8 nm) are both α migrating particles (high negative charge), whereas pre-β HDL (5.4-7 nm) are β migrating particles for which they are defined. To quantify pre-β HDL particles, 2-D gel electrophoresis is often used to separate pre-β from mature HDL (766). HDL-P numbers can be quantified by either nuclear magnetic resonance spectroscopy or calibrated ion mobility assays. HDL can also be quantified and qualified by other methods, including vertical rotor ultracentrifugation, and transmission electron microscopy.

 

HDL are very dynamic and should be acknowledged as a heterogeneous pool of sub-classes with differing sizes, shapes, densities, protein compositions, and lipid diversity. Lipid-free apoA-I is secreted from the liver and small intestine as an amphipathic helix, and it quickly becomes lipidated by ABCA1 to form pre-β HDL, which then becomes discoidal after accepting phospholipid and free cholesterol from hepatocytes and peripheral cells. Upon further lipidation and cholesterol accumulation and esterification, nascent spherical HDL forms that range 7-12 nm in diameter. Mature HDL contains 3-4 apoA-I molecules of which 1 remains on the particle and the other apoA-I are free to (dis)associate (exchange) on and off the particle with other HDL. This is predominantly associated with rearrangement of HDL’s aqueous phase and surface area (767). As such, HDL are in a constant state of remodeling and interconversion. Each spherical HDL particle has approximately 50-130 phospholipids, 10-50 free cholesterol molecules, 30-90 CE molecules, and 10-20 triglyceride (TG) molecules (536). Phosphatidylcholine makes up the largest amount of lipid on HDL (approximately 90%); however, over 200 species of lipids have been reported, including sphingolipids, acylglycerols, isoprenoids, glycerophospholipids, and vitamins (768,769). The HDL proteome has been extensively studied and there is a general consensus of approximately 80 proteins (770,771). In addition to apoA-I and apoA-II, HDL transports over a dozen other apolipoproteins, as well as many enzymes and other factors. HDL have also been found to transport small RNAs, namely microRNAs (miRNA), which were found to be altered in hypercholesterolemia and atherosclerosis (772,773). Most interestingly, HDL have been demonstrated to transport a wide-variety of exogenous non-host small RNAs, including rRNA and tRNA fragments derived from bacterial and fungal species present in the microbiome and environment (774).The size of HDL is determined by the amount of CE and triglyceride (TG) in the hydrophobic core, and HDL is generally separated into 5 sub-classes based on size. Distinct HDL sub-species have been associated with CVD risk, and the sub-species have differential biological functions, e.g. large HDL are less anti-inflammatory (775-777). Many of the cargo or components of HDL are enriched in the small HDL sub-class which provides many of the alternative functions to the total HDL pool (778,779). The concentration of all HDL particles in plasma is approximately 20 umol/L; however, small HDL particles are the most abundant sub-class at approximately 10 umol/L. HDL are heterogeneous particles that transport a wide-variety of proteins, lipids, and nucleic acids, which confer many of HDL’s biological properties and beneficial functions in health and dysfunction in specific diseases.

 

HDL Cell Signaling

 

Many of HDL’s cellular functions – cell survival, proliferation, vasodilation -- are mediated by HDL-induced cell signaling cascades (780). As such, HDL can be characterized as hormone-like agonists. Although substantial work still remains in identifying HDL binding proteins and receptors on the cell surface, HDL have been found to activate many signaling cascades through various receptors. The most studied example of this is HDL’s ability to bind to the plasma membrane and through cell signaling mobilize cholesterol from intracellular stores in organelles to the plasma membrane for efflux. This has been attributed to HDL-induced activation of protein kinase C (PKC) (781). Specifically, apoA-I binds to ABCA1 and activates phosphatidylcholine lipases, which activate PKC leading to the movement of cellular cholesterol from intracellular stores to the plasma membrane for efflux, as well as PKC-mediated phosphorylation of ABCA1, which increases the transporter’s stability and efflux activity (782-784). This is a prime example of HDL-induced cell signaling that contributes to HDL cholesterol efflux capacity, which reduces the cholesterol burden for macrophages in the lesion, prevents foam cell formation, and antagonizes atherogenesis. Other HDL-induced signaling pathways that result in increased cholesterol and lipid efflux include protein kinase A (PKA) (785,786), cell division control protein 42 (Cdc42) (787), and Janus kinases-2 (JAK2) (788,789)cascades. HDL (i.e. apoA-I)-induced cell signaling through ABCA1 also suppresses macrophage M1 phenotype activation and pro-inflammatory cytokine production (Figure 10), and promotes M2 phenotype anti-inflammatory cytokine secretion (e.g. interleukin 10 (IL-10)) through JAK2 signaling and activation of signal transducer and activator of transcription 3 (STAT3) (75). In addition, the apoA-I:ABCA1:JAK2 axis was reported to suppress inflammation in endothelial cells through cyclooxygenase-2 (COX-2) activation leading to increased prostaglandins (PGI2), which also suppresses atherogenesis (790). HDL have also been reported to induce cell signaling through SR-BI. HDL binding to SR-BI’s extracellular loop was reported to trigger activation of SR-BI’s cytoplasmic C terminal domain leading to the phosphorylation of protein kinase Src and activation of both liver kinase B1 (LKB1) and calmodulin-dependent protein kinase (CAMK) (791,792). This results in cell signaling through downstream kinases – AMP-activated protein kinases (AMPK) (792), protein kinase Akt (791), and mitogen-activated protein kinase (MAPK)(791)– which ultimately regulates angiogenesis (ubiquitin ligase Siah (Siah1/2) and hypoxia-inducible factor 1α (HIF1α) (793)), insulin sensitivity (glucose transporter 4 (Glut4)(794)), re-vascularization (Rac1(795)), and vasodilation (COX(796), endothelial nitric oxide synthase (eNOS)(797,798)). Interestingly, macrophage SR-BI has recently been shown to mediate efferocytosis (phagocytosis of dead cells) in the setting of atherosclerosis via a Src/Akt/Rac1 signaling pathway, reducing necrosis in lesions (185). All of these downstream effects contribute to HDL function, and to a lesser degree atherogenesis.

 

The most robust HDL signaling activation is mediated by bioactive lipids on HDL, namely the lysosphingolipid sphingosine-1-phosphate (S1P). A majority of S1P in circulation is associated with HDL, and HDL-S1P activates the G-coupled S1P receptors (S1P1-5) on the surface of many vascular cell types, including macrophages, endothelial cells, and smooth muscle cells. Activation of S1P1and S1P2 receptors turns on a host of signaling cascades and factors that directly contribute to the many anti-atherogenic properties of HDL, including increasing endothelial barrier function (799)and angiogenesis (800,801)while decreasing inflammation (802)and apoptosis (803). HDL were also found to inhibit smooth muscle migration through S1P signaling, a key factor in restenosis and plaque development (804). All of these are critical processes to atherogenesis. In support of these studies, subjects with CAD were found to have decreased HDL-S1P levels (805). The key terminal effector factors in these G-protein receptor signaling cascades are focal adhesion kinase (FAK), nuclear factor κ beta (NF-κB), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, eNOS, STAT3, and B-cell lymphoma-extra-large (Bcl-xl) (780). This HDL-S1P signaling pathway has also been linked to vasorelaxation (806)and cytoprotection (e.g. cardiomyocytes) (807). In addition to these direct pathways, HDL also likely activates cell signaling indirectly through ATP (β-ATPase/P2Y12/13)(808)or toll-like receptors (809). Collectively, HDL induced cell signaling in vascular and inflammatory cells underlies HDL’s anti-atherogenic properties in health, and deficits in HDL signaling likely link HDL dysfunction in metabolic diseases to increased risk of atherosclerosis.

 

Anti-Inflammatory HDL

 

Outside reverse cholesterol transport, HDL’s anti-inflammatory properties have been the most extensively studied HDL function and likely play a large role in HDL’s anti-atherogenicity (Figure 10). HDL’s anti-inflammatory properties are conferred by numerous mechanisms in many types of cells. In addition to providing the vascular barrier, endothelial cells control vascular inflammation through expressing adhesion molecules that aid in monocyte adhesion and ultimate migration into the atherosclerotic lesion. Moreover, activated endothelial cells secrete cytokines and recruit monocytes through chemokine release. The induction of adhesion molecules, cytokines, and chemokines in activated endothelial cells is largely due to NF-B transcriptional activation. In humans, injection of apoA-I resulted in decreased adhesion molecule expression in atherosclerotic plaques (810). One mechanism by which HDL suppresses endothelial cell and monocyte activation is through inhibiting NF-kB activity by attenuating IkB kinase activity (811). Nonetheless, HDL decreases adhesion molecule expression through multiple mechanisms. Cells pre-treated with HDL or apoA-I are protected from TNFα or oxidized LDL (oxLDL)-induced adhesion molecule expression. In addition, HDL binding to SR-BI may also contribute to inhibition of adhesion molecule expression, as SR-BI-mediated Akt activation promoted heme oxygenase-1 expression. In addition, up-regulation of 3-beta-hydroxysteroid-delta 24 (DHCR24) by HDL binding to SR-BI was reported to underlie HDL’s ability to suppress adhesion molecules (812). Furthermore, HDL suppression of intracellular adhesion molecule-1 (ICAM-1) in endothelial cells was found to be mediated, in part, through the transfer of miR-223 to recipient cells (773). Recent studies also suggest that TGFβ and AMPK also contribute to HDL’s suppression of adhesion molecule expression (813).

 

In addition to HDL’s profound effects on vascular endothelium, HDL suppresses myelopoiesis, monocyte recruitment, macrophage activation, proliferation, and emigration from atherosclerotic lesions. Similar to its impact on endothelial cells, HDL also suppresses adhesion molecule expression in monocytes, which inhibits monocyte adhesion and migration to atherosclerotic lesions (814). HDL and apoA-I were demonstrated to suppress CD11b expression on human monocytes through both ABCA1-dependent and independent mechanisms (814). HDL inhibition of monocyte activation, which includes suppression of cytokines and adhesion molecules, is mediated through both peroxisome proliferator-activated receptor gamma (PPARγ) and NF-kB transcription factors (815). Suppression of chemokine and cytokines in myeloid cells inhibits infiltration and migration of circulating monocytes, and thus antagonizes atherosclerosis. HDL have also been reported to mediate macrophage reprogramming through the transcription factor ATF3 that reduces Toll-like receptor signaling (816). Importantly, much of HDL’s (and apoA-I’s) inhibition of macrophage activation is mediated through altering cholesterol levels in plasma membrane lipid rafts through cholesterol efflux mediated by ABCG1/SR-BI and ABCA1; however, apoA-I induced signaling through ABCA1 and the JAK/STAT pathway independent of cholesterol efflux may also contribute to HDL’s effect, as described above (75,817)(814,818). HDL have also been demonstrated to promote macrophage emigration through removing excess cholesterol and induction of signaling pathways (208). In addition to HDL’s impact on monocytes and macrophages, HDL also strongly suppresses neutrophil activation and vascular smooth muscle cell secretion of monocyte chemoattractant protein-1 (MCP1) (819).

 

In addition to HDL’s roles in innate immunity, recent evidence suggests that HDL play multiple roles in adaptive immunity (820). Mice lacking apoA-I develop autoimmunity when challenged with a high cholesterol (diet and background, Ldlr-/-), which includes T cell activation and production of autoantibodies (821,822). This phenotype was rescued by apoA-I injections. HDL have also been reported to repress both antigen-presenting cell (APC) activation of T cells and T cell activation of monocytes, thus preventing the secretion of proinflammatory cytokines and chemokines (Figure 10) (823,824). ApoA-I also prevents the phenotypic switching of T-regs into pro-inflammatory follicular helper T cells during atheroprogression (92). Moreover, cholesterol efflux to HDL and apoA-I have been reported to suppress myelopoiesis and proliferation of myelopoietic stem and progenitor cells, as loss of function for both Abca1and Abcg1in mice resulted in increased myelopoiesis (820). Injection of apoA-I was also found to rescue this phenotype (825). In addition, HDL and cholesterol efflux were reported to suppress megakaryocyte progenitor proliferation, platelet levels, and thrombocytosis (826). Collectively, HDL and apoA-I inhibit circulating levels of hematopoietic progenitor cells, monocytes, neutrophils, and platelets all of which contribute to HDL’s capacity to limit inflammation and atherosclerosis.

 

Antithrombotic HDL

 

Another anti-atherogenic function of HDL is the capacity to directly and indirectly inhibit platelet activation, aggregation, and thrombus formation (Figure 10). HDL-C levels were found to be inversely associated with thrombus formation in humans (827). HDL is required to remove excess cholesterol from the plasma membrane of platelets for proper function, and platelets isolated from mice lacking SR-BI to mediate cholesterol efflux to HDL were found to be more susceptible to activation (828,829). Both HDL and cyclodextrin-mediated cholesterol efflux were found to inhibit platelet aggregation (828). However, HDL-induced cell signaling through binding to glycoprotein IIb/IIIa on the surface of platelets was reported to activate phospholipase C (PLC) and PKC, thus leading to flux through the Na+/H+ antiport system (717). This pathway can result in alkalization of the cytoplasm and calcium release, which can reduce platelet activation (830). Furthermore, HDL dose-dependently inhibits stimulated platelet activation, which leads to reduced platelet aggregation, granule secretion and fibrinogen binding. In rats, apoA-I injections inhibited thrombus formation and reduced thrombus mass (831). HDL’s anti-thrombotic effects are also mediated, in part, through HDL’s ability to inhibit tissue factor and factors X, Va, and VIIIa (Figure 10)(832). HDL also prevents thrombus formation through cell signaling and nitric oxide (NO) production in endothelial cells (828), and suppression of tissue factor and platelet-activating factor expression in endothelial cells (833,834). HDL also reduces erythrocyte influence on thrombus formation (835). Collectively, HDL has multiple biological mechanisms that inhibit thrombus formation, and thus, contribute to HDL’s anti-atherogenic properties.

 

Pro-Vasodilatory HDL

 

The endothelium significantly contributes to vascular tone, and HDL confer protection against endothelial cell activation, apoptosis, and loss of barrier function, which is critical to atherogenesis. HDL have been reported to induce endothelium-dependent vasodilation in aortic rings (806), and individuals with low HDL have reduced endothelium-dependent vasorelaxation (Figure 10) (741). HDL’s benefit to endothelial cells is largely mediated by cell signaling through phosphatidylinositol 3-kinase (PI3K) and Akt and is induced by bioactive lipids and associated proteins on HDL, including lysosulfatide, S1P, and sphingosylphosphorylcholine (SPC) (791,798,806). A key outcome of HDL-induced cell signaling is the production of NO (Figure 10) through both signaling induced phosphorylation of eNOS and increased eNOS expression (791,836). HDL can trigger eNOS-phosphorylation through SR-BI, S1P receptor (S1P1-5), and ABCG1-mediated cholesterol efflux (806,837). HDL-induced NO underlies many of HDL’s beneficial properties to endothelial cells, including HDL-induced vasodilation, tightening of cell-to-cell junctions and increased barrier function, differentiation of endothelial progenitor cells, cell survival and proliferation, cell migration, inhibition of apoptosis, and suppression of adhesion molecule expression. In addition, HDL also has NO-independent properties on endothelial cells, including induced proliferation, increased barrier function, suppressed inflammation and decreased apoptosis (838). These studies clearly define a beneficial role for HDL in vascular integrity, which underlies HDL protection against atherosclerosis.

 

Anti-Apoptotic HDL

 

HDL have multiple anti-apoptotic properties that enhance cell survival (Figure 10). By various metrics, HDL support mitochondrial function and prevent the release of apoptotic signals, including cytochrome C (205,839). Moreover, HDL drives the expression of Bcl-xl, which is a strong anti-apoptotic factor and suppresses Bid, which is a pro-apoptic protein (839,840). HDL mediates these gene expression changes through cell signaling and NO production through activation of surface receptors by HDL-associated proteins and bioactive lipids, including apolipoprotein J (apoJ) and S1P (803,840). In addition, there are likely alternative anti-apoptotic mechanisms resulting from HDL-induced signaling. Nonetheless, HDL has been demonstrated to suppress apoptosis in endothelial cells (Figure 10) activated with tumor necrosis factor (TNFα) and oxLDL (839,841,842). HDL proteins (apolipoprotein M, apoM) and apoM-binding lipids (S1P) contribute to HDL’s ability to increase tight junctions and endothelial cell survival (843). Mice deficient in apoM have reduced S1P levels and loss of endothelium barrier function (843). HDL’s ability to support the endothelium barrier function is a key feature of its anti-atherosclerosis properties and represents a classic example of HDL’s control of cellular gene expression and phenotype that are beneficial to vascular health. However, HDL also have many capacities in the extracellular space (e.g. plasma) that protect against atherosclerosis.

 

Anti-Oxidative HDL

 

A key factor in monocyte activation and chemotaxis in the vascular wall is the accumulation of oxLDL, which is more pro-inflammatory and pro-atherogenic than unmodified LDL. LDL can become oxidized by a variety of endogenous mechanisms (844). In the vascular wall, LDL can be modified (oxidized) by many cell types, including vascular smooth muscle cells, endothelial cells, and macrophages (776). Remarkably, HDL prevents the oxidation of LDL (Figure 10) and recent evidence suggests that this may occur through 4 distinct proteins circulating on HDL – apoA-I (845,846), LCAT (847), lipoprotein-associated phospholipase A2 (Lp-PLA2)(848,849), and paraoxonase 1 (PON1) (430,846). First, HDL can simply soak up oxidized lipids or oxidizing factors from cells preventing their association with LDL and their modification of LDL lipids and proteins. In addition, HDL removes lipid hydroperoxides from LDL particles (846). Specifically, small apoAI containing HDL particles are the most efficient at accepting lipid hydroperoxides, which are reduced to their inactive lipid hydroxides via oxidation of the methionine residues in apoA-I (850). Compared to apoA-II the methionine residues in apoA-I are more conformationally conducive to reducing lipid hydroperoxides (851,852). In addition, HDL with low surface free cholesterol and sphingomyelin are more efficient at accepting lipid hydroperoxides (745,853). The capacity of HDL to prevent oxidation via this mechanism is also maintained by the selective removal of HDL lipid hydroperoxides and hydroxides by hepatocyte SR-BI (854). In addition, ApoA-I methionine sulfoxide is reduced to methionine by methionine sulfoxide reductases.(850). LCAT circulates on HDL and has also been reported to block LDL oxidation, as LCAT over-expression in mice reduced LDL oxidation as determined by reduced LDL autoantibodies (855). Lp-PLA2appears to be pro-atherogenic on LDL and anti-atherogenic on HDL (856). Its activity on HDL likely contributes to HDL’s anti-oxidative capacity, as inhibition of HDL-associated Lp-PLA2attenuated HDL’s ability to block LDL oxidation (848). The strongest anti-oxidative HDL protein is likely PON1. Over-expression of PON1 in mice confers enhanced HDL anti-oxidative capacity, and PON1 itself prevents LDL oxidation in vitro (432). Most importantly, HDL isolated from mice lacking PON1 have reduced ability to prevent LDL oxidation. HDL’s anti-oxidative capacity likely plays a large role in preventing inflammation and atherogenesis, and like many of the other alternative functions, confer HDL’s beneficial role in health.

