Abstract 5281: Novel Role of p66shc in ROS-dependent VEGF Signaling and Cell-Cell Adhesions in Endothelial Cells

p66shc is an adaptor protein and functions as a major regulator of reactive oxygen species (ROS) production and cardiovascular oxidative stress responses. We demonstrated that VEGF stimulation rapidly increases ROS via activation of NADPH oxidase, which are involved in VEGF receptor 2 (VEGFR2) autophosphorylation as well as endothelial cell (EC) proliferation and migration. However, the role of p66shc in VEGF signaling has not been investigated. Here we show that VEGF stimulation increases serine phosphorylation of p66shc at Ser36 in ECs (3.1-fold at 5mins, 5.4-fold at 15mins). Adenovirus-mediated transfer of mutant p66shc (S36A) or knockdown of p66shc with siRNA decreases both basal- and VEGF-stimulated ROS levels, which results in inhibiting VEGF-induced VEGFR2 autophosphorylation (43.6% and 64.9%, respectively) as well as phosphorylation of p38MAPK (20.2% and 58.5%) and Akt (57.7% and 76.8%). Functionally, p66shc siRNA or overexpression of p66shc (S36A) inhibits VEGF-induced EC migration (58.6% and 50.9%) and proliferation (86.2% and 100%). Of note, immunofluorescence analysis reveals that p66shc is colocalized with and binds to VE-cadherin/beta-catenin complex at cell-cell contacts in confluent ECs, which is required for stabilizing cell-cell adhesions. Depletion of p66shc with siRNA disrupts VE-cadherin-beta-catenin complex, thereby reducing VE-cadherin localization at adherens junction and increasing endothelial permeability (40.8% increase), as measured by transendothelial electric resistance. Moreover, p66shc siRNA markedly reduces basal- and VEGF-stimulated stress fiber formation, which may contribute to a mislocalization of VE-cadherin. In summary, p66shc functions as a key regulator for ROS-dependent VEGF signaling linked to angiogenesis and actin reorganization as well as stabilizing cell-cell junctions. Thus, p66shc is a potential therapeutic target for angiogenesis-dependent cardiovascular diseases. This research has received full or partial funding support from the American Heart Association, Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).