VEGFR2-dependent VEGFR3 expression leads to CXCL1/2 and CXCR2 induction in the development of retinal neovascularization

Elevated levels of IL-8 have been detected in the vitreous samples of patients with diabetic retinopathy. However, its role in retinal neovascularization remains unknown. We hypothesized that IL-8 and its murine orthologues, CXCL1/2, are key mediators of oxygen-induced retinopathy (OIR)-driven retinal angiogenesis. To this end, we found that VEGFA stimulates CXCL1/2 and their receptor CXCR2 expression in mouse retinal microvascular endothelial cells (MRMVECs) in a time-dependent manner. Recombinant CXCL1/2 induced MRMVEC migration, proliferation, sprouting, and tube formation alone and in combination. Neutralizing CXCL1/2 or CXCR2 antibodies (Abs) attenuated the angiogenic effects of CXCL1/2 as well as VEGFA. In line with these observations in mice, VEGFA induced IL-8 and its receptor CXCR2 expression in human retinal microvascular endothelial cells (HRMVECs), and neutralizing IL-8 or CXCR2 Abs suppressed their angiogenic effects. Furthermore, OIR induced CXCL1/2 and CXCR2 expression in the mouse retina in vivo, and blockade of their function using their neutralizing Abs reduced retinal neovascularization. Mechanistically, depletion of ATF6 or GRP78 levels abrogated VEGFA-induced CXCL1/2 and CXCR2 expression in MRMVECs and IL-8 and CXCR2 expression in HRMVECs in vitro and OIR-induced CXCL1/2 and CXCR2 expression in vivo in mice. Furthermore, silencing VEGFR2 or VEGFR3 levels inhibited VEGFA/OIR-induced ATF6-GRP78 activation and their downstream CXCL1/2, IL-8 and CXCR2 expression both in vitro and in vivo. Together, these findings identify IL-8 as potential therapeutic target for treating retinal neovascular diseases.