 

HDL Intercellular Communication

 

HDL also likely participate in intercellular communication through the transfer of nucleic acids between tissues. Recently, HDL have been reported to transport miRNA (Figure 10), which are small non-coding RNAs that suppress gene expression through binding to complimentary target sites in the 3’ untranslated region of mRNAs, and thus inhibit translation and induce mRNA degradation (772). Most interestingly, the HDL-miRNA profile is significantly altered in hypercholesterolemia and atherosclerosis (772). miRNAs have been reported to be exported from macrophages to HDL, and HDL has been demonstrated to transfer specific miRNAs to recipient hepatoma cells (Huh7) and endothelial cells, likely through HDL’s receptor SR-BI (773). In endothelial cells, HDL was found to deliver miR-223 to recipient cells, where it directly targeted intracellular adhesion molecule-1 (ICAM-1) expression (Figure 10), and thus inhibited neutrophil adhesion to the cells (773). miR-223 is not transcribed or processed in endothelial cells and HDL delivery of mature miR-223 to endothelium likely confers, in part, HDL’s anti-inflammatory capacity associated with adhesion molecule suppression. Future studies are needed to determine the physiological relevance and functional impact of HDL-miRNAs in humans and animal models in the context of atherosclerosis and other inflammatory diseases.

 

Anti-Infectious HDL

 

HDL also contributes to innate immunity by modulating immune cell function. However, this hypothesis has not been extensively studied in the context of atherosclerosis. HDL are anti-infectious, anti-parasitic, and anti-viral. HDL have the unique capacity to prevent endotoxic shock and readily binds to lipopolysaccharides (LPS) and contributes to removing LPS through biliary excretion thus aiding innate immunity (857-859). Amongst the many proteins that circulate on HDL, apolipoprotein L1 (apo-L1) (also known as trypanosome lytic factor) is present in specific sub-classes of HDL (860,861). This factor kills Trypanosome bruceiand Trypanosome brucei rhdesiense, parasites that cause sleeping sickness, through creating ionic pores in endosomes (860-862). Although promising, future studies are required to define how HDL regulation of innate immunity contributes to the inhibition of atherogenesis.

 

HDL Dysfunction

 

HDL confer many anti-atherogenic properties that are lost in atherosclerosis and other inflammatory and metabolic diseases. These include 9 key processes –

  • Loss of cholesterol efflux capacity from macrophages
  • Reduced ability to inhibit LDL oxidation
  • Decreased vasodilation through reduced NO production in endothelial cells
  • Reduced ability to inhibit monocyte chemotactic activity
  • Loss of the ability to metabolize hydroperoxides on erythrocyte membranes
  • Reduced ability to suppress TNFα-induced NF-κB activation and adhesion molecule expression
  • Loss of anti-apoptotic capacity in endothelial cells
  • Decreased capacity to block TNFα-induced NADPH oxidase activity and superoxide production
  • Suppression of cytokine inhibition in activated inflammatory cells.

 

Many of these defects are due to changes in HDL cargo, e.g. decreased PON1 levels or increased serum amyloid A (SAA) levels. Moreover, changes in the content of bioactive lipids or increased oxidative modifications to HDL’s lipids and protein cargo likely confer dysfunction. HDL-miRNAs have been shown to be significantly altered in hypercholesterolemia and atherosclerosis (772). It is unknown how these changes contribute to HDL’s loss of anti-atherogenic properties, but they hold great potential for future studies. In CHD, acute coronary syndrome (ACS), and ischemic cardiomyopathy, HDL have reduced ability to inhibit oxidation of LDL, likely through reduced PON1 levels as reported in CHD (845,863,864). Loss of PON1 also reduces HDL’s ability to prevent oxidation of its own lipids and proteins, which has been reported in metabolic syndrome as oxidation of apoA-I impairs HDL’s RCT and anti-inflammatory functions (865). Reduced HDL-PON1 levels are also found in other cardiometabolic diseases, including type 2 diabetic mellitus (T2DM) (866,867), type 1 diabetes mellitus (T1DM) (868), rheumatoid arthritis (RA) (869,870), dyslipidemia (e.g. hyperalphalipoproteinemia (HALP) (871)), and patients after cardiac surgery (872). In subjects with ACS and CAD, HDL have been reported to have decreased ability to prevent endothelial cell apoptosis likely through decreased activation (phosphorylation) of Bcl-xl and increased activation of Bcl-2, which are anti-apoptotic and pro-apoptotic proteins, respectively (840). Loss of HDL’s anti-apoptotic capacity has been proposed to be due to increased apoCIII and possibly decreased clusterin levels on HDL (840). HDL from subjects with CHD also have decreased ability to prevent monocyte adhesion to endothelial cells and recruitment in arterial wall co-cultures, which could be associated with reduced PON1 levels amongst other cargo (845,873,874). HDL from ischemic cardiomyopathy and CAD subjects also have reduced cholesterol efflux acceptance capacity, which likely leads to increased foam cell formation in the atherosclerotic lesion and increased atherogenesis (746,863). Although the molecular basis for all of HDL’s loss of anti-atherogenicity in CHD is not known, other functions of HDL are compromised in these subjects, including the ability to reduce hydroperoxides on erythrocyte membranes (875). This loss of HDL’s anti-oxidant capacity is also found in T2DM (875)and T1DM (868,876). HDL in metabolic syndrome have been reported to have decreased capacity to prevent oxidation of LDL and inhibit endothelial cell apoptosis (877,878). This loss of anti-atherogenic properties is also found in hypertension(879), T2DM (867,880,881), end-stage renal disease (ESRD) (882,883), RA (869,870,884,885), systemic erythematosus lupus (SLE) (884), obstructive sleep apnea (886), and dyslipidemia (HALP) (871). Reduced ability of HDL to stimulate NO production from endothelial cells and decreased vasorelaxation properties are reported for T2DM (887,888), T1DM (889), mild chronic kidney disease (CKD) (890), and rare forms of autoimmunity (ALPS) (891). Loss of HDL-mediated cholesterol efflux capacity has been found in patients with hyperhomocysteinemia (892), sepsis (893), psoriasis (894), SLE (895), RA (885,896), ESRD (897,898), and T2DM (899,900). Not only does HDL dysfunction result from loss of key proteins and cargo, HDL can gain pro-atherogenic cargo and properties in cardiometabolic diseases. Due to loss of PON1, HDL accumulate malonaldehydes, which inhibits NO production through increased phosphorylation of eNOS through LOX-1 receptor signaling (901).

 

HDL Summary

 

Years of sound epidemiological studies have clearly established an inverse relationship between HDL-C levels and risk of CVD. Nevertheless, recent GWAS studies suggest that individuals with high HDL-C levels are not protected from CVD. Furthermore, clinical studies aimed at raising HDL-C levels through niacin and CETP inhibitors have failed to reduce risk of cardiovascular events and have been stopped prematurely due to lack of efficacy or increased number of events. Although they’re often lumped together, HDL-C levels do not represent HDL particle numbers or HDL function (e.g. cholesterol efflux capacity); both of which have been reported to be better indicators of CVD risk than HDL-C. In addition to HDL’s transport of cholesterol and lipids in the RCT pathway, HDL transports a wide-variety of cargo, including a diverse group of proteins, small RNAs, bioactive lipids, and many other small molecules. These alternative cargos may confer many of HDL’s alternative functions outside of RCT. In fact, HDL have many beneficial properties, including anti-inflammatory, anti-oxidative, anti-thrombotic, anti-infectious, anti-apoptotic, intercellular communication, and pro-vasodilatory capacities. Recently, HDL dysfunction has been reported in many cardiometabolic diseases, including CAD, T2D, and CKD. Current and future challenges include the need to better define HDL anti-atherogenic properties in health and pro-atherogenic influences in disease to better control HDL function to potentially prevent and treat CVD.

 

ACKNOWLEGEMENTS

:

This work was supported in part by National Institutes of Health grants HL116263 and HL127173.

 

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Role Of Glucose And Lipids In The Cardiovascular Disease Of Patients With Diabetes

ABSTRACT

Cardiovascular disease is a major cause of morbidity and mortality in both men and women with Type 1 and Type 2 diabetes. In patients with Type 1 diabetes, intensive glycemic control results in a reduction in cardiovascular disease. However, intensive glycemic control does not have a major impact in reducing cardiovascular disease in patients with Type 2 diabetes. Metformin, pioglitazone, SGLT2 inhibitors, and certain GLP-1 receptor agonists have been shown to decrease cardiovascular disease in patients with Type 2 diabetes to a greater extent than other treatment modalities. In patients with Type 2 diabetes other risk factors including, hypertension and dyslipidemia, play a major role in inducing cardiovascular disease, and control of these risk factors is paramount. In patients with Type 1 diabetes in good glycemic control, the lipid profile is very similar to the general population. In contrast, in patients with Type 2 diabetes, even with good glycemic control, there are frequently lipid abnormalities (elevated triglycerides and non-HDL cholesterol, decreased HDL cholesterol, and an increase in small dense LDL). In both Type 1 and Type 2 diabetes, poor glycemic control increases triglyceride levels and decreases HDL cholesterol levels with only modest effects on LDL cholesterol levels.  Extensive studies have demonstrated that statins decrease cardiovascular disease in patients with diabetes. Treatment with high doses of potent statins reduces cardiovascular events to a greater extent than low dose statin therapy. Adding fibrates or niacin to statin therapy has not been shown to further decrease cardiovascular events. In contrast, recent studies have shown that the combination of a statin and ezetimibe, a PCSK9 inhibitor, or EPA, an omega-3-fatty acid, does result in a greater decrease in cardiovascular events than statins alone. Current recommendations state that most patients with diabetes should be on statin therapy.  For complete coverage of all related areas of Endocrinology, please visit our on-line FREE web-text,  WWW.ENDOTEXT.ORG.

 

INTRODUCTION

 

Cardiovascular disease is the major cause of morbidity and mortality in both men and women with diabetes (approximately 50-70% of deaths) [1-5]. The risk of cardiovascular disease is increased approximately 2 fold in men and 3-4 fold in women [2-4, 6, 7]. In the Framingham study, the annual rate of cardiovascular disease was similar in men and women with diabetes, emphasizing that woman with diabetes need as aggressive preventive treatment as men with diabetes [2, 6]. In addition, several but not all studies, have shown that patients with diabetes who have no history of cardiovascular disease have approximately the same risk of having a myocardial infarction as non-diabetic patients who have a history of cardiovascular disease, i.e., diabetes is an equivalent risk factor as a history of a previous cardiovascular event [8, 9]. The duration of diabetes and the presence of other risk factors likely determine whether a patient with diabetes has a risk equivalent to patients with a history of previous cardiovascular events [10, 11]. Moreover, numerous studies have shown that patients with diabetes who have cardiovascular disease are at a very high risk of having another event, indicating that this population of patient’s needs especially aggressive preventive measures [1, 8]. This increased risk for the development of cardiovascular disease in patients with diabetes is seen both in populations where the prevalence of cardiovascular disease is high (Western societies) and low (for example, Japan) [2]. However, in societies where the prevalence of cardiovascular disease is low, the contribution of cardiovascular disease as a cause of morbidity and mortality in patients with diabetes is reduced[2]. While the database is not as robust, the evidence indicates that patients with Type 1 diabetes are also at high risk for the development of cardiovascular disease [1, 12-14]. Interestingly, women with type 1 diabetes have twice the excess risk of fatal and nonfatal vascular events compared to men with type 1 diabetes [15, 16]. Additionally, developing type 1 diabetes at a young age increases the risk of cardiovascular disease to a greater degree than late onset type 1 diabetes [16]. While the development of diabetes at a young age increases the risk of cardiovascular disease in patients with both Type 1 and Type 2 diabetes the deleterious impact is greater in patients with Type 2 diabetes [17]. Lastly, in patients with both Type 1 and Type 2 diabetes the presence of renal disease increases the risk of cardiovascular disease [4, 13]. Of note is that the risk of developing  cardiovascular events in patients with diabetes has decreased recently, most likely due to better lipid and blood pressure control, which again reinforces the need to aggressively treat these risk factors in patients with diabetes [5, 7, 18].

 

ROLE OF GLYCEMIC CONTROL

 

Epidemiological studies have shown an association between the level of glycemic control and the development of cardiovascular disease [1, 4, 5]. However, the association of glycemic control with cardiovascular disease is considerably weaker than the association of glycemic control with the microvascular complications of diabetes, such as retinopathy and nephropathy [4]. It must be recognized that epidemiological studies can only demonstrate associations and that confounding variables could account for the association between poor glycemic control and cardiovascular disease. For example, patients with poor glycemic control may not undertake other preventive measures that could reduce cardiovascular disease such as exercise, healthy diet, etc. Furthermore, the patients with poor glycemic control may have less compliance with therapies that reduce lipids and blood pressure. Therefore, randomized studies are essential in determining the role of glycemic control on cardiovascular disease.

 

Early randomized studies, such as the UGDP and VA cooperative study, did not demonstrate a reduction in cardiovascular events in patients who were aggressively treated for glucose control [19-21]. In fact, the data suggested that improvements in glycemic control (VA cooperative study) or the use of certain drugs to treat diabetes (oral sulfonylureas in UGDP) may actually increase the risk of cardiovascular disease.

 

DCCT and Kumamoto Studies

 

More recent studies, the DCCT in patients with Type 1 diabetes and the Kumamoto study in patients with Type 2 diabetes, while finding a decrease in cardiovascular events in the subjects randomized to improved glycemic control did not have enough cardiovascular disease events to demonstrate a statistically significant reduction (DCCT studied a population at low risk for cardiovascular disease and the Kumamoto study had a very small number of subjects) [22-24]. In contrast, both the DCCT and the Kumamoto study clearly demonstrated that improvements in glycemic control resulted in a reduction in microvascular disease [22-24]. However the long term follow-up of the DCCT has demonstrated that those in the intensive glycemic control group had a decrease in cardiovascular disease in subsequent years [25, 26]. The initial DCCT compared intensive vs. conventional therapy for a mean of 6.5 years. At the end of the study, a very large proportion of subjects agreed to participate in a follow-up observational study (Epidemiology of Diabetes Interventions and Complications- EDIC). During this follow-up period, glycemic control was relatively similar between the intensive therapy and conventional therapy group (glycosylated hemoglobin 7.9% vs. 7.8%) but during the trial there was a large difference in glycosylated hemoglobin levels (7.4% vs. 9.1%). After a mean 17 years of observation, the risk of any cardiovascular event was reduced by 42% and the risk of nonfatal myocardial infarction, stroke, or death from cardiovascular disease was reduced by 57% in the intensive control group. This study demonstrates that being in the intensive glycemic control group (for 6.5 of the 17 years of observation) is sufficient to have long term beneficial effects on the risk of developing cardiovascular disease in patients with Type 1 diabetes. This beneficial effect was not entirely due to the prevention of microvascular complications as the differences between the intensive and conventional treatment groups for cardiovascular disease persisted after adjusting for microalbuminuria and albuminuria. When an outcome of improved glycemic control is seen, or persists for years after the trial is over the phenomenon is called a “metabolic memory” effect.

 

UK Prospective Diabetes Study (UKPDS)

 

A similar finding has been reported with regard to Type 2 diabetes. The UKPDS studied a large number of newly diagnosed patients with Type 2 diabetes at risk for cardiovascular disease. In this study improved glycemic control, with either insulin or sulfonylureas, reduced cardiovascular disease by 16%, which just missed being statistically significant (p=0.052) [27]. In the UKPDS, the improvement in glycemic control was modest (HbA1c reduced by approximately 0.9%) and the 16% reduction in cardiovascular disease was in the range predicted based on epidemiological studies. The results of a 10 year follow-up of the UKPDS study have been reported (total duration of observation 25 years) [28]. After termination of the study, glycosylated hemoglobin levels became very similar between the control and treatment groups. Nevertheless, risk reductions for MI became statistically significant for the insulin and the sulfonylurea group compared to controls (15% decrease, p=0.01).

 

DiGami Studies

 

Similarly, the DiGami study, which used insulin infusion during the peri-MI period to improve glycemic control followed by long-term glycemic control, demonstrated that survival post MI was significantly improved by good glycemic control [29]. While this study focused on a highly-selected population and time period (patients undergoing a MI), the results are consistent with the hypothesis that improvements in glycemic control will reduce cardiovascular disease. However, the DiGami 2 study did not confirm the benefits of tight glucose control beginning in the peri-MI period on outcomes [30]. It must be noted though that the differences in glucose control achieved in DiGami 2 were much smaller than planned and the number of patients recruited was less than anticipated. Together these deficiencies could account for the failure to demonstrate significant differences in cardiovascular disease events in this study.

 

ACCORD Study

 

Because of the need for more definitive data on the effect of glycemic control on cardiovascular disease in Type 2 diabetes, three large randomized trials, the ACCORD, ADVANCE, and VA Diabetes Trial, have been carried out. Much to everyone’s surprise and disappointment, improvement in glycemic control did not clearly result in a reduction in cardiovascular disease in these trials.

 

The ACCORD study randomized 10,251 subjects with Type 2 diabetes in the US and Canada with either a history of cardiovascular disease or at increased risk for the development of cardiovascular disease [31]. Multiple different treatment protocols were used with the goal of achieving an A1c level < 6% in the intensive group and between 7-7.9% in the standard glycemic control group. During the trial the A1c levels were 6.4% in the intensive group and 7.5% in the standard group. As expected, the use of insulin therapy was much greater in the intensive group, as was the occurrence of hypoglycemia and weight gain. After a mean duration of 3.5 years this study was stopped early by the data safety monitoring board due to an increased all-cause mortality in the intensive treatment group (1.41 vs. 1.14% per year; hazard ratio 1.22 CI 1.01- 1.46). The primary outcome (MI, stroke, cardiovascular disease death) was reduced by 10% in the intensive control group but this was not statistically significant (p=0.16). Of note, intensive glycemic control reduced the incidence of any myocardial infarction (i.e. fatal or non-fatal) by 16%, nonfatal myocardial infarction by 19%, coronary revascularization by 16%, and unstable angina by 19% [32].The explanation for the increased death rate in the intensive treatment arm remains unknown, but it has been speculated that the increased deaths might have been due to hypoglycemia, weight gain, too rapidly lowering A1c levels, or unrecognized drug toxicity. Long term follow-up of the ACCORD study did not reveal any beneficial effects on the primary outcome (nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death), death from any cause, and an expanded composite outcome that included all-cause death in the intensive glycemic control group [33].

 

ADVANCE Study

 

The ADVANCE study randomized 11,140 subjects with Type 2 diabetes in Europe, Asia, Australia/New Zealand and Canada who either had cardiovascular disease or at least one other risk factor for cardiovascular disease [34]. In the intensive group the goal A1c was <6.5%. The achieved A1c levels during the trial were 6.3% in the intensive group and 7.3% in the standard treatment group. Of note is that compared to the ACCORD study, less insulin use was required to achieve these A1c levels. With regard to macrovascular disease (MI, stroke, and cardiovascular death), no significant differences were observed between the intensive and standard treatment groups (HR 0.94, CI 0.84-1.06, p=0.32). In contrast to the ACCORD trial, no increase in overall or cardiovascular mortality in the intensive treatment group was observed in the ADVANCE study. Long term follow-up did not demonstrate a decrease in the risk of death from any cause or major macrovascular events between the intensive-glucose-control group and the standard-glucose-control group [35].

 

VA Diabetes Trial

 

The VA Diabetes Trial randomized 1,791 subjects with poor glycemic control on maximal oral agent therapy or insulin (entry A1c 9.4%) [36]. In the intensive group, the goal A1c was <6.0%. The achieved A1c levels during the trial were 6.9% in the intensive group and 8.5% in the standard treatment group. Similar to the other trials, a significant reduction in cardiovascular disease was not observed in the intensive glycemic control group (HR 0.88, CI 0.74-1.05, p=0.12). Notably there were more cardiovascular disease deaths and sudden deaths in the intensive treatment group, but this increase was not statistically significant. With long-term follow-up, the intensive-therapy group had a significantly lower risk of heart attack, stroke, congestive heart failure, amputation for ischemic gangrene, or cardiovascular-related death than did the standard-therapy group (hazard ratio, 0.83; P=0.04 [37]. However, there was no reduction in cardiovascular or total mortality.

