Comparative vitreous proteomic profiling of proliferative diabetic retinopathy and diabetic with no-retinopathy subjects implicates impaired autophagy in DR pathogenesis

Abstract

PurposeDiabetic retinopathy (DR) is a neurovascular complication of diabetes (DM) causing the loss of neurons (ganglion cells) in the retina. The disease etiology and potential pathogenic mechanisms in this disease remains unclear. In the present study, we aimed to further understand the key and novel pathogenic mechanisms involved in DR pathogenesis by taking cues from our global proteomics data. MethodologyThe study was approved by the institutional review board (IRB) of LVPEI, Hyderabad, India. Vitreous humour samples (PDR; n=3, DM; n=3, Control; n=3) were collected from patients undergoing vitrectomy and subjected to LC-MS-MS analysis. The acquired raw data were searched against the human vitreous proteome and was further analysed by various bioinformatic and proteomic tools. Western blotting and IHC was performed to validate crucial pathways. Blood samples from patients (DM, PDR & NPDR) and controls (n=50); cadaveric retinas from diabetic and non-diabetic donors (n=10) and epiretinal membranes (ERM, n=10) from DR cases and controls were collected and RNA was isolated. Quantitative expression of genes involved in autophagy were performed. CT was compared across different categories and significance estimated using a student t-test. ResultA total of 1079 proteins were identified with 16 completely novel proteins in eye proteome. Top pathways identified were autophagy, inflammation, LXR/RXR activation (lipid metabolism), ROS generation by macrophages, apoptosis and protein degradation. Regulatory proteins identified were associated with cell death, phagocytic activation, angiogenesis and apoptosis. Autophagy inducers such as ROS was found to be accumulated in the DR vitreous. TREM2, microglial receptor was identified as a novel protein in PDR vitreous. The expression of TREM2, an autophagy-associated gene was significantly (p-value = 0.05) upregulated in all categories as compared to control (NDM and/or NDM/No-DR). TREM2 protein also seemed to colocalise with microglial marker F4/80 in retinal tissues and intense expression was observed near the blood vessels in case of PDR retina. Other autophagy-associated markers were also differentially regulated in DR as compared to controls. ConclusionThis study emphasises on the strong role of autophagy pathways and its associated genes in the development of DR.