Inhibition of VEGF expression and corneal neovascularization by siRNA targeting cytochrome P450 4B1.
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.
Seta
Francesca
F
Department of Pharmacology, New York Medical College, Valhalla, NY 10595, USA.
Patil
Kiran
K
Bellner
Lars
L
Mezentsev
Alexandre
A
Kemp
Rowena
R
Dunn
Michael W
MW
Schwartzman
Michal Laniado
ML
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*
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