 

Summary

 

Thus, while the epidemiological data strongly suggests that glycemic control would favorably impact cardiovascular disease the recent randomized trials that were designed specifically to prove this hypothesis have failed to definitively demonstrate a clear link. There are several explanations for why these trials may not have worked as planned.

 

First, in the ACCORD, ADVANCE, and VA Diabetes Trial, other cardiovascular risk factors were aggressively treated (lipid and BP lowering, ASA therapy). As a result of these treatments, the actual number of cardiovascular events was considerably less than expected in these trials. The lower event rate may have reduced the ability to see a beneficial effect of glucose control. Additionally, the beneficial effects of glucose control maybe more robust if other risk factors are not aggressively controlled. In this regard, it is worth noting that in the earlier UKPDS, which showed that improved glycemic control reduced cardiovascular events, both BP and lipids were not aggressively treated by current standards (systolic BP 135-140mm Hg, LDL cholesterol 135-142mg/dl), which could be why this older trial demonstrated a small benefit of improving glycemic control on cardiovascular disease.

 

Second, these three recent trials were comparing relatively low A1c levels in both the intensive and usual control groups (A1c in intensive from ~6.4-6.9% and usual control group from ~7.0-8.4%). It is likely that both levels are on the “flatter” portion of the glycemic control-cardiovascular risk curve and that if one compared patients with higher A1c values one would see more impressive results.

 

Third, all three trials were carried out by initiating tight control in patients with long standing diabetes who either had pre-existing cardiovascular disease or were at high risk for cardiovascular disease. It is possible that patients with a different clinical profile would be more likely to benefit from intensive glucose control. Subgroup analysis from these trials have suggested that patients with a shorter duration of diabetes, less severe diabetes, or the absence of pre-existing cardiovascular disease actually benefited from intensive control. It may be that glycemic control is most important prior to the development of significant atherosclerosis. Clearly additional studies on different types of patients (i.e. newly diagnosed without evidence of cardiovascular disease) will be necessary to definitively determine the role of glycemic control in different diabetic populations.

 

Fourth, the duration of these studies was relatively short and it is possible that a much longer duration of glycemic control is required to show benefits on cardiovascular disease. In the UKPDS study the beneficial effects of intensive glucose control was not statistically significant at the end of the study but with an extended duration of follow-up (15-25 years) became statistically significant.

 

Fifth, it may be that glycemic control will be more important in patients with Type 1 diabetes where abnormalities in glucose metabolism are the major reason for the increased risk of atherosclerosis. In contrast, patients with Type 2 diabetes have multiple risk factors for atherosclerosis (dyslipidemia, hypertension, inflammation, insulin resistance, coagulation disorders) and glucose may play only a minor role in the increased risk. The differences in other cardiovascular risk factors could account for why intensive glycemic control produced a marked reduction in cardiovascular disease in the DCCT (Type 1 trial) and had only minimal effects in the trials carried out in patients with Type 2 diabetes.

 

Finally, it is possible that our current treatments have side effects that mask the beneficial effects of glucose control. For example, hypoglycemia and weight gain could counterbalance the beneficial effects of improvements in glycemic control. It is possible that different treatment strategies could lead to more profound benefits (see below).

 

Thus, the currently available data do not definitively indicate that glycemic control will have major effects on reducing cardiovascular disease in patients with Type 2 diabetes. Furthermore, there are concerns that too tight control in patients with advanced disease could be harmful. The reduction of A1c to below 7% may be detrimental in patients at high risk of cardiovascular events. In contrast, in patients with Type 1 diabetes intensive glucose control appears to be very beneficial based on the results of the DCCT.

 

THE EFFECT OF GLUCOSE LOWERING DRUGS ON CARDIOVASCULAR DISEASE

 

Metformin

 

In the UKPDS, metformin, while producing a similar improvement in glycemic control as insulin or sulfonylureas, markedly reduced cardiovascular disease by approximately 40% [38]. In the ten year follow-up the patients randomized to metformin in the UKPDS continued to show a reduction in MI and all-cause mortality [28]. Two other randomized controlled trials have also demonstrated cardiovascular benefits with metformin therapy.

 

A study by Kooy et al compared the effect of adding metformin or placebo in overweight or obese patients already on insulin therapy [39]. After a mean follow-up of 4.3 years this study observed a reduction in macrovascular events (HR 0.61 CI- 0.40-0.94, p=0.02), which was partially accounted for by metformin’s beneficial effects on weight. In this study the difference in A1c between the metformin and placebo group was only 0.3%.

 

Hong et al randomized non-obese patients with coronary artery disease to glipizide vs. metformin therapy for three years[40]. A1c levels were similar, but there was a marked reduction in cardiovascular events in the metformin treated group (HR 0.54 CI 0.30- 0.90, p=0.026).

 

Further support for the beneficial effects of metformin on atherosclerosis comes from long term follow-up of the Diabetes Prevention Program, which compared the effect of lifestyle changes or metformin in patients at high risk of developing diabetes [41]. Coronary artery calcium scores were measured on average 13-14 years after randomization [41]. There were no differences in coronary artery calcium scores between the lifestyle and placebo groups. However, in males, coronary artery calcium scores were significantly lower in the metformin group vs. the placebo group. In females treated with metformin coronary artery calcium scores were similar to the placebo group. The absence of a beneficial effect of metformin in women could be due to the lower baseline coronary artery calcium scores making it more difficult to demonstrate a beneficial effect. In HIV-infected patients with the metabolic syndrome metformin similarly reduced the progression of coronary artery calcium scores [42].

 

Together, these results suggest that metformin may reduce cardiovascular disease and that this effect is not due to improving glucose control. Metformin decreases weight or prevents weight gain and lowers lipid levels and these or other non-glucose effects may account for the beneficial effects on cardiovascular disease.

 

Thiazolidinediones

 

Studies with pioglitazone have also suggested a beneficial effect on cardiovascular disease. The ProActive study was a randomized controlled trial that examined the effect of pioglitazone vs. placebo over a 3 year period in type 2 diabetics with pre-existing macrovascular disease [43]. With regard to the primary endpoint (a composite of all-cause mortality, non-fatal myocardial infarction including silent MI, stroke, acute coronary syndrome, endovascular or surgical intervention in the coronary or leg arteries, and amputation above the ankle), there was a 10% reduction in events in the pioglitazone group but this difference was not statistically significant (p=0.095). It should be noted that both leg revascularization and leg amputations are not typical primary end points in cardiovascular disease trials and these could be affected by pioglitazone induced edema. When one focuses on standard cardiovascular disease endpoints, the pioglitazone treated group did demonstrate a 16% reduction in the main secondary endpoint (composite of all-cause mortality, non-fatal myocardial infarction, and stroke) that was statistically significant (p=0.027). In the pioglitazone treated group, blood pressure, A1c, triglyceride, and HDL cholesterol levels were all improved compared to the placebo group making it very likely that the mechanism by which pioglitazone decreased vascular events was multifactorial.

 

Recently a multicenter, double-blind trial (IRIS Trial), randomly assigned 3876 patients with insulin resistance (defined as score of more than 3.0 on the homeostasis model assessment  of insulin resistance [HOMA-IR] index) but without diabetes and a recent ischemic stroke or TIA to treatment with either pioglitazone (target dose, 45 mg daily) or placebo [44]. After 4.8 years, the primary outcome of fatal or nonfatal stroke or myocardial infarction occurred in 9.0% of the pioglitazone group and 11.8% of the placebo group (hazard ratio 0.76; P=0.007). All components of the primary outcome were reduced in the pioglitazone treated group. Fasting glucose, fasting triglycerides, and systolic and diastolic blood pressure were lower while HDL cholesterol and LDL cholesterol levels were higher in the pioglitazone group than in the placebo group. Although this study excluded patients with diabetes the results are consistent with and support the results of a protective effect of pioglitazone observed in the ProActive study.

 

In contrast, a recent study compared the effect of pioglitazone vs. sulfonylurea on cardiovascular disease and did not observe a reduction in events with pioglitazone treatment [45]. Patient with type 2 diabetes (n= 3028), inadequately controlled with metformin monotherapy (2-3 g per day), were randomized to pioglitazone or sulfonylurea and followed for a median of 57 months. Only 11% of the participants had a previous cardiovascular event. The primary outcome, was a composite of first occurrence of all-cause death, non-fatal myocardial infarction, non-fatal stroke, or urgent coronary revascularization and occurred in 6.8% of the patients treated with pioglitazone and 7.2% of the patients treated with a sulfonylurea (HR 0.96; NS). Limitations of this study are the small number of events likely due to low risk population studied and the relatively small number of participants. Additionally, 28% of the subjects randomized to pioglitazone prematurely discontinued the medication. Thus, the results of this study should be interpreted with caution.

 

Further support for the beneficial effects of pioglitazone on atherosclerosis is provided by studies that have examined the effect of pioglitazone on carotid intima-medial thickness. Both the Chicago and Pioneer studies demonstrated favorable effects on carotid intima-medial thickness in patients treated with pioglitazone compared to patients treated with sulfonylureas [46, 47]. Similarly, Periscope, a study that measured atheroma volume by intravascular ultrasonography, also demonstrated less atherosclerosis in the pioglitazone treated group compared to patients treated with sulfonylureas [48].

 

While the data from a variety of different types of studies strongly suggests that pioglitazone is anti-atherogenic, the results with rosiglitazone are different. Several meta-analyses of small and short-duration rosiglitazone trials suggested that rosiglitazone was associated with an increased risk of adverse cardiovascular outcomes [49, 50]. However, the final results of the RECORD study, a randomized trial that was specifically designed to compare the effect of rosiglitazone vs. either metformin or sulfonylurea therapy as a second oral drug in those receiving either metformin or a sulfonylurea on cardiovascular events, have been published and did not reveal a difference in cardiovascular disease death, myocardial infarctions, or stroke [51-53]. Similarly, an analysis of patients on rosiglitazone in the BARI 2D trial also did not suggest an increase or decrease in cardiovascular events in the patients treated with rosiglitazone [54]. Thus, while the available data suggests that pioglitazone is anti-atherogenic, the data for rosiglitazone suggests a neutral effect. Whether these differences between pioglitazone and rosiglitazone are accounted for by their differential effects on lipid levels are unknown (see below for information on the effects of these drugs on lipid levels).

 

DPP4 Inhibitors

 

Because of the importance of cardiovascular disease in patients with diabetes the FDA is requiring manufacturers of new drugs to treat diabetes to carry out studies addressing cardiovascular endpoints. Recently, the effect of the DPP4 inhibitors saxagliptin, alogliptin, sitagliptin, and linagliptin on cardiovascular endpoints has been reported. In the saxagliptin study, 16,492 patients with Type 2 diabetes who had a history of cardiovascular events or who were at high risk were randomized to saxagliptin or placebo for 2.1 years [55]. Saxagliptin did not increase or decrease cardiovascular death, myocardial infarction, or ischemic stroke. Interestingly more patients treated with saxagliptin were admitted to the hospital for heart failure. In the alogliptin trial, 5380 patients with either an acute myocardial infarction or unstable angina within the previous 15-90 days were randomized to alogliptin or placebo and followed for a median of 18 months [56]. As seen in the saxagliptin study the rates of cardiovascular events were similar in the alogliptin and placebo groups. The risk of hospitalization for heart failure was not statistically increased in the entire subset of patients treated with alogliptin [57]. However, the hazard ratio for the subgroup of patients without heart failure at baseline was 1.76, p=0.026) [57]. The trend for an increase in heart failure led to the Federal Drug Administration to add it to the label.  In the sitagliptin trial, 14,671 patients with established cardiovascular disease were randomized to sitagliptin or placebo for 3 years [58]. Sitagliptin did not decrease the risk of major adverse cardiovascular events or increase hospitalization for heart failure. Finally, in the linagliptin trial, 6979 patients at high risk for cardiovascular disease were randomized to linagliptin or placebo for a median follow-up of 2.2 years [59]. As in the other DPP4 inhibitor studies, linagliptin did not have a beneficial effect on cardiovascular events. Additionally, linagliptin did not increase the risk of hospitalization for heart failure. Thus, these results indicate that DPP4 inhibitors do not reduce cardiovascular disease. The extent to which specific DPP4 inhibitors affect heart failure needs further investigation.

 

SGLT2 Inhibitors

 

The effects of empagliflozin, an inhibitor of sodium–glucose cotransporter 2 (SGLT2 inhibitor), on cardiovascular morbidity and mortality in patients with Type 2 diabetes has been reported [60]. In this study, 7020 patients at high risk for cardiovascular disease were randomly assigned to receive 10 mg or 25 mg of empagliflozin or placebo once daily and were followed for 3.1 years. In the combined empagliflozin treated groups there was a statistically significant 14% reduction in the primary outcome (death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke). As compared with placebo, empagliflozin treatment did not result in a significant difference in the occurrence of non-fatal myocardial infarction or strokes. However, empagliflozin resulted in a significantly lower risk of death from cardiovascular causes (hazard ratio, 0.62), death from any cause (hazard ratio, 0.68), and hospitalization for heart failure (hazard ratio, 0.65). The beneficial effects of empagliflozin were noted to occur very rapidly and the beneficial effects on congestive heart failure appeared to be the dominant effect compared to effects on atherosclerotic events.

 

The effects of placebo vs. canagliflozin, another inhibitor of SGLT2, were determined in two combined trials involving a total of 10,142 participants with type 2 diabetes and high cardiovascular risk [61]. The primary outcome was a composite of death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke and the mean follow-up was 188 weeks. The primary outcome was reduced in the canagliflozin group (hazard ratio, 0.86; P=0.02). Death from any cause (hazard ratio 0.87; 95% CI 0.74-1.01) and death from cardiovascular disease (hazard ratio 0.87; 95% CI 0.72-1.06) were reduced but were not statistically significant. Similarly, canagliflozin treatment did not result in a significant difference in non-fatal strokes or non-fatal myocardial infarctions (hazard ratio 0.90 for stroke and 0.85 for myocardial infarction). As seen with empagliflozin, hospitalization for heart failure was markedly reduced (hazard ratio 0.67; 95% CI 0.52-0.87) and this beneficial effect occurred rapidly. Notably, there was an increased risk of amputation (hazard ratio, 1.97; 95% CI, 1.41 to 2.75), which were primarily at the level of the toe or metatarsal. The basis for the increase in amputations is unknown. In the empagliflozin study an increase in amputations was not noted.

 

Recently the effect of a 3rdSGLT2 inhibitor on cardiovascular events has been reported [62]. 17,160 patients, including 10,186 without atherosclerotic cardiovascular disease were randomized to dapagliflozin or placebo and followed for a median of 4.2 years. The primary outcome was a composite of major adverse cardiovascular events, defined as cardiovascular death, myocardial infarction, or ischemic stroke. The primary efficacy outcomes were MACE and a composite of cardiovascular death or hospitalization for heart failure. Dapagliflozin did not result in a lower rate of major adverse cardiovascular events (8.8% in the dapagliflozin group and 9.4% in the placebo group; hazard ratio, 0.93; P=0.17) but did result in a lower rate of cardiovascular death or hospitalization for heart failure (4.9% vs. 5.8%; hazard ratio, 0.83; P=0.005), which reflected a lower rate of hospitalization for heart failure (hazard ratio, 0.73; 95% CI, 0.61 to 0.88). Additionally, there was not an increase in lower extremities amputations in the dapagliflozin treated group.

 

Thus, all three SGLT2 inhibitor studies demonstrated a decrease in congestive heart failure with SGLT2 inhibitor therapy. In a meta-analysis of these three trials it was observed that SGLT2 inhibitors reduced the risk of cardiovascular death or hospitalization for heart failure by 23% (p<0·0001), with a similar benefit in patients with and without atherosclerotic cardiovascular disease and with and without a history of heart failure [63]. Additionally, greater reductions in hospitalizations for heart failure was observed in patients with more severe kidney disease at baseline.

 

The mechanisms accounting for the beneficial effects of SGLT2 inhibitors on heart failure are uncertain [64]. Glycemic control was better in the SGLT2 inhibitor treated patients but it is doubtful that this could account for the observed results. SGLT2 inhibitor treatment was associated with small reductions in weight, waist circumference, uric acid level, and systolic and diastolic blood pressure, with no increase in heart rate and small increases in both LDL and HDL cholesterol. Whether these changes played a role in reducing events remains to be determined. It is possible that hemodynamic changes secondary to the osmotic diuresis induced by SGLT2 inhibitors contributed to the beneficial effects. SGLT2 inhibitors increase glucagon secretion, which promotes the production of ketone bodies such as beta-hydroxybutyrate that are utilized by the heart for energy production. It is possible that this alternative source of energy could be protective for heart function. Finally, there may be direct effects of SGLT2 inhibition on myocardial and renal metabolism [64, 65]. Additional cardiovascular outcome studies with other SGLT2 inhibitors and in different patient populations are being carried out and it will be of great interest to see if these studies also demonstrate a reduction in cardiovascular events, particularly heart failure or an increase in amputations [64, 66].

 

GLP-1 Receptor Agonists

 

The effect of five GLP-1 receptor agonists on cardiovascular disease has been reported. 6068 patients with Type 2 diabetes and who recently had a myocardial infarction or been hospitalized for unstable angina were randomized to placebo or lixisenatide, a once-daily GLP-1 receptor agonist, and followed for a median of 25 months [67]. The primary end point of cardiovascular death, myocardial infarction, stroke, or hospitalization for unstable angina was similar in the placebo or lixisenatide groups.

 

In contrast, a study has shown that liraglutide decreased cardiovascular events [68]. In this trial 9340 patients at high cardiovascular risk were randomly assigned to receive liraglutide or placebo. After median time of 3.5 years, the primary outcome of death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke occurred in significantly fewer patients in the liraglutide group (13.0%) than in the placebo group (14.9%) (hazard ratio, 0.87, P=0.01). Additionally, deaths from cardiovascular causes (hazard ratio 0.78, P=0.007) or any cause was lower in the liraglutide group than in the placebo group (hazard ratio, 0.85; P=0.02). Interestingly patients with established cardiovascular disease or decreased renal function (eGFR < 60) appeared to derive the greatest benefit of liraglutide treatment. As expected, weight and blood pressure were decreased in the liraglutide treated group and A1c levels were also decreased by 0.4%.

 

In support of the beneficial effects of some GLP1 receptor agonists to reduce cardiovascular events, semaglutide, a long acting GLP-1 receptor agonist, has been shown to also reduce cardiovascular events [69]. In this trial, 3297 patients with Type 2 diabetes with established cardiovascular disease, chronic heart failure, chronic kidney disease, or age >60 with at least one cardiovascular risk factor were randomized to receive once-weekly semaglutide (0.5 mg or 1.0 mg) or placebo for 104 weeks. The primary outcome of cardiovascular death, nonfatal myocardial infarction, or nonfatal stroke occurred in 6.6% of the semaglutide group and 8.9% of the placebo group (hazard ratio, 0.74;P = 0.02). In this study, both body weight and A1c levels were decreased in the patients treated with semaglutide.

