Differential Gene Expression in Human Retinal Pericytes in Response to Glucose and Hypoxia

Abstract

Abstract: : Purpose: Diabetic retinopathy is one of the most frequent causes of new blindness in the working age population. There is strong and consistent relationship between hyperglycemia and the incidence and progression of diabetic retinopathy. Early features of non-proliferative diabetic retinopathy include occlusion of small retinal capillaries, resulting in localised ischemia which induces expression of VEGF. Retinal pericytes are smooth muscle-like cells that are associated with the microvascular endothelium. We have investigated differential gene expression in an in vitro model of diabetic retinopathy: a culture of human retinal pericytes in high (30mM) glucose relative to control conditions (5mM glucose) and a combination of glucose and hypoxia (1% O₂). Methods: Human retinal pericytes were cultured in MCDB 131, 5% FBS, 2mM L-glutamine, 100U/ml penicillin and 0.1mg/ml streptomycin. To generate a model of diabetic retinopathy, human retinal pericytes were cultured in 5mM or 30mM glucose for 7 days. Cells were also cultured in 30mM glucose for 5 days, and then exposed to the combination of hypoxia (1% O₂) and 30mM glucose for a further 48 hours, using cells cultured in 5mM glucose as control. RNA was extracted and reverse-transcribed using standard methods. This cDNA was used for TaqMan analysis of VEGF expression. For Affymetrix gene chip analysis samples were prepared exactly as described in Affymetrix protocols and hybridised to the U133A chip. Results: Using real-time PCR to analyse gene expression, exposure of retinal pericytes to 30mM glucose alone for 7 days had no significant effect on VEGF expression. However, exposure of retinal pericytes to hypoxia for 48 hours induced VEGF expression. This increase in VEGF expression was also observed by the combination of 30mM glucose and hypoxia. This model was then subject to microarray analysis to assay for patterns of gene expression. Triplicate biological replicates were analysed. Conclusions: Differentially expressed genes could be classified into functional groups, including, growth factors, apoptosis, angiogenesis, transcriptional regulation, extracellular matrix proteins and cell cycle proteins.

Keywords: diabetic retinopathy • gene/expression • gene microarray