ABSTRACT
Atherosclerosis is promoted by a combination of hypercholesterolemia and vascular inflammation. The function of Angiopoietin (Ang)-2, a key regulator of angiogenesis, in the maintenance of large vessels is unknown. A single systemic administration of Ang-2 adenovirus (AdAng-2) to apoE(-/-) mice fed a Western diet significantly reduced atherosclerotic lesion size ( approximately 40%) and oxidized LDL and macrophage content of the plaques. These beneficial effects were abolished by the inhibition of nitric oxide synthase (NOS). In endothelial cells, endothelial NOS activation per se inhibited LDL oxidation and Ang-2 stimulated NO release in a Tie2-dependent manner to decrease LDL oxidation. These findings demonstrate a novel atheroprotective role for Ang-2 when endothelial cell function is compromised and suggest that growth factors, which stimulate NO release without inducing inflammation, could offer atheroprotection.
Subject(s)
Angiopoietin-2/metabolism , Apolipoproteins E , Atherosclerosis/metabolism , Lipoproteins, LDL/metabolism , Receptor, TIE-2/metabolism , Adenoviridae , Angiopoietin-2/genetics , Animals , Atherosclerosis/genetics , Atherosclerosis/prevention & control , Endothelial Cells/metabolism , Endothelium, Vascular/metabolism , Female , Lipoproteins, LDL/genetics , Male , Mice , Mice, Knockout , Neovascularization, Physiologic/genetics , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III/biosynthesis , Nitric Oxide Synthase Type III/genetics , Oxidation-Reduction , Receptor, TIE-2/genetics , Transduction, Genetic , Vasculitis/genetics , Vasculitis/metabolism , Vasculitis/prevention & controlABSTRACT
INTRODUCTION: Hypercholesterolemia causes a decrease in normal corporal tissue vasoreactivity in a preclinical model of erectile dysfunction. Previous studies have shown that intracorporal injection (ICI) of basic fibroblast growth factor (bFGF) reverses some of the detrimental vasoreactivity effects of hypercholesterolemia and increases vascular endothelial growth factor (VEGF) expression. AIM: We sought to determine whether the beneficial effects of bFGF are VEGF-mediated. METHODS: A total of 32 New Zealand white rabbits were fed a 1% cholesterol diet for 6 weeks and randomly divided into four groups (N = 8/group). Group 1 received a 2.5 microg bFGF ICI and 2.5 x 10(11) viral particle unit (vpu) of adenovirus encoding beta-galactosidase (Ad beta-gal) ICI, 10 days later. Group 2 received a 2.5 microg bFGF ICI and 2.5 x 10(11) vpu of adenovirus encoding soluble VEGF receptor (VEGFR) (AdsVEGFR, a VEGF trap) ICI, 10 days later. Group 3 received phosphate buffered saline solution (PBS) ICI and 2.5 x 10(11) vpu Ad beta-gal ICI, 10 days later. Group 4 received PBS ICI and 2.5 x 10(11) vpu AdsVEGFR ICI, 10 days later. MAIN OUTCOME MEASURES: The corpus cavernosum was harvested for vasoreactivity studies 10 days post viral injection. The effective dose of 50% maximum relaxation was determined. VEGF levels were assessed by enzyme-linked immunosorbent assay. Total and phosphorylated Akt and endothelial nitric oxide were analyzed by Western blot. RESULTS: Endothelium-dependent vasoreactivity was significantly greater in Group 1 vs. all other groups. The VEGF trap eliminated the beneficial effects of bFGF on endothelium-dependent vasoreactivity and decreased Akt and nitric oxide phosphorylation. CONCLUSIONS: These data demonstrate that VEGF activity contributes much of the therapeutic modulation of bFGF-mediated vasoreactivity in corporal tissue.
