Pro‐inflammatory cytokines induce apoptosis of human retinal capillary endothelial cells through downregulation of Hsp27

Abstract

AbstractPurpose Retinal capillary cells undergo apoptosis in diabetes, but the mechanism is not clear. Pro‐inflammatory cytokines are upregulated in the diabetic retina of humans and rodents. In this study, we have investigated the effect if pro‐inflammatory cytokines on a small heat shock protein, Hsp27 in human retinal endothelial cells (HREC)Methods HREC were cultured in the presence of pro‐inflammatory cytokines, interferon ‐γ (IFN‐γ, 50 and 100 units/ml), interleukin‐1β (IL‐1β, 10 and 20 ng/ml) and tumor necrosis factor‐α (TNF‐α, 10 and 20 ng/ml) for 48 hrs and in the presence or absence of high glucose (25 mM, HG). The roles of the kynurenine pathway and NOS were determined by adding 20 μM 1‐methyl tryptophan (MT) and 500 μM L‐Nω‐nitroarginine methyl ester hydrochloride (L‐NAME), respectively to the culture medium.Results HREC cultured in the presence of mixed cytokines showed a significant downregulation of Hsp27 at the protein and mRNA levels. This downregulation was not due to downregulation of the transcription factor, HSF‐1. Mixed cytokines activated indoleamine 2,3‐dioxygenase (IDO), enhanced kynurenine production and increased the ROS content in HREC. MT inhibited the effects of mixed cytokines on Hsp27. Mixed cytokines upregulated NOS2 and increased levels of NO. A peroxynitrite donor and exogenous peroxynitrite drastically reduced Hsp27. The cytokine and HG‐mediated downregulation of Hsp27 was accompanied by increased apoptosis of HRECConclusion Our data suggest that pro‐inflammatory cytokines induce ROS and NO formation, which through peroxynitrite production reduce Hsp27 and bring about apoptosis of HREC. These results suggest a novel mechanism for capillary cell death in diabetic retinopathy.