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

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Abstract

Purpose

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 HREC

Conclusion

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.

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