Activation of AMP-activated protein kinase by vascular endothelial growth factor mediates endothelial angiogenesis independently of nitric-oxide synthase.
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 Ca(2+)/calmodulin-dependent protein kinase kinase beta (CaMKKbeta). 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 CaMKKbeta. VEGF-induced AMPK activation is potentiated under conditions of energy deprivation induced by 2-deoxyglucose. To investigate the role of AMPK in endothelial function, CaMKKbeta, AMPKalpha1, or AMPKalpha2 was down-regulated by RNA interference, and studies in AMPKalpha1(-/-) 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 AMPKalpha1- and AMPKalpha2-dependent manner. Our results show that AMPKalpha1 plays an essential role in VEGF-induced angiogenesis in vitro (tube formation and sprouting from spheroids) and in vivo (Matrigel plug assay). In contrast, AMPKalpha2 was not involved in VEGF-triggered sprouting. The data suggest that AMPKalpha1 promotes VEGF-induced angiogenesis independently of eNOS, possibly by providing energy via inhibition of acetyl-CoA carboxylase.
Institute for Molecular Cell Biology, Center for Molecular Biomedicine, Friedrich Schiller University of Jena, 07745 Jena, Germany.
Nadine
N
Stahmann
Angela
A
Woods
Katrin
K
Spengler
Amanda
A
Heslegrave
Reinhard
R
Bauer
Siegfried
S
Krause
Benoit
B
Viollet
David
D
Carling
Regine
R
Heller
D055372:AMP-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D003847:Deoxyglucose / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D008168:Lung / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D018919:Neovascularization, Physiologic; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction; D018874:Spheroids, Cellular / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
D000963:Antimetabolites; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; D009569:Nitric Oxide; D003847:Deoxyglucose; D052250:Nitric Oxide Synthase Type III; D040301:Vascular Endothelial Growth Factor Receptor-2; C548179:Prkaa1 protein, mouse; C534978:Prkaa2 protein, mouse; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D055372:AMP-Activated Protein Kinases; D010738:Type C Phospholipases; D002118:Calcium