title: Pro-inflammatory cytokines downregulate Hsp27 and cause apoptosis of human retinal capillary endothelial cells.
abstract: 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.
authors.affiliation: Department of Ophthalmology and Visual Sciences, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
authors.forename: Rooban B
authors.initials: RB
authors.lastname: Nahomi
authors.affiliation: Department of Ophthalmology and Visual Sciences, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
authors.forename: Allison
authors.initials: A
authors.lastname: Palmer
authors.affiliation: Kellogg Eye Center, University of Michigan, Ann Arbor, MI 48105, USA.
authors.forename: Katelyn M
authors.initials: KM
authors.lastname: Green
authors.affiliation: Kellogg Eye Center, University of Michigan, Ann Arbor, MI 48105, USA.
authors.forename: Patrice E
authors.initials: PE
authors.lastname: Fort
authors.affiliation: Department of Ophthalmology and Visual Sciences, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA. Electronic address: ram.nagaraj@case.edu.
authors.forename: Ram H
authors.initials: RH
authors.lastname: Nagaraj
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology; D055551:HSP27 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins; D006801:Humans; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase / Q000037:antagonists & inhibitors / Q000378:metabolism; D018836:Inflammation Mediators / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D053583:Interleukin-1beta / Q000494:pharmacology; D018832:Molecular Chaperones; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000378:metabolism / Q000494:pharmacology; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism; D012160:Retina / Q000166:cytology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014364:Tryptophan / Q000031:analogs & derivatives / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D016207:Cytokines; D055551:HSP27 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D018836:Inflammation Mediators; D053583:Interleukin-1beta; D018832:Molecular Chaperones; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; C028129:tryptophan methyl ester; D007371:Interferon-gamma; D014364:Tryptophan; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; D005947:Glucose; D019331:NG-Nitroarginine Methyl Ester
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