title: Inhibition of VEGF expression and corneal neovascularization by siRNA targeting cytochrome P450 4B1.
abstract: Injury to the cornea leads to formation of mediators that initiate and amplify inflammatory responses and neovascularization. Among these are lipid mediators generated by a cytochrome P450 (CYP) enzyme identified as CYP4B1. Increased corneal CYP4B1 expression increases limbal angiogenic activity through the production of 12-hydroxyeicosatrienoic acid (12-HETrE), a potent inflammatory and angiogenic eicosanoid. We used siRNA duplexes targeting CYP4B1 to substantiate the link between CYP4B1 expression, 12-HETrE production and angiogenesis in a model of suture-induced corneal neovascularization. Intrastromal sutures induced a time-dependent neovascular response which was significantly attenuated by CYP4B1-specific siRNAs but not by nonspecific siRNA. CYP4B1 mRNA was reduced by 60% and 12-HETrE's levels were barely detected in corneal homogenates from eyes treated with the CYP4B1-specific siRNA. The decreased neovascular response in CYP4B1 siRNA-treated eyes was associated with a 75% reduction in corneal VEGF mRNA levels. Transfection of rabbit corneal epithelial cells with CYP4B1 cDNA induced VEGF expression. Conversely, treatment with CYP4B1 siRNA or addition of a CYP4B1 inhibitor significantly decreased VEGF mRNA levels; addition of 12-HETrE potently increased them. The results strongly implicate the corneal CYP4B1 as a component of the inflammatory and neovascular cascade initiated by injury and further suggest that CYP4B1-12-HETrE is a proximal regulator of VEGF expression.
authors.affiliation: Department of Pharmacology, New York Medical College, Valhalla, NY 10595, USA.
authors.forename: Francesca
authors.initials: F
authors.lastname: Seta
authors.forename: Kiran
authors.initials: K
authors.lastname: Patil
authors.forename: Lars
authors.initials: L
authors.lastname: Bellner
authors.forename: Alexandre
authors.initials: A
authors.lastname: Mezentsev
authors.forename: Rowena
authors.initials: R
authors.lastname: Kemp
authors.forename: Michael W
authors.initials: MW
authors.lastname: Dunn
authors.forename: Michal Laniado
authors.initials: ML
authors.lastname: Schwartzman
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000172:deficiency* / Q000235:genetics* / Q000378:metabolism; D003315:Cornea / Q000166:cytology; D016510:Corneal Neovascularization / Q000201:enzymology* / Q000235:genetics*; D015536:Down-Regulation; D004847:Epithelial Cells / Q000201:enzymology; D006893:Hydroxyeicosatetraenoic Acids / Q000096:biosynthesis; D016133:Polymerase Chain Reaction; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D006893:Hydroxyeicosatetraenoic Acids; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; D001189:Aryl Hydrocarbon Hydroxylases; C095029:cytochrome P-450 CYP4B1