Napoli, Claudio

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.

Lower-limb ischemia is a major health problem. Because of the absence of effective treatment in the advanced stages of the disease, amputation is undertaken to alleviate unbearable symptoms. Novel therapeutic approaches include the intramuscular use of autologous bone marrow cells (BMCs). Because tissue ischemia is associated with an overwhelming generation of oxygen radicals and negative effects due to perturbed shear-stress, metabolic intervention with antioxidants and l -arginine could potentially induce beneficial effects beyond those achieved by BMCs. The protective effect of autologous BMCs and vascular protection by metabolic cotreatment (1.0% vitamin E added to the chow and 0.05% vitamin C and 6% l -arginine added to the drinking water) were examined in ischemia-induced angiogenesis in the mouse hindlimb, a model of extensive acute peripheral arterial occlusion. i.v. BMC therapy improved blood flow and increased capillary densities and expression of Ki-67, a proliferation-associated protein. This beneficial effect was amplified by metabolic cotreatment, an intervention inducing vascular protection, at least in part, through the nitric oxide pathway, reduction of systemic oxidative stress, and macrophage activation. Therefore, although a cautious approach is mandatory when experimental findings are extended to human diseases, autologous BMCs together with metabolic intervention could be an effective clinical treatment for peripheral arterial disease.

Atherosclerosis is enhanced in arterial segments exposed to disturbed flow. Perturbed shear stress increases the expression of oxidation-sensitive responsive genes (such as ELK-1 and p -JUN) in the endothelium. Evidence suggests that polyphenolic antioxidants contained in the juice derived from the pomegranate can contribute to the reduction of oxidative stress and atherogenesis. The aim of the present study was to evaluate the effects of intervention with pomegranate juice (PJ) on oxidation-sensitive genes and endothelial NO synthase (eNOS) expression induced by high shear stress in vitro and in vivo . Cultured human coronary artery endothelial cells (EC) exposed to high shear stress in vitro and hypercholesterolemic mice were used in this study. PJ concentrate reduced the activation of redox-sensitive genes (ELK-1 and p -JUN) and increased eNOS expression (which was decreased by perturbed shear stress) in cultured EC and in atherosclerosis-prone areas of hypercholesterolemic mice. Moreover, oral administration of PJ to hypercholesterolemic mice at various stages of disease reduced significantly the progression of atherosclerosis. This experimental study indicates that the proatherogenic effects induced by perturbed shear stress can be reversed by chronic administration of PJ. This approach may have implications for the prevention or treatment of atherosclerosis and its clinical manifestations.

The pathogenic mechanisms by which physical exercise influences atherosclerotic lesion formation remain poorly understood. Because vigorous physical training increases oxidative stress, this study tested the hypothesis that graduated and moderate physical exercise together with metabolic intervention ( l -arginine and antioxidants) may contribute to increased vascular protection. Exercise training in mice was induced by graduated swimming. In hypercholesterolemic male mice on an atherogenic high-cholesterol diet, graduated and moderate exercise lowered plasma cholesterol and decreased atherosclerotic lesions compared with sedentary control mice. Antioxidants (1.0% vitamin E added to the chow and 0.05% vitamin C added to the drinking water) and l -arginine (6% in drinking water) supplementation to exercising hypercholesterolemic mice further and synergistically reduced atherosclerosis compared with untreated exercised mice. Arterial oxidation-specific epitopes and systemic oxidative stress were reduced by metabolic intervention. Graduated chronic exercise elicited an increase in production of nitric oxide through increased endothelial nitric oxide synthase expression and ameliorated scavenger activities. Thus, metabolic intervention with l -arginine and antioxidants together with graduated and moderate exercise training reduce atherosclerotic lesion formation.

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.

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.

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.

The massive increase in information on the human DNA sequence and the development of new technologies will have a profound impact on the diagnosis and treatment of cardiovascular diseases. The microarray is a micro-hybridisation based assay. The filter, called microchip or chip, is a special kind of membrane in which are spotted several thousands of oligonucleotides of cDNA fragments coding for known genes or expressed sequence tags. The resulting hybridisation signal on the chip is analysed by a fluorescent scanner and processed with a software package utilising the information on the oligonucleotide or cDNA map of the chip to generate a list of relative gene expression. Microarray technology can be used for many different purposes, most prominently to measure differential gene expression, variations in gene sequence (by analysing the genome of mutant phenotypes), or more recently, the entire binding site for transcription factors. Measurements of gene expression have the advantage of providing all available sequence information for any given experimental design and data interpretation in pursuit of biological understanding. This research tool will contribute to radically changing our understanding of cardiovascular diseases.

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.

Atherogenesis is enhanced in arterial segments exposed to disturbed blood flow, indicating the active participation of the hemodynamic environment in lesion formation. Turbulent shear stress selectively regulates responsive genes in the endothelium and increases the damage induced by free radicals. The purpose of the present study was to evaluate the effects of intervention with antioxidants and l -arginine on endothelial NO synthase (eNOS) and oxidation-sensitive gene perturbation induced by disturbed flow in vitro and in vivo . Both human endothelial cells exposed to shear stress and high atherosclerosis-prone areas of hypercholesterolemic low-density lipoprotein receptor knockout (LDLR −/− ) mice showed increased activities of redox-transcription factors (ELK-1, p -Jun, and p -CREB) and decreased expression of eNOS. Intervention with antioxidants and l -arginine reduced the activation of redox-transcription factors and increased eNOS expression in cells and in vivo . These results demonstrate that atherogenic effects induced by turbulent shear stress can be prevented by cotreatment with antioxidants and l -arginine. The therapeutic possibility to modulate shear stress-response genes may have important implications for the prevention of atherosclerosis and its clinical manifestations.