Activation of cytosolic phospholipase A2 downstream to Src-PLD1-PKCγ signaling axis is required for hypoxia-induced pathological retinal angiogenesis

Abstract

Towards understanding the mechanisms of retinal neovascularization, previously we have reported that VEGF-induced pathological retinal angiogenesis requires activation of Src-PLD1-PKCγ signaling. In the present work, we have identified cytosolic phospholipase A2 (cPLA2) as an effector molecule of Src-PLD1-PKCγ signaling in the mediation of VEGF-induced pathological retinal angiogenesis based on the following observations: VEGF induced cPLA2 phosphorylation in a time-dependent manner in human retinal microvascular endothelial cells (HRMVECs). VEGF also induced arachidonic acid (AA) release in a dose-, time- and cPLA2-dependent manner. Depletion of cPLA2 levels inhibited VEGF-induced HRMVEC DNA synthesis, migration and tube formation. In addition, exogenous addition of AA rescued VEGF-induced HRMVEC DNA synthesis, migration and tube formation from inhibition by downregulation of cPLA2. Inhibition of Src, PLD1 or PKCγ attenuated VEGF-induced cPLA2 phosphorylation and AA release. Consistent with these findings, hypoxia induced cPLA2 phosphorylation and activity in VEGF-Src-PLD1-PKCγ -dependent manner in a mouse model of oxygen-induced retinopathy (OIR). In addition, siRNA-mediated downregulation of cPLA2 levels in retina abrogated hypoxia-induced retinal endothelial cell proliferation and neovascularization. These observations suggest that cPLA2-dependent AA release is required for VEGF-induced Src-PLD1-PKCγ -mediated pathological retinal angiogenesis.