Prediman K. Shah

Coronary Plaque Disruption

Coronary atherosclerosis is by far the most frequent cause of ischemic heart disease, and plaque disruption with superimposed thrombosis is the main cause of the acute coronary syndromes of unstable angina, myocardial infarction, and sudden death.1 2 3 4 5 Therefore, for event-free survival, the vital question is not why atherosclerosis develops but rather why, after years of indolent growth, it suddenly becomes complicated by life-threatening thrombosis. The composition and vulnerability of plaque rather than its volume or the consequent severity of stenosis produced have emerged as being the most important determinants for the development of the thrombus-mediated acute coronary syndromes; lipid-rich and soft plaques are more dangerous than collagen-rich and hard plaques because they are more unstable and rupture-prone and highly thrombogenic after disruption.6 This review will explore potential mechanisms responsible for the sudden conversion of a stable atherosclerotic plaque to an unstable and life-threatening atherothrombotic lesion—an event known as plaque fissuring, rupture, or disruption.7 8 Atherosclerosis is the result of a complex interaction between blood elements, disturbed flow, and vessel wall abnormality, involving several pathological processes: inflammation, with increased endothelial permeability, endothelial activation, and monocyte recruitment9 10 11 12 13 14 ; growth, with smooth muscle cell (SMC) proliferation, migration, and matrix synthesis15 16 ; degeneration, with lipid accumulation17 18 ; necrosis, possibly related to the cytotoxic effect of oxidized lipid19 ; calcification/ossification, which may represent an active rather than a dystrophic process20 21 ; and thrombosis, with platelet recruitment and fibrin formation.1 22 23 Thrombotic factors may play a role early during atherogenesis, but a flow-limiting thrombus does not develop until mature plaques are present, which is why thrombosis often is classified as a complication rather than a genuine component of atherosclerosis. ### Mature Plaques: Atherosis and Sclerosis As the name atherosclerosis implies, mature …

Effects of Dalcetrapib in Patients with a Recent Acute Coronary Syndrome

BACKGROUND In observational analyses, higher levels of high-density lipoprotein (HDL) cholesterol have been associated with a lower risk of coronary heart disease events. However, whether raising HDL cholesterol levels therapeutically reduces cardiovascular risk remains uncertain. Inhibition of cholesteryl ester transfer protein (CETP) raises HDL cholesterol levels and might therefore improve cardiovascular outcomes. METHODS We randomly assigned 15,871 patients who had had a recent acute coronary syndrome to receive the CETP inhibitor dalcetrapib, at a dose of 600 mg daily, or placebo, in addition to the best available evidence-based care. The primary efficacy end point was a composite of death from coronary heart disease, nonfatal myocardial infarction, ischemic stroke, unstable angina, or cardiac arrest with resuscitation. RESULTS At the time of randomization, the mean HDL cholesterol level was 42 mg per deciliter (1.1 mmol per liter), and the mean low-density lipoprotein (LDL) cholesterol level was 76 mg per deciliter (2.0 mmol per liter). Over the course of the trial, HDL cholesterol levels increased from baseline by 4 to 11% in the placebo group and by 31 to 40% in the dalcetrapib group. Dalcetrapib had a minimal effect on LDL cholesterol levels. Patients were followed for a median of 31 months. At a prespecified interim analysis that included 1135 primary end-point events (71% of the projected total number), the independent data and safety monitoring board recommended termination of the trial for futility. As compared with placebo, dalcetrapib did not alter the risk of the primary end point (cumulative event rate, 8.0% and 8.3%, respectively; hazard ratio with dalcetrapib, 1.04; 95% confidence interval, 0.93 to 1.16; P=0.52) and did not have a significant effect on any component of the primary end point or total mortality. The median C-reactive protein level was 0.2 mg per liter higher and the mean systolic blood pressure was 0.6 mm Hg higher with dalcetrapib as compared with placebo (P<0.001 for both comparisons). CONCLUSIONS In patients who had had a recent acute coronary syndrome, dalcetrapib increased HDL cholesterol levels but did not reduce the risk of recurrent cardiovascular events. (Funded by F. Hoffmann-La Roche; dal-OUTCOMES ClinicalTrials.gov number, NCT00658515.).

