Daniele Versari

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.

Chronic Proteasome Inhibition Contributes to Coronary Atherosclerosis

The proteasome is responsible for the degradation of oxidized proteins, and proteasome inhibition has been shown to generate oxidative stress in vitro. Atherosclerosis is thought to be initiated as a consequence of increased endogenous oxidative stress. The current study was designed to assess whether chronic proteasome inhibition is associated with early coronary atherosclerosis. Female pigs, 3 months of age, were randomized to a normal (N) or high-cholesterol (HC) diet (2% cholesterol, 15% lard) without or with twice weekly subcutaneous injections of the proteasome inhibitor (PSI) MLN-273 (0.08 mg/kg, N+PSI and HC+PSI) for a period of 12 weeks (n=5 per group). Coronary vasorelaxation to bradykinin (10−10.5 to 10−6.5 mol/L) and sodium nitroprusside (10−9 to 10−5 mol/L) was assessed by in vitro organ chamber experiments, intima–media ratio by morphometric analysis of Elastica–van Gieson–stained slides, and intima superoxide production by dihydroethidium fluorescence. Vasorelaxation to 10−6.5 mol/L bradykinin was reduced in HC compared with N (69±7 versus 90±2%, P<0.05) and further reduced in N+PSI and HC+PSI (57±6 and 48±13%, P<0.05 versus N and HC for each). Compared with N (0.03±0.01), intima–media ratio was higher in N+PSI (0.09±0.04, P<0.01) and HC+PSI (0.15±0.06, P<0.05). Compared with N (0.6±0.9% of intima area), dihydroethidium fluorescence was higher in HC, N+PSI, and HC+PSI (8.9±1.6, 6.0±3.5, and 7.2±3.9% of intima area, P<0.05 for all). Thus, chronic proteasome inhibition is associated with increased coronary artery oxidative stress and early atherosclerosis. These findings support the significance of the proteasome and related protein quality control for vascular biology and pathology.

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.

Segmental Heterogeneity of Vasa Vasorum Neovascularization in Human Coronary Atherosclerosis

OBJECTIVES Our aim was to investigate the role of coronary vasa vasorum (VV) neovascularization in the progression and complications of human coronary atherosclerotic plaques. BACKGROUND Accumulating evidence supports an important role of VV neovascularization in atherogenesis and lesion location determination in coronary artery disease. VV neovascularization can lead to intraplaque hemorrhage, which has been identified as a promoter of plaque progression and complications like plaque rupture. We hypothesized that distinctive patterns of VV neovascularization and associated plaque complications can be found in different stages of human coronary atherosclerosis. METHODS Hearts from 15 patients (age 52+/-5 years, mean+/-SEM) were obtained at autopsy, perfused with Microfil (Flow Tech, Inc., Carver, Massachusetts), and subsequently scanned with micro-computed tomography (CT). The 2-cm segments (n=50) were histologically classified as either normal (n=12), nonstenotic plaque (<50% stenosis, n=18), calcified (n=10) or noncalcified (n=10) stenotic plaque. Micro-CT images were analyzed for VV density (number/mm2), VV vascular area fraction (mm2/mm2), and VV endothelial surface fraction (mm2/mm3). Histological sections were stained for Mallorys (iron), von Kossa (calcium), and glycophorin-A (erythrocyte fragments) as well as endothelial nitric oxide synthase, vascular endothelial growth factor, and tumor necrosis factor-alpha. RESULTS VV density was higher in segments with nonstenotic and noncalcified stenotic plaques as compared with normal segments (3.36+/-0.45, 3.72+/-1.03 vs. 1.16+/-0.21, p<0.01). In calcified stenotic plaques, VV spatial density was lowest (0.95+/-0.21, p<0.05 vs. nonstenotic and noncalcified stenotic plaque). The amount of iron and glycophorin A was significantly higher in nonstenotic and stenotic plaques as compared with normal segments, and correlated with VV density (Kendall-Tau correlation coefficient 0.65 and 0.58, respectively, p<0.01). Moreover, relatively high amounts of iron and glycophorin A were found in calcified plaques. Further immunohistochemical characterization of VV revealed positive staining for endothelial nitric oxide synthase and tumor necrosis factor-alpha but not vascular endothelial growth factor. CONCLUSIONS Our results support a possible role of VV neovascularization, VV rupture, and intraplaque hemorrhage in the progression and complications of human coronary atherosclerosis.

The Importance of Reendothelialization After Arterial Injury

Atherosclerosis is still the principal cause of morbidity and mortality in Western countries and although a significant progress has been made in the understanding of its pathophysiology, the determinants of atherosclerotic plaque instability are still poorly understood. The endothelium plays a pivotal role for the development, progression, and complication of atherosclerosis. Endothelial dysfunction is widely recognized as one of the early alteration in the vessel wall preceding the development of the plaque. However, considering the plethora of vascular functions which are regulated by endothelium, it plays a pivotal role throughout the atherosclerotic process and indeed the loss of endothelial cells, leading to plaque denudation, is one of the main causes of plaque complication. It is therefore conceivable that the maintenance of the endothelial layer physical continuity and function is crucial for the prevention of atherosclerosis. In the presence of cardiovascular risk factors, endothelial cells are continuously injured and repaired by the proliferation of resident cells and circulating endothelial progenitor cells. Indeed the number of circulating endothelial progenitor cells has been identified as an predictor of cardiovascular events. The increase in bone marrow release of endogenous progenitor cells or the enhancement of their homing in arterial denuded sites or in intravascular stent surface, are currently pursued to reduce atherosclerosis development/complication and intrastent restenosis, respectively. However, some challenges may arise from procedures enhancing endothelialization, including unwanted angiogenesis which may favor neoplasia progression and paradoxically atherosclerotic plaque expansion and complication.

