title: Direct sensing of endothelial oxidants by vascular endothelial growth factor receptor-2 and c-Src.
abstract: BACKGROUND
  ADPH oxidase-derived reactive oxygen species (ROS) play important roles in redox homeostasis and signal transduction in endothelial cells (ECs). We previously demonstrated that c-Src plays a key role in VEGF-induced, ROS-dependent selective activation of PI3K-Akt but not PLCγ-1-ERK1/2 signaling pathways. The aim of the present study was to understand how VEGFR-2-c-Src signaling axis 'senses' NADPH oxidase-derived ROS levels and couples VEGF activation of c-Src to the redox state of ECs.
  
  
  RESULTS
  Using biotinylated probe that detects oxidation of cysteine thiol (cys-OH) in intracellular proteins, we demonstrate that VEGF induced oxidative modification in c-Src and VEGFR-2, and that reduction in ROS levels using siRNA against p47(phox) subunit of Rac1-dependent NADPH oxidase inhibited this phenomenon. Co-immunoprecipitation studies using human coronary artery ECs (HCAEC) showed that VEGF-induced ROS-dependent interaction between VEGFR-2 and c-Src correlated with their thiol oxidation status. Immunofluorescence studies using antibodies against internalized VEGFR-2 and c-Src demonstrated that VEGF-induced subcellular co-localization of these tyrosine kinases were also dependent on NADPH oxidsase-derived ROS.
  
  
  CONCLUSIONS
  These results demonstrate that VEGF induces cysteine oxidation in VEGFR-2 and c-Src in an NADPH oxidase-derived ROS-dependent manner, suggesting that VEGFR-2 and c-Src can 'sense' redox levels in ECs. The data also suggest that thiol oxidation status of VEGFR-2 and c-Src correlates with their ability to physically interact with each other and c-Src activation. Taken together, these findings suggest that prior to activating downstream c-Src-PI3K-Akt signaling pathway, VEGFR-2-c-Src axis requires an NADPH oxidase-derived ROS threshold in ECs.
authors.affiliation: Division of Cardiothoracic Surgery, Cardiovascular Research Center, Brown University Warren Alpert Medical School, Rhode Island Hospital, Providence, Rhode Island, United States of America.
authors.forename: Monica
authors.initials: M
authors.lastname: Lee
authors.forename: Wing C
authors.initials: WC
authors.lastname: Choy
authors.forename: Md Ruhul
authors.initials: MR
authors.lastname: Abid
mesh_terms: D015153:Blotting, Western; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D016391:Genes, src / Q000502:physiology*; D006801:Humans; D047468:Immunoprecipitation; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*
chemical_list: D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D019869:Phosphatidylinositol 3-Kinases; D040301:Vascular Endothelial Growth Factor Receptor-2; D051057:Proto-Oncogene Proteins c-akt