 

The effect of once weekly exenatide vs. placebo on cardiovascular outcomes was tested in 14,752 patients, 73% who had cardiovascular disease [70]. The primary outcome was the occurrence of death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke. After a median follow-up of 3.2 years (duration of drug exposure 2.4 years) the primary outcome was reduced in the exenatide treated group but this difference just missed achieving statistical significance (hazard ratio 0.91; 95% CI 0.83-1.00; p=0.06). While not statistically significant these results are consistent with the results observed with liraglutide and semaglutide treatment. It should be recognized that a high percentage of patients discontinued exenatide therapy in this trial (>40%) and this could have adversely affected the ability of exenatide treatment to favorably effect cardiovascular outcomes.

 

Finally, the effect of once weekly albiglutide vs. placebo was tested in 9,463 patients with cardiovascular disease. The primary outcome was first occurrence of cardiovascular death, myocardial infarction, or stroke. After a median follow-up of 1.6 years a 22% decrease in the primary endpoint was observed in the albiglutide group (hazard ratio 0·78, p<0·0001). It should be noted that albiglutide is no longer available as it was removed from the market due to commercial considerations by Glaxo.

 

Thus, three studies have clearly demonstrated that treatment with GLP-1 receptor agonists reduces cardiovascular events, one study has provided data consistent with these results, and one study failed to demonstrate benefit. Why the differences in results between these studies is unknown but could be due to differential effects of the GLP-1 receptor agonists, differences in the patient populations studied, or other unrecognized variables. The mechanism accounting for this decrease in cardiovascular disease is uncertain but could be related to reductions in glycated hemoglobin, body weight, systolic blood pressure, or the direct effect of activation of GLP-1 receptors on the atherosclerotic process. Cardiovascular studies using long acting dulaglutide are in-progress and will provide additional information on the effect of GLP-1 agonists on cardiovascular events [66].

 

Acarbose

 

In the STOP-NIDDM trial 1429 subjects with impaired glucose tolerance were randomized to placebo vs. acarbose and followed for 3.3 years [71]. In the acarbose group a 49% relative risk reduction in the development of cardiovascular events (hazard ratio 0.51; P =0.03) was observed. Among cardiovascular events, the major reduction was in the risk of myocardial infarction (HR, 0.09; P =.02). In a smaller trial, 135 patients hospitalized for the acute coronary syndrome who were newly diagnosed with IGT were randomly assigned to acarbose or placebo [72]. During a mean follow-up of 2.3 years the risk of recurrent major adverse cardiovascular event was decreased significantly in the acarbose group compared with that in control group (26.7% versus 46.9%, P < 0.05).

 

Despite these favorable observations a large recently published trial failed to demonstrate a beneficial effect of acarbose in Chinese patients with impaired glucose tolerance [73]. In a randomized trial acarbose vs. placebo was compared in 6522 patients with coronary heart disease and impaired glucose tolerance. The primary outcome was cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, hospital admission for unstable angina, and hospital admission for heart failure and patients were followed up for a median of 5 years. The primary outcome was similar in the acarbose and placebo groups (hazard ratio 0·98; 95% CI 0·86-1·11, p=0·73). No significant differences were seen for death from any cause, cardiovascular death, fatal or non-fatal myocardial infarction, fatal or non-fatal stroke, hospital admission for unstable angina, hospital admission for heart failure, or impaired renal function.

 

Thus, whether acarbose favorably affects cardiovascular disease in patients at high risk for developing diabetes is uncertain. Moreover, the effect of acarbose on cardiovascular disease in patients with diabetes is unknown. 

 

Cycloset

 

Cycloset is a quick-release bromocriptine formulation (bromocriptine-QR) that activates the D2 dopamine receptor and is approved for the treatment of diabetes. A 52 week, randomized, double-blind, multicenter trial evaluated cardiovascular safety in 3,095 patients with type 2 diabetes treated with bromocriptine-QR or placebo [74].  The composite end point of first myocardial infarction, stroke, coronary revascularization, or hospitalization for angina or congestive heart failure occurred in 1.8% of the bromocriptine-QR treated vs. 3.2% of the placebo-treated patients resulting in a 40% decrease in cardiovascular events (HR 0.60; CI 0.37– 0.96). Clearly further studies to confirm this finding and to elucidate the mechanism of this beneficial effect are required.

 

Insulin

 

In patients with Type 1 diabetes the DCCT trial demonstrated that intensive insulin therapy reduced cardiovascular events [25, 26]. With regard to patients with Type 2 diabetes, in the Origin Trial 12,537 people with cardiovascular risk factors plus impaired fasting glucose, impaired glucose tolerance, or Type 2 diabetes were randomized to receive insulin glargine or standard care [75]. The cardiovascular outcomes, which included nonfatal myocardial infarction, nonfatal stroke, death from cardiovascular causes, revascularization, or hospitalization for heart failure, were similar in the glargine and placebo groups. Extended follow-up also did not demonstrate favorable effects on cardiovascular events in the glargine treated patients [76]. Additionally, in patients with type 2 diabetes at high risk for cardiovascular events the occurrence ofmajor cardiovascular events was similar in patients treated with degludec insulin or glargine insulin [77]. These studies demonstrate that insulin does not accelerate atherosclerosis and by lowering glucose levels may decrease atherosclerosis, although the protective effects are mainly observed in patients with Type 1 diabetes over a protracted period of time.

 

Other Studies

 

Finally, the Bari 2D study compared the effect of insulin sensitizers (metformin/TZD- mostly rosiglitazone) vs. insulin provision therapy (sulfonylureas/insulin) on cardiovascular outcomes in patients with Type 2 diabetes and coronary artery disease (> 50% stenosis and positive stress test or > 70% stenosis and classic angina) [78, 79]. In this study, no differences in survival or cardiovascular endpoints were observed between metformin/TZD therapy vs. sulfonylurea/insulin therapy for the entire study. However, in the group with more severe coronary artery disease who were selected for coronary artery bypass surgery, the combination of coronary artery bypass and treatment with insulin sensitizers was associated with a lower rate of cardiovascular events. Why the metformin/TZD group only derived an enhanced benefit in the coronary artery bypass patients in this study is unknown. It should be noted that the vast majority of patients on TZD therapy were treated with rosiglitazone and, as discussed above, the effects of rosiglitazone on cardiovascular disease do not appear to be as beneficial as pioglitazone.

 

Summary

 

These studies demonstrate that the method by which one improves glycemic control may be very important with different drugs having effects in addition to glucose lowering that could reduce cardiovascular events. While previous treatment algorithms have primarily focused on the effect of drugs on glycemic control, current treatment recommendations for patients with diabetes are using the results of these cardiovascular disease trials to decide which drugs should be employed. For example, the ADA/EASD is recommending that in patients with cardiovascular disease an SGLT inhibitor or GLP1 agonist with proven cardiovascular benefit should be part of the treatment regimen [80].

 

ROLE OF OTHER RISK FACTORS IN CARDIOVASCULAR DISEASE

 

Numerous studies have demonstrated that the traditional risk factors for cardiovascular disease play an important role in patients with diabetes [2, 4, 5, 81]. Patients with diabetes without other risk factors have a relatively low risk of cardiovascular disease (albeit higher than similar non-diabetic patients), whereas the increasing prevalence of other risk factors markedly increases the risk of developing cardiovascular disease [2]. The major reversible traditional risk factors are hypertension, cigarette smoking, and lipid abnormalities [2, 4, 5, 13, 82]. Other risk factors include obesity (particularly visceral obesity), insulin resistance, small dense LDL, elevated triglycerides, procoagulant state (increased PAI-1, fibrinogen), homocystine, Lp (a), renal disease, microalbuminuria, and inflammation (C-reactive protein, SAA, cytokines) [2, 4, 5, 81, 82]. In the last decade, it has become clear that to reduce the risk of cardiovascular disease in patients with diabetes, one will not only need to improve glycemic control but also address these other cardiovascular risk factors. In the remainder of this chapter we will focus on the dyslipidemia that occurs in patients with diabetes.

 

ROLE OF LIPIDS IN CARDIOVASCULAR DISEASE

 

As in the non-diabetic population, epidemiological studies have shown that increased LDL cholesterol and non-HDL cholesterol levels and decreased HDL cholesterol levels are associated with an increased risk of cardiovascular disease in patients with diabetes [2, 4, 81, 82]. While it is universally accepted that elevated levels of LDL cholesterol and non-HDL cholesterol cause atherosclerosis and cardiovascular disease the role of HDL cholesterol is uncertain. Genetic studies and studies of drugs that raise HDL cholesterol have not supported a causative role of low HDL cholesterol levels as a causative factor for atherosclerosis [83]. Rather it is currently thought that HDL function is associated with atherosclerosis risk and that this does not precisely correlate with HDL cholesterol levels [83]. In patients with diabetes, elevations in serum triglyceride levels also are associated with an increased risk of cardiovascular disease [4, 82, 84]. With regard to triglycerides, it is not clear whether they are an independent risk factor for cardiovascular disease or whether the elevation in triglycerides is a marker for other abnormalities, such as decreased HDL cholesterol levels or increased non-HDL cholesterol levels [4, 82, 84]. Recent Mendelian Randomization studies have provided strong support for the hypothesis that elevated triglyceride levels play a causal role in atherosclerosis [85].

 

LIPID ABNORMALITIES IN PATIENTS WITH DIABETES

 

In patients with Type 1 diabetes in good glycemic control, the lipid profile is very similar to lipid profiles in the general population [81]. In contrast, in patients with Type 2 diabetes, even when in good glycemic control, there are abnormalities in lipid levels [86-89]. It is estimated that 30-60% of patients with Type 2 diabetes have dyslipidemia [5, 90]. Specifically, patients with Type 2 diabetes often have an increase in serum triglyceride levels, increased VLDL and IDL, and decreased HDL cholesterol levels. Non-HDL cholesterol levels are increased due to the increase in VLDL and IDL. LDL cholesterol levels are typically not different than in normal subjects but there is an increase in small dense LDL, a lipoprotein particle that may be particularly pro-atherogenic. As a consequence there are more LDL particles, which coupled with the increases in VLDL and IDL, leads to an increase in Apo B [86-89]. Studies have shown that the anti-oxidant and anti-inflammatory functions of HDL isolated from patients with diabetes are reduced, indicating that HDL levels per se may not fully reflect risk [91]. Additionally, the postprandial increase in serum triglycerides is accentuated and elevations in postprandial lipids may increase the risk of cardiovascular disease [86-89]. It should be recognized that these lipid changes are characteristic of the alterations in lipid profile seen in obesity and the metabolic syndrome (insulin resistance syndrome) [92]. Additionally, the ability of HDL to facilitate cholesterol efflux is reduced in patients with Type 2 diabetes [93]. Since a high percentage of patients with Type 2 diabetes are obese, insulin resistant and have the metabolic syndrome, it is not surprising that the prevalence of increased triglycerides and small dense LDL and decreased HDL cholesterol is common in patients with Type 2 diabetes even when these patients are in good glycemic control.

 

In both Type 1 and Type 2 diabetes, poor glycemic control increases serum triglyceride levels, VLDL and IDL, and decreases HDL cholesterol levels [87]. Poor glycemic control can also result in a modest increase in LDL cholesterol, which because of the elevation in triglycerides is often in the small dense LDL subfraction. It is therefore important to optimize glycemic control in patients with diabetes because this will have secondary beneficial effects on lipid levels.

 

Lp (a) levels are usually within the normal range in patients with Type 2 diabetes and do not appear to be greatly affected by glycemic control [94-96]. In patients with Type 1 diabetes Lp(a) levels are also within the normal range but improvements in glycemic control result in decreases in Lp (a) levels [97]. The development of microalbuminuria and the onset of renal disease are associated with an increase in Lp (a) levels [98].

 

Table 1: Lipid Abnormalities in Patients with Diabetes
Type 1 Diabetes Lipid profile is similar to controls if glycemic control is good
Type 2 Diabetes Increased triglycerides, VLDL, IDL, and non-HDLc. Decreased HDLc. Normal LDLc but increase in small dense LDL, LDL particle number, and apolipoprotein B.
Poor glycemic control Increased triglycerides, VLDL and IDL and decreased HDLc. Modest increase in LDLc with increase in small dense LDL and particle number.

 

 

EFFECT OF GLUCOSE LOWERING DRUGS ON LIPIDS

 

Some therapies used to improve glycemic control may have an impact on lipid levels above and beyond their effects on glucose metabolism. Specifically, insulin, sulfonylureas, meglinitides, DPP4 inhibitors, and alpha-glucosidase inhibitors do not markedly alter fasting lipid profiles other than by improving glucose control (there are data indicating that DPP4 inhibitors and acarbose decrease postprandial triglyceride excursions, but they do not alter fasting lipid levels) [66]. In contrast, metformin, thiazolidinediones, GLP1 agonists, and SGLT2 inhibitors have effects independent of glycemic control on serum lipid levels. Metformin decreases serum triglyceride levels and may modestly decrease LDL cholesterol without altering HDL cholesterol [66]. The effect of thiazolidinediones appears to depend on which agent is used. Rosiglitazone increases serum LDL cholesterol levels, increases HDL cholesterol levels, and only decreases serum triglycerides if the baseline triglyceride levels are high [66]. In contrast, pioglitazone has less impact on LDL cholesterol levels, but increases HDL cholesterol levels, and decreases serum triglyceride levels [66]. It should be noted that reductions in the small dense LDL subfraction and an increase in the large buoyant LDL subfraction are seen with both thiazolidinediones [66]. In a randomized head to head trial it was shown that pioglitazone decreased serum triglyceride levels and increased serum HDL cholesterol levels to a greater degree than rosiglitazone treatment [99, 100]. Additionally, pioglitazone increased LDL cholesterol levels less than rosiglitazone. In contrast to the differences in lipid parameters, both rosiglitazone and pioglitazone decreased A1c and C-reactive protein to a similar extent. The mechanism by which pioglitazone induces more favorable changes in lipid levels than rosiglitazone is unclear, but differential actions of ligands for nuclear hormone receptors are well described. Treatment with SGLT2 inhibitors results in a small increase in LDL cholesterol and HDL cholesterol levels [66]. Finally, GLP-1 receptor agonists, such as exenatide and liraglutide, can favorably affect the lipid profile by inducing weight loss (decreasing triglycerides and increasing HDL cholesterol levels) [66]. Additionally, GLP-1 receptor agonists reduce postprandial triglycerides [66].

 

Table 2: Effect of Glucose Lowering Drugs on Lipid Levels
Metformin Decrease triglycerides and modestly decrease LDLc
Sulfonylureas No effect
DPP4 inhibitors Decrease postprandial triglycerides
GLP1 analogues Decrease fasting and postprandial triglycerides and increase HDLc
Acarbose Decrease postprandial triglycerides

Pioglitazone

Rosiglitazone

Decrease triglycerides and increase HDLc. Small increase LDLc but a decrease in small dense LDL
SGLT2 inhibitors Small increase in LDLc and HDLc
Insulin No effect

 

PATHOPHYSIOLOGY OF THE DYSLIPIDEMIA OF DIABETES

Figure 1. Pathophysiology of the Dyslipidemia of Diabetes

Increase in Triglycerides

 

There are a number of different abnormalities that contribute to the dyslipidemia seen in patients with Type 2 diabetes and obesity (figure 1) [87-90, 101-103]. A key abnormality is the overproduction of VLDL by the liver, which is a major contributor to the elevations in serum triglyceride levels. The rate of secretion of VLDL is highly dependent on triglyceride availability, which is determined by the levels of fatty acids available for the synthesis of triglycerides in the liver. An abundance of triglycerides prevents the intra-hepatic degradation of Apo B-100 allowing for increased VLDL formation and secretion. There are three major sources of fatty acids in the liver all of which may be altered in patients with type 2 diabetes. First, the flux of fatty acids from adipose tissue to the liver is increased. An increased mass of adipose tissue, particularly visceral stores, results in increased fatty acid delivery to the liver. Additionally, insulin suppresses the lipolysis of triglycerides to free fatty acids in adipose tissue; thus, in patients with either poorly controlled diabetes due to a decrease in insulin or a decrease in insulin activity due to insulin resistance, the inhibition of triglyceride lipolysis is blunted and there is increased triglyceride breakdown leading to increased fatty acid deliver to the liver. A second source of fatty acids in the liver is de novofatty acid synthesis from glucose. Numerous studies have shown that fatty acid synthesis is increased in the liver in patients with type 2 diabetes. This increase may be mediated by the hyperinsulinemia seen in patients with insulin resistance. While the liver is resistant to the effects of insulin on carbohydrate metabolism, the liver remains sensitive to the effects of insulin stimulating lipid synthesis. Specifically, insulin stimulates the activity of SREBP-1c, a transcription factor that increases the expression of the enzymes required for the synthesis of fatty acids. Thus, while the liver is resistant to the effects of insulin on carbohydrate metabolism the liver remains sensitive to the effects of insulin stimulating lipid synthesis. Additionally, in the presence of hyperglycemia, glucose can induce another transcription factor, carbohydrate responsive element binding protein (ChREBP), which also stimulates the transcription of the enzymes required for fatty acid synthesis. The third source of fatty acids is the uptake of triglyceride rich lipoproteins by the liver. Studies have shown an increase in intestinal fatty acid synthesis and the enhanced secretion of chylomicrons in animal models of type 2 diabetes. This increase in chylomicrons leads to the increased delivery of fatty acids to the liver. The increase in hepatic fatty acids produced by these three pathways results in an increase in the synthesis of triglycerides in the liver and the protection of Apo B-100 from degradation resulting in the increased formation and secretion of VLDL. Finally, insulin stimulates the post translational degradation of Apo B-100 in the liver and a decrease in insulin activity in patients with Type 2 diabetes also allows for the enhanced survival of Apo B-100 promoting increased VLDL formation.

 

While the overproduction of triglyceride rich lipoproteins by the liver and intestine are the main contributors to the elevations in serum triglyceride levels in patients with Type 2 diabetes, there are also abnormalities in the metabolism of these triglyceride rich lipoproteins. First, there is a modest decrease in lipoprotein lipase activity, the key enzyme that metabolizes triglyceride rich lipoproteins. The expression of lipoprotein lipase is stimulated by insulin and decreased insulin activity in patients with Type 2 diabetes results in a decrease in lipoprotein lipase, which plays a key role in the hydrolysis of the triglycerides carried in chylomicrons and VLDL. Additionally, patients with Type 2 diabetes have an increase in Apo C-III levels. Glucose stimulates and insulin suppresses Apo C-III expression. Apo C-III is an inhibitor of lipoprotein lipase activity and thereby reduces the clearance of triglyceride rich lipoproteins. In addition, Apo C-III also inhibits the cellular uptake of lipoproteins. Recent studies have shown that loss of function mutations in Apo C-III lead to lower serum triglyceride levels and a reduced risk of cardiovascular disease [104, 105]. Interestingly, inhibition of Apo C-III expression results in a decrease in serum triglyceride levels even in patients deficient in lipoprotein lipase, indicating that the ability of Apo C-III to modulate serum triglyceride levels is not dependent solely on regulating lipoprotein lipase activity [106]. Thus, in patients with diabetes, a decrease in clearance of triglyceride rich lipoproteins also contributes to the elevation in serum triglyceride levels.