Pravastatin Treatment Increases Collagen Content and Decreases Lipid Content, Inflammation, Metalloproteinases, and Cell Death in Human Carotid Plaques Implications for Plaque Stabilization

Background —The clinical benefits of lipid lowering with statins are attributed to changes in plaque composition leading to lesion stability, but supporting clinical data from human studies are lacking. Therefore, we investigated the effect of 3 months of pravastatin treatment on composition of human carotid plaques removed during carotid endarterectomy. Methods and Results —Consecutive patients with symptomatic carotid artery stenosis received 40 mg/d pravastatin (n=11) or no lipid-lowering therapy (n=13; control subjects) for 3 months before scheduled carotid endarterectomy. Carotid plaque composition was assessed with special stains and immunocytochemistry with quantitative image analysis. Plaques from the pravastatin group had less lipid by oil red O staining (8.2±8.4% versus 23.9±21.1% of the plaque area, P <0.05), less oxidized LDL immunoreactivity (13.3±3.6% versus 22.0±6.5%, P <0.001), fewer macrophages (15.0±10.2% versus 25.3±12.5%, P <0.05), fewer T cells (11.2±9.3% versus 24.3±13.4%, P <0.05), less matrix metalloproteinase 2 (MMP-2) immunoreactivity (3.6±3.9% versus 8.4±5.3%, P <0.05), greater tissue inhibitor of metalloproteinase 1 (TIMP-1) immunoreactivity (9.0±6.2% versus 3.1±3.9%, P <0.05), and a higher collagen content by Sirius red staining (12.4±3.1% versus 7.5±3.5%, P <0.005). Cell death by TUNEL staining was reduced in the pravastatin group (17.7±7.8% versus 32.0±12.6%, P <0.05). Conclusions —Pravastatin decreased lipids, lipid oxidation, inflammation, MMP-2, and cell death and increased TIMP-1 and collagen content in human carotid plaques, confirming its plaque-stabilizing effect in humans.

Mechanisms of plaque vulnerability and rupture

Rupture of atherosclerotic plaque has been identified as the proximate event in the majority of cases of acute ischemic syndromes. Plaque rupture exposes thrombogenic components of the plaque, activating the clotting cascade and promoting thrombus formation. Future culprit lesions are difficult to identify, however, and angiographic assessment of stenosis severity is prone to underestimation. Compared with plaques that cause severe luminal stenosis, vulnerable plaques may cause relatively minor stenosis, although they account for more cases of rupture and thrombosis. Such unstable, vulnerable plaques may be associated with outward remodeling of the vessel. Because severely stenotic plaques are more likely to stimulate collateral circulation to the post-stenotic segment, plaque rupture and thrombosis at such sites may be clinically silent. Characteristic histomorphologic features of vulnerable plaques include a high lipid content, increased numbers of inflammatory cells, and extensive adventitial and intimal neovascularity. The fibrous cap of an atherosclerotic plaque may become thin and rupture as a result of the depletion of matrix components through the activation of enzymes, such as matrix-degrading proteinases and cystine and aspartate proteases, and through the reduction in the number of smooth muscle cells. Activated T cells may also inhibit matrix synthesis through the production of interferon-gamma. A number of triggers of plaque rupture have been identified. Also, some thrombi may occur without rupture of the fibrous cap. Reducing the lipid component and inflammation in atherosclerotic plaques may help reduce the risk of plaque rupture. This may account for the clinical benefit of risk-factor reduction gained from changes in lifestyle and from drug therapy.

Efficacy and safety of a microsomal triglyceride transfer protein inhibitor in patients with homozygous familial hypercholesterolaemia: a single-arm, open-label, phase 3 study

BACKGROUND Patients with homozygous familial hypercholesterolaemia respond inadequately to existing drugs. We aimed to assess the efficacy and safety of the microsomal triglyceride transfer protein inhibitor lomitapide in adults with this disease. METHODS We did a single-arm, open-label, phase 3 study of lomitapide for treatment of patients with homozygous familial hypercholesterolemia. Current lipid lowering therapy was maintained from 6 weeks before baseline through to at least week 26. Lomitapide dose was escalated on the basis of safety and tolerability from 5 mg to a maximum of 60 mg a day. The primary endpoint was mean percent change in levels of LDL cholesterol from baseline to week 26, after which patients remained on lomitapide through to week 78 for safety assessment. Percent change from baseline to week 26 was assessed with a mixed linear model. FINDINGS 29 men and women with homozygous familial hypercholesterolaemia, aged 18 years or older, were recruited from 11 centres in four countries (USA, Canada, South Africa, and Italy). 23 of 29 enrolled patients completed both the efficacy phase (26 weeks) and the full study (78 weeks). The median dose of lomitapide was 40 mg a day. LDL cholesterol was reduced by 50% (95% CI -62 to -39) from baseline (mean 8·7 mmol/L [SD 2·9]) to week 26 (4·3 mmol/L [2·5]; p<0·0001). Levels of LDL cholesterol were lower than 2·6 mmol/L in eight patients at 26 weeks. Concentrations of LDL cholesterol remained reduced by 44% (95% CI -57 to -31; p<0·0001) at week 56 and 38% (-52 to -24; p<0·0001) at week 78. Gastrointestinal symptoms were the most common adverse event. Four patients had aminotransaminase levels of more than five times the upper limit of normal, which resolved after dose reduction or temporary interruption of lomitapide. No patient permanently discontinued treatment because of liver abnormalities. INTERPRETATION Our study suggests that treatment with lomitapide could be a valuable drug in the management of homozygous familial hypercholesterolaemia. FUNDING FDA Office of the Orphan Product Development, Aegerion Pharmaceuticals.

