VASCULAR BIOLOGY Both Kdr and Flt1 play a vital role in hypoxia-induced Src-PLD1-PKCg-cPLA2 activation and retinal neovascularization

Ischemic retinal diseases such as diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity may precipitate retinal neovascularization. Pathological retinal angiogenesis, in turn, may cause vitreous hemorrhage, retinal detachment, and/or neovascular glaucoma affecting vision. Among the many molecules produced by the hypoxic retina, vascular endothelial growth factor A (VEGFA) is a potent angiogenic and vascular permeability factor. VEGFA induces angiogenesis via its ability to stimulate growth, migration, and capillary-like structure formation of endothelial cells (ECs) and enhance their permeability. Among the 5 VEGF molecules identified, VEGFA and VEGFD are reported to be the most potent angiogenic factors. Similarly, among the 3 VEGF receptors characterized thus far, namely Flt1, Kdr, and Flt4 (also known as VEGFR1, VEGFR2, and VEGFR3, respectively), VEGFA binds to both Flt1 and Kdr. On the other hand, VEGFB and VEGFD bind only to Flt1, and VEGFC and VEGFE bind to Kdr as well as Flt4. Although VEGFA binds to both Flt1 and Kdr, its cellular effects appear to be predominantly mediated by Kdr in ECs. It was further suggested that even though VEGFA binds to Flt1 with high affinity, activation might not occur because the receptor shows only weak kinase activity. Despite its weak kinase activity in response to VEGFA, Flt1 has been shown to suppress tip-cell formation and its deletion led to embryonic lethality from excessive vessel overgrowth, suggesting that this receptor negatively affects angiogenesis, particularly developmental angiogenesis, although some investigations dispute this assertion. Recent studies, however, have demonstrated that Flt1 still could mediate some cellular effects of VEGFA such as EC migration. In addition, the development of antiangiogenic therapies targeting Flt1 for the treatment of tumors and ischemic diseases suggests that Flt1 does play a role in angiogenesis, at least in pathological angiogenesis. However, due to functional redundancies among these various VEGF molecules and the complexities in their receptor selectivity, antiangiogenic therapies targeting VEGFA or its receptors, Kdr or Flt1, though effective significantly, appear to be inadequate, because resistance to these therapies develops in both tumors and ischemic diseases. Thus, these inadequacies in current antiangiogenic therapies necessitate further studies to identify the comprehensive mechanisms by which these VEGF molecules and other factors modulate pathological angiogenesis. We have previously demonstrated that VEGFA activates SrcPLD1-PKCg-cPLA2 signaling in human retinal microvascular endothelial cells (HRMVECs), facilitating their growth, migration, and tube formation and, in the mouse retina, mediating hypoxiainduced neovascularization. Because our previous findings showed that activation of Src-PLD1-PKCg-cPLA2 signaling is required for VEGFA-induced angiogenic effects both in vitro and in vivo, we questioned which VEGF receptor(s) mediates this signaling axis in ECs and retina. In this communication, we report