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P. C. Pereira, C. Bento, R. Fernandes, A. Fernandes, A. Taylor, F. Shang; Methylglyoxal-Induced Degradation of HIF-1a: Implications to Diabetic Retinopathy. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1378.
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Intracellular production and accumulation of glycating agents such as methylglyoxal (MGO) is a main feature of many pathological complications associated with diabetes including diabetic retinopathy.This study is designed to assess the role of methylglyoxal in degradation of HIF-1a and implications on production of VEGF by human retinal-pigmented epithelial cells (RPE)
ARPE-19 cells were submitted to hypoxic conditions (CoCl2 or 2%O2) in the presence or absence of MGO. The role of the proteasome on MGO-induced degradation of HIF-1a was assessed by incubating the cells with proteasome inhibitors (MG132, PS-341). To assess whether degradation of HIF-1a requires prior ubiquitinylation of the protein, HIF-1a was immunoprecipitated and Western blots were probed for ubiquitin conjugates. To assess the requirement for prolyl hydroxylation and ligase Von Hippel-Lindau (VHL) protein, RPE cells were transfected either with the wild type (wt-HIF-1a) or the prolyl hydroxylation mutant (P402/564A) and treated as described above. Levels of VEGF released into the medium were determined by ELISA.
In RPE, MGO strongly destabilizes HIF-1a by promoting increased degradation of HIF-1a by an ubiquitin-proteasome dependent mechanism. Significantly, the MGO mediated degradation of HIF-1a is independent on the ubiquitin ligase Von Hippel-Lindau (VHL) protein and does not require prior hydroxylation of P402 and P564 as revealed by the sensitivity of the P402/564A-HIF-1a to MGO-dependent degradation. Increased degradation of HIF-1a resulted in decreased production of VEGF by RPE.
Increased degradation of HIF-1a in the presence of MGO is likely to compromise retina ability to cope with hypoxia at early stages of diabetic retinopathy thus leading to increased endothelial cell death and pericytes dropout.
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