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Ram Nagaraj, Allison Palmer, Rooban Nahomi; Pro-inflammatory cytokines induce apoptosis of human retinal endothelial cells by downregulating Hsp27. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1588.
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© ARVO (1962-2015); The Authors (2016-present)
The biochemical mechanisms for retinal capillary cell death in diabetes are not clear. In the diabetic retina of humans and rodents, pro-inflammatory cytokines are upregulated. In this study, we have investigated the effect if pro-inflammatory cytokines on a small heat shock protein, Hsp27 in primary human retinal endothelial cells (HREC).
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. The mRNA and protein levels of Hsp27 and heat shock factor-1 (HSF-1) were determined by PCR and Western blotting. Apoptosis was assessed by Hoechst staining.
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. The presence of high glucose (25 mM) further increased the effect of mixed cytokines. Mixed cytokines activated indoleamine 2,3-dioxygenase (IDO), enhanced kynurenine production and increased the ROS content in HREC. An inhibitor of IDO (MT) inhibited the effects of mixed cytokines on Hsp27. Mixed cytokines upregulated NOS2 and consequently increased levels of nitric oxide. A peroxynitrite donor and exogenous peroxynitrite drastically reduced Hsp27 in HREC. The cytokine and high glucose-mediated downregulation of Hsp27 was accompanied by increased apoptosis of HREC, which was largely inhibited by treatment with MT or a NOS inhibitor, L-NAME. Downregulation of Hsp27 by a siRNA promoted apoptosis in HREC.
Together our data suggest that pro-inflammatory cytokines induce formation of ROS through the IDO-mediated kynurenine pathway, which through peroxynitrite production reduces Hsp27 and brings about apoptosis of capillary endothelial cells. Our results suggest a novel mechanism for capillary cell death in diabetic retinopathy.
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