Activation of AMP-activated Protein Kinase by Vascular Endothelial Growth Factor Mediates Endothelial Angiogenesis Independently of Nitric-oxide Synthase

• Published in: The Journal of biological chemistry Vol. 285; no. 14; pp. 10638 - 10652
• Main Authors: Stahmann, Nadine, Woods, Angela, Spengler, Katrin, Heslegrave, Amanda, Bauer, Reinhard, Krause, Siegfried, Viollet, Benoit, Carling, David, Heller, Regine
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02.04.2010; American Society for Biochemistry and Molecular Biology
• Subjects: AMP-activated Protein Kinase (AMPK); AMP-Activated Protein Kinases - antagonists & inhibitors; AMP-Activated Protein Kinases - genetics; AMP-Activated Protein Kinases - metabolism; Animals; Antimetabolites - pharmacology; Blotting, Western; Calcium - metabolism; Calcium-Calmodulin-Dependent Protein Kinase Kinase - antagonists & inhibitors; Calcium-Calmodulin-Dependent Protein Kinase Kinase - genetics; Calcium-Calmodulin-Dependent Protein Kinase Kinase - metabolism; Cell Biology; Cells, Cultured; Deoxyglucose - pharmacology; Endothelium; Endothelium, Vascular - cytology; Endothelium, Vascular - drug effects; Endothelium, Vascular - metabolism; Energy Metabolism; Female; Fluorescent Antibody Technique; Humans; Lung - cytology; Lung - drug effects; Lung - metabolism; Mice; Mice, Knockout; Neovascularization, Physiologic; Nitric Oxide; Nitric Oxide - metabolism; Nitric Oxide Synthase Type III - metabolism; Nitric-oxide Synthase; Phosphorylation; RNA, Small Interfering - pharmacology; Serine Threonine Protein Kinase; Signal Transduction; Spheroids, Cellular - metabolism; Type C Phospholipases - metabolism; Umbilical Veins - cytology; Umbilical Veins - drug effects; Umbilical Veins - metabolism; Vascular Endothelial Growth Factor; Vascular Endothelial Growth Factor A - metabolism; Vascular Endothelial Growth Factor Receptor-2 - metabolism; Energy Metabolism; AMP-activated Protein Kinase (AMPK); Nitric-oxide Synthase; Vascular Endothelial Growth Factor; Nitric Oxide; Serine Threonine Protein Kinase; Endothelium
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.108688

AMP-activated protein kinase (AMPK) is a sensor of cellular energy state and a regulator of cellular homeostasis. In endothelial cells, AMPK is stimulated via the upstream kinases LKB1 and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ). Previously, AMPK has been reported to activate endothelial nitric-oxide synthase (eNOS). Using genetic and pharmacological approaches, we show that vascular endothelial growth factor (VEGF) stimulates AMPK in human and mice endothelial cells via CaMKKβ. VEGF-induced AMPK activation is potentiated under conditions of energy deprivation induced by 2-deoxyglucose. To investigate the role of AMPK in endothelial function, CaMKKβ, AMPKα1, or AMPKα2 was down-regulated by RNA interference, and studies in AMPKα1−/− mice were performed. We demonstrate that AMPK does not mediate eNOS phosphorylation at serine residue 1177 or 633, NO- dependent cGMP generation, or Akt phosphorylation in response to VEGF. Using inhibitors of eNOS or soluble guanylyl cyclase and small interfering RNA against eNOS, we show that NO does not act upstream of AMPK. Taken together, these data indicate that VEGF-stimulated AMPK and eNOS pathways act independently of each other. However, acetyl-CoA carboxylase, a key enzyme in the regulation of fatty acid oxidation, was phosphorylated in response to VEGF in an AMPKα1- and AMPKα2-dependent manner. Our results show that AMPKα1 plays an essential role in VEGF-induced angiogenesis in vitro (tube formation and sprouting from spheroids) and in vivo (Matrigel plug assay). In contrast, AMPKα2 was not involved in VEGF-triggered sprouting. The data suggest that AMPKα1 promotes VEGF-induced angiogenesis independently of eNOS, possibly by providing energy via inhibition of acetyl-CoA carboxylase.