Versari, Daniele

Background and Purpose— Circulating lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) has emerged as a novel biomarker for cardiovascular diseases. However, the correlation between the plaque expression of Lp-PLA 2 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-PLA 2 expression is higher in symptomatic than in asymptomatic carotid plaques of patients undergoing carotid endarterectomy. Methods— The expression of Lp-PLA 2 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-PLA 2 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-PLA 2 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-PLA 2 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.

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.

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.

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 α-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.

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.