VEGFR2 signalling contributes to increased endothelial susceptibility to TNF-α under chronic non-uniform shear stress

ObjectivesVascular endothelial growth factor receptor 2 (VEGFR2), a tyrosine kinase receptor activated by VEGF and shear stress, is critically involved in endothelial mechanotransduction. We investigated the role of VEGFR2 in non-uniform shear stress-induced endothelial susceptibility to inflammatory stimuli.MethodsEndothelial cells (ECs) were exposed to non-uniform shear stress, followed by stimulation with TNF-α. ECs were transfected with siRNAs against VEGFR2. Alternatively, ECs were treated with blocking antibody against VEGFR2, or with inhibitors of VEGFR2 (ZM 323881), PI3K (LY 294002), or Src-kinase (PP2). THP-1 monocytes were used for dynamic adhesion assays. Endothelial protein expression was determined by immunofluorescence.ResultssiRNA against VEGFR2 decreased VEGFR2 protein expression by 40% as determined by Western blotting. In endothelial cells exposed to non-uniform shear stress, VEGFR2 knockdown inhibited TNF-α-induced NF-κB translocation to the nucleus, and the upregulation of VCAM-1 and E-selectin. Consequently, monocytic cell recruitment to endothelium under non-uniform shear stress conditions was reduced. Similar effects were observed by blocking VEGFR2 activity using a specific inhibitor ZM 323881, or an antibody against VEGFR2 before TNF-α stimulation. Inhibition of PI3K with LY 294002 significantly reduced non-uniform shear stress-induced endothelial susceptibility to TNF-α, whereas blocking Src-kinase with PP2 was ineffective.ConclusionVEGFR2 is critically involved in adhesion molecule induction and monocytic cell recruitment to endothelium in response to non-uniform shear stress and TNF-α. Targeting the mechanosensory cascade can prevent endothelial activation in atherosclerosis-prone regions.