Lipid lowering in patients 75 years and older.

More than twenty years ago, knowledge about the importance of cholesterol absorption and the potential therapeutic effect of its inhibition led to the discovery and clinical application of the first and only cholesterol absorption inhibitor to date - ezetimibe. Since then, ezetimibe has become a well-recognized player in lipid-lowering therapy. Recent findings of IMPROVE-IT and EWTOPIA 75 imply that elderly patients over the age of 75 years in particular benefit from ezetimibe. This review summarizes the evidence, discusses the possible underlying pathophysiological mechanisms and calls for a change in future dyslipidemia guidelines.

APOL1 Genetic Variants Are Associated With Increased Risk of Coronary Atherosclerotic Plaque Rupture in the Black Population.

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Progress and perspectives in plant sterol and plant stanol research.

Current evidence indicates that foods with added plant sterols or stanols can lower serum levels of low-density lipoprotein cholesterol. This review summarizes the recent findings and deliberations of 31 experts in the field who participated in a scientific meeting in Winnipeg, Canada, on the health effects of plant sterols and stanols. Participants discussed issues including, but not limited to, the health benefits of plant sterols and stanols beyond cholesterol lowering, the role of plant sterols and stanols as adjuncts to diet and drugs, and the challenges involved in measuring plant sterols and stanols in biological samples. Variations in interindividual responses to plant sterols and stanols, as well as the personalization of lipid-lowering therapies, were addressed. Finally, the clinical aspects and treatment of sitosterolemia were reviewed. Although plant sterols and stanols continue to offer an efficacious and convenient dietary approach to cholesterol management, long-term clinical trials investigating the endpoints of cardiovascular disease are still lacking.

Community-based statins and advanced carotid plaque: Role of CD163 positive macrophages in lipoprotein-associated phospholipase A2 activity in atherosclerotic plaque.

BACKGROUND AND AIMS: Lipoprotein-associated phospholipase A2 (Lp-PLA2), an enzymatic inflammatory biomarker primarily bound to low-density lipoprotein cholesterol, is associated with an approximate twofold increased risk of cardiovascular disease and stroke. Despite indications that circulating Lp-PLA2 is sensitive to statins, it remains largely unknown whether statin usage exerts local effects on Lp-PLA2 expression at the site of atheromatous plaque. METHODS: Carotid plaques (n = 38) were prospectively collected from symptomatic (n = 18) and asymptomatic (n = 20) patients with (n = 20) or without (n = 18) documented statin history. In all cases, endarterectomy was performed where the primary stenosis was removed in an undisturbed manner. Serial cryosections of the presenting lesion were assessed histologically for macrophages, Lp-PLA2, and cell death (apoptotic index). RESULTS: Symptomatic lesions exhibited less calcification, with greater inflammation characterized by increased expression of CD68+ and CD163+ macrophage subsets, and Lp-PLA2. Symptomatic plaques also exhibited greater necrotic core area and increased apoptosis, as compared with asymptomatic lesions. In contrast, statin treatment did not appear to influence any of these parameters, except for the extent of apoptosis, which was less in statin treated as compared with statin naïve lesions. Overall, Lp-PLA2 expression correlated positively with necrotic core area, CD68+ and CD163+ macrophage area, and cell death. Finally, in vitro assays and dual immunofluorescence staining confirmed CD163-expressing monocytes/macrophages are also a major source of Lp-PLA2. CONCLUSIONS: Statin treatment has no effect on local atherosclerotic lesion Lp-PLA2 activity, therefore, the addition of anti-inflammatory treatments to further decrease macrophage Lp-PLA2 expression in atherosclerotic lesions may reduce lesional inflammation and cell death, and prevent necrotic core expansion and lesion progression.

Pathophysiology of native coronary, vein graft, and in-stent atherosclerosis.

