Inflammatory markers in diabetic macular

Abstract

Purpose Complications of diabetic retinopathy, such as macular edema, appear to be generated by multiple inflammatory factors that affect the retinal microcirculation. To elucidate the cell types and mechanisms underlying diabetic complications, human retinal microvascular pericytes (HRMP), monocytes (THP‐1), and retinal endothelial cells (HREC) were treated with either high glucose, TNF‐_‐ or IL‐1_, and protein secretion was measured in the presence or absence of dexamethasone (DEX). In addition, retinal levels of several inflammatory proteins were measured in an animal model of diabetes over a 3 month study period. Methods Cells were incubated for 5 hr (THP‐1) or 24 hr (HRMP, HREC) with medium, TNF‐_‐ (10 ng/ml) or IL‐1_ (10 ng/ml), high glucose (25 mM) in the presence or absence of dexamethasone (10 nM–1 µM). Sprague Dawley rats were rendered diabetic by intraperitoneal STZ administration (65 mg/kg; blood glucose >220 mg/dl after 48h) and age‐matched control rats were sacrificed 7 days, 4 weeks, and 1 month after STZ injection. Eyes were enucleated, snap frozen in liquid nitrogen and stored at ‐70°C. In both cell and retinal assays, protein levels (89 for human, 68 for rat) were measured by Rules Based Medicine using their Luminex‐baed human and rodent antigen panels. Results Compared to control responses, TNF‐_ or IL‐1_ induced a five‐fold or more increase in several inflammation‐associated proteins in each cell type. The number of mediators and extent of increased secretion were greatest in HRMP (≥ five‐fold increase in 33 proteins with TNF‐_ and 29 proteins with IL‐1_). In HRMP and THP‐1 cells, DEX inhibited the secretion of several inflammation‐associated proteins in a dose‐dependent manner. The IC50 for DEX inhibition ranged from 2 nM for some proteins to 1 μM for others, and this differential effect was dependent on cell type and inflammatory stimulator. Of 68 proteins measured in diabetic rat retinae, 9 were significantly elevated at 3 months including beta‐2 macroglobulin, eotaxin, FGF‐2, MCP‐1, MCP‐3, M‐CSF, NGAL, osteopontin, and TIMP‐1. At this time, there was a reproducible but not significant decrease in VEGF expression. Conclusion Our results support the hypothesis that the early stages of diabetic retinopathy are associated with a subclinical inflammatory response and point to microvascular pericytes as a primary source of these mediators.