Abstract 4343242: CXCL1 and CXCL2 act downstream to ATF6 and GRP78 in hypoxia-induced retinal neovascularization

Abstract

Background: Retinal neovascularization is a significant contributor of vision loss. One of the chemokines found to be elevated in the vitreous samples of diabetic retinopathy patients is IL-8. However, its role in retinal neovascularization has never been investigated. Hypothesis: Based on these clues, we hypothesize that IL-8 could be one the major factors in the mediation of retinal neovascularization. As mice express CXCL1 and CXCL2, the orthologues of human IL-8, the objective of this study is to test the role of CXCL1/2 in hypoxia-induced retinal neovascularization. Methods: Cellular and molecular approaches such as cell migration, proliferation, sprouting and tube formation assays in vitro and oxygen-induced retinopathy (OIR) mouse model in vivo were used. Results: We found that VEGFA induces CXCL1/2 expression both at mRNA and protein levels in mouse retinal microvascular endothelial cells (MRMVECs) in a time-dependent manner. CXCL1 and CXCL2 while alone were able to trigger MRMVEC migration, proliferation, sprouting and tube formation, in combination, they showed additive effects. Neutralizing CXCL1 or CXCL2 antibodies blunted CXCL1 and CXCL2-induced angiogenic responses of MRMVECs, alone or in a combinatorial manner. Neutralizing CXCL1/2 antibodies also blocked VEGFA-induced MRMVEC migration, proliferation, sprouting and tube formation. In line with these observations, hypoxia induced CXCL1/2 expression in the mouse retina and blockade of their function with their neutralizing antibodies inhibited hypoxia-induced retinal neovascularization. Besides, VEGFA induced IL-8 expression in human retinal microvascular endothelial cells (HRMVECs) and neutralizing IL-8 antibodies suppressed these effects. In exploring the mechanisms, we found that depletion of ATF6 or GRP78 levels blocked VEGFA-induced CXCL1/2 expression in MRMVECs and IL-8 induction in HRMVECs in vitro and hypoxia-induced CXCL1/2 expression in the retina in vivo. In addition, we observed that downregulation of either VEGFR2 or VEGFR3 levels by their respective siRNAs inhibited VEGFA-induced ATF6-GRP78-mediated CXCL1/2 expression in MRMVECs and IL-8 expression in HRMVECs. Conclusion: Our findings reveal that CXCL1/2 and their human orthologue IL-8 play an important role in the regulation of retinal neovascularization and suggest that their neutralizing antibodies could offer as potential therapeutic molecules against this ocular disease.