Effect of Immunization With Homologous LDL and Oxidized LDL on Early Atherosclerosis in Hypercholesterolemic Rabbits

Although the existence of an immune response against modified lipoproteins in atherosclerosis has been observed in experimental animals as well as in humans, the precise pathophysiological relevance of these findings remains unclear. In this study we determined the effect of an immunization with homologous LDL and copper-oxidized LDL on the formation of atherosclerotic plaque in hypercholesterolemic rabbits. Immunizations were performed at the start of a cholesterol-rich diet and 3 weeks later. After 16 weeks, antibodies against oxidized LDL had developed in rabbits given hypercholesterolemic diet alone, but the titers were increased by twofold in rabbits immunized with oxidized LDL as well as in rabbits immunized with LDL, suggesting that the LDL had also become oxidized during the preparation and/or immunization procedure. Immunization with LDL and oxidized LDL reduced atherosclerotic lesions in the proximal aorta by 74% (P < .05) and 48% (P = NS), respectively. The cellular composition of the lesions was not affected by the immunizations. These results support the hypothesis that an immune response against modified LDL has a protective effect against the development of early atherosclerotic lesions.

Calcification in atherosclerosis: Bone biology and chronic inflammation at the arterial crossroads

Dystrophic or ectopic mineral deposition occurs in many pathologic conditions, including atherosclerosis. Calcium mineral deposits that frequently accompany atherosclerosis are readily quantifiable radiographically, serve as a surrogate marker for the disease, and predict a higher risk of myocardial infarction and death. Accelerating research interest has been propelled by a clear need to understand how plaque structure, composition, and stability lead to devastating cardiovascular events. In atherosclerotic plaque, accumulating evidence is consistent with the notion that calcification involves the participation of arterial osteoblasts and osteoclasts. Here we summarize current models of intimal arterial plaque calcification and highlight intriguing questions that require further investigation. Because atherosclerosis is a chronic vascular inflammation, we propose that arterial plaque calcification is best conceptualized as a convergence of bone biology with vascular inflammatory pathobiology.

Membrane Type 1 Matrix Metalloproteinase Expression in Human Atherosclerotic Plaques Evidence for Activation by Proinflammatory Mediators

BACKGROUND Matrix metalloproteinases (MMPs) are expressed in atherosclerotic plaques, where in their active form, they may contribute to vascular remodeling and plaque disruption. In this study, we tested the hypothesis that membrane type 1 MMP (MT1-MMP), a novel transmembrane MMP that activates pro-MMP-2 (gelatinase A), is expressed in human atherosclerotic plaques and that its expression is regulated by proinflammatory molecules. METHODS AND RESULTS MT1-MMP expression was examined in normal and atherosclerotic human arteries by immunocytochemistry with specific antibodies. MT1-MMP expression in human saphenous vein-derived smooth muscle cells (SMCs) maintained in tissue culture was determined under basal conditions and in response to proinflammatory molecules (interleukin [IL]-1alpha, tumor necrosis factor [TNF]-alpha, and oxidized LDL [ox-LDL]) by use of Northern blot and ribonuclease protection assays for mRNA, Western blot and immunoprecipitation for protein, and gelatin zymography for catalytic activity. Medial SMCs of normal vessel wall expressed MT1-MMP. In atherosclerotic arteries, MT1-MMP expression was noted within the complex atheroma colocalizing with SMCs and macrophages (Mphi). Cultured SMCs constitutively expressed MT1-MMP mRNA and protein, which increased 2- to 4-fold over control in a time-dependent manner within 4 to 8 hours of exposure to IL-1alpha, TNF-alpha, and ox-LDL (thiobarbituric acid-reactive substances, 13.4 nmol/mg LDL protein), whereas native LDL had no effect. Flow cytometry revealed MT1-MMP expression by human monocyte-derived Mphi, which increased 3.8-fold over baseline within 6 hours after exposure to 10 ng/mL TNF-alpha. CONCLUSIONS This study demonstrates that MT1-MMP, an activator of pro-MMP-2, is expressed by SMCs and Mphi in human atherosclerotic plaques. Furthermore, proinflammatory molecules upregulate MT1-MMP expression in vascular SMCs and Mphi. Thus, activation of SMCs and Mphi by proinflammatory molecules may influence extracellular matrix remodeling in atherosclerosis by regulating MT1-MMP expression.

