Alain Rivard

Inhibition of hypoxia-induced angiogenesis by cigarette smoke exposure: impairment of the HIF-1α/VEGF pathway

Smoking is a major risk factor for atherosclerotic diseases. However, the impact of cigarette smoke exposure on neovascularization that develops in response to tissue ischemia is unknown. Here we demonstrate that cigarette smoke extracts inhibit hypoxia‐induced in vitro angiogenesis (matrigel assay) in human umbilical vascular endothelial cells. In vivo, mice exposed to cigarette smoke (MES) were shown to have a significant impairment of angiogenesis following surgically induced hindlimb ischemia. The reduced angiogenic response in MES was documented by Laser Doppler flow perfusion studies and capillary density analyses in ischemic hindlimbs. Inhibition of angiogenesis by cigarette smoke in vitro and in vivo was associated with a reduced expression of hypoxia‐inducible factor‐1α (HIF‐1α) and vascular endothelial growth factor (VEGF) in hypoxic conditions. Administration of an adenoviral vector encoding for HIF‐1α/VP16, a hybrid transcription factor that is stable in hypoxic and normoxic conditions, restored VEGF expression and completely reversed the cigarette smoke inhibition of angiogenesis in hypoxic conditions. Taken together, these results suggest that cigarette smoke exposure impairs angiogenesis by inhibiting VEGF through decreased expression of HIF‐1α in hypoxic conditions.

Moderate consumption of red wine (cabernet sauvignon) improves ischemia-induced neovascularization in ApoE-deficient mice: effect on endothelial progenitor cells and nitric oxide

Moderate consumption of red wine is associated with a decreased incidence of cardiovascular diseases in populations with relatively high amount of fat in the diet. However, the mechanisms involved in this protective effect are not completely understood. Here we show that moderate consumption of red wine (equivalent to 2 glasses/day in humans) but not ethanol only, improves blood flow recovery by 32% after hind‐limb ischemia in hypercholesterolemic ApoE‐deficient mice. In ischemic tissues, red wine consumption reduces oxidative stress and increases capillary density by 46%. Endothelial progenitor cells (EPCs) have been shown to have an important role in postnatal neovascu‐larization. We found that the number of EPCs is increased by 60% in ApoE mice exposed to red wine. Moreover, the migratory capacity of EPCs is significantly improved in red wine‐drinking mice. The wine used in our study is a cabernet sauvignon from Langue‐doc‐Roussillon, France, which contains a relatively high concentration (4–6 mg/L) of the polyphenolic antiox‐idant resveratrol. We demonstrate that resveratrol can rescue oxidized low‐density lipoprotein (oxLDL)‐in‐duced impairment of in vitro angiogenic activities in human umbilical vein endothelial cells (HUVECs). Res‐veratrol exposure is also associated with increased activation of Akt/eNOS together with a restoration of nitric oxide production in HUVECs exposed to oxLDL. Our study suggests that moderate consumption of red wine improves ischemia‐induced neovascularization in high‐cholesterol conditions by increasing the number and the functional activities of EPCs and by restoring the Akt‐eNOS‐NO pathway.— Lefevre J., Michaud, S.‐E., Haddad, P., Dussault, S., Ménard C., Groleau, J., Turgeon, J., Rivard A. Moderate consumption of red wine (cabernet sauvignon) improves ischemia‐induced neovascularization in ApoE‐deficient mice: effect on endothelial progenitor cells and nitric oxide. FASEB J. 21, 3845–3852 (2007)

Age-Dependent Impairment of Reendothelialization After Arterial Injury Role of Vascular Endothelial Growth Factor

Background—The mechanisms responsible for the association between advanced age and atherosclerotic diseases are not clear. Because atherosclerosis develops in response to local endothelial injuries, we investigated the effect of aging on vascular healing and reendothelialization. Methods and Results—Endothelium denudation was performed by balloon angioplasty of the iliac arteries in young and old New Zealand White rabbits. Planimetric analysis after Evans Blue staining at 28 days after injury showed a significant decrease in reendothelialization in old versus young animals, which was associated with an important increase in neointimal formation in old rabbits. Vascular endothelial growth factor (VEGF) was rapidly induced after balloon injury. However, arterial VEGF expression was significantly reduced in old versus young animals. To confirm the role of VEGF in the age-dependent impairment of reendothelialization, an adenoviral vector encoding for VEGF165 (adeno-VEGF) was locally delivered at the time of iliac artery angioplasty. Compared with animals treated with the control vector (adeno-&bgr;Gal), reendothelialization was significantly improved and neointimal formation reduced in old rabbits treated with adeno-VEGF. Conclusions—These results document for the first time an age-dependent impairment of reendothelialization after arterial injury. Our study indicates that VEGF supplementation may represent a useful strategy to accelerate reendothelialization and improve vascular healing in the context of aging.