Plaque rupture, usually of a precursor lesion known as a 'vulnerable plaque' or 'thin-cap fibroatheroma', is the leading cause of thrombosis. Less-frequent aetiologies of coronary thrombosis are erosion, observed with greatest incidence in women aged <50 years, and eruptive calcified nodules, which are occasionally identified in older individuals. Various treatments for patients with coronary artery disease, such as CABG surgery and interventional therapies, have led to accelerated atherosclerosis. These processes occur within months to years, compared with the decades that it generally takes for native disease to develop. Morphological identifiers of accelerated atherosclerosis include macrophage-derived foam cells, intraplaque haemorrhage, and thin fibrous cap. Foam-cell infiltration can be observed within 1 year of a saphenous vein graft implantation, with subsequent necrotic core formation and rupture ensuing after 7 years in over one-third of patients. Neoatherosclerosis occurs early and with greater prevalence in drug-eluting stents than in bare-metal stents and, although rare, complications of late stent thrombosis from rupture are associated with high mortality. Comparison of lesion progression in native atherosclerotic disease, atherosclerosis in saphenous vein grafts, and in-stent neoatherosclerosis provides insight into the pathogenesis of atheroma formation in natural and iatrogenic settings.

CDKN2B Regulates TGFß Signaling and Smooth Muscle Cell Investment of Hypoxic Neovessels.

RATIONALE: Genetic variation at the chromosome 9p21 cardiovascular risk locus has been associated with peripheral artery disease, but its mechanism remains unknown. OBJECTIVE: To determine whether this association is secondary to an increase in atherosclerosis, or it is the result of a separate angiogenesis-related mechanism. METHODS AND RESULTS: Quantitative evaluation of human vascular samples revealed that carriers of the 9p21 risk allele possess a significantly higher burden of immature intraplaque microvessels than carriers of the ancestral allele, irrespective of lesion size or patient comorbidity. To determine whether aberrant angiogenesis also occurs under nonatherosclerotic conditions, we performed femoral artery ligation surgery in mice lacking the 9p21 candidate gene, Cdkn2b. These animals developed advanced hindlimb ischemia and digital autoamputation, secondary to a defect in the capacity of the Cdkn2b-deficient smooth muscle cell to support the developing neovessel. Microarray studies identified impaired transforming growth factor ß (TGFß) signaling in cultured cyclin-dependent kinase inhibitor 2B (CDKN2B)-deficient cells, as well as TGFß1 upregulation in the vasculature of 9p21 risk allele carriers. Molecular signaling studies indicated that loss of CDKN2B impairs the expression of the inhibitory factor, SMAD-7, which promotes downstream TGFß activation. Ultimately, this manifests in the upregulation of a poorly studied effector molecule, TGFß1-induced-1, which is a TGFß-rheostat known to have antagonistic effects on the endothelial cell and smooth muscle cell. Dual knockdown studies confirmed the reversibility of the proposed mechanism, in vitro. CONCLUSIONS: These results suggest that loss of CDKN2B may not only promote cardiovascular disease through the development of atherosclerosis but may also impair TGFß signaling and hypoxic neovessel maturation.

Glucagon receptor antagonism induces increased cholesterol absorption.

Glucagon and insulin have opposing action in governing glucose homeostasis. In type 2 diabetes mellitus (T2DM), plasma glucagon is characteristically elevated, contributing to increased gluconeogenesis and hyperglycemia. Therefore, glucagon receptor (GCGR) antagonism has been proposed as a pharmacologic approach to treat T2DM. In support of this concept, a potent small-molecule GCGR antagonist (GRA), MK-0893, demonstrated dose-dependent efficacy to reduce hyperglycemia, with an HbA1c reduction of 1.5% at the 80 mg dose for 12 weeks in T2DM. However, GRA treatment was associated with dose-dependent elevation of plasma LDL-cholesterol (LDL-c). The current studies investigated the cause for increased LDL-c. We report findings that link MK-0893 with increased glucagon-like peptide 2 and cholesterol absorption. There was not, however, a GRA-related modulation of cholesterol synthesis. These findings were replicated using structurally diverse GRAs. To examine potential pharmacologic mitigation, coadministration of ezetimibe (a potent inhibitor of cholesterol absorption) in mice abrogated the GRA-associated increase of LDL-c. Although the molecular mechanism is unknown, our results provide a novel finding by which glucagon and, hence, GCGR antagonism govern cholesterol metabolism.

Natural progression of atherosclerosis from pathologic intimal thickening to late fibroatheroma in human coronary arteries: A pathology study.

