PMID- 25477319 OWN - NLM STAT- MEDLINE DCOM- 20150302 LR - 20240322 IS - 1552-5783 (Electronic) IS - 0146-0404 (Print) IS - 0146-0404 (Linking) VI - 55 IP - 12 DP - 2014 Dec 4 TI - Proinflammatory responses induced by CD40 in retinal endothelial and Muller cells are inhibited by blocking CD40-Traf2,3 or CD40-Traf6 signaling. PG - 8590-7 LID - 10.1167/iovs.14-15340 [doi] AB - PURPOSE: The cell surface receptor CD40 is required for the development of retinopathies induced by diabetes and ischemia/reperfusion. The purpose of this study was to identify signaling pathways by which CD40 triggers proinflammatory responses in retinal cells, since this may lead to pharmacologic targeting of these pathways as novel therapy against retinopathies. METHODS: Retinal endothelial and Muller cells were transduced with vectors that encode wild-type CD40 or CD40 with mutations in sites that recruit TNF receptor associated factors (TRAF): TRAF2,3 (DeltaT2,3), TRAF6 (DeltaT6), or TRAF2,3 plus TRAF6 (DeltaT2,3,6). Cells also were incubated with CD40-TRAF2,3 or CD40-TRAF6 blocking peptides. We assessed intercellular adhesion molecule-1 (ICAM-1), CD40, monocyte chemoattractant protein-1 (MCP-1), VEGF, and prostaglandin E(2) (PGE(2)) by fluorescence-activated cell sorting (FACS), ELISA, or mass spectrometry. Mice (B6 and CD40(-/-)) were made diabetic using streptozotocin. The MCP-1 mRNA was assessed by real-time PCR. RESULTS: The CD40-mediated ICAM-1 upregulation in endothelial and Muller cells was markedly inhibited by expression of CD40 DeltaT2,3 or CD40 DeltaT6. The CD40 was required for MCP-1 mRNA upregulation in the retina of diabetic mice. The CD40 stimulation of endothelial and Muller cells enhanced MCP-1 production that was markedly diminished by CD40 DeltaT2,3 or CD40 DeltaT6. Similar results were obtained in cells incubated with CD40-TRAF2,3 or CD40-TRAF6 blocking peptides. The CD40 ligation upregulated PGE(2) and VEGF production by Muller cells, that was inhibited by CD40 DeltaT2,3 or CD40 DeltaT6. All cellular responses tested were obliterated by expression of CD40 DeltaT2,3,6. CONCLUSIONS: Blockade of a single CD40-TRAF pathway was sufficient to impair ICAM-1, MCP-1, PGE(2), and VEGF upregulation in retinal endothelial and/or Muller cells. Blockade of CD40-TRAF signaling may control retinopathies. CI - Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc. FAU - Portillo, Jose-Andres C AU - Portillo JA AD - Division of Infectious Diseases and HIV Medicine, Department of Medicine, Case Western Reserve University, Cleveland, Ohio, United States. FAU - Schwartz, Isaac AU - Schwartz I AD - Division of Infectious Diseases and HIV Medicine, Department of Medicine, Case Western Reserve University, Cleveland, Ohio, United States. FAU - Zarini, Simona AU - Zarini S AD - Department of Pharmacology, University of Colorado Denver, Aurora, Colorado, United States. FAU - Bapputty, Reena AU - Bapputty R AD - Department of Pediatrics, Case Western Reserve University/Rainbow Babies and Children's Hospital, Cleveland, Ohio, United States. FAU - Kern, Timothy S AU - Kern TS AD - Division of Clinical and Molecular Endocrinology, Department of Medicine, Case Western Reserve University, Cleveland, Ohio, United States. FAU - Gubitosi-Klug, Rose A AU - Gubitosi-Klug RA AD - Department of Pediatrics, Case Western Reserve University/Rainbow Babies and Children's Hospital, Cleveland, Ohio, United States. FAU - Murphy, Robert C AU - Murphy RC AD - Department of Pharmacology, University of Colorado Denver, Aurora, Colorado, United States. FAU - Subauste, M Cecilia AU - Subauste MC AD - Veterans Administration Medical Center, Research Service 151, Cleveland, Ohio, United States Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Case Western Reserve University, Cleveland, Ohio, United States. FAU - Subauste, Carlos S AU - Subauste CS AD - Division of Infectious Diseases and HIV Medicine, Department of Medicine, Case Western Reserve University, Cleveland, Ohio, United States Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, Ohio, United States Department of Pathology, Case Western Reserve University, Cleveland, Ohio, United States. LA - eng GR - P30 EY011373/EY/NEI NIH HHS/United States GR - EY019250/EY/NEI NIH HHS/United States GR - R01 EY019250/EY/NEI NIH HHS/United States GR - UL1 TR000439/TR/NCATS NIH HHS/United States GR - P30 EY11373/EY/NEI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20141204 PL - United States TA - Invest Ophthalmol Vis Sci JT - Investigative ophthalmology & visual science JID - 7703701 RN - 0 (Biomarkers) RN - 0 (CD40 Antigens) RN - 0 (Chemokine CCL2) RN - 0 (Tumor Necrosis Factor Receptor-Associated Peptides and Proteins) RN - 0 (Vascular Endothelial Growth Factor A) RN - 126547-89-5 (Intercellular Adhesion Molecule-1) RN - K7Q1JQR04M (Dinoprostone) SB - IM MH - Analysis of Variance MH - Animals MH - Biomarkers/metabolism MH - CD40 Antigens/antagonists & inhibitors/*immunology MH - Cells, Cultured MH - Chemokine CCL2/metabolism MH - Diabetes Mellitus, Experimental/*immunology/metabolism MH - Dinoprostone/metabolism MH - Endothelial Cells/*immunology/metabolism MH - Ependymoglial Cells/*immunology/metabolism MH - Humans MH - Intercellular Adhesion Molecule-1/metabolism MH - Mice MH - Rats MH - Retina/*immunology/metabolism MH - Signal Transduction/immunology/physiology MH - Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/antagonists & inhibitors/*immunology MH - Vascular Endothelial Growth Factor A/metabolism PMC - PMC4280881 OTO - NOTNLM OT - CD40 OT - chemokine OT - diabetes OT - intercellular adhesion molecules EDAT- 2014/12/06 06:00 MHDA- 2015/03/03 06:00 PMCR- 2015/06/01 CRDT- 2014/12/06 06:00 PHST- 2014/12/06 06:00 [entrez] PHST- 2014/12/06 06:00 [pubmed] PHST- 2015/03/03 06:00 [medline] PHST- 2015/06/01 00:00 [pmc-release] AID - iovs.14-15340 [pii] AID - 10.1167/iovs.14-15340 [doi] PST - epublish SO - Invest Ophthalmol Vis Sci. 2014 Dec 4;55(12):8590-7. doi: 10.1167/iovs.14-15340.