Glyoxalase I Plays a Critical Role in the Prevention of High Glucose‐Induced Apoptosis in Human Retinal Pericytes

Abstract

Objective We investigated whether the glyoxalase system protects human retinal pericytes (HRP) against cell death when cultured in high concentrations of glucose. This hypothesis was probed using a glyoxalase I inhibitor, bromobenzylglutathione cyclopentyl diester (BBGC) and a nitric oxide donor (DETANONOATE). Methods HRP were isolated and cultured in low glucose DMEM, 10% FBS and 1% PenStrep, with the appropriate treatment added. Apoptosis was measured by flow cytometry (Annexin-FITC and Propidium Iodide staining). Methylglyoxal (MGO) was quantified by HPLC. Nitric oxide (NO) was quantified using Griess reagent. Results A slight 1.4 fold increase in apoptosis of 25 mM D-glucose (D-glu)-treated HRP was observed relative to the controls. BBGC inclusion caused a dramatic increase in cell death (4.4 fold, P = 0.05). Methylglyoxal (a substrate of glyoxalase I) increased in D-glu-treated HRP (P = 0.026). BBGC inclusion exacerbated this increase (121 vs 89.5 pmol MGO/mg protein, P = 0.05). Treatment of HRP with D-glu resulted in elevated NO compared to the control (1.77 vs 0.91 nmol/mg protein, P = 0.008). Studies with DETANONOATE demonstrated both a down-regulation of transcription and a decrease in glyoxalase I activity in HRP. Furthermore, incubation of HRP with 1 mM DETANONOATE resulted in elevated apoptosis over the control (9.05 vs 6.14%, P = 0.0028). Co-incubation with D-glu increased this value (11.02%, P = 0.0021). Conclusion Under high glucose conditions, glyoxalase I is essential for protecting against HRP death. Nitrosative stress downregulates its expression, and may have significant consequences for glyoxalase I-mediated protection of HRP.