Kevin D. O’Brien

Neovascular Expression of E-Selectin, Intercellular Adhesion Molecule-1, and Vascular Cell Adhesion Molecule-1 in Human Atherosclerosis and Their Relation to Intimal Leukocyte Content

BACKGROUND Leukocyte recruitment is an early event in atherogenesis, and the leukocyte adhesion molecules E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) recently have been detected in human atherosclerosis. However, no previous study has evaluated either the distribution of these three molecules at different sites within the arterial intima or their relation to plaque leukocyte content. METHODS AND RESULTS Immunohistochemistry was performed on 99 coronary artery segments (34 controls and 65 with atherosclerotic plaque) to identify E-selectin, ICAM-1, VCAM-1, macrophages, smooth muscle cells, and T lymphocytes. For each segment, the presence or absence of adhesion molecule was determined at the arterial lumen, on intimal neovasculature, and on intimal nonendothelial cells. Each segment was scored for intimal macrophage and T-lymphocyte densities on a semiquantitative scale of 0 to 3. In atherosclerotic plaques, the prevalences of E-selectin, ICAM-1, and VCAM-1 on plaque neovasculature were twofold higher than their prevalences on arterial luminal endothelium. E-selectin was the only adhesion molecule for which expression on arterial luminal endothelial cells was more prevalent in plaques than in control segments. Increased plaque intimal macrophage density was associated with expression of VCAM-1 on neovasculature (P < .01) and on nonendothelial cells (P < .01). Increased plaque intimal T-lymphocyte density was associated with the presence of both ICAM-1 and VCAM-1 on neovasculature (both P < .01) and on nonendothelial cells (both P < .01). CONCLUSIONS In atherosclerotic plaques, the expression of all three leukocyte adhesion molecules was more prevalent on intimal neovasculature than on arterial luminal endothelium. Further, the presence on neovasculature and nonendothelial cells of VCAM-1 and ICAM-1 was strongly associated with increased intimal leukocyte accumulation. These findings suggest that leukocyte recruitment through and/or activation of intimal neovasculature may play important roles in the pathogenesis of human atherosclerosis.

Hemorrhage in the Atherosclerotic Carotid Plaque: A High-Resolution MRI Study

Background and Purpose— High-resolution, multicontrast magnetic resonance imaging (MRI) has developed into an effective tool for the identification of carotid atherosclerotic plaque components, such as necrotic core, fibrous matrix, and hemorrhage/thrombus. Factors that may lead to plaque instability are lipid content, thin fibrous cap, and intraplaque hemorrhage. Determining the age of intraplaque hemorrhage can give insight to the history and current condition of the biologically active plaque. The aim of this study was to develop criteria for the identification of the stages of intraplaque hemorrhage using high-resolution MRI. Methods— Twenty-seven patients, scheduled for carotid endarterectomy (CEA), were imaged on a 1.5-T GE SIGNA scanner (sequences: 3-dimensional time of flight, double-inversion recovery, T1-weighted (T1W), PDW and T2W). Two readers, blinded to histology, reviewed MR images and grouped hemorrhage into fresh, recent, and old categories using a modified cerebral hemorrhage criteria. The CEA specimens were serially sectioned and graded as to presence and stage of hemorrhage. Results— Hemorrhage was histologically identified and staged in 145/189 (77%) of carotid artery plaque locations. MRI detected intraplaque hemorrhage with high sensitivity (90%) but moderate specificity (74%). Moderate agreement in classifying stages occurred between MRI and histology (Cohen κ = 0.7, 95% CI: 0.5 to 0.8 for reviewer 1 and 0.4, 95% CI: 0.2 to 0.6 for reviewer 2), with moderate agreement between the 2 MRI readers (κ = 0.4, 95% CI: 0.3 to 0.6). Conclusion— Multicontrast MRI can detect and classify carotid intraplaque hemorrhage with high sensitivity and moderate specificity.

Features of the Metabolic Syndrome and Diabetes Mellitus as Predictors of Aortic Valve Calcification in the Multi-Ethnic Study of Atherosclerosis