Oxidized Low-Density Lipoprotein Regulates Matrix Metalloproteinase-9 and Its Tissue Inhibitor in Human Monocyte-Derived Macrophages

BACKGROUND Macrophages in human atherosclerotic plaques produce a family of matrix metalloproteinases (MMPs), which may influence vascular remodeling and plaque disruption. Because oxidized LDL (ox-LDL) is implicated in many proatherogenic events, we hypothesized that ox-LDL would regulate expression of MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) in monocyte-derived macrophages. MWRHOSA AND RESULTS: Mononuclear cells were isolated from normal human subjects with Ficoll-Paque density gradient centrifugation, and adherent cells were allowed to differentiate into macrophages during 7 days of culture in plastic dishes. On day 7, by use of serum-free medium, the macrophages were incubated with various concentrations of native LDL (n-LDL) and copper-oxidized LDL. Exposure to ox-LDL (10 to 50 microg/mL) increased MMP-9 mRNA expression as analyzed by Northern blot, protein expression as measured by ELISA and Western blot, and gelatinolytic activity as determined by zymography. The increase in MMP-9 expression was associated with increased nuclear binding of transcription factor NF-kappaB and AP-1 complex on electromobility shift assay. In contrast, ox-LDL (10 to 50 microg/mL) decreased TIMP-1 expression. Ox-LDL-induced increase in MMP-9 expression was abrogated by HDL (100 microg/mL). n-LDL had no significant effect on MMP-9 or TIMP-1 expression. CONCLUSIONS These data demonstrate that unlike n-LDL, ox-LDL upregulates MMP-9 expression while reducing TIMP-1 expression in monocyte-derived macrophages. Furthermore, HDL abrogates ox-LDL-induced MMP-9 expression. Thus, ox-LDL may contribute to macrophage-mediated matrix breakdown in the atherosclerotic plaques, thereby predisposing them to plaque disruption and/or vascular remodeling.

Effects of Recombinant Apolipoprotein A-IMilano on Aortic Atherosclerosis in Apolipoprotein E–Deficient Mice

BACKGROUND We previously reported marked inhibitory effects of recombinant apolipoprotein (apo) A-I(Milano)/phospholipid complex (A-I[Milano]/PC) on neointimal lesions in balloon-injured iliofemoral arteries of hypercholesterolemic rabbits. In this study, we tested the hypothesis that apo A-I(Milano)/PC would inhibit aortic atherosclerosis in apo E-deficient mice. METHODS AND RESULTS Thirty-five apo E-deficient mice fed a high-cholesterol diet were included in the study. Control mice were killed at 20 (n=8) or 25 (n=7) weeks. Treated mice received 18 injections of either 40 mg/kg apo A-I(Milano)/PC (n=15) or PC only (n=5) intravenously every other day from 20 weeks until death at 25 weeks. Aortic atherosclerosis was identified with Sudan IV staining. Lipid and macrophage contents of the aortic sinus plaques were measured after oil-red O and Mac-1 antibody staining, respectively, and quantified with computed morphometry. In control mice, from 20 to 25 weeks, aortic atherosclerosis increased by 59% (11 +/- 1% versus 17 +/- 5% of the aortic surface, P=.002), and lipid content increased by 45% (22 +/- 8% versus 32 +/- 6% of plaque area, P=.02) without a significant change in macrophage content (10.8 +/- 2% versus 13.2 +/- 6%). Compared with 20-week-old untreated control mice, PC only-treated mice at 25 weeks demonstrated a 32% increase in aortic atherosclerosis (11 +/- 1% versus 15 +/- 4%, P=.01) and an increase in lipid content (22 +/- 8% versus 47 +/- 3%, P<.0001) without a change in macrophage content (10.8 +/- 2% versus 11 +/- 2%). In comparison with 20-week-old untreated control mice, 25-week-old apo A-I(Milano)/PC-treated mice demonstrated no increase in aortic atherosclerosis (11 +/- 1% versus 10 +/- 4%, P=NS), a 40% reduction in lipid content (22 +/- 8% versus 13 +/- 8%, P=.01), and a 46% reduction in macrophage content (10.8 +/- 2% versus 5.8 +/- 2.9%; P=.03). Serum cholesterol levels were markedly elevated in all groups and did not change significantly with apo A-I(Milano)/PC or PC only. In vitro, apo A-I(Milano)/PC stimulated cholesterol efflux from cholesterol-loaded FU5AH hepatoma cell lines in a dose-dependent manner, whereas PC only or PC-free apo A-I(Miano) had no effect. CONCLUSIONS Recombinant A-I(Milano)/PC prevented progression of aortic atherosclerosis and reduced lipid and macrophage content of plaques in apo E-deficient mice despite severe hypercholesterolemia. Thus, A-I(Milano)/PC may have a role in inhibiting progression and promoting stabilization of atherosclerosis.