title: Chronic hypoxia attenuates VEGF signaling and angiogenic responses by downregulation of KDR in human endothelial cells.
abstract: Coronary artery disease results in progressive vascular stenosis associated with chronic myocardial ischemia. Vascular endothelial growth factor (VEGF) stimulates endothelial cell angiogenic responses to revascularize ischemic tissues; however, the effect of chronic hypoxia on the responsiveness of endothelial cells to VEGF remains unclear. We, therefore, investigated whether hypoxia alters VEGF-stimulated signaling and angiogenic responses in primary human coronary artery endothelial (HCAE) cells. Exposure of HCAE cells to hypoxia (1% O(2)) for 24 h decreased VEGF-stimulated endothelial cell migration ( approximately 82%), proliferation ( approximately 30%), and tube formation. Hypoxia attenuated VEGF-stimulated activation of endothelial nitric oxide (NO) synthase (eNOS) ( approximately 72%) and reduced NO production in VEGF-stimulated cells from 237 +/- 38.8 to 61.3 +/- 28.4 nmol/l. Moreover, hypoxia also decreased the ratio of phosphorylated eNOS to total eNOS in VEGF-stimulated cells by approximately 50%. This effect was not observed in thrombin-stimulated cells, suggesting that hypoxia specifically inhibited VEGF signaling upstream of eNOS phosphorylation. VEGF-induced activation of Akt, ERK1/2, p38, p70S6 kinases, and S6 ribosomal protein was also attenuated in hypoxic cells. Moreover, VEGF-stimulated phosphorylation of VEGF receptor-2 (KDR) at Y996 and Y1175 was decreased by hypoxia. This decrease correlated with a 70 +/- 12% decrease in KDR protein expression. Analysis of mRNA from these cells showed that hypoxia reduced steady-state levels of KDR mRNA by 52 +/- 16% and decreased mRNA stability relative to normoxic cells. Our findings demonstrate that chronic hypoxia attenuates VEGF-stimulated signaling in HCAE cells by specific downregulation of KDR expression. These data provide a novel explanation for the impaired angiogenic responses to VEGF in endothelial cells exposed to chronic hypoxia.
authors.affiliation: Dept. of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, TX 77555-0645, USA.
authors.forename: Barbara
authors.initials: B
authors.lastname: Olszewska-Pazdrak
authors.forename: Travis W
authors.initials: TW
authors.lastname: Hein
authors.forename: Paulina
authors.initials: P
authors.lastname: Olszewska
authors.forename: Darrell H
authors.initials: DH
authors.lastname: Carney
mesh_terms: D000328:Adult; D002196:Capillaries / Q000166:cytology / Q000378:metabolism; D002455:Cell Division / Q000502:physiology; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D002908:Chronic Disease; D003331:Coronary Vessels / Q000166:cytology; D015536:Down-Regulation / Q000502:physiology; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D000860:Hypoxia / Q000378:metabolism* / Q000473:pathology; D017202:Myocardial Ischemia / Q000378:metabolism* / Q000473:pathology; D018919:Neovascularization, Physiologic / Q000502:physiology*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D040301:Vascular Endothelial Growth Factor Receptor-2; D051057:Proto-Oncogene Proteins c-akt; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases