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 (20 mu m cubic-voxel-size). After scanning, randomly selected cross-sectional histological sections were stained for nuclear-factor kappaB (NF-kappa B), hypoxia-inducible factor-1alpha (HIF-1 alpha), 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-kappa B (r = 0.73 and 0.70), HIF-1 alpha (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. (C) 2009 Published by Elsevier Ireland Ltd.

Hypercholesterolemia (HC), a major risk factor for onset and progression of renal disease, is associated with increased oxidative stress, potentially causing endothelial dysfunction. One of the sources of superoxide anion is xanthine oxidase (XO), but its contribution to renal endothelial function in HC remains unclear. We tested the hypothesis that XO modulates renal hemodynamics and endothelial function in HC pigs. Four groups (n = 23) of female domestic pigs were studied 12 wk after either normal (n = 11) or HC diet (n = 12). Oxidative stress was assessed by plasma isoprostanes and oxidized LDL, and the XO system by plasma uric acid, urinary xanthine, and renal XO expression (by immunoblotting and immunohistochemistry). Renal hemodynamics and function were studied with electron beam-computed tomography before and after endothelium-dependent (ACh) and -independent (sodium nitroprusside) challenge, during a concurrent intrarenal infusion of either oxypurinol or saline (n = 5-6 in each group). HC showed elevated oxidative stress, higher plasma uric acid (23.8 +/- 3.8 vs. 6.2 +/- 0.8 microM/mM creatinine, P = 0.001), lower urinary xanthine, and greater renal XO expression compared with normal. Inhibition of XO in HC significantly improved the blunted responses to ACh of cortical perfusion (13.5 +/- 12.1 and 37.2 +/- 10.6%, P = 0.01 and P = not significant vs. baseline, respectively), renal blood flow, and glomerular filtration rate; restored medullary perfusion; and improved the blunted cortical perfusion response to sodium nitroprusside. This study demonstrates that the endogenous XO system is activated in swine HC. Furthermore, it suggests an important role for XO in regulation of renal hemodynamics, function, and endothelial function in experimental HC.

Patients with chronic kidney disease (CKD) have increased risk for cardiovascular events. However, the association between these pathophysiological processes is unclear. Therefore, this study was designed to determine the association between early CKD and coronary microvascular disease in patients with normal or mildly diseased coronary arteries. A total of 605 patients with normal or mildly diseased coronary arteries based on angiography underwent coronary flow reserve (CFR) evaluation using intracoronary adenosine. Patients were divided based on glomerular filtration rate (GFR). CKD was defined as calculated GFR<60 ml/min/1.73 m(2). Patients with normal GFR (>= 60 ml/ min/1.73 m(2), n = 481) had higher CFR compared to those with CKD (n = 124, CFR 3.0 +/- 0.8 vs 2.6 +/- 0.6, P<0.001, respectively). Patients with abnormal GFR were more likely to be older and of female gender, with greater prevalence of hypertension. Multiple logistic regression analysis adjusted for the aforementioned risk factors further supported the observed relationship. The current study shows that reduced renal function is associated with attenuated coronary vasodilator capacity in patients without obstructive coronary artery disease. The correlation between low GFR and reduced CFR may suggest parallel alterations in the renal and coronary microcirculation at the early stage of disease. Impairment in both microcirculatory beds may reflect an unmeasured risk factor induced by blunted renal function and add a burden to the increased propensity for cardiovascular events in CKD.

The main goal in the treatment of obstructive atherosclerotic renovascular disease (ARVD) is to preserve or recover renal function. The ARVD kidney continues to deteriorate in 20-40% of cases despite restoration of blood flow. Holden et al. report that renal function stabilized or improved in up to 97% of patients with the use of a distal embolic protection device.

Hypertension is a multifactorial disease involving complex interactions between genetic and environmental factors. Development of experimental models of hypertension allowed dissection and isolation of various factors associated with regulation of blood pressure, inheritance of hypertensive traits, and cellular responses to injury. The phenotype-driven approach is taking advantage of selective breeding of animals (primarily rats) that exhibit a desired phenotype, like the useful SHR. Genotype-driven models include transgenic techniques, in which mice are the most successful for selective deletion or overexpression of target genes. Notably, a combination of comparative genomics strategies and phenotypic correlates enhances the utility of hypertension models and their clinical relevance. Indeed, experimental models enabled development of targeted interventions aimed at decreasing not only blood pressure but also target organ injury. Continued utilization of experimental models simulating human hypertension, particularly those that combine other clinically relevant comorbidities like obesity or hypercholesterolemia, may afford development of effective strategies to address this common disease. Nevertheless, a cautious approach is mandatory when experimental findings in these models are extrapolated to human hypertension.

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.

Hypercholesterolemia (HC) and atherosclerosis often accompany and aggravate renal disease. Proteasome inhibitors (PSI) can decrease proliferation and inflammation, likely by reducing activation of the proinflammatory NF-kappaB. However, chronic proteasome inhibition has never been demonstrated in the HC kidney. Four groups of pigs (n = 7 each) were studied after a 12-wk normal (N) or 2% HC diet alone or supplemented (N+PSI and HC+PSI) with MLN-273 (0.08 mg/kg subcutaneously twice weekly). Renal hemodynamics and function were quantified in vivo using electron-beam computed tomography at baseline and after vasodilator challenge using acetylcholine. Renal tissue was studied ex vivo using immunoblotting, PCR, and immunohistochemistry. Serum cholesterol was similarly elevated in HC and HC+PSI. Basal renal blood flow was similar among the groups, whereas GFR was decreased in both N+PSI and HC+PSI. The blunted renovascular and functional responses to acetylcholine in HC were normalized in HC+PSI (suggesting renal endothelial function improvement), which was accompanied by decreased renal endothelin, NF-kappaB, and augmented endothelial nitric oxide synthase expression. In parallel, HC+PSI animals also showed elevated NAD(P)H oxidase expression and circulating oxidized LDL, suggesting a potential for increased oxidative stress. This study shows that chronic PSI intervention in HC improves renal endothelial functional responses to challenge, possibly by modulating nitric oxide availability and endothelin. Furthermore, PSI may decrease intrarenal inflammation through modulation of the NF-kappaB pathway but may potentially increase oxidative stress, which warrants further investigation. This study may support a role for the ubiquitin/proteasome system in the kidney in HC and early atherosclerosis.

Experimental hypercholesterolemia (HC) may lead to microvascular neovascularization, but the underlying pathogenic mechanism remains unclear. We tested the hypothesis that HC-induced intra-renal neovascularization is associated with inflammation and increased oxidative stress, and would be prevented by chronic antioxidant intervention. Kidneys were excised from pigs after a 12-wk normal (n = 10) or HC diet (n = 8), or HC diet supplemented daily with antioxidant vitamins C (1 g) and E (100 IU/kg) (HC + vitamins, n = 7). Renal cortical samples were then scanned three dimensionally with micro-computed tomography, and microvessels were counted in situ. Blood and tissue samples were removed for measurements of superoxide dismutase (SOD) activity, protein expression of the NADP(H)-oxidase subunits gp91phox, p47phox, and p67phox, vascular endothelial growth factor (VEGF) levels and mRNA, VEGF receptors (Flt-1 and FIk-1), the proinflammatory transcription factor NFkappaB, and the oxidized LDL receptor LOX-1. Microvascular spatial density was significantly elevated in HC compared with normal kidneys but preserved in HC + vitamins. Expression of gp91phox and p67phox was decreased in HC pigs after antioxidant intervention, and SOD improved. The increased renal expression of VEGF and Flk-1 in HC was blunted in HC + vitamins, as were the significant increases in LOX-1, NFkappaB, and interstitial fibrosis. This study shows that renal cortical neovascularization elicited by diet-induced HC is associated with renal inflammation, fibrosis, and upregulation of VEGF and its receptor Flk-1, likely mediated by increased endogenous oxidative stress. Chronic antioxidant supplementation may preserve the kidney in HC.

Atherosclerotic renovascular disease (RVD) amplifies damage in a stenotic kidney by inducing pro-inflammatory mechanisms and disrupting tissue remodeling. Oxidative stress is increased in RVD, but its direct contribution to renal injury has not been fully established. The authors hypothesized that chronic antioxidant intervention in RVD would improve renal function and attenuate tissue injury. Single-kidney hemodynamics and function at baseline and during vasoactive challenge were quantified using electron-beam computed tomography in pigs after 12 wk of experimental RVD (simulated by concurrent hypercholesterolemia and renal artery stenosis, n = 7), RVD daily supplemented with antioxidant vitamins C (1 g), and E (100 IU/kg) (RVD+Vitamins, n = 7), or controls (normal, n = 7). Renal tissue was studied ex vivo using Western blot analysis and immunohistochemistry. Basal renal blood flow (RBF) and glomerular filtration rate (GFR) were similarly decreased in the stenotic kidney of both RVD groups. RBF and GFR response to acetylcholine was blunted in RVD, but significantly improved in RVD+Vitamins (P < 0.05 versus RVD). RVD+Vitamins also showed increased renal expression of endothelial nitric oxide synthase (eNOS) and decreased expression of NAD(P)H-oxidase, nitrotyrosine, inducible-NOS, and NF-kappaB, suggesting decreased superoxide abundance and inflammation. Furthermore, decreased expression of pro-fibrotic factors in RVD+Vitamins was accompanied by augmented expression of extracellular (matrix metalloproteinase-2) and intracellular (ubiquitin) protein degradation systems, resulting in significantly attenuated glomerulosclerosis and renal fibrosis. In conclusion, chronic antioxidant intervention in early experimental RVD improved renal functional responses, enhanced tissue remodeling, and decreased structural injury. This study supports critical pathogenic contribution of increased oxidative stress to renal injury and scarring in RVD and suggests a role for antioxidant strategies in preserving the atherosclerotic and ischemic kidney.

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)-1alpha 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-1alpha, 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-1alpha, 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-1alpha 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.

Reactive oxygen species (ROS) can modulate renal hemodynamics and function both directly, by leading to vasoconstriction, and indirectly, by inducing renal inflammation and tissue growth. The involvement of oxidative stress in the pathogenesis of renovascular disease (RVD) is increasingly recognized, but the relative contribution of long-term tissue injury to renal dysfunction remains unclear. We hypothesized that functional and structural alterations elicited by oxidative stress in RVD would be more effectively modulated by chronic than by acute antioxidant intervention. Renal hemodynamics and function were quantified in vivo in pigs using electron-beam computed tomography at baseline and after vasoactive challenge (ACh and sodium nitroprusside); after 12 wk of RVD (simulated by concurrent hypercholesterolemia and renal artery stenosis, n = 7); RVD acutely infused with the SOD-mimetic tempol (RVD+tempol, n = 7); RVD chronically supplemented with antioxidant vitamins C (1 g) and E (100 IU/kg; RVD+vitamins, n = 7); or control (normal, n = 7). Renal tissue was studied ex vivo using immunoblotting and immunohistochemistry. Basal renal blood flow (RBF) and glomerular filtration rate were similarly decreased in all RVD groups. ACh-stimulated RBF remained unchanged in RVD, increased in RVD+tempol, but further increased (similarly to normal) in RVD+vitamins (P < 0.05 vs. RVD). Furthermore, RVD+vitamins also showed a decreased presence of superoxide anion, decreased NAD(P)H-oxidase and nitrotyrosine expression, increased endothelial nitric oxide synthase expression, and attenuated renal fibrosis. Chronic antioxidant intervention in early RVD improved renal hemodynamic responses more effectively than acute intervention, likely due to increased nitric oxide bioavailability and decreased structural injury. These suggest that chronic tissue changes play an important role in renal compromise mediated by oxidative stress in RVD.

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 mu m) 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)-1 alpha, 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-1 alpha mRNA expression. In contrast, expression of HIF-1 alpha, 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.