 

Effect on HDL and LDL

 

The elevation in triglyceride rich lipoproteins in turn has effects on other lipoproteins. Specifically, cholesterol ester transfer protein (CETP) mediates the exchange of triglycerides from triglyceride rich VLDL and chylomicrons to LDL and HDL. The increase in triglyceride rich lipoproteins per seleads to an increase in CETP mediated exchange, increasing the triglyceride content of both LDL and HDL. The triglyceride on LDL and HDL is then hydrolyzed by hepatic lipase and lipoprotein lipase leading to the production of small dense LDL and small HDL. Notably hepatic lipase activity is increased in patients with Type 2 diabetes, which will also facilitate the removal of triglyceride from LDL and HDL resulting in small lipoprotein particles. The affinity of Apo A-I for small HDL particles is reduced, leading to the disassociation of Apo A-I, which in turn leads to the accelerated clearance and breakdown of Apo A-I by the kidneys. Additionally, the production of Apo A-I may be reduced in patients with diabetes. High glucose levels can activate ChREBP and this transcription factor inhibits Apo A-I expression. Furthermore, insulin stimulates Apo A-I expression and a reduction in insulin activity due to insulin resistance or decreased insulin levels may also lead to a decrease in ApoA-I expression. The net result is lower levels of Apo A-I and HDL cholesterol levels in patients with Type 2 diabetes.

 

Role of Glucose and Insulin

 

The above described changes lead to the typical dyslipidemia observed in patients with Type 2 diabetes (increased triglycerides, decreased HDL cholesterol, and an abundance of small dense LDL and small HDL). In patients with both Type 1 and Type 2 diabetes, poor glycemic control can further adversely affect lipid and lipoprotein metabolism. As noted above the expression of lipoprotein lipase is stimulated by insulin. If insulin activity is very low the expression of lipoprotein lipase is severely suppressed and the metabolism of triglyceride rich lipoproteins is markedly impaired. This leads to the delayed clearance of both chylomicrons and VLDL and elevations of triglyceride rich lipoproteins. Additionally, insulinopenia results in a marked increase in lipolysis in adipose tissue, leading to the release of free fatty acids into the circulation. This increase in serum fatty acids results in the increased delivery of fatty acids to the liver, enhanced triglyceride synthesis in the liver, and the increased production and secretion of VLDL. Whereas patients with Type 1 diabetes who are well controlled typically have normal serum lipid profiles, if their control deteriorates, they will develop hypertriglyceridemia. In patients with Type 2 diabetes deterioration of glycemic control will further exacerbate their underlying dyslipidemia resulting in greater increases in serum triglyceride levels. If the synthesis of new VLDL is increased sufficiently this can result in an increase in LDL levels. HDL levels may decrease due to the formation of small HDL that are more susceptible to accelerated clearance. Improvements in glycemic control can markedly lower serum triglyceride levels and may increase serum HDL levels. In patients with very poorly controlled diabetes improvements in glycemic control may also lower LDL levels.

 

Role of Inflammation

 

Many if not most patients with Type 2 diabetes are obese. Obesity is a pro-inflammatory state due to the macrophages that infiltrate adipose tissue. The cytokines produced by these macrophages and the adipokines that are produced by fat cells also alter lipid metabolism [107, 108]. The pro-inflammatory cytokines, TNF and IL-1, decrease the expression of lipoprotein lipase and increase the expression of angiopoietin like protein 4, an inhibitor of lipoprotein lipase. Together these changes decrease lipoprotein lipase activity, thereby delaying the clearance of triglyceride rich lipoproteins. In addition, pro-inflammatory cytokines stimulate lipolysis in adipocytes increasing circulating free fatty acid levels, which will provide substrate for hepatic triglyceride synthesis. In the liver, pro-inflammatory cytokines stimulate de novo fatty acid and triglyceride synthesis. These alterations will lead to the increased production and secretion of VLDL. Thus, increases in the levels of pro-inflammatory cytokines will stimulate the production of triglyceride rich lipoproteins and delay the clearance of triglyceride rich lipoproteins, which together will contribute to the increase in serum triglycerides that occurs in obese patients.

 

Adipokines, such as leptin and adiponectin, also regulate lipid metabolism. Obesity increases serum leptin levels and leptin stimulates lipolysis in adipocytes which will increase serum free fatty acid levels. Obesity decreases adiponectin serum levels and studies have shown that the administration of adiponectin to mice decreases serum triglyceride levels. Adiponectin increases lipoprotein lipase and improves the clearance of an exogenous fat load. One would therefore anticipate that the decrease in adiponectin that occurs with obesity would have adverse effects on triglyceride metabolism.

 

Pro-inflammatory cytokines also affect HDL metabolism [109, 110]. First, they decrease the production of Apo A-I, the main protein constituent of HDL. Second, in many tissues pro-inflammatory cytokines decrease the expression of ABCA1 and ABCG1, which will lead to a decrease in the efflux of phospholipids and cholesterol from the cell to HDL. Third, pro-inflammatory cytokines decrease the production and activity of LCAT, which will limit the conversion of cholesterol to cholesterol esters in HDL. This step is required for the formation of a normal spherical HDL particle and facilitates the ability of HDL to transport cholesterol. Fourth, pro-inflammatory cytokines decrease CETP levels, which will decrease the movement of cholesterol from HDL to Apo B containing lipoproteins. Pro-inflammatory cytokines decrease the expression of SR-B1 in the liver. SR-B1 plays a key role in the uptake of cholesterol from HDL particles into hepatocytes. Finally, pro-inflammatory cytokines decrease the expression of ABCG5 and ABCG8 in the liver, which reduces the secretion of cholesterol into the bile, providing more cholesterol for the formation and secretion of VLDL into the circulation. Together these changes induced by pro-inflammatory cytokines result in a decrease in reverse cholesterol transport. Reverse cholesterol transport plays a key role in preventing cholesterol accumulation in macrophages and thereby reduces atherosclerosis. Inflammation also decreases other important functions of HDL, such as its ability to prevent LDL oxidation [111]. In parallel inflammation increases the oxidation of LDL and the small dense LDL that occurs in patients with diabetes is more susceptible to oxidation.

 

EFFECT OF LIPID LOWERING DRUGS ON CARDIOVASCULAR EVENTS

 

Monotherapy Studies

 

STATINS

 

As shown in Table 3, statin trials, including both primary and secondary prevention trials, have consistently shown the beneficial effect of statins on cardiovascular disease including patients with diabetes, primarily by lowering LDL cholesterol levels. The Cholesterol Treatment Trialists analyzed data from 18,686 subjects with diabetes (mostly Type 2 diabetes) from 14 randomized trials [112]. In the statin treated group there was a 9% decrease in all-cause mortality, a 13% decrease in vascular mortality, and a 21% decrease in major vascular events per 39mg/dl reduction in LDL cholesterol. The beneficial effect of statin therapy was seen in both primary and secondary prevention patients. The effect of statin treatment on cardiovascular events in patients with diabetes was similar to that seen in non-diabetic subjects. Thus, these studies indicate that statins are beneficial in reducing cardiovascular disease in patients with diabetes. Because of the large number of patients with diabetes included in the Heart Protection Study (HPS) and CARDS these two studies will be discussed in greater depth.

 

Table 3: Effect of Monotherapy with Statins on Cardiovascular Outcomes
Study Drug % Decrease
   Controls Diabetics
2º Prevention
4S Simvastatin 32 55
CARE Pravastatin 23 25
LIPID Pravastatin 25 19
LIPS Fluvastatin 20 43
HPS Simvastatin 24 26
1º Prevention
AFCAPS Lovastatin 37 42
HPS Simvastatin 24 24
ASCOT Atorvastatin 44 16
CARDS Atorvastatin -- 37

 

The HPS was a double blind randomized trial that focused on patients at high risk for the development of cardiovascular events, including patients with a history of myocardial infarctions, other atherosclerotic lesions, diabetes, and/or hypertension [113, 114]. Patients were between 40 and 80 years of age and had to have total serum cholesterol levels greater than 135mg/dl (thus very few patients were excluded because they did not have a high enough cholesterol level). The major strength of this trial was the large number of patients studied (>20,000). The diabetes subgroup included 5,963 subjects and thus was as large as many other prevention trials. The study was a 2x2 study design comparing simvastatin 40mg a day vs. placebo and anti-oxidant vitamins (vitamin E 600mg, vitamin C 250mg, and beta-carotene 20mg) vs. placebo and lasted approximately 5 years. Analysis of the group randomized to the anti-oxidant vitamins revealed no beneficial or harmful effects. In contrast, simvastatin therapy (40mg per day) reduced cardiovascular events, including myocardial infarctions and strokes, by approximately 25% in all participants and to a similar degree in the diabetic subjects (total cardiovascular disease reduced 27%, coronary mortality 20%, myocardial infarction 37%, stroke 24%). Further analysis of the subjects with diabetes revealed that the reduction in cardiovascular events with statin therapy was similar in individuals with diabetes diagnosed for a short duration (<6 years) and for a long duration (>13 years). Similarly, subjects with diabetes in good control (HbA1c <7%) and those not in ideal control (HbA1c >7%) also benefited to a similar degree with statin therapy. Moreover, both Type 1 and Type 2 diabetic patients had a comparable reduction in cardiovascular disease with simvastatin therapy. The decrease in cardiovascular events in patients with Type 1 diabetes was not statistically significant because of the small number of subjects. Nevertheless, this is the only trial that included Type 1 diabetics and suggests that patients with Type 1 will benefit from statin therapy similar to Type 2 diabetics. In general, statin therapy reduced cardiovascular disease in all subgroups of subjects with diabetes (females, males, older age, renal disease, hypertension, high triglycerides, low HDL, ASA therapy, etc.) i.e. statin therapy benefits all patients with diabetes. Of particular note, even subjects with diabetes whose baseline LDL cholesterol levels were less than 116mg/dl had a reduction in cardiovascular events when treated with simvastatin. Moreover, analysis of all study patients similarly demonstrated that subjects with LDL cholesterol levels less than 100mg/dl benefited from statin therapy. These results were of particular clinical importance because they demonstrated that in high-risk patients with LDL cholesterol levels < 100mg/dl statin therapy would nevertheless result in benefit.

 

The CARDS trial specifically focused on subjects with diabetes [115]. The subjects in this trial were males and females with Type 2 diabetes between the ages of 40 to 75 years of age who were at high risk of developing cardiovascular disease based on the presence of hypertension, retinopathy, renal disease, or current smoking. Of particular note, the subjects did not have any evidence of clinical atherosclerosis (myocardial disease, stroke, peripheral vascular disease) at entry and hence this study is a primary prevention trial. Inclusion criteria included LDL cholesterol levels less than 160mg/dl and triglyceride levels less than 600mg/dl. It is important to recognize that the average LDL cholesterol in this trial was approximately 118mg/dl, indicating relatively low LDL cholesterol levels. A total of 2838 Type 2 diabetic subjects were randomized to either placebo or atorvastatin 10mg a day. Atorvastatin therapy resulted in a 40% decrease in LDL cholesterol levels with over 80% of patients achieving LDL cholesterol levels less than 100mg/dl. Most importantly, atorvastatin therapy resulted in a 37% reduction in cardiovascular events. In addition, strokes were reduced by 48% and coronary revascularization by 31%. As seen in the HPS, subjects with relatively low LDL cholesterol levels (LDL <120mg/dl) benefited to a similar extent as subjects with higher LDL cholesterol levels (>120mg/dl). CARDS, in combination with the other statin trials, provide conclusive evidence that statin therapy will reduce cardiovascular events in patients with diabetes. Importantly, the benefits of statin therapy are seen in patients with diabetes in both primary and secondary prevention trials.

 

A few studies have compared the effect of different magnitudes of LDL cholesterol lowering with statins on the reduction in cardiovascular events in patients with diabetes. The Post-CABG study compared very low dose lovastatin (2.5-5.0mg per day) vs. high dose lovastatin (40-80mg per day) in 1,351 subjects post bypass surgery [116]. Approximately 10% of patients in this trial had diabetes. Baseline LDL cholesterol levels were between 130-174mg/dl. As expected, the high dose of lovastatin reduced LDL cholesterol levels to a much greater degree than the low dose lovastatin (low dose achieved LDL cholesterol levels of approximately 135mg/dl vs. high dose achieved LDL cholesterol levels of approximately 95mg/dl). The main comparison in this trial was the change in atherosclerosis in the grafts measured by comparing baseline angiography to angiography after an average of 4.3 years. In the entire population, the mean percentage of grafts with progression of atherosclerosis was 27 percent in the high dose lovastatin group and 39 percent in the low dose lovastatin group. Additionally, the rate of revascularization was reduced by 29 percent in the high dose lovastatin group. When the patients with diabetes were analyzed separately, similar beneficial effects were observed. These results indicate that lowering LDL cholesterol levels to less than 100mg/dl would slow the angiographic changes to a greater extent than lowering the LDL cholesterol levels to 135mg/dl. Of note though is that even with LDL cholesterol levels less than 100mg/dl progression of atherosclerosis still occurred.

 

Studies have also compared reductions of LDL cholesterol to approximately 100mg/dl to more aggressive reductions in LDL cholesterol. The Reversal Trial studied 502 symptomatic coronary artery disease patients with an average LDL cholesterol of 150mg/dl [117]. Approximately 19% of the patients in this trial had diabetes. Patients were randomized to moderate LDL lowering therapy with pravastatin 40mg per day or to aggressive lipid lowering with atorvastatin 80mg per day. As expected, LDL cholesterol levels were considerably lower in the atorvastatin treated group (pravastatin LDL= 110mg/dl vs. atorvastatin LDL= 79mg/dl). Most importantly, when one analyzed the change in atheroma volume determined after 18 months of therapy using intravascular ultrasound, the group treated aggressively with atorvastatin had a much lower progression rate than the group treated with pravastatin. Compared with baseline values, patients treated with atorvastatin had no change in atheroma burden (there was a very slight regression of lesions), whereas patients treated with pravastatin showed progression of lesions. When one compares the extent of the reduction in LDL cholesterol to the change in atheroma volume, a 50% reduction in LDL (LDL cholesterol levels of approximately 75mg/dl) resulted in the absence of lesion progression. This study suggests that lowering the LDL cholesterol to levels well below 100mg/dl is required to prevent disease progression as measured by intravascular ultrasound. Other studies, such as Asteroid, have shown that marked reductions in LDL cholesterol (in Asteroid the mean LDL cholesterol levels were 61mg/dl) can even result in the regression of coronary artery atherosclerosis determined by intravascular ultrasound measurements [118]. Recently the Saturn trial demonstrated that aggressive lipid lowering with either atorvastatin 80mg or rosuvastatin 40mg would induce regression of coronary artery atherosclerosis to a similar degree in patients with and without diabetes if the LDL cholesterol levels were reduced to less than 70mg/dl [119]. Together these trials indicate that aggressive lowering of LDL cholesterol levels to below 70mg/dl can induce regression of atherosclerotic lesions.

 

The Prove-It trial determined in patients recently hospitalized for an acute coronary syndrome whether aggressively lowering of LDL cholesterol with atorvastatin 80mg per day vs. moderate LDL cholesterol lowering with pravastatin 40mg per day would have a similar effect on cardiovascular end points such as death, myocardial infarction, documented unstable angina requiring hospitalization, revascularization, or stroke [120, 121]. In this trial, approximately 18% of the patients were diabetic. As expected, the on-treatment LDL cholesterol levels were significantly lower in patients aggressively treated with atorvastatin compared to the moderate treated pravastatin group (atorvastatin LDL cholesterol = approximately 62 vs. pravastatin LDL cholesterol = approximately 95mg/dl). Of great significance, death or major cardiovascular events was reduced by 16% over the two years of the study in the group aggressively treated with atorvastatin. Moreover, the risk reduction in the patients with diabetes in the aggressive treatment group was similar to that observed in non-diabetics.

 

In the treating to new targets trial (TNT) patients with stable coronary heart disease and LDL cholesterol levels less than 130mg/dl were randomized to either 10mg or 80mg atorvastatin and followed for an average of 4.9years[122, 123]. Approximately 15% of the patients had diabetes. As expected, LDL cholesterol levels were lowered to a greater extent in the patients treated with 80mg atorvastatin than with 10mg atorvastatin (77mg/dl vs. 101mg/dl). Impressively, the occurrence of major cardiovascular events was reduced by 22% in the group treated with atorvastatin 80mg (p<0.001). In the patients with diabetes events were reduced by 25% in the high dose statin group. Once again, the risk reduction in the patients with diabetes randomized to the aggressive treatment group was similar to that observed in non-diabetics.

 

Finally, the IDEAL trial was a randomized study that compared atorvastatin 80mg vs. simvastatin 20-40mg in 8,888 patients with a history of cardiovascular disease [124]. Approximately 12% of the patients had diabetes.As expected, LDL cholesterol levels were reduced to a greater extent in the atorvastatin treated group than the simvastatin treated group (approximately 104mg/dl vs. 81mg/dl). Once again, the greater reduction in LDL cholesterol levels was associated with a greater reduction in cardiovascular events. Specifically, major coronary events defined as coronary death, nonfatal myocardial infarction, or cardiac arrest was reduced by 11% (p=0.07), while nonfatal acute myocardial infarctions were reduced by 17% (p=0.02).

 

Combining the results of the Heart Protection Study, CARDS, Reversal, Prove-It, TNT, and IDEAL leads one to the conclusion that aggressive lowering of LDL cholesterol with statin therapy will be beneficial and suggests that in high risk patients lowering the LDL to levels well below 100mg/dl is desirable. Recently, the Cholesterol Treatment Trialists reviewed five trials with 39,612 subjects that were designed to determine the effect of usual vs. aggressive reductions in LDL cholesterol [125]. They reported that intensive control (approximately a 19mg/dl difference in LDL cholesterol) resulted in a 15% decrease in major vascular events, a 13% reduction in coronary death or non-fatal MI, a 19% decrease in coronary revascularization, and a 16% decrease in strokes. As will be discussed below most treatment guidelines reflect the results of these studies. Additionally, as described in detail below, recent studies of the addition of either ezetimibe or PCSK9 inhibitors to statins further demonstrates that aggressive lowering of LDL cholesterol levels further reduces cardiovascular events

 

FIBRATES

 

The beneficial effect of monotherapy with fibrates (e.g. gemfibrozil, fenofibrate) on cardiovascular disease in patients with diabetes is shown in Table 4. While the data are not as strong as with statins, the results of these randomized trials suggest that this class of drug also reduces cardiovascular events in patients with diabetes. The largest trial was the Field Trial [126]. In this trial, 9795 patients with Type 2 diabetes between the ages of 50 and 75 not taking statin therapy were randomized to fenofibrate or placebo and followed for approximately 5 years. Fenofibrate therapy resulted in a 12% decrease in LDL cholesterol, a 29% decrease in triglycerides and a 5% increase in HDL cholesterol levels. The primary outcome was coronary events (coronary heart disease death and non-fatal MI), which were reduced by 11% in the fenofibrate group but did not reach statistical significance (p= 0.16). However, there was a 24% decrease in non-fatal MI in the fenofibrate treated group (p=0.01) and a non-significant increase in coronary heart disease mortality. Total cardiovascular disease events (coronary events plus stroke and coronary or carotid revascularization) were reduced 11% (p=0.035). These beneficial effects of fenofibrate therapy on cardiovascular disease were observed in patients without a previous history of cardiovascular disease. In patients with a previous history of cardiovascular disease no benefits were observed. Additionally, the beneficial effect of fenofibrate therapy was seen only in those subjects less than 65 years of age. The beneficial effects of fenofibrate in this study may have been muted by the increased use of statins in the placebo group, which reduced the differences in lipid levels between the placebo and fenofibrate groups. If one adjusted for the addition of lipid-lowering therapy, fenofibrate reduced the risk of coronary heart disease events by 19% (p=0·01) and of total cardiovascular disease events by 15% (p=0·004).

 

While the results of fibrate trials have been very heterogeneous it should be noted that fibrate trials in patients with elevated triglyceride levels have reported a greater reduction of cardiovascular events [127]. Additionally, subgroup analysis of several fibrate trials has also suggested that the benefit of fibrates was greatest in patients with elevated triglyceride levels [127, 128].

