title: 12/15-Lipoxygenase-derived lipid metabolites induce retinal endothelial cell barrier dysfunction: contribution of NADPH oxidase.
abstract: The purpose of the current study was to evaluate the effect of 12/15-lipoxygenase (12/15-LOX) metabolites on retinal endothelial cell (REC) barrier function. FITC-dextran flux across the REC monolayers and electrical cell-substrate impedance sensing (ECIS) were used to evaluate the effect of 12- and 15-hydroxyeicosatetreanoic acids (HETE) on REC permeability and transcellular electrical resistance (TER). Effect of 12- or 15-HETE on the levels of zonula occludens protein 1 (ZO-1), reactive oxygen species (ROS), NOX2, pVEGF-R2 and pSHP1 was examined in the presence or absence of inhibitors of NADPH oxidase. In vivo studies were performed using Ins2(Akita) mice treated with or without the 12/15-LOX inhibitor baicalein. Levels of HETE and inflammatory mediators were examined by LC/MS and Multiplex Immunoassay respectively. ROS generation and NOX2 expression were also measured in mice retinas. 12- and 15- HETE significantly increased permeability and reduced TER and ZO-1 expression in REC. VEGF-R2 inhibitor reduced the permeability effect of 12-HETE. Treatment of REC with HETE also increased ROS generation and expression of NOX2 and pVEGF-R2 and decreased pSHP1 expression. Treatment of diabetic mice with baicalein significantly decreased retinal HETE, ICAM-1, VCAM-1, IL-6, ROS generation, and NOX2 expression. Baicalein also reduced pVEGF-R2 while restored pSHP1 levels in diabetic retina. Our findings suggest that 12/15-LOX contributes to vascular hyperpermeability during DR via NADPH oxidase dependent mechanism which involves suppression of protein tyrosine phosphatase and activation of VEGF-R2 signal pathway.
authors: Department of Oral Biology/Anatomy, College of Dental Medicine, Georgia Regents University (GRU), Augusta, Georgia, United States of America.
authors: Amira
authors: A
authors: Othman
authors: Saif
authors: S
authors: Ahmad
authors: Sylvia
authors: S
authors: Megyerdi
authors: Rene
authors: R
authors: Mussell
authors: Karishma
authors: K
authors: Choksi
authors: Krishna Rao
authors: KR
authors: Maddipati
authors: Ahmed
authors: A
authors: Elmarakby
authors: Nasser
authors: N
authors: Rizk
authors: Mohamed
authors: M
authors: Al-Shabrawey
mesh_terms: D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / Q000494:pharmacology*; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000378:metabolism*; D001093:Arachidonate 15-Lipoxygenase / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D003911:Dextrans; D003930:Diabetic Retinopathy / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D017097:Electric Impedance; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D044950:Flavanones / Q000494:pharmacology*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D015870:Gene Expression / Q000187:drug effects; D006893:Hydroxyeicosatetraenoic Acids / Q000494:pharmacology*; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics* / Q000378:metabolism; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D012160:Retina / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D015398:Signal Transduction / Q000187:drug effects; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D062826:Zonula Occludens-1 Protein / Q000235:genetics / Q000378:metabolism
chemical_list: C475064:12-15-lipoxygenase; D003911:Dextrans; D004791:Enzyme Inhibitors; D044950:Flavanones; D006893:Hydroxyeicosatetraenoic Acids; D008562:Membrane Glycoproteins; D017382:Reactive Oxygen Species; C569043:Tjp1 protein, mouse; D062826:Zonula Occludens-1 Protein; C015219:fluorescein isothiocyanate dextran; C006680:baicalein; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; D001092:Arachidonate 12-Lipoxygenase; D001093:Arachidonate 15-Lipoxygenase; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D040301:Vascular Endothelial Growth Factor Receptor-2; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; C509248:Ptpn6 protein, mouse; D016650:Fluorescein-5-isothiocyanate