Abstract 18457: p66shc Mediates VEGF Receptor Signaling Linked to Production of ROS from Mitochondria and NO from AMPK-eNOS Involved in Endothelial Cell Migration and Proliferation
p66shc is an adaptor protein that is implicated as a major regulator of reactive oxygen species (ROS) involved in aging and cardiovascular diseases. VEGF stimulation increases ROS and nitric oxide (NO), both of which are involved in VEGF receptor2 (VEGFR2) downstream signaling involved in endothelial cell (EC) migration and proliferation. However, the role of p66shc in VEGF signaling and ROS/NO production remain unknown. Here we show that VEGF increases Ser36 phosphorylation of p66shc in human ECs within 5 min (3.1-fold). Adenovirus-mediated transfer of mutant p66shc (S36A) or knockdown of p66shc with siRNA reduces VEGF-stimulated increase in ROS (43.6%), which results in inhibiting VEGF-induced VEGFR2 autophosphorylation (64.9%). VEGF stimulation promotes p66shc translocation from cytosol to the mitochondria where it binds to cytochrome C, which may contribute to ROS production from mitochondria. Of note, overexpression of mitochondria-targeted catalase and p22phox siRNA significantly inhibit VEGF- VEGFR2 autophosphorylation. Moreover, p66shc siRNA, but not p66shc(S36A), significantly inhibits VEGF-induced NO production (73.8%), which is not involved in VEGFR2 autophosphorylation. Thus, pSer36-p66shc is involved in VEGF-induced mitochondrial ROS, but not NO production. Mechanistically, knockdown of p66shc, but not p66shc(S36A), inhibits VEGF-induced phosphorylation of AMPK(pT172)(68.8%) and eNOS(pS1177)(70.5%), which are upstream kinases mediating NO production. Subcellular fractionation reveals that VEGF increases pY-VEGFR2, p-AMPK and p-eNOS at both caveoale/lipid rafts and non-lipid rafts microdomains, and p66shc siRNA inhibits them in non-caveolae/lipid rafts. Functionally, p66shc siRNA or overexpression of p66shc (S36A) inhibits VEGF-induced EC migration (58.6% and 50.9%) and proliferation (86.2% and 90%). In vivo, p66shc expression is increased (1.5-fold) in a mouse hindlimb ischemia model. In summary, p66shc functions as a positive regulator for VEGF-induced angiogenic responses in ECs by mediating production of ROS from mitochondria and NO from AMPK-eNOS localized at non-lipid rafts signaling domains. Thus, p66shc is a potential therapeutic target for angiogenesis-dependent cardiovascular diseases.
- © 2010 by American Heart Association, Inc.