 

The mechanism by which fibrates reduce cardiovascular events is unclear. These drugs lower serum triglyceride levels and increase HDL cholesterol, but it should be recognized that the beneficial effects of fibrates could be due to other actions of these drugs. Specifically, these drugs activate PPAR alpha, which is present in the cells that comprise the atherosclerotic lesions, and it is possible that these compounds directly affect lesion formation and development. In addition, fibrates are anti-inflammatory. In fact, analysis of the VA-HIT study suggested that much of the benefit of fibrate therapy was not due to changes in serum lipoprotein levels [129, 130].

 

To summarize, while in general the studies to date suggest that monotherapy with fibrates reduce cardiovascular disease in patients with diabetes, the results are not as robust or consistent as seen in the statin trials. Of note fibrate therapy was most effective in patients with increased triglyceride levels and decreased HDL levels, a lipid profile typically seen in patients with type 2 diabetes.

 

Table 4: Effect of Fibrate Monotherapy on Cardiovascular Outcomes
Study Drug #Diabetic subjects %Decrease % Decrease
      controls diabetics
 
Helsinki Heart Study Gemfibrozil 135 34 60*
VA-HIT Gemfibrozil 620 24 24
DIAS Fenofibrate 418 - 33*
Sendcap Bezafibrate 164 - 70
Field Fenofibrate 9795 - 11*
             

* Not statistically significant

 

NIACIN

 

A single randomized trial, the Coronary Drug Project, has examined the effect of niacin monotherapy on cardiovascular outcomes [131]. This trial was carried out from 1966 to 1974 (before the introduction of statin therapy) in men with a history of a prior myocardial infarction and demonstrated that niacin therapy reduced cardiovascular events. The results of this study were re-analyzed to determine the effect of niacin therapy in subjects with varying baseline fasting and 1-hour post meal glucose levels [132]. It was noted that 6 years of niacin therapy reduced the risk of coronary heart disease death or nonfatal MI by approximately 15-25% regardless of baseline fasting or 1-hour post glucose challenge glucose levels. Particularly notable is that reductions in events were seen in the subjects who had a fasting glucose levels >126mg/dl or 1-hour glucose levels >220mg/dl (i.e. patients with diabetes). Thus, based on this single study, niacin monotherapy reduces cardiovascular events both in normal subjects and patients with diabetes.

 

OTHER DRUGS

 

With regard to ezetimibe, PCSK9 inhibitors, and bile acid sequestrants, there have been no randomized monotherapy studies that have examined the effect of these drugs on cardiovascular end points in subjects with diabetes. In non-diabetic subjects bile acid sequestrants have reduced cardiovascular events [133, 134]. Since bile acid sequestrants have a similar beneficial impact on serum lipid levels in diabetic and non-diabetic subjects one would anticipate that these drugs would also result in a reduction in events in the diabetic population. However, bile acid sequestrants can raise triglyceride levels and therefore must be used with caution in hypertriglyceridemic patients. There are no outcome studies with ezetimibe monotherapy or PCSK9 inhibitor monotherapy in patients with diabetes but given that these drugs reduce LDL cholesterol levels and in combination with statins reduce cardiovascular events one would anticipate that ezetimibe and PCSK9 inhibitor monotherapy will also reduce cardiovascular events.

 

Combination Therapy

 

The studies with statins have been so impressive that most patients with diabetes over the age of 40 are routinely treated with statin therapy and younger patients with diabetes at high risk for cardiovascular disease are also typically on statin therapy (see Current Guidelines Section). Therefore, a key issue is whether the addition of other lipid lowering drugs to statins will result in a further reduction in cardiovascular events. A difficulty with such studies is that the reduction in cardiovascular events induced by statin therapy is so robust that very large trials may be required to see additional benefit.

 

STATINS + FIBRATES

 

The ACCORD-LIPID trial was designed to determine if the addition of fenofibrate to aggressive statin therapy would result in a further reduction in cardiovascular disease in patients with Type 2 diabetes [33]. In this trial, 5,518 patients on statin therapy were randomized to placebo or fenofibrate therapy. The patients had diabetes for approximately 10 years and either had pre-existing cardiovascular disease or were at high risk for developing cardiovascular disease. During the trial, LDL cholesterol levels were approximately 80mg/dl. There was only a small difference in HDL cholesterol with the fenofibrate groups having a mean HDL cholesterol of 41.2mg/dl while the control group had an HDL cholesterol of 40.5mg/dl. Differences in triglyceride levels were somewhat more impressive with the fenofibrate group having a mean triglyceride level of 122mg/dl while the control group had a triglyceride level of 144mg/dl. First occurrence of nonfatal myocardial infarction, nonfatal stroke, or death from cardiovascular causes was the primary outcome and there was no statistical difference between the fenofibrate treated group and the placebo group. Additionally, there were also no statistically significant differences between the groups with regards to any of the secondary outcome measures of cardiovascular disease. Of note, the addition of fenofibrate to statin therapy did not result in an increase in either muscle or liver side effects. On further analysis, there was a possible benefit of fenofibrate therapy in the patients in whom the baseline triglyceride levels were elevated (>204mg/dl) and HDL cholesterol levels decreased (<34mg/dl). In the fibrate monotherapy trials, this same group of patients also derived the greatest benefit of fibrate therapy. Future fibrate statin combination therapy trials will need to focus on patients with high triglycerides and low HDL cholesterol levels. Finally, similar to what has been reported in other trials, fenofibrate had beneficial effects on the progression of microvascular disease [135, 136]. While this was a negative study, it must be recognized that most of the patients included in this study did not have the lipid profile that would typically lead to treatment with fibrates.

 

STATIN + NIACIN

 

The AIM-HIGH trial was designed to determine if the addition of Niaspan to aggressive statin therapy would result in a further reduction in cardiovascular events in patients with pre-existing cardiovascular disease [137]. In this trial 3,314 patients were randomized to Niaspan vs. placebo. Approximately 33% of the patients had diabetes. On trial, LDL cholesterol levels were in the 60-70mg/dl range in both groups. As expected, HDL cholesterol levels were increased in the Niaspan treated group (approximately 44mg/dl vs. 38mg/dl), while triglycerides were decreased (approximately 121mg/dl vs. 155mg/dl). However, there were no differences in the primary endpoint between the control and Niaspan treated groups (Primary endpoint consisted of the first event of death from coronary heart disease, nonfatal myocardial infarction, ischemic stroke, hospitalization for an acute coronary syndrome, or symptom-driven coronary or cerebral revascularization). There were also no differences in secondary endpoints except for a possible increase in strokes in the Niaspan treated group. The addition of Niaspan to statin therapy did not result in a significant increase in either muscle or liver toxicity. Thus, this study does not provide support for the addition of niacin to statins. However, it should be recognized that this was a relatively small study and a considerable number of patients stopped taking the Niaspan during the course of the study (25.4% of patients discontinued Niaspan therapy). In addition, most of the patients included in this study did not have a lipid profile that one would typically consider treating with niacin therapy. In the subset of patients with TG > 198mg/dl and HDL cholesterol < 33mg/dl niacin showed a trend towards benefit (hazard ratio 0.74; p=0.073) in this study, suggesting that if the appropriate patient population was studied the results may have been positive [138].

 

HPS 2 Thrive also studied the effect of niacin added to statin therapy [139]. This trial utilized extended release niacin combined with laropiprant, a prostaglandin D2receptor antagonist that reduces the flushing side effect of niacin treatment. HPS 2 Thrive was a very large trial with over 25,000 patients randomized to either niacin therapy or placebo. Approximately 32% of the patients in this trial had diabetes. The LDL cholesterol level was 63mg/dl, the HDL cholesterol 44mg/dl, and the triglycerides 125mg/dl at baseline. As expected, niacin therapy resulted in a modest reduction in LDL cholesterol (10mg/dl), a modest increase in HDL cholesterol (6mg/dl), and a marked reduction in triglycerides (33mg/dl). However, despite these lipid changes there were no significant differences in major cardiovascular events between the niacin and control group (risk ratio 0.96 CI 0.90- 1.03). It is unknown whether laropiprant, the prostaglandin D2receptor antagonist, might have effects that worsen atherosclerosis and increase event rates. Similar to the ACCORD-LIPID and AIM-HIGH studies, the group of patients included in the HPS 2 Thrive trial were not the ideal patient population to test for the beneficial effects of niacin treatment added to statin therapy. Ideally, patients with high triglycerides and high non-HDL cholesterol levels coupled with low HDL cholesterol levels should be studied.

 

STATIN + EZETIMIBE

 

The IMPROVE-IT trial tested whether the addition of ezetimibe to statin therapy would provide an additional beneficial effect in patients with the acute coronary syndrome [140]. This was a large trial with over 18,000 patients randomized to statin therapy vs. statin therapy + ezetimibe. Approximately 27% of the patients in this trial had diabetes. On treatment LDL cholesterol levels were 70mg/dl in the statin alone group vs. 53mg/dl in the statin + ezetimibe group. There was a small but significant 6.4% decrease in major cardiovascular events (Cardiovascular death, MI, documented unstable angina requiring re-hospitalization, coronary revascularization, or stroke) in the statin + ezetimibe group (HR 0.936 CI (0.887, 0.988) p=0.016). Cardiovascular death, non-fatal MI, or non-fatal stroke were reduced by 10% (HR 0.90 CI (0.84, 0.97) p=0.003). These beneficial effects were particularly pronounced in the patients with diabetes and other risk factors [141].

 

STATIN + PCSK9 INHIBITORS

 

The FOURIER trial was a randomized, double-blind, placebo-controlled trial of evolocumab vs. placebo in 27,564 patients with atherosclerotic cardiovascular disease and an LDL cholesterol level of 70 mg/dl or higher who were on statin therapy [142]. Approximately 40% of the patients had diabetes [143]. The primary end point was cardiovascular death, myocardial infarction, stroke, hospitalization for unstable angina, or coronary revascularization and the key secondary end point was cardiovascular death, myocardial infarction, or stroke. The median duration of follow-up was 2.2 years. Baseline LDL cholesterol levels were 92mg/dl and evolocumab resulted in a 59% decrease in LDL cholesterol levels (LDL cholesterol level on treatment approximately 30mg/dl). Evolocumab treatment significantly reduced the risk of the primary end point (hazard ratio, 0.85; 95% confidence interval [CI], 0.79 to 0.92; P<0.001) and the key secondary end point (hazard ratio, 0.80; 95% CI, 0.73 to 0.88; P<0.001). The results were consistent across key subgroups, including the subgroup of patients in the lowest quartile for baseline LDL cholesterol levels (median, 74 mg/dl). Of note, a similar decrease in cardiovascular events occurred in patients with diabetes treated with evolocumab and glycemic control was not altered [144]. Further analysis has shown that in the small number of patients with a baseline LDL cholesterol level less than 70mg/dl, evolocumab reduced cardiovascular events to a similar degree as in the patients with an LDL cholesterol greater than 70mg/dl [145]. Finally, the lower the on-treatment LDL cholesterol levels (down to levels below 20mg/dl), the lower the cardiovascular event rate, suggesting that greater reductions in LDL cholesterol levels will result in greater reductions in cardiovascular disease [146].

 

It should be noted that that the duration of the FOURIER trial was very short and it is well recognized from previous statin trials that the beneficial effects of lowering LDL cholesterol levels takes time with only modest effects observed during the first year of treatment. In the FOURIER trial the reduction of cardiovascular death, myocardial infarction, or stroke was 16% during the first year but was 25% beyond 12 months. Thus, long-term benefit may be greater than observed during the study.

 

Two recent trials examined the effects of bococizumab, another PCSK9 inhibitor, on cardiovascular outcomes [147]. In one trial patients with cardiovascular disease or at high risk for cardiovascular disease with LDL cholesterol levels greater than 70mg/dl on statin therapy were randomized to bococizumab or placebo (SPIRE 1; n= 16,817)). In the second trial, similar patients were studied except LDL cholesterol levels were greater than 100mg/dl and some patients were statin intolerant (SPIRE 2; n= 10,621). Almost 50% of the patients in these trials had diabetes. The primary end point was nonfatal myocardial infarction, nonfatal stroke, hospitalization for unstable angina requiring urgent revascularization, or cardiovascular death. The trials were stopped early after a median follow-up of 7months in SPIRE 1 and 12 months in SPIRE 2 due to high rates of development of antidrug antibodies, which markedly reduced the magnitude and durability of the decrease in LDL cholesterol levels [148]. In SPIRE 1 baseline LDL cholesterol levels were 94mg/dl while in SPIRE 2 LDL cholesterol levels were 133mg/dl. At 14 weeks LDL cholesterol levels were reduced by approximately 55% in the bococizumab treated groups at 14 weeks. In patients with lower baseline LDL cholesterol levels (SPIRE 1) bococizumab treatment did not reduce cardiovascular events (hazard ratio, 0.99; 95% CI 0.80 to 1.22; P=0.94). However, in patients with higher LDL cholesterol levels (SPIRE 2) cardiovascular disease was reduced by bococizumab treatment (hazard ratio, 0.79; 95% CI, 0.65 to 0.97; P=0.02). Thus, in patients with higher LDL cholesterol levels who were treated for 12 months lowering LDL cholesterol levels with a PCSK9 inhibitor decreased cardiovascular outcomes.

 

The ODYSSEY trial was a multicenter, randomized, double-blind, placebo-controlled trial involving 18,924 patients who had an acute coronary syndrome 1 to 12 months earlier, an LDL cholesterol level of at least 70 mg/dl, a non-HDL cholesterol level of at least 100 mg/dl, or an apolipoprotein B level of at least 80 mg/dl while on high intensity statin therapy or the maximum tolerated statin dose [149]. Approximately 29% of the patients had diabetes. Patients were randomly assigned to receive alirocumab 75 mg every 2 weeks or matching placebo. The dose of alirocumab was adjusted to target an LDL cholesterol level of 25 to 50 mg/dl. The primary end point was a composite of death from coronary heart disease, nonfatal myocardial infarction, fatal or nonfatal ischemic stroke, or unstable angina requiring hospitalization. During the trial LDL cholesterol levels in the placebo group was 93-103mg/dl while in the alirocumab group LDL cholesterol levels were 40mg/dl at 4 months, 48mg/dl at 12 months, and 66mg/dl at 48 months (the increase with time was due to discontinuation of alirocumab or a decrease in dose). The primary endpoint was reduced by 15% in the alirocumab group (HR 0.85; 95% CI 0.78 to 0.93; P<0.001). In addition, total mortality was reduced by 15% in the alirocumab group (HR 0.85; 95% CI 0.73 to 0.98). The absolute benefit of alirocumab was greatest in patients with a baseline LDL cholesterol level greater than 100mg/dl. In patients with an LDL cholesterol level > than 100mg/dl the number needed to treat with alirocumab to prevent an event was only 16. It should be noted that similar to the other PCSK9 outcome trials the duration of this trial was very short (median follow-up 2.8 years) which may have minimized the beneficial effects. Additionally, because alirocumab 75mg every 2 weeks was stopped if the LDL cholesterol level was < 15mg/dl on two consecutive measurements the beneficial effects may have been blunted (7.7% of patients randomized to alirocumab were switched to placebo).

 

It should be noted that that the duration of the PCSK9 outcome trials were relatively short and it is well recognized from previous statin trials that the beneficial effects of lowering LDL cholesterol levels takes time with only modest effects observed during the first year of treatment. In the FOURIER trial the reduction of cardiovascular death, myocardial infarction, or stroke was 16% during the first year but was 25% beyond 12 months. In the ODYSSEY trial the occurrence of cardiovascular events was similar in the alirocumab and placebo group during the first year of the study with benefits of alirocumab appearing after year one. Thus, the long-term benefits of treatment with a PCSK9 inhibitor may be greater than that observed during these relatively short-term studies.

 

Support for the benefits of further lowering of LDL cholesterol levels with a PCSK9 inhibitor added to statin therapy is seen in the GLAGOV trial [150]. This trial was a double-blind, placebo-controlled, randomized trial of evolocumab vs. placebo in 968 patients presenting for coronary angiography. Approximately 20-21% of the patients had diabetes. The primary efficacy measure was the change in percent atheroma volume (PAV) from baseline to week 78, measured by serial intravascular ultrasonography (IVUS) imaging. Secondary efficacy measures included change in normalized total atheroma volume (TAV) and percentage of patients demonstrating plaque regression. As expected, there was a marked decrease in LDL cholesterol levels in the evolocumab group (Placebo 93mg/dl vs. evolocumab 37mg/dl; p<0.001). PAV increased 0.05% with placebo and decreased 0.95% with evolocumab (P < .001) while TAV decreased 0.9 mm3 with placebo and 5.8 mm3 with evolocumab (P < .001). There was a linear relationship between achieved LDL cholesterol and change in PAV (i.e. the lower the LDL cholesterol the greater the regression in atheroma volume down to an LDL cholesterol of 20mg/dl). Additionally, evolocumab induced plaque regression in a greater percentage of patients than placebo (64.3% vs 47.3%; P < .001 for PAV and 61.5% vs 48.9%; P < .001 for TAV). The results in the patients with diabetes were similar to the non-diabetic patients.

 

Taken together these trials demonstrate that further lowering LDL cholesterol levels with PCSK9 inhibitors in patients taking statins will have beneficial effects on atherosclerosis and cardiovascular outcomes.

 

The results of the ezetimibe and PCSK9 trials have several important implications. First, it demonstrates that combination therapy may have benefits above and beyond statin therapy alone. Second, it provides further support for the hypothesis that lowering LDL per se will reduce cardiovascular events. Third, it suggests that lowering LDL levels to much lower levels than usual will have significant benefits. These new results have implications for determining goals of therapy.

 

STATINS + LOW DOSE OMEGA-3-FATTY ACIDS

 

Origin was a double-blind study in 12,536 patients at high risk for cardiovascular disease who had impaired fasting glucose, impaired glucose tolerance, or diabetes [151]. Patients were randomized to receive a 1-gram capsule containing at least 900mg of ethyl esters of omega-3 fatty acids (EPA 465mg and DHA 375mg) or placebo for approximately 6 years. Greater than 50% of the patients were on statin therapy. The primary outcome was death from cardiovascular causes. Triglyceride levels were reduced by 14.5mg/dl in the group receiving omega-3-fatty acids compared to the placebo group (P<0.001), without a significant effect on other lipids. The incidence of the primary outcome was not significantly decreased among patients receiving omega-3-fatty acids as compared with those receiving placebo. The use of omega-3-fatty acids also had no significant effect on the rates of major vascular events, death from any cause, or death from arrhythmia.

 

A Study of Cardiovascular Events in Diabetes (ASCEND)was a randomized, placebo controlled, double blind trial of 1-gram omega-3-fattys acids (400mg EPA and 300mg DHA ethyl esters) vs. olive oil placebo in 15,480 patients with diabetes without a history of cardiovascular disease (primary prevention trial) [152]. Approximately 75% of patients were on statin therapy. The primary end point was serious vascular events (non-fatal myocardial infarction, non-fatal stroke, transient ischemic attack, or vascular death). Total cholesterol, HDLc, and non-HDLc levels were not significantly altered by omega-3-fatty acid treatment (changes in triglyceride levels were not reported). After a mean follow-up of 7.4 years the composite outcome of a serious vascular event or revascularization occurred in 882 patients (11.4%) on omega-3-fatty acids and 887 patients (11.5%) on placebo (rate ratio, 1.00; 95% CI, 0.91 to 1.09). Serious adverse events were similar in placebo and omega-3-fatty acid treated groups.

