Lerman, Amir

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.

Objective— We tested the hypothesis that in early hypertension (HT), increased oxidative stress leads to myocardial microvascular remodeling. Methods and Results— Pigs were studied after a 12-week observation: normal (n=8), untreated renovascular HT (n=8), or HT+chronic antioxidant supplementation (HT+A, n=6). Left ventricular muscle mass (LVMM) and myocardial blood flow (MBF) reserve were determined using electron beam computer tomography (CT), and the spatial density and tortuousity of myocardial microvessels (<500 μm) was then measured in myocardial samples with micro-CT. Myocardial microvascular morphology, oxidative stress, inflammation, and growth factor expression were determined in vitro. HT and HT+A had similarly increased arterial pressure and LVMM, but only HT showed impaired MBF response to adenosine. Compared with normal, HT had increased spatial density of myocardial microvessels, which was preserved in HT+A (111.8±7.8, 166.3±15.7, and 106.4±6.1 vessels per cm 2 , respectively). HT also showed microvascular wall thickening, increased systemic and tissue oxidative stress, inflammation, and expression of vascular endothelial growth factor and its receptor Flk-1, most of which were attenuated by antioxidants. Conclusions— Myocardial microvascular remodeling in early HT is accompanied by tissue oxidative stress, inflammation, and altered growth factor expression, and attenuated by antioxidant intervention. This study underscores a role of increased oxidative stress in modulating myocardial microvascular architecture in early HT.

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.

Dyslipidemia often accompanies and accelerates renal disease, partly by promoting fibrosis. However, the mechanisms mediating this effect are unclear. We hypothesized that hypercholesterolemia modulates several interlinked pathways that promote deposition and blunt degradation of extracellular matrix, and that these could be manipulated by reversal of hypercholesterolemia. Fourteen pigs were fed a 16-week 2% high-cholesterol diet (HC-HC; n=7) or normal diet (n=7), whereas in 7 others, a 10-week HC was followed by a 6-week normal diet (HC-N). Renal endothelial function was assessed in vivo with electron-beam computed tomography, and renal tissue was then studied ex vivo using Western blot, real-time quantitative polymerase chain reaction, gelatin zymography, and immunostaining. HC-HC kidneys showed endothelial dysfunction, accompanied by increased intrarenal oxidative stress, inflammation, activation of the endothelin and transforming-growth factor-β systems, and decreased matrix metalloproteinase expression and activity. Accordingly, HC-HC kidneys showed increased collagen IV expression and fibrosis. A lipid-lowering dietary intervention reversed most of these changes. In conclusion, this study indicates that renal fibrosis in early atherosclerosis is a result of a simultaneous increase in extracellular matrix deposition and blunted matrix metalloproteinase–mediated degradation, overall promoting perivascular and tubulointerstitial fibrosis. Notably, many of these pathways may be reversible in hypercholesterolemia, and crucial targets could potentially be identified for early interventions to preserve the kidney.

Atherosclerosis represents one of the major causes of premature death in the United States today, and it is frequently associated with, exacerbates, and is aggravated by chronic kidney disease (CKD). Atherosclerosis integrates the response to a number of insults, and consequently, the accelerated atherosclerosis found in CKD patients is associated with activation of a variety of humoral and tissue mechanisms. Hypertension, diabetes, dyslipidemia, obesity, metabolic syndrome, and additional nontraditional risk factors can damage the kidney directly and by promoting intrarenal atherogenesis, even in the absence of obstructive lesions in the renal artery. Evidence indicates that increased oxidative stress and inflammation may mediate a large part of the effects of risk factors on the kidney. In turn, progressive deterioration of renal function in CKD may lead to dyslipidemia or accumulation of uremic toxins, which can induce production of free radicals and activate proinflammatory and fibrogenic factors, leading to vascular endothelial cell dysfunction and injury, and favoring development of atherosclerosis. Therefore, the kidney can be a villain or a victim during atherogenesis. The purpose of this review is to provide new insights into the mechanisms by which atherogenic factors may instigate early renal injury.

Objective— Intima-media remodeling, as frequently assessed by changes in the external elastic lamina-to-lumen area (EELLA), is well-described in coronary artery disease in contrast to adventitial remodeling, especially in the early disease stage. Method and Results— Female domestic pigs were randomized to one of the following 12-week treatment groups: normal diet (N; n=6), high-cholesterol diet (HC; n=6), or renovascular hypertension (HT; n=4). Low-density lipoprotein (LDL) cholesterol serum concentration was higher in HC than in N and HT (395.5±106 versus 38.6±14 and 37.2±6.8 mg/dL; P <0.05 for both). Mean arterial pressure was higher in HT than in N and HC (141.3±21 versus 107.4±8.9 and 109.4±7.8 mm Hg; P <0.05 for both). EELLA ratio, as assessed by morphometry, was similar in N, HC, and HTN (1.03±0.32 versus 0.95±0.29 and 1.01±0.09; P <0.05 for both). Coronary vasa vasorum density, as assessed by 3-dimensional micro-computed tomography, was higher in HC than in N and HT (3.4±1.0 versus 1.9±0.3 and 2.0±1.2; P <0.05 for both). In contrast, immunostaining showed a higher collagen III content and the presence of adventitial myofibroblasts in HT compared with N and HC. Conclusions— The current study suggests that adventitial remodeling precedes intima and media remodeling of coronary arteries early after exposure to hypercholesterolemia and hypertension, with distinct qualitative differences between them.

Background and Purpose— The local renin-angiotensin system (RAS) and cyclooxygenase-2 contribute to the activation of nuclear factor κB (NFκB) and C-reactive protein (CRP). We hypothesized that the combination of RAS blockers (RASb) and ASA reduces NFκB and CRP within atherosclerotic plaques. Methods— Patients undergoing carotid endarterectomy were divided into groups according to treatment (RASb–acetylsalicylic acid [ASA], ASA, RASb, and control). The expression of NFκB, CRP, and CD40L was analyzed through Western blots in the obtained plaques. Results— Plaques from patients treated with the combination of RASb and ASA showed lower expression of NFκB (25.4±9.8 densitometric units [DU]) than those of the control group (57.6±13.2 DU, P =0.03) as well as lower expression of CRP (20.9±9.6 DU) than those of the other treatment groups (ASA 86.1±13 DU, RASb 88.4±31 DU, controls 67.8±18.6, P =0.004). A negative expression of NFκB was associated with a reduced incidence of symptoms compared with a positive expression (5/33 [15.1%] versus 14/35 [40%], P =0.031). Conclusions— The combined treatment with RASb and ASA decreases the expression of inflammatory markers in atherosclerosis in humans. This study supports the role of the local RAS and cyclooxygenase-2 in the progression of atherosclerosis.