Background— Calcific aortic valve disease is common in the elderly, is correlated with common cardiovascular risk factors, and is associated with increased cardiovascular event risk; however, whether metabolic syndrome is associated with an increased prevalence of aortic valve calcium (AVC) is not known. Methods and Results— The prevalence of AVC, as assessed by computed tomography, was compared in 6780 Multi-Ethnic Study of Atherosclerosis (MESA) participants with metabolic syndrome (n=1550; National Cholesterol Education Program’s Adult Treatment Panel III [ATP III] criteria), diabetes mellitus (n=1016), or neither condition (n=4024). The prevalence of AVC for those with neither condition, metabolic syndrome, or diabetes mellitus was, respectively, 8%, 12%, and 17% in women (P<0.001) and 14%, 22%, and 24% in men (P<0.001). Compared with those with neither condition, the adjusted relative risks for the presence of AVC were 1.45 (95% CI 1.11 to 1.90) for metabolic syndrome and 2.12 (95% CI 1.54 to 2.92) for diabetes mellitus in women and 1.70 (95% CI 1.32 to 2.19) for metabolic syndrome and 1.73 (95% CI 1.33 to 2.25) for diabetes mellitus in men. There was a graded, linear relationship between AVC prevalence and the number of metabolic syndrome components in both women and men (both P<0.001). Similar results were obtained when the International Diabetes Federation metabolic syndrome definition was used. Conclusions— In the MESA cohort, the metabolic syndrome and diabetes mellitus are associated with increased risk of AVC, and AVC prevalence is increased with increasing number of metabolic syndrome components.

Increase in serum amyloid a evoked by dietary cholesterol is associated with increased atherosclerosis in mice.

Background—Elevated serum amyloid A (SAA) levels are associated with increased cardiovascular risk. SAA levels can be increased by dietary fat and cholesterol. Moreover, SAA can cause lipoproteins to bind extracellular vascular proteoglycans, a process that is critical in atherogenesis. Therefore, we hypothesized that diet-induced increases in SAA would increase atherosclerosis independent of their effect on plasma cholesterol levels. Methods and Results—Female LDL-receptor–null (LDLR−/−) mice were fed high–saturated fat diets (21%, wt/wt), with or without added cholesterol (0.15%, wt/wt), for 10 weeks. Compared with chow-fed controls, the high-fat diets increased plasma SAA levels. Addition of cholesterol further increased SAA levels 2-fold (P<0.05) without further increasing plasma cholesterol levels. Addition of dietary cholesterol also increased atherosclerosis (P<0.05). Four lines of evidence suggest that SAA actually might cause atherosclerosis: (1) SAA levels when mice were euthanized correlated with the extent of atherosclerosis (r=0.49; P<0.02); (2) SAA levels after 5 weeks of diet correlated with the extent of atherosclerosis at 10 weeks (r=0.66; P<0.01); (3) binding of HDL from these animals to proteoglycans in vitro was related to the HDL-SAA content (r=0.65; P<0.01); and (4) immunoreactive SAA was present in lesion areas enriched with both proteoglycans and apolipoprotein A-I, the major HDL apolipoprotein. Conclusions—Addition of cholesterol to a high-fat diet increased plasma SAA levels and atherosclerosis independent of an adverse effect on plasma lipoproteins, consistent with the hypothesis that SAA may promote atherosclerosis directly by mediating retention of SAA-enriched HDL to vascular proteoglycans.

Dietary cholesterol worsens adipose tissue macrophage accumulation and atherosclerosis in obese LDL receptor-deficient mice.

Objective—Chronic systemic inflammation accompanies obesity and predicts development of cardiovascular disease. Dietary cholesterol has been shown to increase inflammation and atherosclerosis in LDL receptor–deficient (LDLR−/−) mice. This study was undertaken to determine whether dietary cholesterol and obesity have additive effects on inflammation and atherosclerosis. Methods and Results—LDLR−/− mice were fed chow, high-fat, high-carbohydrate (diabetogenic) diets without (DD) or with added cholesterol (DDC) for 24 weeks. Effects on adipose tissue, inflammatory markers, and atherosclerosis were studied. Despite similar weight gain between DD and DDC groups, addition of dietary cholesterol increased insulin resistance relative to DD. Adipocyte hypertrophy, macrophage accumulation, and local inflammation were observed in intraabdominal adipose tissue in DD and DDC, but were significantly higher in the DDC group. Circulating levels of the inflammatory protein serum amyloid A (SAA) were 4.4-fold higher in DD animals and 15-fold higher in DDC animals than controls, suggesting chronic systemic inflammation. Hepatic SAA mRNA levels were similarly elevated. Atherosclerosis was increased in the DD-fed animals and further increased in the DDC group. Conclusions—Obesity-induced macrophage accumulation in adipose tissue is exacerbated by dietary cholesterol. These local inflammatory changes in adipose tissue are associated with insulin resistance, systemic inflammation, and increased atherosclerosis in this mouse model.