OBJECTIVE: Smooth muscle cells, macrophage infiltration and accumulation of lipids, proteoglycans, collagen matrix and calcification play a central role in atherosclerosis. The early histologic changes of plaque progression from pathologic intimal thickenings (PIT) to late fibroatheroma lesions have not been fully characterized. METHODS: A total of 151 atherosclerotic coronary lesions were collected from 67 sudden death victims. Atherosclerotic plaques were classified as PIT without macrophage infiltration, PIT with macrophages, and early and late fibroatheromas. Presence of macrophages and proteoglycans (versican, decorin and biglycan) were recognized by specific antibodies while hyaluronan was detected by affinity histochemistry. Lipid deposition was identified by oil-red-O, and calcification was assessed following von Kossa and alizarin red staining. RESULTS: Lesion progression from PIT to late fibroatheroma was associated with increase in macrophage accumulation (p < 0.001) and decreasing apoptotic body clearance by macrophages (ratio of engulfed-to-total apoptotic bodies) (p < 0.001). Lipid deposition in lipid pool of PIT had a microvesicular appearance whereas those in the necrotic core were globular in nature. Overall, the accumulation of hyaluronan (p < 0.001), and proteoglycan versican (p < 0.001) and biglycan (p = 0.013) declined along with lesion progression from PIT to fibroatheromas. Microcalcification was first observed only within areas of lipid pools and its presence and size increased in lesions with necrotic core. CONCLUSIONS: PIT to fibroatheroma lesions are accompanied by early lipid accumulation, followed by macrophage infiltration with defective clearance of apoptotic bodies along with decrease in proteoglycan and hyaluronan in lipid pools that convert to necrotic cores. Calcification starts in PIT and increases with plaque progression.

Sex differences in coronary artery disease: pathological observations.

Cardiovascular disease (CVD) remains the most frequent cause of death in both men and women. Many studies on CVD have included mostly men, and the knowledge about coronary artery disease (CAD) in women has largely been extrapolated from studies primarily focused on men. The influence of various risk factors is different between men and women; untoward effects of smoking of CAD are greater in women than men. Furthermore, the effect of the menopause is important in women, with higher incidence of plaque erosion in young women versus greater incidence of plaque rupture in older women. This review focuses on differences in plaque morphology in men and women presenting with sudden coronary death and acute myocardial infarction.

Inverse relationship between LDL cholesterol and PCSK9 plasma levels in dyslipidemic cynomolgus monkeys: effects of LDL lowering by ezetimibe in the absence of statins.

OBJECTIVES: To assess the lipid-lowering efficacy of ezetimibe in dyslipidemic cynomolgus monkeys comparing two dosing methods, and to evaluate PCSK9 plasma levels during dyslipidemia induction by feeding a high-fat/high-cholesterol diet (HFD), ezetimibe (Zetia(®), Ezetrol(®)) treatment, ezetimibe washout, and HFD washout. METHODS AND RESULTS: Twenty dyslipidemic cynomolgus monkeys on HFD for seven months (LDL cholesterol 100-400 mg/dL) were randomized into two groups and treated with ezetimibe for two weeks, either by oral gavage or by using food treats. The lipid-lowering effects of ezetimibe were identical between the two groups. After treatment, mean LDL cholesterol was decreased by 58% (174-72 mg/dL), total cholesterol by 42% (241-138 mg/dL), and PCSK9 levels were increased by 137% (147-314 ng/mL). PCSK9 levels on regular diet before and after HFD were also inversely correlated to LDL cholesterol. CONCLUSIONS: In a cynomolgus dyslipidemia model, PCSK9 levels are inversely correlated with LDL cholesterol in the absence of statin treatment, regardless whether lipid changes are modulated by diet or ezetimibe treatment.

Intestinal sterol transporters and cholesterol absorption inhibition.