Chronic Antioxidant Supplementation Impairs Coronary Endothelial Function and Myocardial Perfusion in Normal Pigs

Experimental studies have shown the beneficial effects of antioxidant supplementation on endothelial function in the presence of increased endogenous oxidative stress, whereas limited data are available under normal conditions. The present study tested the hypothesis that in normal pigs long-term antioxidants would have deleterious effects on the cardiovascular system. Normal domestic pigs (V, n=6) were studied 12 weeks after dietary supplementation with vitamin E (100 IU/kg per day) and vitamin C (1 g/day) and compared with normal controls (C, n=7). Myocardial perfusion and permeability index were evaluated by electron beam computed tomography after intravenous adenosine and dobutamine. Coronary endothelial function was evaluated in vitro by organ chamber and coronary tissue studied by immunoblotting and staining. Myocardial perfusion response was lower in V than in C after adenosine (10.1±4.5 versus 53.4±5.2%; P<0.01) and dobutamine (V, 78.4±8.1; C, 193.0±39.0%; P<0.05). The permeability index increased in V after adenosine (48.8±5.1%) and dobutamine (59.9±13.6%) and did not change in C. Coronary vasodilation to bradykinin and substance P was lower in V than in C. Moreover, in V, coronary nitrotyrosine and superoxide content was significantly higher than in C. The groups had similar total monomer expression of endothelial nitric oxide synthase, whereas the dimerized form, reflecting coupled enzyme, was lower in V. These findings suggest that long-term experimental antioxidant vitamin supplementation in normal pigs impairs myocardial perfusion and coronary endothelial function via an increased level of oxidative stress in the arterial wall, which may be partly related to the uncoupling of endothelial nitric oxide synthase and/or the direct prooxidant effect of vitamin radicals.

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.

Low vasa vasorum densities correlate with inflammation and subintimal thickening: Potential role in location-Determination of atherogenesis

OBJECTIVES To assess the role of coronary vasa vasorum (VV) spatial distribution in determining the location of early atherosclerotic lesion development. METHODS AND RESULTS Six, 3-month-old, female, crossbred swine were fed 2% high-cholesterol (HC) diet for 3 months prior to euthanasia. Six other pigs were fed normal diet (N) for the entire 6 months. Right coronary arteries were harvested and scanned intact with micro-CT (20mum cubic-voxel-size). After scanning, randomly selected cross-sectional histological sections were stained for nuclear-factor kappaB (NF-kappaB), hypoxia-inducible factor-1alpha (HIF-1alpha), macrophages, von-Willebrand-factor, dihydroethidium (DHE), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). The number of positive stained cells, as well as intima-to-media ratio, were compared with VV density (#/mm(2)) obtained from micro-CT images (which closely matched the location of the histological sections) in each of four equal quadrants of the coronary vessel wall. In normal, as well as HC pigs, the number of NF-kappaB (r=0.73 and 0.70), HIF-1alpha (r=0.74 and 0.77), TNF-alpha (r=0.58 and 0.72) and IL-6 (r=0.70 and 0.72) positive cells as well as the expression of DHE (Kendall tau coefficient -0.64 and -0.63) inversely correlated with VV density. In HC the VV density also inversely correlated with intima/media ratios (r=0.65). CONCLUSIONS Our data suggest that low VV density territories within the coronary vessel wall are susceptible to hypoxia, oxidative stress and microinflammation and may therefore be starting points of early atherogenesis.

Myocardial microvascular function during acute coronary artery stenosis: effect of hypertension and hypercholesterolaemia

AIMS Coronary collateral arteries (CCA) reduce cardiovascular events. We tested the hypothesis that new microvessels that proliferate in early atherosclerosis may be associated with myocardial protection during acute subtotal coronary artery obstruction (CAO). METHODS AND RESULTS Acute left anterior descending CAO was induced by a balloon catheter in pigs after 12 weeks of high-cholesterol (HC) diet, renovascular hypertension (HTN), or normal control. Cardiac structure, myocardial perfusion, and functional response to iv adenosine and CAO were studied in vivo using electron beam computed tomography (CT). The intra-myocardial microvessels were subsequently evaluated ex vivo using micro-CT, and myocardial expression of growth factors using immunoblotting. Basal myocardial perfusion and microvascular permeability were similar among the groups, whereas their responses to adenosine were attenuated in HC and HTN. A significant decline in myocardial perfusion in normal pigs during acute CAO was attenuated in HC and abolished in HTN. CAO also elicited an increase in normal anterior wall microvascular permeability (+202 +/- 59%, P < 0.05), which was attenuated in HC and HTN (+55 +/- 9 and +31 +/- 8%, respectively, P < 0.05 vs. normal). Microvascular (<200 microm) spatial density was significantly elevated in HC and HTN, accompanied by increased myocardial growth factor expression. CONCLUSION This study demonstrates that early exposure to the cardiovascular risk factors HC and HTN protects the heart from decreases in myocardial perfusion during acute subtotal CAO. This protective effect is associated with and potentially mediated by pre-emptive development of intra-myocardial microvessels that might serve as recruitable CCA.

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.