Inhibition of Vascular Smooth Muscle Cell Proliferation and Neointimal Formation in Injured Arteries by a Novel, Oral Mitogen-Activated Protein Kinase/Extracellular Signal–Regulated Kinase Inhibitor

Background—Mitogen-activated protein kinases (MAPKs) are rapidly induced after arterial injury in different animal models. However, their precise role in vascular smooth muscle cell (VSMC) proliferation and neointimal formation in vivo remains to be determined. Methods and Results—We investigated the properties of a novel, selective inhibitor of the upstream kinase, MAPK/extracellular signal–regulated kinase, that is orally active (PD0185625). In vitro, PD0185625 was shown to abrogate p44/p42 MAPK activation in VSMCs after serum stimulation. This was associated with a dose-dependent inhibition of VSMC proliferation. In vivo, PD0185625 was administered orally to rats (200 mg · kg−1 · d−1) beginning 2 days before balloon injury of the left carotid artery and for 2 weeks thereafter. Treatment with PD0185625 led to nearly complete inhibition of p44/p42 MAPK activation after balloon injury. This resulted in a significant decrease in VSMC proliferation (BrdU incorporation) at day 7 after injury. Moreover, neointimal formation was significantly reduced in PD0185625-treated animals at 14 and 28 days after arterial injury. We found that PD0185625 did not increase the rate of apoptotic cell death but prevented cell cycle progression and induced a G1 block. Conclusions—PD0185625 reduced neointimal formation after arterial injury. The mechanism involved inhibition of VSMC proliferation via a G1 block of the cell cycle. Orally active selective MAPK inhibitors could represent a novel therapeutic approach for vascular diseases.

COMPARE-AMI Trial: Comparison of Intracoronary Injection of CD133+ Bone Marrow Stem Cells to Placebo in Patients After Acute Myocardial Infarction and Left Ventricular Dysfunction: Study Rationale and Design

Stem cell therapy has emerged as a promising approach to improve healing of the infarcted myocardium, to treat or prevent cardiac failure, and to restore lost cardiac function. Despite initial excitement, recent clinical trials using nonhomogenous human stem cells preparations showed variable results, raising concerns about the best cell type to transplant. Selected CD133+ hematopoietic stem cells are promising candidate cells with great potential. COMPARE-acute myocardial infarction (AMI) study is a phase II, randomized, double-blind, placebo-controlled trial evaluating the safety and effectiveness of intracoronary CD133+-enriched hematopoietic bone marrow stem cells in patients with acute myocardial infarction and persistent left ventricular dysfunction. Patients who underwent successful percutaneous coronary intervention and present a persistent left ventricular ejection fraction <50% will be eligible to have bone marrow aspiration and randomized for intracoronary injection of selected CD 133+ bone marrow cells vs placebo. The primary end point is a composite of a safety and efficacy end points evaluating the change at 4xa0months in the coronary atherosclerotic burden progression proximal and distal to the coronary stent in the infarct related artery; and the change in global left ventricular ejection fraction at 4xa0months relative to baseline as measured by magnetic resonance imaging. The secondary end point will be the occurrence of a major adverse cardiac event. To date, 14 patients were successfully randomized and treated without any protocol-related complication. COMPARE-AMI trial will help identify the effect of a selected population of the bone marrow stem cells on cardiac recovery of infarcted myocardium.

