Dallit Mannheim

Enhanced Expression of Lp-PLA2 and Lysophosphatidylcholine in Symptomatic Carotid Atherosclerotic Plaques

Background and Purpose— Circulating lipoprotein-associated phospholipase A2 (Lp-PLA2) has emerged as a novel biomarker for cardiovascular diseases. However, the correlation between the plaque expression of Lp-PLA2 and plaque oxidative stress, inflammation, and stability as well as the clinical presentation remains poorly defined, especially for cerebrovascular disease. Therefore, this study was performed to test the hypothesis that Lp-PLA2 expression is higher in symptomatic than in asymptomatic carotid plaques of patients undergoing carotid endarterectomy. Methods— The expression of Lp-PLA2 in 167 carotid artery plaques was determined by immunoblotting and immunostaining. Plaque oxidative stress, inflammation, and stability were quantified by NAD(P)H oxidase p67phox and MMP-2 immunoblotting, oxidized LDL (oxLDL) immunoreactivity, macrophage and Sirius red collagen staining. Lysophosphatidylcholine 16:0 (lysoPC) concentration was measured in 55 plaques using liquid chromatography tandem mass spectrometry. Results— Lp-PLA2 expression was significantly higher in plaques of symptomatic patients than asymptomatic patients (1.66±0.19 versus 1.14±0.10, P<0.05) and localized mainly to shoulder and necrotic lipid core areas in colocalization with oxLDL and macrophage content. Similarly, Lp-PLA2 expression was related to collagen content, which was lower in plaques from symptomatic patients than in plaques from asymptomatic patients (9.1±2.2 versus 18.5±1.7% of staining/field, P<0.001). LysoPC plaque concentration was significantly higher in plaques of symptomatic than asymptomatic patients (437.0±57.91 versus 228.84±37.00 mmol/L, P<0.05). Conclusions— Symptomatic carotid artery plaques are characterized by increased levels of Lp-PLA2 and its product lysoPC in correlation with markers of tissue oxidative stress, inflammation, and instability. These findings strongly support a role for Lp-PLA2 in the pathophysiology and clinical presentation of cerebrovascular disease.

Dysregulation of the Ubiquitin-Proteasome System in Human Carotid Atherosclerosis

Objective—The ubiquitin-proteasome system is the principal degradation route of intracellular and oxidized proteins, thus regulating many cellular processes conceivably important for atherosclerosis. The aim of this study was to evaluate the activity of ubiquitin-proteasome system in human carotid artery plaques in relation to oxidative stress and clinical manifestation. Methods and Results—In carotid endarterectomy specimens from 83 asymptomatic and 94 symptomatic patients, content of ubiquitin, ubiquitin conjugates, matrix metalloproteases, and NADPH-oxidase-p67 was evaluated by immunoblotting; proteolytic proteasome activity by fluorometric assay; single and double immunostaining for ubiquitin conjugates, 3-nitrotyrosine, apoptosis, smooth muscle &agr;-actin, and macrophage CD-68, as well as Sirius Red staining for collagen were performed. Compared with asymptomatic patients, symptomatic patients showed a more unstable plaque phenotype, an increased degree of apoptosis, a significantly higher ubiquitin conjugates content (17.72±1.36 versus 10.99±1.04; P<0.001), and lower proteasome activity (5.01±0.70 versus 9.41±1.19 nmol AMC/mg protein/min; P<0.01). Ubiquitin conjugates content was directly correlated to NADPH-p67 and degree of apoptosis. Immunostaining revealed colocalization of ubiquitin conjugates and 3-nitrotyrosine, and accumulation of ubiquitin conjugates in smooth muscle cells and macrophages. Conclusions—In human carotid plaques increased oxidative stress is associated with inhibition of the proteasome activity and accumulation of ubiquitin conjugates, particularly in symptomatic patients. These results suggest a possible role of the ubiquitin-proteasome system in influencing plaque stability.

Expression of lipoprotein-associated phospholipase A2 in carotid artery plaques predicts long-term cardiac outcome