Relationship of Apolipoproteins A-1 and B, and Lipoprotein (a) to Cardiovascular Outcomes in the AIM-HIGH Trial

OBJECTIVES This study sought to examine the relationship between baseline and on-study apolipoproteins (apo) A-1 and B and lipoprotein(a) [Lp(a)] levels and the development of subsequent cardiovascular (CV) events in the AIM-HIGH (Atherothrombosis Intervention in Metabolic Syndrome with Low HDL/High Triglyceride and Impact on Global Health Outcomes) trial. BACKGROUND Niacin has been reported to lower apoB and Lp(a) and to raise apoA-1. METHODS Individuals with CV disease and low baseline levels of high-density lipoprotein cholesterol were randomized to simvastatin plus placebo or simvastatin, plus extended-release niacin ([ERN], 1,500 to 2,000 mg/day), with ezetimibe added as needed, in both groups, to maintain an on-treatment low-density lipoprotein cholesterol in the range of 40 to 80 mg/dl. Hazard ratios (HRs) were used to evaluate the relationship between levels of apoA-1, apoB, and Lp(a), and CV events in each treatment group. RESULTS Baseline apoB and the apoB/apoA-I ratio were significantly predictive of CV events only for the placebo group (HR: 1.17 [p = 0.018] and HR: 1.19 [p = 0.016]). Baseline and on-study Lp(a) were predictive of CV events in both simvastatin plus placebo (baseline HR: 1.24 [p = 0.002] and on-study HR: 1.21 [p = 0.017]) and the simvastatin plus ERN group (baseline HR: 1.25 [p = 0.001] and on-study HR: 1.18 [p = 0.028]). The ERN modestly increased 1-year apoA-1 (7%), decreased apoB (13%), decreased the ApoB/ApoA-1 ratio (19%), and decreased Lp(a) 21%, but did not reduce CV events. CONCLUSIONS Lp(a) was associated with increased CV risk in both treatment groups indicating that it contributes to residual CV risk. However, there was no evidence that ERN reduced CV risk, despite favorable lipoprotein changes.

Serum Amyloid A and Lipoprotein Retention in Murine Models of Atherosclerosis

Objective—Elevated serum amyloid A (SAA) levels are associated with increased cardiovascular risk in humans. Because SAA associates primarily with lipoproteins in plasma and has proteoglycan binding domains, we postulated that SAA might mediate lipoprotein retention on atherosclerotic extracellular matrix. Methods and Results—Immunohistochemistry was performed for SAA, apolipoprotein A-I (apoA-I), apolipoprotein B (apoB), and perlecan on proximal aortic lesions from chow-fed low-density lipoprotein receptor (LDLR)−/− and apoE−/− mice euthanized at 10, 50, and 70 weeks. SAA was detected on atherosclerotic lesion extracellular matrix at all time points in both strains. SAA area correlated highly with lesion areas (apoE−/−, r=0.76; LDLR−/−, r=0.86), apoA-I areas (apoE−/−, r=0.88; LDLR−/−, r=0.80), apoB areas (apoE−/−, r=0.74; LDLR−/−, r=0.89), and perlecan areas (apoE−/−, r=0.83; LDLR−/−, r=0.79) (all P<0.0001). In vitro, SAA enrichment increased high-density lipoprotein (HDL) binding to heparan sulfate proteoglycans, and immunoprecipitation experiments using plasma from apoE−/− and LDLR−/− mice demonstrated that SAA was present on both apoA-I–containing and apoB-containing lipoproteins. Conclusions—In chow-fed apoE−/− and LDLR−/− mice, SAA is deposited in murine atherosclerosis at all stages of lesion development, and SAA immunoreactive area correlates highly with lesion area, apoA-I area, apoB area, and perlecan area. These findings are consistent with a possible role for SAA-mediated lipoprotein retention in atherosclerosis.

Relationship of Lipoproteins to Cardiovascular Events in the Atherothrombosis Intervention in Metabolic Syndrome with Low HDL/High Triglycerides and Impact on Global Health Outcomes (AIM-HIGH) Trial