Nox2-Containing NADPH Oxidase Deficiency Confers Protection From Hindlimb Ischemia in Conditions of Increased Oxidative Stress

Objective—Because Nox2-containing NADPH oxidase is a major source of ROS in the vasculature, we investigated its potential role for the modulation of ischemia-induced neovascularization in conditions of increased oxidative stress. Methods and Results—To mimic a clinical situation of increased oxidative stress, mice were exposed to cigarette smoke before and after the surgical induction of hindlimb ischemia. Nox2 expression and oxidative stress in ischemic tissues were significantly increased in wild-type mice, but not in mice deficient for the Nox2-containing NADPH oxidase (Nox2−/−). Nox2−/− mice demonstrated faster blood flow recovery, increased capillary density in ischemic muscles, and improved endothelial progenitor cell functional activities compared to Nox2+/+ mice. In addition, Nox2 deficiency was associated with increased antioxidant and nitrite concentrations in plasma, together with a preserved expression of eNOS in ischemic tissues. In vitro, Nox2−/− endothelial cells exhibit resistance against superoxide induction and improved VEGF-dependent angiogenic activities compared to Nox2+/+ endothelial cells. Importantly, the beneficial effects of Nox2 deficiency on neovascularization in vitro and in vivo were lost after treatment with the NO inhibitor L-NAME. Conclusions—Nox2-containing NADPH oxidase deficiency protects against ischemia in conditions of increased oxidative stress. The mechanism involves improved neovascularization through a reduction of ROS formation, preserved activation of the VEGF/NO angiogenic pathway, and improved functional activities of endothelial progenitor cells.

Role of p44/p42 MAP Kinase in the Age-Dependent Increase in Vascular Smooth Muscle Cell Proliferation and Neointimal Formation

Objective—Age-dependent increase in vascular smooth muscle cell (VSMC) proliferation is thought to contribute to the pathology of atherosclerotic diseases. In this study, we investigated the role of mitogen-activated protein kinases (MAPKs) on VSMC proliferation and neointimal formation in the context of aging. Methods and Results—VSMCs were isolated from the aorta of young and old rabbits. The proliferative index after serum stimulation was significantly increased in old versus young VSMCs. This was associated with a significant and specific age-dependent increase in p44/p42 MAPK activation. Treatment with MEK inhibitor PD98059 successfully inhibited p44/p42 MAPK activities and VSMC proliferation. These results were confirmed in vivo using a model of balloon injury in rabbit iliac arteries. p44/p42 MAPK activities were rapidly induced by angioplasty in young and old animals. However, the levels of p44/p42 MAPK activities achieved in arteries of old rabbits were significantly higher than those of young rabbits. This was associated with a higher cellular proliferative index and a significant increase in neointimal formation in old animals. Local delivery of PD98059 in old rabbits successfully inhibited p44/p42 MAPK activities after angioplasty, which led to a significant reduction in cellular proliferation and neointimal formation in treated animals. Conclusions—Our study suggests for the first time that increased p44/p42 MAPK activation contributes to augmented VSMC proliferation and neointimal formation with aging. p44/p42 MAPK inhibition could represent a novel therapeutic avenue against atherosclerotic diseases.

Nox2-derived reactive oxygen species contribute to hypercholesterolemia-induced inhibition of neovascularization: Effects on endothelial progenitor cells and mature endothelial cells

BACKGROUNDnHypercholesterolemia has been associated with impaired angiogenesis and reduced blood flow recuperation after ischemia. However, the precise mechanisms involved are unknown. Here we investigated the role of Nox2-derived reactive oxygen species (ROS) in the modulation of neovascularization by hypercholesterolemia.nnnMETHODS AND RESULTSnMice deficient for the Nox2-containing NADPH oxidase (Nox2(-/-)) and control mice (Nox2(+/+)) were put on a high cholesterol diet (HCD) for a total of 15 weeks. After three months, hindlimb ischemia was surgically induced by femoral artery removal. Nox2 expression and oxidative stress levels in ischemic tissues were significantly increased by HCD in control mice, but not in Nox2(-/-) mice. Nox2(-/-) mice were also protected against hypercholesterolemia-induced impairment of neovascularization, as demonstrated by faster blood flow recovery after ischemia and increased capillary density in ischemic muscles. Nox2 deficiency was associated with preserved activity of eNOS in ischemic tissues, and improved activity of endothelial progenitor cells (EPCs). In vitro, HUVECs treated with the NADPH oxidase inhibitor apocynin or endothelial cells isolated from the aorta of Nox2(-/-) mice exhibited reduced ROS formation following exposure to oxLDL. This was associated with improved nitric oxide (NO) bioavailability and protection against oxLDL-induced inhibition of angiogenic activities.nnnCONCLUSIONSnNox2-containing NADPH oxidase deficiency protects against hypercholesterolemia-induced impairment of neovascularization. The potential mechanisms involved include reduced ROS formation, preserved activation of angiogenic signals, and improved functional activities of EPCs and mature endothelial cells.