Subject(s)
Disease Models, Animal , Fibroblast Growth Factor 2/pharmacology , Hypercholesterolemia/physiopathology , Impotence, Vasculogenic/physiopathology , Penis/blood supply , Vascular Endothelial Growth Factor A/physiology , Vasodilation/drug effects , Animals , Endothelium, Vascular/physiopathology , Gene Transfer Techniques , Injections, Intramuscular , Male , Nitric Oxide Synthase Type III/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Rabbits , Receptors, Vascular Endothelial Growth Factor/genetics , Recombinant Proteins/pharmacology , Vasodilation/physiologyABSTRACT
OBJECTIVE: Tie2 and its ligands, the angiopoietins (Ang), are required for embryonic and postnatal angiogenesis. Previous studies have demonstrated that Tie2 is proteolytically cleaved, resulting in the production of a 75-kDa soluble receptor fragment (sTie2). We investigated mechanisms responsible for Tie2 shedding and its effects on Tie2 signaling and endothelial cellular responses. METHODS AND RESULTS: sTie2 bound both Ang1 and Ang2 and inhibited angiopoietin-mediated Tie2 phosphorylation and antiapoptosis. In human umbilical vein endothelial cells, Tie2 shedding was both constitutive and induced by treatment with PMA or vascular endothelial growth factor (VEGF). Constitutive and VEGF-inducible Tie2 shedding were mediated by PI3K/Akt and p38 MAPK. Tie2 shedding was blocked by pharmacological inhibitors of either PI3K or Akt as well as by overexpression of the lipid phosphatase PTEN. In contrast, sTie2 shedding was enhanced by overexpression of either dominant negative PTEN, which increased Akt phosphorylation, or constitutively active, myristoylated Akt. CONCLUSIONS: These findings demonstrate that VEGF regulates angiopoietin-Tie2 signaling by inducing proteolytic cleavage and shedding of Tie2 via a novel PI3K/Akt-dependent pathway. These results suggest a previously unrecognized mechanism by which VEGF may inhibit vascular stabilization to promote angiogenesis and vascular remodeling.
Subject(s)
Endothelial Cells/metabolism , Neovascularization, Physiologic , Peptide Fragments/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Receptor, TIE-2/metabolism , Signal Transduction , Vascular Endothelial Growth Factor A/metabolism , Angiopoietin-1/metabolism , Angiopoietin-2/metabolism , Animals , Apoptosis , Cells, Cultured , Chromones/pharmacology , Dipeptides/pharmacology , Endothelial Cells/drug effects , Endothelial Cells/enzymology , Endothelial Cells/pathology , Fibroblasts/enzymology , Fibroblasts/metabolism , Humans , Imidazoles/pharmacology , Matrix Metalloproteinase Inhibitors , Matrix Metalloproteinases/metabolism , Mice , Morpholines/pharmacology , NIH 3T3 Cells , Neovascularization, Physiologic/drug effects , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , Peptide Fragments/blood , Peptide Fragments/genetics , Peptide Hydrolases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Pyridines/pharmacology , Receptor, TIE-2/blood , Receptor, TIE-2/genetics , Signal Transduction/drug effects , Tetradecanoylphorbol Acetate/pharmacology , Transfection , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
OBJECTIVES: Our purpose was to determine whether factors that regulate angiogenesis are altered in peripheral arterial disease (PAD) and whether these factors are associated with the severity of PAD. BACKGROUND: Alterations in angiogenic growth factors occur in cardiovascular disease (CVD), but whether these factors are altered in PAD or correlate with disease severity is unknown. METHODS: Plasma was collected from patients with PAD (n = 46) and healthy control subjects (n = 23). Peripheral arterial disease patients included those with intermittent claudication (IC) (n = 23) and critical limb ischemia (CLI) (n = 23). Plasma angiopoietin-2 (Ang2), soluble Tie2 (sTie2), vascular endothelial growth factor (VEGF), soluble VEGF receptor 1 (sVEGFR-1), and placenta growth factor (PlGF) were measured by enzyme-linked immunoadsorbent assay. In vitro, endothelial cells (ECs) were treated with recombinant VEGF to investigate effects on sTie2 production. RESULTS: Plasma concentrations of sTie2 (p < 0.01), Ang2 (p < 0.001), and VEGF (p < 0.01), but not PlGF or sVEGFR-1, were significantly greater in PAD patients compared with control subjects. Plasma Ang2 was significantly increased in both IC and CLI compared with control subjects (p < 0.0001), but there was no difference between IC and CLI. Plasma VEGF and sTie2 were similar in control subjects and IC but were significantly increased in CLI (p < 0.001 vs. control or IC). Increased sTie2 and VEGF were independent of CVD risk factors or the ankle-brachial index, and VEGF treatment of ECs in vitro significantly increased sTie2 shedding. CONCLUSIONS: Levels of VEGF and sTie2 are significantly increased in CLI, and sTie2 production is induced by VEGF. These proteins may provide novel biomarkers for CLI, and sTie2 may be both a marker and a cause of CLI.