 

STATINS + HIGH DOSE OMEGA-3-FATTY ACIDS

 

Japan EPA Lipid Intervention Study (JELIS) was an open label study in patients with total cholesterol levels > 254mg/dl with (n= 3664) or without cardiovascular disease (n=14,981) who were randomly assigned to be treated with 1800 mg of EPA (Vascepa) + statin (n=9326) or statin alone (n= 9319) with a 5 year follow-up [153]. Approximately 16% of the patients had diabetes. The mean baseline triglyceride level was 153mg/dl. The primary endpoint was any major coronary event, including sudden cardiac death, fatal and non-fatal myocardial infarction, and other non-fatal events including unstable angina pectoris, angioplasty, stenting, or coronary artery bypass grafting. Total, LDL, and HDL cholesterol levels were similar in the two groups but plasma triglycerides were modestly decreased in the EPA treated group (5% decrease in EPA group compared to controls; p = 0.0001). In the EPA group the primary endpoint occurred in 2.8% of the patients vs. 3.5% of the patients in the statin alone group (19% decrease; p = 0.011). Unstable angina and non-fatal coronary events were also significantly reduced in the EPA group but in this study sudden cardiac death and coronary death did not differ between groups. Unstable angina was the main component contributing to the primary endpoint and this is a more subjective endpoint than other endpoints such as a myocardial infarction, stroke, or cardiovascular death. A subjective endpoint has the potential to be an unreliable endpoint in an open label study and is a limitation of the JELIS Study. The reduction in events was similar in the subgroup of patients with diabetes. In patients with triglyceride levels >150mg/dl and HDL cholesterol levels < 40mg/dl there was a 53% decrease in events [154].

 

The Reduction of Cardiovascular Events with EPA – Intervention Trial (REDUCE-IT)was a randomized, double blind trial of 2 grams twice per day of EPA ethyl ester (icosapent ethyl) (Vascepa) vs. placebo in 8179 patients with hypertriglyceridemia (135mg/dl to 499mg/dl) and established cardiovascular disease or high cardiovascular disease risk (diabetes plus one risk factor) who were on stable statin therapy [155]. Approximately 60% of the patients in this trial had diabetes. The primary end point was a composite of cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, coronary revascularization, or unstable angina. The key secondary end point was a composite of cardiovascular death, nonfatal myocardial infarction, or nonfatal stroke. At baseline, the median LDL cholesterol level was 75.0 mg/dl, HDL cholesterol level was 40.0 mg/dl, and triglyceride level was 216.0 mg/dl. The median change in triglyceride level from baseline to 1 year was a decrease of 18.3% (−39.0 mg/dl) in the EPA group and an increase of 2.2% (4.5 mg/dl) in the placebo group. After a median of 4.9 years the primary end-point occurred in 17.2% of the patients in the EPA group vs. 22.0% of the patients in the placebo group (hazard ratio, 0.75; 95% confidence interval [CI], 0.68 to 0.83; P<0.001), indicating a 25% decrease in events. The beneficial effects were similar in patients with and without diabetes. The number needed to treat to avoid one primary end-point event was 21. The reduction in cardiovascular events was noted after approximately 2 years of EPA treatment. Additionally, the rate of cardiovascular death was decreased by 20% in the EPA group (4.3% vs. 5.2%; hazard ratio, 0.80; 95% CI, 0.66 to 0.98; P=0.03). The cardiovascular benefits of EPA were similar across baseline levels of triglycerides (<150, ≥150 to <200, and ≥200 mg per deciliter). Moreover, the cardiovascular benefits of EPA appeared to occur irrespective of the attained triglyceride level at 1 year (≥150 or <150 mg per deciliter), suggesting that the cardiovascular risk reduction was not associated with attainment of a normal triglyceride level. An increase in hospitalization for atrial fibrillation or flutter (3.1% vs. 2.1%, P=0.004) occurred in the EPA group. In addition, serious bleeding events occurred in 2.7% of the patients in the EPA group and in 2.1% in the placebo group (P=0.06). There were no fatal bleeding events in either group and the rates of hemorrhagic stroke, serious central nervous system bleeding, and serious gastrointestinal bleeding were not significantly higher in the EPA group than in the placebo group.

 

These studies suggest that the addition of EPA to statins in patients with diabetes who have elevated triglyceride levels will reduce cardiovascular events. Whether the decrease in events is due to lowering of triglyceride levels or to other actions of EPA, such as effecting platelet function, remains to be determined.

 

CURRENT GUIDELINES FOR SERUM LIPIDS

 

There are several different guidelines for treating lipids in patients with diabetes. The American College of Cardiology and American Heart Association (ACC/AHA) 2013 guidelines recommend that patients with both type 1 and type 2 diabetes between 40 and 75 years of age be treated with statin therapy[156]. If the estimated 10-year risk of developing a cardiovascular event is > 7.5% they recommend intensive statin therapy (atorvastatin 40-80mg or rosuvastatin 20-40mg). If the 10-year cardiovascular risk is < 7.5% they recommend moderate statin therapy (for example atorvastatin 10-20mg, simvastatin 20-40mg, pravastatin 40mg). Cardiovascular risk can be determined using a calculator that is available at http://my.americanheart.org/cvriskcalculatoror can be downloaded as an app for a smart phone or tablet. If a patient with diabetes has clinical ASCVD they should be treated with intensive statin therapy if less than 75 years of age. Patients with diabetes and clinical ASCVD over 75 years of age should be treated with either intensive or moderate statin therapy depending upon the risks of developing drug toxicity. The ACC/AHA do not recommend any specific LDL goal but rather to just treat with statin therapy. The ACC/AHA guidelines do not recommend the treatment with drugs other than statins, but these guidelines were published before the results of the IMPROVE-IT trial and PCSK9 inhibitor trials were known. The ACC in more recent recommendations acknowledges that one may consider absolute LDL-C or non-HDL-C levels for patients on statin therapy and where appropriate use additional drugs, such as ezetimibe and PCSK9 inhibitors, to lower lipid levels [157].

 

The 2018 ACC/AHA guidelines recommend the following [158]. “In patients 40 to 75 years of age with diabetes mellitus and LDL-C ≥70 mg/dL (≥1.8 mmol/L), start moderate-intensity statin therapy without calculating 10-year ASCVD risk. In patients with diabetes mellitus at higher risk, especially those with multiple risk factors or those 50 to 75 years of age, it is reasonable to use a high-intensity statin to reduce the LDL-C level by ≥50%.” In patients with cardiovascular disease they recommend “In patients with clinical ASCVD, reduce low-density lipoprotein cholesterol (LDL-C) with high-intensity statin therapy or maximally tolerated statin therapy. The more LDL-C is reduced on statin therapy, the greater will be subsequent risk reduction. Use a maximally tolerated statin to lower LDLC levels by ≥50%. In very high-risk ASCVD, use a LDL-C threshold of 70 mg/dL (1.8 mmol/L) to consider addition of nonstatins to statin therapy. Very high-risk includes a history of multiple major ASCVD events or 1 major ASCVD event and multiple high-risk conditions. In very high-risk ASCVD patients, it is reasonable to add ezetimibe to maximally tolerated statin therapy when the LDL-C level remains ≥70 mg/dL (≥1.8 mmol/L). In patients at very high risk whose LDL-C level remains ≥70 mg/dL (≥1.8 mmol/L) on maximally tolerated statin and ezetimibe therapy, adding a PCSK9 inhibitor is reasonable, although the long-term safety (>3 years) is uncertain and cost effectiveness is low at mid-2018 list prices.” With regards to testing they recommend “Assess adherence and percentage response to LDL-C–lowering medications and lifestyle changes with repeat lipid measurement 4 to 12 weeks after statin initiation or dose adjustment, repeated every 3 to 12 months as needed”.

 

The 2019 American Diabetes Association (ADA) recommends that adult patients with diabetes have their lipid profile determined at the time of diabetes diagnosis and at least every 5 years thereafter or more frequently if indicated [159]. This profile includes total cholesterol, HDL cholesterol, triglycerides, and calculated LDL cholesterol. A lipid panel should be obtained immediately prior to initiating statin therapy. Once a patient is on statin therapy testing should be carried out 4-12 weeks after initiating therapy and annually thereafter to monitor adherence and efficacy. Lifestyle modification including a reduction in saturated fat, trans fat, and cholesterol intake, weight loss if indicated, an increase in omega-3-fatty acids, viscous fiber, and plant stanols /sterol intake, and increased physical activity is indicated in all patients with diabetes. In patients with elevated triglyceride levels glycemic control is beneficial. Intensive statin therapy should be added to lifestyle therapy in diabetic patients with overt cardiovascular disease or a 10-year risk of a cardiovascular event > 20% (see table 5 for recommendations). In patients without cardiovascular disease over age 40 moderate intensity statin therapy should be added to lifestyle changes. If some patients, high intensity therapy may be used if multiple other risk factors are present. In patients less than 40 years of age with additional risk factors one should discuss with the patient the use of moderate statin therapy. If one follows these recommendations almost all patients with diabetes over the age of 40 will be on statin therapy and many under the age of 40 will also be treated with statins. The addition of ezetimibe or a PCSK9 inhibitor should be considered to further lower LDL cholesterol levels in patients with atherosclerotic cardiovascular disease if the LDL cholesterol level on statin therapy is greater than 70mg/dl (table 5). The use of fibrates or niacin with statins was generally not recommended. Finally, in patients with fasting triglyceride levels greater than 500mg/dl an evaluation for secondary causes of hypertriglyceridemia should be initiated and consideration of drug therapy to reduce the risk of pancreatitis.

 

Table 5: ADA Recommendations for Statin and Combination Treatment in Adults with Diabetes
Age ASCVD or 10- year risk > 20% Statin Dose*
<40 No None or moderate intensity if multiple risk factors**
>40 No

Moderate intensity (reduce LDL by 30-50%) or high intensity statin therapy if multiple risk factors**

 

Any age Yes

High Intensity (reduce LDLc by > 50%)

If LDLc > 70mg/dl despite maximally tolerated statin therapy consider adding ezetimibe or PCSK9 inhibitor

*In addition to lifestyle therapy; ** ASCVD risk factors include LDL cholesterol > 100mg/dl, high blood pressure, smoking, chronic kidney disease, albuminuria, and family history of premature ASCVD;

 

The National Lipid Association (NLA) has treatment goals for patients with diabetes [160]. In patients with Type 1 or Type 2 diabetes with pre-existing atherosclerotic cardiovascular disease, two or more risk factors for atherosclerotic cardiovascular disease or evidence of end organ damage, the goal LDL is <70mg/dl and the goal non-HDL cholesterol is < 100mg/dl (Table 6). In patients with diabetes with 0-1 risk factors and no end organ damage, the LDL goal is < 100mg/dl and the non-HDL cholesterol goal is < 130mg/dl. The NLA guidelines recommend considering drug therapy if a patient with diabetes is not at goal.

 

Table 6. National Lipid Association Recommendations
Diabetes with 0-1 risk factors and no end organ damage LDL cholesterol < 100mg/dl; Non-HDL cholesterol < 130mg/dl
Diabetes with 2 or more risk factors or end organ damage LDL cholesterol < 70mg/dl; Non-HDL cholesterol < 100mg/dl

Risk factors- age >45 for males, >55 for females; family history of early coronary heart disease; current cigarette smoking; high blood pressure >140/>90 mm HG; or low HDL < 40mg/dl males, < 50mg/dl females

End Organ Damage- retinopathy, albumin/creatinine ratio > 30mg/g, or chronic kidney disease

 

The American Association of Clinical Endocrinologists and American College of Endocrinology guidelines consider individuals with type 2 diabetes to be at high, very high, or extreme risk for ASCVD [161]. Patients with type 1 diabetes and a duration of diabetes of more than 15 years or two or more risk factors, poorly controlled A1c, or insulin resistance with metabolic syndrome should be considered to have an equivalent risk to patients with type 2 diabetes. The recommended treatment goals are shown in Table 7.

 

Table 7. ASCVD Risk Categories and Treatment Goals
Risk Category Risk Factors/10-year risk LDL-C mg/dl Non-HDL-C mg/dl Apo B mg/dl
Extreme Risk Diabetes and clinical cardiovascular disease <55 <80 <70
Very High Risk Diabetes with one or more risk factors* <70 <100 <80
High Risk Diabetes and no other risk factors <100 <130 <90
Moderate Risk Two or fewer risk factors and 10yr risk < 10%** <100 <130 <90
Low Risk No risk factors <130 <160 NR
           

*Factors are high LDL-C, polycystic ovary syndrome, cigarette smoking, hypertension (blood pressure ≥140/90 mm Hg or on hypertensive medication), low HDL-C (<40 mg/dL), family history of coronary artery disease (in male, first-degree relative younger than 55 years; in female, first-degree relative younger than 65 years), chronic renal disease (CKD)

stage 3/4, evidence of coronary artery calcification and age (men ≥45; women ≥55 years). Subtract 1 risk factor if the person has high HDL-C.

***Calculate risk using Framingham 10-year risk scoring

NR= not recommended

 

Thus, different organizations have proposed somewhat different recommendations for the treatment of lipids in patients with diabetes. Despite these differences it is clear that the vast majority of patients with diabetes will need to be treated with statins regardless of which guidelines one elects to follow.

 

One approach is to combine these recommendations. In patients with diabetes who have pre-existing cardiovascular disease initiate intensive statin therapy. In patients with diabetes 40-75 years of age without pre-existing cardiovascular disease calculate the 10-year risk of developing cardiovascular disease. Initiate intensive statin therapy if the 10-year risk is > 7.5% or if there are multiple risk factors or moderate statin therapy if the risk is < 7.5% without multiple risk factors. Eight to twelve weeks after initiating statin therapy obtain a lipid panel to determine if the LDL and non-HDL cholesterol levels are at goal. In patients with pre-existing cardiovascular disease or multiple risk factors the goal should be an LDL cholesterol < 70mg/dl and a non-HDL cholesterol < 100mg/dl. In patients that are not at high risk the goal should be an LDL cholesterol < 100mg/dl and a non-HDL cholesterol < 130mg/dl. In patients with cardiovascular disease that is progressive or who have multiple risk factors a goal LDL cholesterol of < 55mg/dl and a non-HDL c < 80mg/dl should be strongly considered. If the levels are not at goal either adjust the statin dose or consider adding additional medications.  In patients with diabetes less than 40 years of age initiate statin therapy if the patient has overt cardiovascular disease, long standing diabetes, or risk factors for cardiovascular disease and the LDL and non-HDL cholesterol levels are not at goal.

 

TREATMENT OF LIPID ABNORMALITIES IN PATIENT WITH DIABETES

 

Life Style Changes

 

Initial treatment of lipid disorders should focus on lifestyle changes [162]. There is little debate that exercise is beneficial and that all patients with diabetes should, if possible, exercise for at least 150 minutes per week (for example 30 minutes 5 times per week). Exercise will decrease serum triglyceride levels and increase HDL cholesterol levels (an increase in HDL cholesterol requires vigorous exercise) [92, 162]. It should be noted that many patients with diabetes may have substantial barriers to participating in exercise programs, such as comorbidities that limit exercise tolerance, risk of hypoglycemia, and presence of microvascular complications (visual impairment, neuropathy) that make exercise difficult.

 

Diet is debated to a greater extent. Everyone agrees that weight loss in obese patients is essential [92, 162]. But how this can be achieved is hotly debated with many different "experts" advocating different approaches. The wide diversity of approach is likely due to the failure of any approach to be effective in the long termfor the majority of obese patients with diabetes. If successful, weight loss will decrease serum triglyceride levels, increase HDL cholesterol levels, and modestly reduce LDL cholesterol [92, 162]. To reduce LDL cholesterol levels, it is important that the diet decrease saturated fat, trans fatty acids, and cholesterol intake. Increasing soluble fiber is also helpful.

 

It is debated whether a low fat, high complex carbohydrate diets vs. a high monounsaturated fat diet is ideal for obese patients with diabetes [92]. One can find "experts" in favor of either of these approaches and there are pros and cons to each approach. It is essential to recognize that both approaches reduce simple sugars, saturated fat, trans fatty acids, and cholesterol intake. The high complex carbohydrate diet will increase serum triglyceride levels in some patients and if the amount of fat in the diet is markedly reduced serum HDL cholesterol levels may decrease. In obese patients, it has been postulated that a diet high in monounsaturated fats, because of the increase in caloric density, will lead to an increase in weight gain. Both diets reduce saturated fat and cholesterol intake that will result in reductions in LDL cholesterol levels. Additionally, both diets also reduce trans-fatty acid intake, which will have a beneficial effect on LDL and HDL cholesterol levels and simple sugars, which will have a beneficial effect on triglyceride levels.

 

Recently there has been increased interest in low carbohydrate, increased protein diets. Short-term studies have indicated that weight loss is superior with this diet; however longer studies have demonstrated a similar weight loss to that observed with conventional diets. The major concern with the low carbohydrate, high protein diet is that they tend to be high in saturated fats and cholesterol. Additionally, there may also be an increased risk of progression of kidney disease in patients with pre-existing kidney disease. In the short-term studies during active weight loss this diet has not resulted in major perturbations in serum cholesterol levels, but there is concern that when weight becomes stable these diets might adversely affect serum cholesterol levels.

 

Thus, the available data do not indicate that any particular diet is best for inducing weight loss and it is essential to adapt the diet to fit the food preferences of the patient. Ultimately no weight loss diet will be successful if the patient cannot follow the diet for the long term.

 

While it is widely accepted that lifestyle changes will decrease cardiovascular events it should be recognized that the Look Ahead trial failed to demonstrate a reduction in cardiovascular events [163]. In this trial, over 5000 overweight or obese patients with Type 2 diabetes were randomized to either an intensive lifestyle intervention group that promoted weight loss through decreased caloric intake and increased physical activity or to a group that received diabetes support and education (control group). After a median follow-up of 9.6 years there was no difference in cardiovascular events (hazard ratio in the intervention group, 0.95; 95% CI 0.83 to 1.09; P=0.51). A limitation of this study was that while the weight difference between groups was impressive during the first year of the trial, over time the differences greatly narrowed such that at the end of the trial the intensive group had a 6.0% weight loss while the control group had a 3.5% weight loss. This very modest difference demonstrates the difficulty in sustaining long term lifestyle changes. As noted earlier there were no differences in coronary artery calcium scores between the lifestyle and placebo groups in the Diabetes Prevention Program, which also illustrates the difficulty of reducing cardiovascular disease with lifestyle changes [41]. Thus, while weight loss and diet therapy are likely to be beneficial in reducing cardiovascular events, in clinical practice they are seldom sufficient because long-term life style changes are very difficult for most patients to maintain.

 

In contrast to the failure of lifestyle therapy in the Look Ahead trial to reduce cardiovascular events, the PREDIMED trial employing a Mediterranean diet did reduce the incidence of major cardiovascular disease [164, 165]. In this multicenter trial center trial, carried out in Spain, over 7000 patients at high risk for developing cardiovascular disease were randomized to three diets (primary prevention trial). A Mediterranean diet supplemented with extra-virgin olive oil, a Mediterranean diet supplemented with mixed nuts, or a control diet. Approximately 50% of the patients in this trial had type 2 diabetes. In the patients assigned to the Mediterranean diets there was 29% decrease in the primary end point (myocardial infarction, stroke, and death from cardiovascular disease). Subgroup analysis demonstrated that the Mediterranean diet was equally beneficial in patients with and without diabetes. The Mediterranean diet resulted in a small but significant increase in HDL cholesterol levels and a small decrease in both LDL cholesterol and triglyceride levels [166]. A secondary prevention trial of a Mediterranean diet has also demonstrated a reduction in cardiovascular events. The Lyon Diet Heart Study randomized 584 patients who had a myocardial infarction within 6 months to a Mediterranean type diet vs usual diet [167, 168]. There was a marked reduction in events in the group of patients randomized to the Mediterranean diet (cardiac death and nonfatal myocardial infarction rate was 4.07 per 100 patient years in the control diet vs. 1.24 in the Mediterranean diet p<0.0001). Unfortunately, there is no indication of the number of patients with diabetes in the Lyon Diet Heart Study or whether patients with diabetes responded similar to the entire group. Lipid levels were similar in both groups in this trial [167]. The results of these two trials indicate that we should be encouraging our patients to follow a Mediterranean type diet.

