Nigris, Filomena de

Despite advances in imaging, understanding the underlying pathways, and clinical translation of animal models of disease there remains an urgent need for therapies that reduce brain damage after stroke and promote functional recovery in patients. Blocking oxidant radicals, reducing matrix metalloproteinase-induced neuronal damage, and use of stem cell therapy have been proposed and tested individually in prior studies. Here we provide a comprehensive integrative management approach to reducing damage and promoting recovery by combining biological therapies targeting these areas. In a rat model of transient cerebral ischemia (middle cerebral artery occlusion) gene delivery vectors were used to overexpress tissue inhibitor of matrix metalloproteinase 1 and 2 (TIMP1 and TIMP2) 3 days before ischemia. After occlusion, autologous bone marrow cells alone or in combination with agents to improve NO bioavailability were administered intraarterially. When infarct size, BrdU incorporation, and motor function recovery were determined in the treatment groups the largest beneficial effect was seen in rats receiving the triple combined therapy, surpassing effects of single or double therapies. Our study highlights the utility of combined drug, gene, and cell therapy in the treatment of stroke.

Moderate physical exercise (PE) combined with metabolic treatment (MT) (antioxidants and l -arginine) are well known to reduce atherosclerotic lesion formation in hypercholesterolemic mice. However, the long-term beneficial effects on unstable atheroma remain poorly understood. We started early PE training in large groups of 6-week-old hypercholesterolemic mice (by graduated swimming) alone or in combination with nutritional supplementation (1.0% vitamin E added to the chow and 0.05% vitamin C and 6% l -arginine added to the drinking water). Inactive controls did not receive PE. The spontaneous development of atherosclerotic plaque rupture (associated with advanced atherosclerosis) and survival rates were evaluated. Moderate PE elicited an increase in plasma levels of nitric oxide. Early combined treatment with PE and MT in the hypercholesterolemic mice significantly reduced lesions (also detected noninvasively at 10 months) and spontaneous atherosclerotic plaque rupture and prolonged survival more effectively than each intervention alone. Thus, early concerted actions of MT and PE improve the natural history of atherosclerotic lesions and reduce the plaque instability in hypercholesterolemic mice.

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.

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.

Restenosis after percutaneous transluminal coronary angioplasty is caused by neointimal hyperplasia, which involves impairment of nitric oxide (NO)-dependent pathways, and may be further exacerbated by a concomitant aging process. We compared the effects of NO-releasing-aspirin (NCX-4016) and aspirin (ASA) on experimental restenosis in both adult and elderly rats. Moreover, to ascertain the efficacy of NCX-4016 during vascular aging, we fully characterized the release of bioactive NO by the drug. Sprague–Dawley rats aged 6 and 24 months were treated with NO releasing-aspirin (55 mg/kg) or ASA (30 mg/kg) for 7 days before and 21 days after standard carotid balloon injury. Histological examination and immunohistochemical double-staining were used to evaluate restenosis. Plasma nitrite and nitrate and S -nitrosothiols were determined by a chemiluminescence-based assay. Electron spin resonance was used for determining nitrosylhemoglobin. Treatment of aged rats with NCX-4016 was associated with increased bioactive NO, compared with ASA. NO aspirin, but not ASA, reduced experimental restenosis in old rats, an effect associated with reduced vascular smooth muscle cell proliferation. NCX-4016, but not ASA, was well tolerated and virtually devoid of gastric damage in either adult or old rats. Thus, impairment of NO-dependent mechanisms may be involved in the development of restenosis in old rats. We suggest that an NCX-4016 derivative could be an effective drug in reducing restenosis, especially in the presence of aging and/or gastrointestinal damage.