Sildenafil Increases Endothelial Progenitor Cell Function and Improves Ischemia-Induced Neovascularization in Hypercholesterolemic Apolipoprotein E–Deficient Mice

Hypercholesterolemia is associated with impaired neovascularization in response to ischemia. Potential mechanisms include defective NO bioactivity and a reduction in the number/function of endothelial progenitor cells (EPCs). Here we tested the hypothesis that sildenafil, a phosphodiesterase 5 inhibitor that increases NO-driven cGMP levels, could stimulate EPC function and improve ischemia-induced neovascularization in hypercholesterolemic conditions. Apolipoprotein E–deficient (ApoE−/−) mice were treated (or not treated) with sildenafil (40 mg/kg per day in water), and hindlimb ischemia was surgically induced by femoral artery removal. Sildenafil treatment led to an improved blood flow recovery, an increased capillary density, and a reduction of oxidative stress levels in ischemic muscles at day 7 after surgery. Sildenafil therapy is associated with an increased activation of angiogenic transduction pathways, including Akt, p44/42 mitogen-activated protein kinase, and p38. In vitro, sildenafil increases cellular migration and tubule formation of mature endothelial cells (human umbilical vascular endothelial cells) in a cGMP-dependent manner. In vivo, ApoE−/− mice treated with sildenafil exhibit a significant increase in the number of bone marrow–derived EPCs. Moreover, the angiogenic activities of EPCs (migration and adhesion) are significantly improved in ApoE−/− mice treated with sildenafil. In summary, this study demonstrates that sildenafil treatment is associated with improved ischemia-induced neovascularization in hypercholesterolemic ApoE−/− mice. The mechanisms involve beneficial effects on angiogenic transduction pathways together with an increase in the number and the functional activity of EPCs. Sildenafil could constitute a novel therapeutic strategy to reduce tissue ischemia in atherosclerotic diseases.

Protection against vascular aging in Nox2-deficient mice: Impact on endothelial progenitor cells and reparative neovascularization

BACKGROUNDnAging is associated with increased oxidative stress levels and impaired neovascularization following ischemia. Because Nox2-containing NADPH oxidase is a major source of ROS in the vasculature, we investigated its potential role for the modulation of ischemia-induced neovascularization in the context of aging.nnnMETHODS AND RESULTSnHindlimb ischemia was surgically induced by femoral artery removal in young (2 months) and old (10 months) Nox2-deficient (Nox2(-/-)) and wild type mice. We found that Nox2 expression is increased by aging in ischemic muscles of wild type mice. This is associated with a significant reduction of blood flow recovery after ischemia in old compared to young mice at day 21 after surgery (Doppler flow ratios: 0.51 ± 0.05 vs. 0.72 ± 0.05; p < 0.05). We also demonstrate that capillary and arteriolar densities are significantly reduced in ischemic muscles of old animals, while oxidative stress levels are increased (nitrotyrosine immunostaining). Importantly, Nox2 deficiency reduces oxidative stress levels in ischemic tissues and restores blood flow recuperation and vascular densities in old animals. Endothelial progenitor cells (EPCs) have an important role for postnatal neovascularization. Here we show that the functional activities of EPCs (migration, adhesion to mature endothelial cells) are significantly impaired in old compared to young mice. However, Nox2 deficiency rescues EPC functional activities in old animals. We also demonstrate an age-dependent pathological increase of oxidative stress levels in EPCs (DHE, DCF-DA) that is not present in Nox2-deficient animals.nnnCONCLUSIONnNox2-containing NADPH oxidase deficiency protects against age-dependent impairment of neovascularization. Potential mechanisms include reduced ROS generation in ischemic tissues and preserved angiogenic activities of EPCs.