Involvement of Complement Activation and its Regulation in the Pathogenesis of Diabetic Retinopathy

Abstract

Purpose : Abnormal deposition of complement components in the vitreous and RPE-Choroid have been documented in several neovascular conditions. Proliferative diabetic retinopathy (PDR) shares common features of increased inflammation and abnormal vascularization with these conditions, but the involvement of complement pathways has always been debatable. Based on these lacunae,the present study was designed to comprehensively understand the contributions of complement pathway in the pathogenesis of PDR.

Methods : The study was based on the vitreous humor (n= 100) and serum samples(n= 50) collected from PDR patients and controls comprising subjects with macular holes (MH) and retinal detachments (RD). Proteins in the vitreous were separated on single dimension polyacrylamide gel followed by in-gel trypsin digestion and subjected to label free mass spectroscopy (LC-MS/MS). The complement proteins observed in the vitreous were validated by western blotting. Immunohistochemistry of the diabetic and non-diabetic post-mortem eyes were done to check the deposition of complement components in the retina. Further, a validation of differential expression of complement components in DR was done using microglia subjected to hypoxic and hyperglycemic conditions.

Results : Vitreous proteome analysis revealed a total of 1071 proteins of which,549 were shared among all the three categories (PDR, MH and RD) while 58 proteins were exclusive to PDR. While there were no significant differences in complement activation (based on LC-MS/MS and western blotting of complement C3,C4 and C5 protein fragments), a significant increase in Complement factor H (p<0.05) and down regulation of CFB was noted in PDR. The diabetic retina showed an increased expression of the complements and glial specific proteins (C3, CFH, CD11B, CXCR4 and GFAP).

Conclusions : Co-localization of C3 along with microglial specific marker CD11b in the diabetic retina indicated microglia-mediated complement activation in diabetic eyes. Further, an increased expression of CFH that regulates the alternate complement pathway in both vitreous humor of PDR eyes and diabetic retina suggested its role in regulating the microglia-mediated complement activation.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.