Activation of Prolyl Hydroxylase-2 Prevented High Glucose-Induced Endothelial Barrier Dysfunction

Abstract

Purpose: To investigate the role of prolyl hydroxylase-2 (PHD-2) which catalyzes degradation of HIF-1α in high glucose-induced endothelial barrier dysfunction in human retinal vascular endothelial cells (HRECs).

Methods: Cultured cells were pretreated with PHD2 activator (R59949) or inhibitor (DMOG) for 1 hour followed by treatment with 30mM high glucose for 72 hours. Mannitol at 30mM was used as osmotic control. Paracellular permeability assay was measured by using FITC- dextran 70 kDa. Protein levels and arrangement pattern of tight junctions (TJs) including occludin and zona occludens protein-1 (ZO-1) were observed by Western blot and immunofluorescence. The expression of PHD-2, hypoxia-induced factor (HIF)-1α and vascular endothelial growth factor (VEGF) were detected by Western blot or ELISA.

Results: Compared with control, high glucose increased the paracellular permeability of HRECs by 30.8 % (P<0.05). Expression of occludin and ZO-1 declined by 42.0% (P<0.01) and 50.0% (P<0.01) associated with disrupted continuity in cells exposed to high glucose. High glucose up-regulated the concentration of VEGF in the supernatant and the protein level of HIF-1α by 57.6% (P<0.01) and 123.0% (P<0.01) respectively, while down-regulated the expression of PHD-2 by 53.4% (P<0.01). Mannitol at 30mM also induced significant alterations in paracellular permeability and the expression of TJs, PHD-2, HIF-1α and VEGF (P<0.05). Compared with high glucose group, R59949 ameliorated the paracellular permeability by 60.3% (P<0.01) and moderated the interrupted distribution of occludin and ZO-1 induced by high glucose. Expression of HIF-1α and VEGF were 52.1% and 31.4% less than that of high glucose group, while protein level of PHD-2 increased by 66.1% (P<0.01). In contrast, DMOG substantially aggravated the high glucose-induced permeability by 20.5% with more severely damaged TJs. Additionally, HIF-1α and VEGF were elevated by 27.3% and 108.8% compared with high glucose group (P<0.05), whereas PHD-2 was down-regulated further by 13.5% (P<0.05).

Conclusions: Activation of PHD-2 could prevent high glucose-induced endothelial barrier dysfunction, inhibition of PHD-2 aggravated the destruction, which indicated PHD-2 played an important role. The mechanism might be associated with accumulation of HIF-1α and VEGF resulted from inhibited degradation of HIF-1α by PHD-2.

Keywords: 499 diabetic retinopathy • 666 pump/barrier function