PURPOSE OF REVIEW: Statin therapy is the mainstay of lipid-lowering therapy; however, many patients, particularly those at high risk, do not achieve sufficient LDL-cholesterol (LDL-C) lowering. Thus, there remains an unmet medical need for more effective and well tolerated lipid-lowering agents. Guidelines recommend combining additional lipid-lowering agents with a complementary mode of action for these patients. One approach to complementing statin therapy is combination with inhibitors that block the intestinal absorption of dietary and biliary cholesterol. This review summarizes what is currently known about intestinal sterol transporters and cholesterol absorption inhibitors (CAIs). RECENT FINDINGS: The only lipid-lowering agent currently available that specifically targets an intestinal sterol transporter (Niemann-Pick C1-like 1) is the CAI, ezetimibe. It is effective in lowering LDL-C, both when given alone and when combined with a statin. Clinical outcome data with ezetimibe combined with simvastatin have recently become available, and definitive evidence that the incremental LDL-C lowering attributable to the ezetimibe component reduces cardiovascular events beyond simvastatin alone is currently under study. Other novel CAIs have been evaluated based upon the structure and properties of ezetimibe, but none remain in development. SUMMARY: Additional lipid-lowering agents are needed to fulfill an unmet medical need for those patients who do not achieve optimal LDL-C goals on statin monotherapy. The inhibition of cholesterol absorption is an important therapeutic strategy to reduce cholesterol levels. Based upon the demonstrated lipid-altering efficacy and safety of ezetimibe, several CAIs have been identified; all to date have been discontinued due to limited efficacy.

Effects of ezetimibe on atherosclerosis in preclinical models.

Ezetimibe (Zetia(®), Ezetrol(®), Merck, Whitehouse Station, NJ) is a potent inhibitor of sterol absorption, which selectively blocks the uptake of biliary and dietary cholesterol in the small intestine. Clinical trials have demonstrated the beneficial effects of ezetimibe on the reduction of atherogenic lipoproteins and the attainment of guideline-recommended lipid levels. Direct evidence that these improvements translate to a reduction in atherosclerosis or cardiovascular events is limited, although reductions in major atherosclerotic events that are consistent with the LDL-C lowering achieved have recently been presented for patients with chronic kidney disease treated with ezetimibe/simvastatin 10/20mg in the SHARP trial. Animal models of atherosclerosis have played a central role in defining the mechanisms involved in initiation and development of disease and have been used in drug development to evaluate potential therapeutic efficacy. The effect of ezetimibe on atherosclerosis has been examined in several of these animal model systems. ApoE knockout mice develop severe hypercholesterolemia and premature atherosclerosis with features similar to that seen in humans and techniques ranging from gross visualization of plaque to high-resolution MRI have demonstrated the consistent ability of ezetimibe to significantly inhibit atherosclerosis. sr-b1(-/-)/apoE(-/-) double knockout mice exhibit additional characteristics common to human coronary heart disease (CHD), and the one study of ezetimibe in sr-b1(-/-)/apoE(-/-) mice showed a significant reduction in aortic sinus plaque (57%), coronary arterial occlusion (68%), myocardial fibrosis (57%), and cardiomegaly (12%) compared with untreated controls. The effects of ezetimibe have also been evaluated in ldlr(-/-)/apoE(-/-) double knockout mice, demonstrating that functional LDL receptors were not required for ezetimibe-mediated reduction of plasma cholesterol or atherosclerosis. For the few studies that have been conducted in rabbits, ezetimibe has been shown to significantly inhibit diet and vascular-injury-induced atherosclerosis as measured by intima/media thickness, atherosclerotic lesion composition, and thrombosis. The current body of preclinical evidence consistently demonstrates that ezetimibe reduces atherosclerosis in animals, presumably due primarily to the decrease in circulating levels of atherogenic lipoproteins that the drug produces. Demonstration that ezetimibe-mediated lowering of atherogenic lipoproteins in humans has a similar effect on atherosclerosis and cardiovascular risk awaits additional results from recently completed and ongoing outcomes trials.

Improved efficacy for ezetimibe and rosuvastatin by attenuating the induction of PCSK9.

