Lerman, Amir

Objective— Mechanisms of renal injury distal to renal artery stenosis (RAS) remain unclear. We tested the hypothesis that it involves microvascular remodeling consequent to increased oxidative stress. Methods and Results— Three groups of pigs (n=6 each) were studied after 12 weeks of RAS, RAS+antioxidant supplementation (100 IU/kg vitamin E and 1 g vitamin C daily), or controls. The spatial density and tortuousity of renal microvessels (<500 μm) were tomographically determined by 3D microcomputed tomography. The in situ production of superoxide anion and the expression of vascular endothelial growth factor (VEGF), its receptor VEGFR-2, hypoxia-inducible-factor (HIF)-1α, von Hippel-Lindau (VHL) protein, and NAD(P)H oxidase (p47phox and p67phox subunits) were determined in cortical tissue. RAS and RAS+antioxidant groups had similar degrees of stenosis and hypertension. The RAS group showed a decrease in spatial density of cortical microvessels, which was normalized in the RAS+antioxidant group, as was arteriolar tortuousity. RAS kidneys also showed tissue fibrosis (by trichrome and Sirius red staining), increased superoxide anion abundance, NAD(P)H oxidase, VHL protein, and HIF-1α mRNA expression. In contrast, expression of HIF-1α, VEGF, and VEGFR-2 protein was downregulated. These were all significantly improved by antioxidant intervention. Conclusions— Increased oxidative stress in the stenotic kidney alters growth factor activity and plays an important role in renal microvascular remodeling, which can be prevented by chronic antioxidant intervention.

Background— Hypercholesterolemia (HC) and atherosclerosis can elicit oxidative stress, coronary endothelial dysfunction, and myocardial ischemia, which may induce growth-factor expression and lead to myocardial neovascularization. We tested the hypothesis that chronic antioxidant intervention in HC would attenuate neovascularization and preserve the expression of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF). Methods and Results— Three groups of pigs (n=6 each) were studied after 12 weeks of normal or 2% HC diet or HC+antioxidant supplementation (100 IU/kg vitamin E and 1 g vitamin C daily). Myocardial samples were scanned ex vivo with a novel 3D micro-CT scanner, and the spatial density and tortuosity of myocardial microvessels were determined in situ. VEGF mRNA, protein levels of VEGF and VEGF receptor-1, HIF-1α, nitrotyrosine, and superoxide dismutase (SOD) were determined in myocardial tissue. The HC and HC+antioxidant groups had similar increases in serum cholesterol levels. HC animals showed an increase in subendocardial spatial density of microvessels compared with normal (160.5±11.8 versus 95.3±8.2 vessels/cm 2 , P <0.05), which was normalized in HC+antioxidant (92.5±20.5 vessels/cm 2 , P <0.05 versus HC), as was arteriolar tortuosity. In addition, HC induced upregulation of VEGF, HIF-1α, and nitrotyrosine expression and decreased SOD expression and activity, all of which were preserved by antioxidant intervention. Conclusions— Changes in myocardial microvascular architecture invoked by HC are accompanied by increases in HIF-1α and VEGF expression and attenuated by antioxidant intervention. This underscores a role of increased oxidative stress in modulating myocardial microvascular architecture in early atherogenesis.

Hypertension increases oxidative stress, which can impair myocardial microvascular function and integrity. However, it is yet unclear whether long-term antioxidant intervention in early hypertension would preserve myocardial perfusion and vascular permeability responses to challenge. Pigs were studied after 12 weeks of renovascular hypertension without (n=8) or with daily supplementation of antioxidants (100 IU/kg vitamin E and 1 g vitamin C, n=6), and compared with normal controls (n=7). Myocardial perfusion and microvascular permeability were measured in vivo by electron beam computed tomography before and after 2 cardiac challenges (intravenous adenosine and dobutamine). Basal left ventricular muscle mass was also obtained. Mean arterial pressure was significantly increased in both groups of hypertensive animals (without and with antioxidants, 123±9 and 126±4 mm Hg, respectively, versus normal, 101±4 mm Hg; both P <0.05), but muscle mass was not different among the groups. The impaired myocardial perfusion response to adenosine observed in hypertensives (normal, +51±14%; P <0.05 versus baseline; hypertension, +14±15%; P =0.3 versus baseline) was preserved in hypertensive pigs that received antioxidants (+44±15%; P =0.01 compared with baseline). Long-term antioxidant intervention also preserved subendocardial microvascular permeability responses in hypertension. On the other hand, antioxidant intervention had little effect on the hypertension-induced myocardial vascular dysfunction observed in response to dobutamine. This study demonstrates that the impaired myocardial perfusion and permeability responses to increased cardiac demand in early hypertension are significantly improved by long-term antioxidant intervention. These results support the involvement of oxidative stress in myocardial vascular dysfunction in hypertension and suggest a role for antioxidant strategies to preserve the myocardial microvasculature.

