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R.H. Nagaraj, A. Miller, D.G. Smith; Glyoxalase I Activity is Critical for Human Retinal Capillary Pericyte Survival Under Hyperglycemic Conditions . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4237.
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© ARVO (1962-2015); The Authors (2016-present)
Loss of pericytes in retinal capillaries is an early stage event in diabetic retinopathy. We investigated whether the glyoxalase system, which detoxifies reactive alpha–oxoaldehydes, protects human retinal pericytes (HRP) against apoptosis when cultured in high concentrations of glucose.
HRP were cultured with 25 mM D– or L–glucose in the presence or absence bromobenzyglutathione cyclopentyldiester, a glyoxalase I inhibitor (BBGC, 35 microM) or an NO donor, DETANONOATE (1 mM) for 7 days. Glyoxalase I activity was assessed by monitoring the formation of S–lactoylglutathione at 240 nm, and expression was determined by QPCR. Measurement of methylglyoxal (MGO) was performed by HPLC. Apoptosis was analyzed by flow cytometry.
A dramatic increase in MGO was observed in the 25 mM D–glu + BBGC treated HRP (121 pmol/mg protein) compared either L–glu + BBGC or BBGC only treated cells (87 and 85 pmol/mg protein respectively, P < 0.01). Significant increases in both early and late apoptosis was observed in D–glu + BBGC treated HRP relative to L–glu + BBGC (12 % vs 7%, P 0.0001). D–glu only treated HRP exhibited an increase in MGO but no increase in apoptosis. Incubation of HRP with 1 mM DETANONOATE caused significant decreases in glyoxalase I activity (37%, P < 0.001) and expression (60%, P < 0.0001) and increased early apoptosis (9%, P < 0.01) relative to the untreated control (6%) and was exacerbated on co–incubation with D–glu (11%, 0.002 vs L–glu DETANONOATE). We also report here that NO can be elevated on incubation of HRP with D–glu (3.5 pmol/mg protein) compared to either the untreated or osmotic control (2.4 and 1l.8 pmol/mg protein respectively, P < 0.01).
These results suggest that under high glucose conditions, glyoxalase I activity is decreased by downregulation, and inactivation by nitric oxide and that glyoxalase I activity is essential for protecting HRP against apoptosis.
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