 

With the currently available weight loss drugs only modest effects on weight and lipid levels have been observed [92, 162]. In some patients, weight loss drugs may be a useful adjuvant to diet therapy. Bariatric surgery can have profound effects on weight and can result in improvements in lipid profiles [92, 162]. Additionally, observational studies have shown a decrease in cardiovascular events following bariatric surgery in patients with and without diabetes [169-173]. For additional information see the chapter entitled “Lifestyle Changes: Effect of Diet, Exercise, Functional Food, and Obesity Treatment, on Lipids and Lipoproteins” [162].

 

Ethanol and simple sugars, in particular fructose, increase serum triglyceride levels in susceptible patients. In patients with hypertriglyceridemia efforts should be made to reduce the intake of ethanol, simple sugars, and fructose [162].

 

Lastly, in the past some "experts" advocated the addition of fish oil supplements to reduce cardiovascular events. However, both the Origin Trial and the ASCEND Trial did not demonstrate that fish oil supplements were beneficial in patients with type 2 diabetes or patients at high risk for the development of type 2 diabetes [151, 152](see section oneffect of lipid lowering drugs on cardiovascular events for details). It should be recognized that higher doses of fish oil are required to lower serum triglyceride levels (~ 3-4 grams of DHA/EPA per day) and are useful in treating patients with high triglyceride levels [174]. Most studies of fish oil in patients with diabetes have demonstrated that this is a safe approach and that worsening of glycemic control does not occur in patients with diabetes treated with fish oil supplements [174]. Additionally, in some patient's high dose fish oil increases LDL cholesterol levels, particularly when serum triglyceride levels are very high [174]. For additional information on fish oil see the chapter on Triglyceride Lowering Drugs [175].

 

Drug Therapy

 

The effect of statins, fibrates, niacin, ezetimibe, omega-3-fatty acids, bile acid binders, and PCSK9 inhibitors on lipid levels in patients with diabetes is virtually identical to that seen in the non-diabetic patients (Table 8). For detailed information on lipid lowering drugs see the chapters on Triglyceride Lowering Drugs and Cholesterol Lowering Drugs [175, 176].

 

STATINS

 

Statins are easy to use and generally well tolerated by patients with diabetes. However, statins can adversely affect glucose homeostasis. In patients without diabetes the risk of developing diabetes is increased by approximately 10% with higher doses of statin causing a greater risk than more moderate doses[177, 178]. The mechanism for this adverse effect is unknown but older, obese patients with higher baseline glucose levels are at greatest risk. In patients with diabetes, an analysis of 9 studies with over 9,000 patients with diabetes reported that the patients randomized to statin therapy had a 0.12% higher A1c than the placebo group indicating that statin therapy is associated with only a very small increase in A1c levels in patients with diabetes, which is unlikely to be clinically significant [179]. Individual studies such as CARDS and the Heart Protection Study have also shown only a very modest effect of statins on A1c levels in patients with diabetes [113, 115,180]. Muscle symptoms occur in patients with diabetes similar to what is observed in patients without diabetes.

 

EZETIMIBE

 

Ezetimibe is easy to use and generally well tolerated by patients with diabetes.

 

FIBRATES

 

Fibrates are easy to use and generally well tolerated by patients with diabetes. When combining fibrates with statin therapy it is best to use fenofibrate as the risk of inducing myositis is much less than when statins are used in combination with gemfibrozil, which can inhibit statin metabolism [181]. In the ACCORD-LIPID Trial the incidence of muscle disorders was not increased in the statin + fenofibrate group compared to statin alone [33]. The dose of fenofibrate needs to be adjusted in patients with renal disease and fenofibrate itself can induce a reversible increase in serum creatinine levels. It should be noted that marked reductions in HDL cholesterol levels can occur in some patients treated with both fenofibrate and a TZD [182].

 

BILE ACID SEQUESTRANTS

 

Bile acid sequestrants are relatively difficult to take due to GI toxicity (mainly constipation) [176]. Diabetic subjects have an increased prevalence of constipation, which may be exacerbated by the use of bile acid sequestrants. On the other hand, in diabetic patients with diarrhea, the use of bile acid sequestrants may be advantageous. Bile acid sequestrants may also increase serum triglyceride levels, which can be a problem in patients with diabetes who are already hypertriglyceridemic [176]. An additional difficulty in using bile acid sequestrants is their potential for binding other drugs [176]. Many drugs should be taken either two hours before or four hours after taking bile acid sequestrants to avoid the potential of decreased drug absorption. Diabetic patients are frequently on multiple drugs for glycemic control, hypertension, etc., and it can sometimes be difficult to time the ingestion of bile resin sequestrants to avoid these other drugs. Colesevelam (Welchol) is a bile acid sequestrant that comes in pill or powder form, which causes fewer side effects and has fewer interactions with other drugs than other preparations [183]. The usual dose is 3 pills twice a day with meals or 1 packet of powder in water or other liquids once a day with a meal. Of particular note is that a number of studies have shown that colesevelam improves glycemic control in patients with diabetes resulting in an approximately 0.5% decrease in A1c levels [184].

 

NIACIN

 

Niacin is well known to cause skin flushing and itching and GI upset [185]. Additionally, niacin reduces insulin sensitivity (i.e., causes insulin resistance), which can worsen glycemic control [185]. Studies have shown that niacin is usually well tolerated in diabetic subjects who are in good glycemic control [186, 187]. In patients with poor glycemic control, niacin is more likely to adversely impact glucose levels. In the HPS2-Thrive trial, niacin therapy significantly worsened glycemic control in patients with diabetes and induced new onset diabetes in 1.3% of subjects that were non-diabetic [139]. High doses of niacin are more likely to adversely affect glycemic control. Niacin can also increase serum uric acid levels and induce gout, both of which are already common in obese patients with type 2 diabetes [185]. Additionally recent trials have reported an increased incidence of infection and bleeding with niacin therapy [185]. However, niacin is the most effective drug in increasing HDL cholesterol levels, which are frequently low in patients with diabetes.

 

OMEGA-3-FATTY ACIDS

 

A Cochrane review of fish oil in patients with diabetes have demonstrated that this is a safe approach and does not result in worsening of glycemic control in patients with diabetes [174]. Fish oil effectively lowers triglyceride levels but, in some patients, particularly those with significant hypertriglyceridemia, high dose fish oil increases LDL cholesterol levels [174]. It should be noted that fish oil products that contain just EPA (Vascepa) do not adversely affect LDL cholesterol levels [188]. When using fish oil to lower serum triglyceride levels it is important to recognize that one is aiming to provide 3-4 grams of DHA/EPA per day. The quantity of these active omega-3-fatty acids can vary greatly from product to product. Prescription fish oil products contain large amounts of these active ingredients whereas the amount of DHA/EPA in food supplements can vary greatly and in some levels are very low. Additionally, while prescription omega-3-fatty acid preparations have high levels of quality control, omega-3-fish oil food supplements may have contaminants and the dosage may not be precisely controlled.

 

PCSK9 INHIBITORS

 

In 2015 two monoclonal antibodies that inhibit PCSK9 (proprotein convertase subtilisin kexin type 9) were approved for the lowering of LDL cholesterol levels; Alirocumab (Praluent) and evolocumab (Repatha) [176]. Alirocumab is administered as either 75mg or 150mg subcutaneously every 2 weeks or 300mg subcutaneously every 4 weeks while evolocumab is administered as either 70mg subcutaneously every 2 weeks or 420mg subcutaneously once a month [176]. A meta-analysis of three trials with 413 patients with type 2 diabetes found that in patients with type 2 diabetes evolocumab caused a 60% decrease in LDL cholesterol compared to placebo and a 39% decrease in LDL cholesterol compared to ezetimibe treatment [189]. In addition, in patients with type 2 diabetes, evolocumab decreased non-HDL cholesterol 55% vs. placebo and 34% vs. ezetimibe) and Lp(a) (31% vs. placebo and 26% vs. ezetimibe). These beneficial effects were not affected by glycemic control, insulin use, renal function, and cardiovascular disease status. Thus, PCSK9 inhibitors are effective therapy in patients with type 2 diabetes and the beneficial effects on pro-atherogenic lipoproteins is similar to what is observed in non-diabetic patients. Additionally, except for local reactions at the injection sites PCSK9 inhibitors do not seem to cause major side effects.

 

 

Table 8. Effect of Lipid Lowering Drugs
  LDLc HDLc Triglycerides
 
Statins ↓ 20-60% ↑ 5-15% ↓ 0-35%*
Bile acid sequestrants ↓ 10-30% ↑ 0-10% ↑ 0-10%**
Fibrates ↓ 0-15%*** ↑ 5-15% ↓ 20-50%
Niacin ↓ 10-25% ↑ 10-30% ↓ 20-50%
Ezetimibe ↓ 15-25% ↑ 1-3% ↓ 10-20%
PCSK9 Inhibitors ↓ 50-60% ↑ 5-15% ↓ 5-20%
High Dose Fish Oil ↑ 0- 50%** ↑ 4- 9% ↓ 20- 50%*

 *Patients with elevated TG have largest decrease

** In patients with high TG may cause marked increase

*** In patients with high TG may increase LDL

 

Therapeutic Approach

 

The first priority in treating lipid disorders in patients with diabetes is to lower the LDL cholesterol levels to goal, unless triglycerides are markedly elevated (> 500- 1000mg/dl), which increases the risk of pancreatitis. LDL cholesterol is the first priority because the database linking lowering LDL cholesterol with reducing cardiovascular disease is extremely strong and we now have the ability to markedly decrease LDL cholesterol levels. Dietary therapy is the initial step but, in most patients, will not be sufficient to achieve the LDL cholesterol goals. If patients are willing and able to make major changes in their diet it is possible to achieve significant reductions in LDL cholesterol levels but this seldom occurs in clinical practice [190].

 

Statins are the first-choice drugs to lower LDL cholesterol levels and the vast majority of diabetic patients will require statin therapy. There are several statins currently available in the US and one should be sure to choose a statin that is capable of lowering the LDL cholesterol to goal. The effect of different doses of the various statins on LDL cholesterol levels is shown in Table 9. Currently four statins are available as generic drugs, lovastatin, pravastatin, atorvastatin, and simvastatin, and these statins are relatively inexpensive. The particular statin used may be driven by price, ability to lower LDL cholesterol levels, and potential drug interactions.

 

If a patient is unable to tolerate statins or statins as monotherapy are not sufficient to lower LDL cholesterol to goal the second-choice drug is either ezetimibe or a PCSK9 inhibitor. Ezetimibe can be added to any statin. PCSK9 inhibitors can also be added to any statin and are the drug of choice if a large decrease in LDL cholesterol is required to reach goal (PCSK9 inhibitors will lower LDL cholesterol levels by 50-60% when added to a statin, whereas ezetimibe will only lower LDL cholesterol by approximately 20%).  Bile acidsequestrants are an alternative particularly if a reduction in A1c level is also needed. Ezetimibe, PCSK9 inhibitors, and bile acid sequestrants additively lower LDL cholesterol levels when used in combination with a statin, because these drugs increase hepatic LDL receptor levels by different mechanisms, thereby resulting in a reduction in serum LDL cholesterol levels [176]. Niacin and the fibrates also lower LDL cholesterol levels but are not usually employed to lower LDL cholesterol levels (see table 5).

 

Table 9. Approximate Effect of Different Doses of Statins on LDL Cholesterol Levels
% LDL Reduction

Simvastatin

(Zocor)

Atorvastatin

(Lipitor)

Lovastatin

(Mevacor)

Pravastatin

(Pravachol)

Fluvastatin

(Lescol)

Rosuvastatin

(Crestor)

Pitavastatin

(Livalo)

27 10mg - 20mg 20mg 40mg - -
34 20mg 10mg 40mg 40mg 80mg - 1mg
41 40mg 20mg 80mg 80mg - - 2mg
48 80mg 40mg - - - 10mg 4mg
54 - 80mg - - - 20mg -
60 - - - - - 40mg -

Data modified from package inserts

 

The second priority should be non-HDL cholesterol (non-HDL cholesterol = total cholesterol – HDL cholesterol), which is particularly important in patients with elevated triglyceride levels (>150mg/dl). Non-HDL cholesterol is a measure of all the pro-atherogenic apolipoprotein B containing particles. Numerous studies have shown that non-HDL cholesterol is a strong risk factor for the development of cardiovascular disease [191]. The non-HDL cholesterol goals are 30mg/dl greater than the LDL cholesterol goals. For example, if the LDL goal is <100mg/dl then the non-HDL cholesterol goal would be <130mg/dl. Drugs that reduce either LDL cholesterol or triglyceride levels will reduce non-HDL cholesterol levels.

 

The third priority in treating lipid disorders is to decrease triglyceride levels. Initial therapy should focus on glycemic control and lifestyle changes including a decrease in simple sugars and ethanol intake. Improving glycemic control can have profound effects on serum triglyceride levels. Fibrates, niacin, statins, and omega-3-fatty acids all reduce serum triglyceride levels (see Table 7). Typically, one will target triglyceride levels when one is trying to lower non-HDL cholesterol levels to goal. Patients with very high triglyceride levels (> 500-1000 mg/dl) are at risk of pancreatitis and therefore lifestyle and triglyceride lowering drug therapy should be initiated early. Note that there is limited evidence demonstrating that lowering triglyceride levels reduces cardiovascular events (see section on effect of lipid lowering drugs on cardiovascular events for details of the various studies). With the recent study demonstrating that adding EPA to statins in patients with elevated triglyceride levels reduces cardiovascular events one can anticipate an increased use of omega-3-fatty acids in patients with elevated triglyceride levels and a non-HDL cholesterol level above goal.

 

The fourth priority in treating lipid disorders is to increase HDL cholesterol levels. There is strong epidemiologic data linking low HDL cholesterol levels with cardiovascular disease but whether increasing HDL cholesterol levels with drugs reduces cardiovascular disease has not been demonstrated. Life style changes are the initial step and include increased exercise, weight loss, and stopping cigarette smoking. The role of recommending ethanol, which increases HDL cholesterol levels, is controversial but in patients who already drink moderately there is no reason to recommend that they stop unless they are hypertriglyceridemic. The most effective drug for increasing HDL cholesterol levels is niacin (see Table 8), but studies have not demonstrated a reduction in cardiovascular events when niacin is added to statin therapy (see section on the effect of lipid lowering drugs on cardiovascular events for details). Fibrates and statins also raise HDL cholesterol levels but the increases are modest (usually less than 15%). Unfortunately, given the currently available drugs, it is very difficult to significantly increase HDL cholesterol levels and in many of our diabetic patients we are unable to achieve HDL cholesterol levels in the recommended range. Furthermore, whether this will result in a reduction in cardiovascular events is unknown and studies have not demonstrated a benefit.

 

Many diabetic patients have multiple lipid abnormalities. As discussed in detail above life style changes are the initial therapy. Additionally, improving glycemic control can lead to marked reductions in serum triglyceride levels and modest increases in HDL cholesterol levels. If life style changes are not sufficient in patients with both elevations in LDL cholesterol and triglycerides (and elevations in non-HDL cholesterol), one approach is to base drug therapy on the triglyceride levels (Figure 2). If the serum triglycerides are very high (greater than 500-1000mg/dl), where there is an increased risk for pancreatitis and hyperviscosity syndromes, initial pharmacological therapy is directed at the elevated triglycerides and the initial drug choice is either a fibrate or high dose omega-3-fatty acids (3-4 grams EPA/DHA per day). After lowering triglyceride levels to < 500mg/dl, which may require more than one drug, statin therapy should be initiated if the LDL cholesterol and/or non-HDL cholesterol is not at goal. If the serum triglycerides are less than 500mg/dl, statin therapy to lower the LDL cholesterol level to goal is the initial therapy (see Figure 2). Studies have demonstrated that statins are effective drugs in lowering triglyceride levels in patients with elevated triglycerides [176]. In patients with low triglyceride levels statins do not greatly affect serum triglyceride levels. If the non-HDL cholesterol levels remain above goal after one reaches the LDL cholesterol goal, one should then consider combination therapy to lower triglyceride levels, which will lower non-HDL cholesterol levels.

Figure 2. Combined Hyperlipidemia. Increased LDL Cholesterol and TG

Often monotherapy is not sufficient to completely normalize the lipid profile. For example, with statin therapy one may often lower the LDL cholesterol to goal but the non-HDL cholesterol, HDL cholesterol, and triglycerides remain in the abnormal range. Currently, there are no randomized controlled trials demonstrating that combination therapy with fibrates or niacin reduces cardiovascular disease to a greater extent than statin monotherapy. In fact, as noted above, three outcome studies adding either niacin or fenofibrate to statin therapy failed to demonstrate additional benefit while two trials with omega-3-fatty acids showed benefit (see section on the effect of lipid lowering drugs on cardiovascular events for details). Many experts believe that further improvements in the lipid profile will be beneficial and that the studies completed so far should not be considered definitive as they had flaws such as not treating patients with the appropriate lipid profile. When using combination therapy one must be aware that the addition of either fibrates or niacin to statin therapy may increase the risk of myositis [176]. The increased risk of myositis is greatest when gemfibrozil is used in combination with statins. Fenofibrate has a much more modest risk and the FDA approved the use of fenofibrate in combination with moderate doses of statins. Additionally, in the ACCORD LIPID trial the combination of simvastatin and fenofibrate was well tolerated [33]. The increased risk with niacin appears to be very modest. In the AIM-HIGH trial the risk of myositis was not increased in patients on the combination of Niaspan and statin, whereas in the HPS2-Thrive trial myopathy was increased in the group treated with the combination of statin and niacin [137, 139]. The absolute risks of combination therapy are relatively modest if patients are carefully selected; in many patients at high risk for cardiovascular disease combination therapy may be appropriate. Notably, omega-3-fatty acids do not interact with statins or other drugs and hence do not have an increased risk when used in combination therapy. One should be aware of the steps listed in Table 10 that can reduce the potential for toxicity when one uses combination therapy. As with many decisions in medicine one needs to balance the benefits of therapy with the risks of therapy and determine for the individual patient the best approach. In deciding to use combination therapy a key focus is the non-HDL cholesterol level. When the LDL cholesterol is at goal but the non-HDL cholesterol is still markedly above goal it may be appropriate to resort to combination therapy in patients at high risk.

 

Table 10: When to Use Combination Therapy

·       Clinical Evidence of Arteriosclerosis

·       High Risk Patient

o   Hypertension

o   Family History of CAD

o   Cigarettes

o   Proteinuria

o   Microalbuminuria

o   Central Obesity

o   Inactivity

o   Elevated CRP

·       No Contraindications

o   Renal or Liver Disease

o   Non-compliant patient

o   Use of other drugs that effect statin metabolism

o   Other medical disorders that increase risk of toxicity

 

In summary, modern therapy of patients with diabetes demands that we aggressively treat lipids to reduce the high risk of cardiovascular disease in this susceptible population and in those with very high triglycerides to reduce the risk of pancreatitis.

 

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