Background— Bone marrow–derived cells have been shown to contribute to endothelial replacement after vascular injury. In vitro culture of peripheral blood mononuclear cells produces cells with phenotypic characteristics of endothelium. To test the hypothesis that delivery of autologous culture-modified mononuclear cells (CMMCs) to injured arteries could attenuate the vascular response to injury, a rabbit model was studied. Methods and Results— Rabbit peripheral blood mononuclear cells were cultured in endothelial growth media for 7 to 12 days, yielding highly proliferative cells with distinct endothelial phenotype (expressing CD31 and endothelial nitric oxide synthase and capable of acetylated LDL uptake). A rabbit model of balloon carotid injury was used to evaluate the effect of day 7 CMMC delivery on vascular responses. Animals underwent balloon injury and immediate delivery of autologous CMMCs or buffered saline by 20 minutes of local dwelling. Fluorescence-labeled CMMCs were detected in all vessel layers 4 weeks after delivery. Colonies of cells that localized to the lumen and stained for endothelial markers were also identified. Local CMMC administration at the time of balloon injury accelerated reendothelialization at 4 weeks compared with saline ( P <0.05). Moreover, CMMC delivery markedly improved endothelium-dependent vasoreactivity at 4 weeks compared with saline ( P <0.005). Finally, CMMC treatment reduced neointimal formation by 55% at 4 weeks ( P <0.05). Conclusions— These data demonstrate that delivery of CMMCs to balloon-injured arteries is associated with accelerated reendothelialization, enhanced endothelium-dependent vasoreactivity, and reduced neointimal formation. Thus, delivery of autologous CMMCs represents a novel vasculoprotective approach to attenuate the response to acute vascular injury.

Renal artery stenosis (RAS) may lead to renal injury, partly mediated through increased oxidative stress. However, the potential effects of chronic oral antioxidant intervention on the stenotic kidney remain unknown. This study was designed to test the hypothesis that chronic antioxidant vitamin supplementation in RAS would preserve renal function and structure. Single-kidney hemodynamics and function were quantified in vivo in pigs using electron-beam CT after 12 weeks of unilateral RAS (n=7), a similar degree of RAS orally supplemented with vitamins C (1 g) and E (100 IU/kg) (RAS+Vitamins, n=7), or controls (normal, n=7). Renal tissue was studied ex vivo using Western blotting and immunohistochemistry. Mean arterial pressure was similarly elevated in both RAS groups, while ischemic renal volume and glomerular filtration rate were similarly reduced. Renal blood flow was decreased in RAS compared with normal (326.5±99.9 versus 553.4±48.7 mL/min, respectively, P =0.01), but preserved in RAS+Vitamins (485.2±104.1 mL/min, P =0.3 versus normal). The marked increase in the expression of the NADPH-oxidase subunits p47phox and p67phox, nitrotyrosine, endothelial and inducible nitric oxide synthase, and nuclear factor-κB observed in RAS ( P <0.05 versus normal) was normalized in RAS+Vitamins ( P >0.1). Furthermore, trichrome staining and the expression of transforming growth factor-β and tissue inhibitor of matrix-metalloproteinase-1 were also decreased in RAS+Vitamins. In conclusion, chronic blockade of the oxidative stress pathway in RAS using antioxidant vitamins improved renal hemodynamics and decreased oxidative stress, inflammation, and fibrosis in the ischemic kidney. These observations underscore the involvement of oxidative stress in renal injury in RAS and support a role for antioxidant vitamins in preserving the ischemic kidney.

ABSTRACT The ubiquitin‐proteasome system (UPS) is involved in the removal of damaged proteins and the activation of transcription factors, such as nuclear‐factor‐κB. Recent reports, however, questioned the functional activity of the UPS under conditions of increased oxidative stress, such as experimental hypercholesterolemia, which was the objective of our study. Pigs were placed on a normal chow diet (N) or on a hypercholesterolemic diet without (HC) or with vitamin C and E supplementation (HC+VIT) for 12 weeks. Compared with N, plasma concentration of total cholesterol increased in both HC and HC+VIT [76 ± 21 vs. 400 ± 148 ( P <0.05) and 329 ± 102 ( P <0.05) mg/dL], whereas increase in lipid peroxidation, as assessed by LDL‐malondialdehyde plasma concentration, was found in HC but not in HC+VIT [6.6 ± 0.7 vs. 8.5 ± 0.3 ( P <0.05) and 6.8 ± 0.7 nmol/mg protein]. In comparison with N, the level of ubiquitin conjugates in the coronary artery, as assessed by immunoblotting, increased by 42% in HC but not in HC+VIT and was localized predominantly to media vascular smooth muscle cells by immunostaining. There was no difference in proteasome proteolytic activity among the study groups. These results demonstrate that the UPS is functionally active in early atherogenesis despite increase in oxidative stress with important repercussions in the pathophysiology and therapy of cardiovascular diseases.

