Glucose Induction of Apoptosis in Human Retinal Pericytes

Abstract

Abstract: : Purpose:Diabetic Retinopathy (DR) is among the most common diabetic complications. Currently approximately 4.1 million adults over the age of 40 in the U.S. suffer from DR and by 2020 this is projected to grow to 7.1 million. The first histopathological feature of DR is pericyte loss. Clinical manifestations include microaneurysms, hemorrhages and formation of regions of acceluarity. In the diabetic retina pericytes which die leave behind vacant spaces. We decided to investigate the possibility that this cell death could be induced by exposure to phsyiologicaly relevant high levels of glucose. It is well documented that conformational changes in Bax and Bak can lead to pore formation in the outer mitochondrial membrane, allowing the release of cytochrome C into the cytosol. Apoptosome formation can then take place resulting in activation of caspase–9, cleavage of caspase–3 and ultimately cell death by apoptosis. Methods:Human retinal pericytes were cultured in MCDB 131, 10% FBS, 2mM L–Glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin. On becoming confluent these cells were serum restricted (0.5% FBS) for 24 hrs. Serum restricted cells were then cultured in 5mM to 40mM glucose in increments of 5mM for 24 hrs in media containing 0.5% FBS. Total RNA was extracted using Tri–Reagent^TM and reverse transcribed using standard protocols. Following exposure of the serum restricted cells to the various glucose concentrations for 48 hrs. Annexin V staining and PI (viability) uptake were measured using flow cytometry as indicators of apoptosis. Results:Bax, Bak and Bid mRNA expression was detected with maximal levels of expression at 30mM glucose. Cleaved caspase–3 levels were also seen to increase following treatment with increasing glucose concentrations peaking at approximately 30mM glucose following 48 hrs exposure. Annexin V staining and PI uptake were seen to increase with increasing glucose concentration, peaking at approximately 30 mM gluucose. Conclusions:These results demonstrate that in high glucose conditions pro–apoptotic genes upregulated in human retinal pericytes within a time period of 24 hrs. Following 48 hrs exposure to glucose a rise in cleaved caspase–3 levels are detected which appears to correlate with the rise in the upstream mediators of the caspase–3 cascade. Annexin V staining detects earlier apoptotic changes than PI uptake, explaining why it is seen to peak at slightly lower glucose concentrations.

Keywords: gene/expression • apoptosis/cell death • diabetic retinopathy