AIMS The aim was to test the hypothesis that carotid artery plaque expression of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) predicts cardiac events. METHODS AND RESULTS Prospective cohort study of 162 consecutive patients undergoing elective carotid endarterectomy. Lipoprotein-associated phospholipase A(2) content was quantified by immunoblotting and lysophosphatidylcholine (lysoPC) by liquid chromatography tandem mass spectrometry. Additional biomolecular profiling by immunoblotting included C-reactive protein, p67phox, and matrix metalloproteinase-2 and -9. Macrophage plaque content was determined by quantitative immunostaining, plaque collagen content by quantitative Sirius red staining. Follow-up for cardiac death and non-fatal acute myocardial infarction was accomplished over a period of 48 +/- 14 months. Expression of Lp-PLA(2) and lysoPC was higher in carotid plaques of patients with than without cardiac events [median 1.6 (25th, 75th percentile 0.9, 2.5) vs. 0.8 (0.5, 2.0), P = 0.01 and 413 (281, 443) vs. 226 (96, 351) mmol/L, P = 0.03]. Smoking and point increase in carotid Lp-PLA(2) expression but no other traditional cardiovascular risk factor, histological or molecular marker remained predictive of cardiac events in the multivariate Cox proportional hazard analyses [HR 3.65 (1.36-9.83), P = 0.01 and HR 1.34 (1.01-1.77), P = 0.039]. Carotid plaque Lp-PLA(2) expression above the median constituted a more than three times higher risk for cardiac events [HR 3.39 (1.13-10.17), P = 0.03]. CONCLUSION Lipoprotein-associated phospholipase A(2) expression in carotid artery plaques is a predictor of long-term cardiac outcome. The current study supports the concept of atherosclerosis as a systemic disease with multi-focal complications and personalized medicine.

Simvastatin preserves diastolic function in experimental hypercholesterolemia independently of its lipid lowering effect

OBJECTIVE Isolated diastolic dysfunction is present in 40% of heart failure patients. It has been attributed to myocardial fibrosis and related to cardiovascular risk factor exposure. We hypothesized that simvastatin will improve these dynamics in experimental hypercholesterolemia (HC). METHODS Three groups of pigs were studied after 12 weeks of normal (N) diet, HC diet, or HC diet with simvastatin (80 mg/day) treatment. Cardiac function was assessed by electron beam computed tomography (EBCT) and percentage of myocardium occupied by microvessels (myocardial vascular fraction) was calculated by micro-CT. Collagen content was determined by Sirius red staining and confirmed by a quantitative, hydroxyoproline-based assay. RESULTS Compared with N, LDL serum concentration was higher in HC and HC+simvastatin (1.0±0.1 vs. 7.9±1.7 and 9.6±1.2 mmol/L, p<0.05 for both). Cardiac early diastolic filling was reduced in HC compared with N (102.4±11.3 vs. 151.1±12.1 mL/s; p<0.05) but restored in HC+simvastatin (176.8±21.3 mL/s, p<0.05 vs. HC). Compared with N, myocardial vascular fraction was higher in HC but not in HC+simvastatin (1.98±0.84 vs. 4.48±0.31 and 2.95±0.95%; p<0.05 for HC vs. N). Myocardial collagen content was higher in HC than in HC+simvastatin and N (4.72±1.03 vs. 1.62±0.12 and 1.21±0.24% area staining; p<0.05 for HC vs. N), which was attributable mainly to an increase in collagen III (2.90±0.48 vs. 1.62±0.12 and 1.21±0.24% area staining; p<0.05 for HC vs. N). CONCLUSIONS Simvastatin is able to prevent diastolic dysfunction in experimental HC independent of its lipid lowering effect. This beneficial effect is, at least partially, due to a decrease in myocardial fibrosis and angiogenesis.

Hypertension and Hypercholesterolemia Differentially Affect the Function and Structure of Pig Carotid Artery

The purpose of this work was to compare the effects of hypertension and hypercholesterolemia on carotid endothelial function, structure, and vasa vasorum density. Seventeen pigs were randomized to a 12-week normal diet without (n=5), or with renovascular hypertension (HT; n=6), or to a high cholesterol diet (HC; n=6). Carotid arteries were studied by organ chambers (endothelial function) and microcomputed tomography (vasa vasorum), and tissue was processed for Sirius red staining and immunoblotting (vascular endothelium growth factor, endostatin, matrix metalloproteinase-9, and matrix metalloproteinase-2). HC and HT showed reduced vasodilation to acetylcholine as compared with controls, but HT also had a lower response to sodium nitroprusside. In addition, HT showed a higher content of organized collagen fibers and increased intima-media thickness. Vasa vasorum density was increased in HC but not in HT. Both HT and HC showed a proangiogenetic biochemical milieu (higher vascular endothelium growth factor, matrix metalloproteinases, and lower endostatin), but this was more pronounced in HC. Both hypertension and hypercholesterolemia induce endothelial dysfunction in the carotid artery. However, hypertension is also associated with greater fibrosis and vascular wall thickening, which might impair endothelium-independent vasorelaxation and vasa vasorum growth. Hypercholesterolemia is, in turn, associated with vasa vasorum neovascularization. These data suggest that carotid atherosclerosis can evolve through different mechanisms in relation to different risk factors.