Background— Stroke, mainly attributable to atherothrombotic disease, represents a leading cause of disability and death in the Western world. Endothelial dysfunction, which is considered a key factor in atherogenesis, is associated with an increased risk of cardiovascular events. However, the magnitude of the association between coronary endothelial dysfunction (CED) and cerebrovascular events is unknown. This study was performed to investigate the association between CED and cerebrovascular events. Methods and Results— We studied 503 patients without obstructive coronary artery disease (CAD) who underwent coronary endothelial function testing by intracoronary acetylcholine infusion. Patients were divided according to the presence (n=305) or absence (n=198) of CED, and medical records were examined for the occurrence of ischemic or hemorrhagic stroke or transient ischemic attack either before (prevalent) or after (incident) coronary endothelial function testing. Among the study population, a total of 25 cerebrovascular events were documented, 22 in patients with CED (15 prevalent) and 3 in patients without (all prevalent) ( P =0.008). Multivariable logistic regression, which included traditional cerebrovascular disease–related risk factors, identified the presence of CED as the single strongest factor associated with cerebrovascular events (OR, 4.32; 95% CI, 1.26 to 14.83). Kaplan-Meier analysis indicated that patients with CED had a significantly higher cumulative cerebrovascular event rate than those without ( P =0.04). Conclusions— Presence of CED in patients without obstructive CAD is independently associated with an increased risk of cerebrovascular events. Thus, detection of this early stage of atherosclerosis may provide important information to identify patients who benefit from aggressive preventive strategies.

Objective— Atherosclerotic renovascular disease (ARVD) aggravates renal scarring more than other causes of renal artery stenosis (RAS), but the underlying pathogenic mechanisms of this potential profibrotic effect remain unclear. We tested the hypothesis that coexistence of atherosclerosis and RAS interferes with renal tissue remodeling. Methods and Results— Single-kidney hemodynamics and function were quantified in vivo with electron-beam computed tomography in 3 groups of pigs (n=7 each): normal pigs, pigs 12 weeks after induction of unilateral RAS (RAS group), and pigs with similar-degree RAS fed a 12-week 2% hypercholesterolemic diet (HC+RAS, simulating early ARVD). Kidneys were studied ex vivo by Western blotting and immunohistochemistry. Renal volume, renal blood flow, and glomerular filtration rate were similarly decreased in RAS and HC+RAS ischemic kidneys, accompanied by similar increased expression of profibrotic factors like transforming growth factor-β, tissue inhibitor of metalloproteinase-1, and plasminogen activator inhibitor-1. Nevertheless, HC+RAS kidneys showed increased intrarenal fibrosis compared with RAS-only kidneys. Furthermore, expression of nuclear factor-κB was increased, expression of extracellular (matrix metalloproteinase-2) and intracellular (ubiquitin) protein degradation systems was decreased, and apoptosis was blunted. Conclusions— Diet-induced HC superimposed on RAS accelerates the development of fibrosis in the stenotic kidney by amplifying profibrotic mechanisms and disrupting tissue remodeling. These alterations might contribute to renal disease progression in ARVD and might account for the increased propensity for end-stage renal disease.

Objective— Coronary endothelial dysfunction is associated with an increase in cardiac events. Hypercholesterolemia (HC) and hypertension (HT) are both associated with endothelial dysfunction, and their coexistence is associated with an increased incidence of cardiac events in epidemiological studies. However, pathogenic mechanisms are poorly understood. Here we studied the effects of coexisting HC and HT on coronary endothelial function. Methods and Results— Four groups of pigs were studied after 12 weeks of a normal diet (n=9), a 2% HC diet (n=9), HT (achieved by unilateral renal artery stenosis, n=8), or HC+HT (n=6). Coronary endothelial function was tested, in epicardial arteries and arterioles, by using organ chamber techniques. Oxidative stress was measured in coronary artery tissue. Vasodilatory response to bradykinin and calcium ionophore was significantly impaired in animals with HC+HT compared with each risk factor alone ( P <0.05 for both). In animals with coexistent HC and HT, the increase in oxidative stress was more pronounced compared with each risk factor alone ( P <0.05). Furthermore, chronic antioxidant supplementation significantly improved coronary artery vasoreactivity. Conclusions— These results suggest that HC and HT have a synergistic deleterious effect on coronary endothelial function, associated with increased oxidative stress. This interaction may contribute to the increased incidence of coronary heart disease and cardiac events seen when HC and HT coexist.

Endothelial dysfunction is a systemic disorder and a key variable in the pathogenesis of atherosclerosis and its complications. Current evidence suggests that endothelial status is not determined solely by the individual risk factor burden but rather, may be regarded as an integrated index of all atherogenic and atheroprotective factors present in an individual, including known as well as yet-unknown variables and genetic predisposition. Endothelial dysfunction reflects a vascular phenotype prone to atherogenesis and may therefore serve as a marker of the inherent atherosclerotic risk in an individual. In line with this hypothesis, dysfunction of either the coronary or peripheral vascular endothelium was shown to constitute an independent predictor of cardiovascular events, providing valuable prognostic information additional to that derived from conventional risk factor assessment. Interventions like risk factor modification and treatment with various drugs, including statins and angiotensin-converting enzyme inhibitors, may improve endothelial function and thereby, potentially prognosis. Hence, given its reversibility and granted the availability of a diagnostic tool to identify patients at risk and to control the efficacy of therapy in clinical practice, endothelial dysfunction may be an attractive primary target in the effort to optimize individualized therapeutic strategies to reduce cardiovascular morbidity and mortality.