OBJECTIVES This study sought to examine the relationship between niacin treatment, lipoproteins, and cardiovascular (CV) outcomes in this secondary analysis of the AIM-HIGH (Atherothrombosis Intervention in Metabolic Syndrome With Low HDL/High Triglycerides and Impact on Global Health Outcomes) trial. BACKGROUND During a 3-year follow-up in 3,414 patients with established CV disease and low high-density lipoprotein cholesterol (HDL-C) levels, combined niacin + low-density lipoprotein cholesterol (LDL-C)-lowering therapy did not reduce CV events compared with LDL-C-lowering therapy alone. METHODS Subjects taking simvastatin and/or ezetimibe were randomized to receive extended-release (ER) niacin 1,500 to 2,000 mg or minimal immediate-release niacin (≤ 150 mg) as placebo at bedtime. LDL-C levels in both groups were maintained from 40 to 80 mg/dl. Hazard ratios were estimated by using Cox proportional hazards models for relationships between lipoproteins and the composite endpoint of CV death, myocardial infarction, acute coronary syndrome, ischemic stroke, or symptom-driven revascularization. RESULTS CV outcomes were not associated with ER niacin in any baseline lipoprotein tertile. In a subset of patients in both the highest triglyceride (≥ 198 mg/dl) and lowest HDL-C (<33 mg/dl) tertiles, ER niacin showed a trend toward benefit (hazard ratio: 0.74, p = 0.073). In-trial LDL-C levels, non-HDL-C levels, and the total cholesterol/HDL-C ratio were positively associated with CV events in the control group, but these relationships were absent in the ER niacin group. CONCLUSIONS Baseline lipoprotein tertiles did not predict differential benefit or harm with ER niacin added to LDL-C-lowering therapy, but a small dyslipidemic subgroup may benefit. ER niacin attenuated expected relationships of lipoprotein risk factors with CV events, raising the possibility that nonlipoprotein actions of niacin could affect risk. (Niacin Plus Statin to Prevent Vascular Events [AIM-HIGH]; NCT00120289).

Cell-Associated and Extracellular Phospholipid Transfer Protein in Human Coronary Atherosclerosis

Background Phospholipid transfer protein (PLTP) plays an important role in HDL particle metabolism and may modulate hepatic secretion of apolipoprotein B‐containing lipoproteins. However, whether PLTP might participate directly in human atherosclerotic lesion formation is unknown. Methods and Results The cellular and extracellular distributions of PLTP were determined in normal and atherosclerotic human coronary lesions with a monoclonal antibody to human PLTP. Cell types (smooth muscle cells [SMCs] or macrophages), apolipoproteins (apoA‐I, apoB, and apoE), and extracellular matrix proteoglycans (biglycan and versican) were identified on adjacent sections with monospecific antibodies. Minimal extracellular PLTP was detected in nonatherosclerotic coronary arteries, but extracellular and cellular PLTP immunostaining was widespread in atherosclerotic lesions. PLTP was detected in foam cell SMCs and in foam cell macrophages, which suggests that cellular cholesterol accumulation might increase PLTP expression in both cell types. This was confirmed by in vitro studies demonstrating that cholesterol loading of macrophages leads to 2‐ to 3‐fold increases in PLTP steady‐state mRNA levels, protein expression, and activity. PLTP also was detected in an extracellular distribution, colocalizing with apoA‐I, apoB, apoE, and the vascular proteoglycan biglycan. In gel mobility shift assays, both active and inactive recombinant PLTP markedly increased HDL binding to biglycan, which suggests that PLTP may mediate lipoprotein binding to proteoglycans independent of its phospholipid transfer activity. Conclusions PLTP is present in human atherosclerotic lesions, and its distribution suggests roles for PLTP in both cellular cholesterol metabolism and lipoprotein retention on extracellular matrix. (Circulation. 2003;108:270‐274.)

Cholesterol Feeding Increases C-Reactive Protein and Serum Amyloid A Levels in Lean Insulin-Sensitive Subjects

Background—Inflammatory markers associated with elevated cardiovascular risk are increased by cholesterol feeding in animal models. However, whether dietary cholesterol increases inflammatory marker levels in humans is not known. Methods and Results—C-reactive protein (CRP), serum amyloid A (SAA), and lipoprotein levels were compared in 201 healthy subjects on an American Heart Association–National Cholesterol Education Program step 1 diet at baseline and after addition of 4 eggs per day for 4 weeks. Subjects were classified a priori into 3 groups based on their body mass index (BMI) and insulin sensitivity index (SI): lean insulin sensitive (LIS), mean±SEM BMI, 23.2±0.3 kg/m2, and SI, 6.7±0.3×10−4min−1/(&mgr;U/mL), n=66; lean insulin resistant (LIR), BMI, 24.5±0.2 kg/m2 and SI, 2.9±0.1×10−4min−1/(&mgr;U/mL), n=76; or obese insulin resistant (OIR), BMI, 31.4±0.5 kg/m2 and SI, 2.1±0.1×10−4min−1/(&mgr;U/mL), n=59. Insulin resistance and obesity each were associated with increased baseline levels of both CRP (P for trend, <0.001) and SAA (P for trend=0.015). Egg feeding was associated with significant increases in both CRP and SAA in the LIS group (both P<0.01) but not in the LIR or OIR groups. Egg feeding also was associated with a significant increase in non-HDL cholesterol (P<0.001) in LIS subjects; however, there was no correlation between the change in non-HDL cholesterol or changes in either CRP or SAA in this group. Conclusions—A high-cholesterol diet leads to significant increases in both inflammatory markers and non-HDL cholesterol levels in insulin-sensitive individuals but not in lean or obese insulin-resistant subjects.