Reducing circulating LDL-cholesterol (LDL-c) reduces the risk of cardiovascular disease in people with hypercholesterolemia. Current approaches to reduce circulating LDL-c include statins, which inhibit cholesterol synthesis, and ezetimibe, which blocks cholesterol absorption. Both elevate serum PCSK9 protein levels in patients, which could attenuate their efficacy by reducing the amount of cholesterol cleared from circulation. To determine whether PCSK9 inhibition could enhance LDL-c lowering of both statins and ezetimibe, we utilized small interfering RNAs (siRNAs) to knock down Pcsk9, together with ezetimibe, rosuvastatin, and an ezetimibe/rosuvastatin combination in a mouse model with a human-like lipid profile. We found that ezetimibe, rosuvastatin, and ezetimibe/rosuvastatin combined lower serum cholesterol but induce the expression of Pcsk9 as well as the Srebp-2 hepatic cholesterol biosynthesis pathway. Pcsk9 knockdown in combination with either treatment led to greater reductions in serum non-HDL with a near-uniform reduction of all LDL-c subfractions. In addition to reducing serum cholesterol, the combined rosuvastatin/ezetimibe/Pcsk9 siRNA treatment exhibited a significant reduction in serum APOB protein and triglyceride levels. Taken together, these data provide evidence that PCSK9 inhibitors, in combination with current therapies, have the potential to achieve greater reductions in both serum cholesterol and triglycerides.

Atorvastatin increases intestinal expression of NPC1L1 in hyperlipidemic men.

Inhibition of cholesterol synthesis by 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoAR) inhibitors has been associated with an increase in intestinal cholesterol absorption. This study examined how HMG-CoAR inhibition by atorvastatin modulates expression of key genes involved in intestinal cholesterol metabolism. A crossover study was conducted in which 22 hyperlipidemic men received atorvastatin, 40 mg/day, or placebo, each for 12 weeks. Gene expression was assessed by real-time PCR using duodenal biopsy samples obtained at the end of each phase of treatment. Treatment with atorvastatin was associated with a 76% reduction in lathosterol and significant increases in sitosterol (70%). Atorvastatin significantly increased intestinal mRNA levels of HMG-CoAR (59%), LDL receptor (LDLR) (52%), PCSK9 (187%), SREBP-2 (44%), and HNF-4α (13%). Furthermore, atorvastatin significantly increased intestinal mRNA levels of NPC1L1 by 19% and decreased mRNA levels of both ABCG5 and ABCG8 by 14%. Positive correlations were observed between changes in SREBP-2 and HNF-4α expression and concurrent changes in the intestinal mRNA levels of HMG-CoAR, LDLR, and NPC1L1. These results indicate that HMG-CoAR inhibition with atorvastatin stimulates the intestinal expression of NPC1L1, LDLR, and PCSK9; increases cholesterol absorption; and reduces expression of ABCG5/8; these effects are most likely mediated by upregulation of the transcription factors SREBP-2 and HNF-4α.

Gender-dependent effect of Gpbar1 genetic deletion on the metabolic profiles of diet-induced obese mice.

G-protein-coupled bile acid receptor 1 (GPBAR1/TGR5/M-Bar/GPR131) is a cell surface receptor involved in the regulation of bile acid metabolism. We have previously shown that Gpbar1-null mice are resistant to cholesterol gallstone disease when fed a lithogenic diet. Other published studies have suggested that Gpbar1 is involved in both energy homeostasis and glucose homeostasis. Here, we examine the functional role of Gpbar1 in diet-induced obese mice. We found that body weight, food intake, and fasted blood glucose levels were similar between Gpbar1-null mice and their wild-type (WT) littermates when fed a chow or high-fat diet (HFD) for 2 months. However, insulin tolerance tests revealed improved insulin sensitivity in male Gpbar1(-/-) mice fed chow, but impaired insulin sensitivity when fed a HFD. In contrast, female Gpbar1(-/-) mice exhibited improved insulin sensitivity when fed a HFD compared with their WT littermates. Female Gpbar1(-/-) mice had significantly lower plasma cholesterol and triglyceride levels than their WT littermates on both diets. Male Gpbar1(-/-) mice on HFD displayed increased hepatic steatosis when compared with Gpbar1(+)(/)(+) males and Gpbar1(-/-) females on HFD. These results suggest a gender-dependent regulation of Gpbar1 function in metabolic disease.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) affects gene expression pathways beyond cholesterol metabolism in liver cells.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) induces degradation of low-density lipoprotein receptor (LDLR) in the liver. It is being pursued as a therapeutic target for LDL-cholesterol reduction. Earlier genome-wide gene expression studies showed that PCSK9 over-expression in HepG2 cells resulted in up-regulation of genes in cholesterol biosynthesis and down-regulation of genes in stress response pathways; however, it was not known whether these changes were directly regulated by PCSK9 or were secondary to PCSK9-induced changes to the intracellular environment. In order to further understand the biological function of PCSK9 we treated HepG2 cells with purified recombinant wild type (WT) and D374Y gain-of-function PCSK9 proteins for 8, 24, and 48 h, and used microarray analysis to identify genome-wide expression changes and pathways. These results were compared to the changes induced by culturing HepG2 cells in cholesterol-free medium, mimicking the intracellular environment of cholesterol starvation. We determined that PCSK9-induced up-regulation of cholesterol biosynthesis genes resulted from intracellular cholesterol starvation. In addition, we identified novel pathways that are presumably regulated by PCSK9 and are independent of its effects on cholesterol uptake. These pathways included "protein ubiquitination," "xenobiotic metabolism," "cell cycle," and "inflammation and stress response." Our results indicate that PCSK9 affects metabolic pathways beyond cholesterol metabolism in HepG2 cells.

