The formation of acellular capillaries in the retina, a hallmark feature of diabetic retinopathy, is caused by apoptosis of endothelial cells and pericytes. The biochemical mechanism of such apoptosis remains unclear. Small heat shock proteins play an important role in the regulation of apoptosis. In the diabetic retina, pro-inflammatory cytokines are upregulated. In this study, we investigated the effects of pro-inflammatory cytokines on small heat shock protein 27 (Hsp27) in human retinal endothelial cells (HREC). In HREC cultured in the presence of cytokine mixtures (CM), a significant downregulation of Hsp27 at the protein and mRNA level occurred, with no effect on HSF-1, the transcription factor for Hsp27. The presence of high glucose (25mM) amplified the effects of cytokines on Hsp27. CM activated indoleamine 2,3-dioxygenase (IDO) and enhanced the production of kynurenine and ROS. An inhibitor of IDO, 1-methyl tryptophan (MT), inhibited the effects of CM on Hsp27. CM also upregulated NOS2 and, consequently, nitric oxide (NO). A NOS inhibitor, L-NAME, and a ROS scavenger blocked the CM-mediated Hsp27 downregulation. While a NO donor in the culture medium did not decrease the Hsp27 content, a peroxynitrite donor and exogenous peroxynitrite did. The cytokines and high glucose-induced apoptosis of HREC were inhibited by MT and L-NAME. Downregulation of Hsp27 by a siRNA treatment promoted apoptosis in HREC. Together, these data suggest that pro-inflammatory cytokines induce the formation of ROS and NO, which, through the formation of peroxynitrite, reduce the Hsp27 content and bring about apoptosis of retinal capillary endothelial cells.
Keywords: (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate; 1-methyl-dl-tryptophan; 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine; 5-(and-6)-chloromethyl-2′,7′-dichlorodihydro-fluorescein diacetate; Ac-DEVD-AFC; Apoptosis; CM; CM-H(2)DCFDA; COX2; Cytokine; DETA NONOate; HG; HREC; HSF-1; Hsp27; ICAM-1; IDO; IFN-γ; IL-1β; L-NAME; MCP-1; MT; N(ω)-nitro-l-arginine methyl ester hydrochloride; N-acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoromethylcoumarin; NO; NOS; ONOO; ROS; Retinal endothelial cell; TEMPOL; TNF-α; VCAM-1; cyclooxygenase-2; cytokine mixture; heat shock factor-1; high glucose; human retinal endothelial cells; indoleamine 2,3-dioxygenase; interferon-γ; interleukin-1β; intracellular adhesion molecule-1; monocyte chemotactic protein-1; nitric oxide; nitric oxide synthase; peroxynitrite; reactive oxygen species; tumor necrosis factor-α; vascular cell adhesion molecule-1.
Copyright © 2013 Elsevier B.V. All rights reserved.