Niemann-Pick C1 Like 1 (NPC1L1) an intestinal sterol transporter.

Niemann-Pick C1 Like 1 (NPC1L1) has been identified and characterized as an essential protein in the intestinal cholesterol absorption process. NPC1L1 localizes to the brush border membrane of absorptive enterocytes in the small intestine. Intestinal expression of NPC1L1 is down regulated by diets containing high levels of cholesterol. While otherwise phenotypically normal, Npc1l1 null mice exhibit a significant reduction in the intestinal uptake and absorption of cholesterol and phytosterols. Characterization of the NPC1L1 pathway revealed that cholesterol absorption inhibitor ezetimibe specifically binds to an extracellular loop of NPC1L1 and inhibits its sterol transport function. Npc1l1 null mice are resistant to diet-induced hypercholesterolemia, and when crossed with apo E null mice, are completely resistant to the development of atherosclerosis. Intestinal gene expression studies in Npc1l1 null mice indicated that no exogenous cholesterol was entering enterocytes lacking NPC1L1, which resulted in an upregulation of intestinal and hepatic LDL receptor and cholesterol biosynthetic gene expression. Polymorphisms in the human NPC1L1 gene have been found to influence cholesterol absorption and plasma low density lipoprotein levels. Therefore, NPC1L1 is a critical intestinal sterol uptake transporter which influences whole body cholesterol homeostasis.

Lack of FFAR1/GPR40 does not protect mice from high-fat diet-induced metabolic disease.

OBJECTIVE: FFAR1/GPR40 is a G-protein-coupled receptor expressed predominantly in pancreatic islets mediating free fatty acid-induced insulin secretion. However, the physiological role of FFAR1 remains controversial. It was previously reported that FFAR1 knockout (Ffar1(-/-)) mice were resistant to high-fat diet-induced hyperinuslinemia, hyperglycemia, hypertriglyceridemia, and hepatic steatosis. A more recent report suggested that although FFAR1 was necessary for fatty acid-induced insulin secretion in vivo, deletion of FFAR1 did not protect pancreatic islets against fatty acid-induced islet dysfunction. This study is designed to investigate FFAR1 function in vivo using a third line of independently generated Ffar1(-/-) mice in the C57BL/6 background. RESEARCH DESIGN AND METHODS: We used CL-316,243, a beta3 adrenergic receptor agonist, to acutely elevate blood free fatty acids and to study its effect on insulin secretion in vivo. Ffar1(+/+) (wild-type) and Ffar1(-/-) (knockout) mice were placed on two distinct high-fat diets to study their response to diet-induced obesity. RESULTS: Insulin secretion was reduced by approximately 50% in Ffar1(-/-) mice, confirming that FFAR1 contributes significantly to fatty acid stimulation of insulin secretion in vivo. However, Ffar1(+/+) and Ffar1(-/-) mice had similar weight, adiposity, and hyperinsulinemia on high-fat diets, and Ffar1(-/-) mice showed no improvement in glucose or insulin tolerance tests. In addition, high-fat diet induced comparable levels of lipid accumulation in livers of Ffar1(+/+) and Ffar1(-/-) mice. CONCLUSIONS: FFAR1 is required for normal insulin secretion in response to fatty acids; however, Ffar1(-/-) mice are not protected from high-fat diet-induced insulin resistance or hepatic steatosis.