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.

Figure 1

Inhibition of 12-HETrE production by CYP4B1-specific siRNA in RCE cells. A) EGFP fluorescence in RCE cells transfected with the pIRES2-EGFP (upper panel) and pIRES2-EGFP-4B1 without (middle panel) and with (lower panel) CYP4B1 specific siRNA (X40). B) Representative HPLC elution profiles of arachidonic acid metabolites in RCE cells transfected with the pIRES2-EGFP (upper panel) and pIRES2-EGFP-4B1 (lower panel). The major metabolite co-eluted with 12-HETrE authentic standatd; other radioactive peaks correspond to: I, unknown polar metabolites; II, 12,20-diHETE; 12-HETE; and 12-oxo-ETrE as previously reported, C) Quantitative analysis of 12-HETrE production in RCE cells treated with siRNA duplexes targeting CYP4B1 (4B1-a, 4B1-b, 4B1-c) and non-specific siRNA (control) and incubated with arachidonic acid. Results are mean±SE, n=3, *p<0.05 from control siRNA.

Figure 2

Effect of siRNA treatment on corneal neovascularization. A) Representative pictures depicting corneal neovascularization in control eyes (treated with the vehicle saline) and in eyes receiving subconjunctival injections of CYP4B1-specific (4B1-siRNA) or nonspecific siRNAs at days 2, 4 and 7 after suture placement (X16). B) Quantitative analysis of corneal neovascularization 4 and 7 days after suture placement. Results are mean±SE; n=6; *p<0.05 from control siRNA-treated eyes; ^‡p<0.05 from day 4.

Figure 3

Effect of siRNA treatment on corneal CYP4B1 mRNA and 12-HETrE levels 7 days after intrastromal suture placement. A) Representative amplification plots for CYP4B1 (left) and 28S rRNA mRNA (right) by real time PCR analysis in RNA from corneas treated with control (blue line) and CYP4B1-specific (red line) siRNA. B) Quantitative analysis of mRNA levels. Results are presented as relative expression and are mean±SE, n=5, *p<0.05 from control siRNA. C) HPLC analysis of 12-HETrE levels in corneal homogenates from control and CYP4B1 (4B1) specific siRNA treated eyes; upper panel, elution profile of 12-HETrE standard (st); lower panel, representative analyses (n=3) of corneal homogenates from eyes treated with control (blue line) and CYP4B1-specific (red line) siRNAs.

Figure 4

Effect of siRNA treatment on corneal VEGF mRNA 7 days after intrastromal suture placement. A) Representative amplification plots for VEGF in corneas treated with control siRNA and CYP4B1 (4B1)-specific siRNA. B) Quantitative analysis of mRNA levels. Results are presented as relative expression and are mean±SE, n=5, *p<0.05 from control siRNA-treated eyes.

Figure 5

Effect of CYP4B1 expression and 12-HETrE on VEGF mRNA levels in RCE cells. Levels of mRNA were measured by real time PCR. A) VEGF mRNA levels in non transfected (control) and cells transfected with the control plasmid pIRES and pIRES plasmid containing the CYP4B1 cDNA (4B1) in the absence and presence of CYP4B1-specific siRNA (4B1+siRNA). Results are expressed as relative expression normalized to 28S levels and are mean±SE; n=3; *p<0.05 from control or pIRES transfected cells; ^‡p<0.05 from 4B1 transfected cells. B) VEGF mRNA levels in cells transfected with pIRES-GFP-CYP4B1 (4B1) and treated with the CYP4B1 inhibitor 17-ODYA (20 μM). Results are expressed as relative expression normalized to 28S levels and are mean±SE; n=3; *p<0.05 from pIRES transfected cells; ^‡p<0.05 from 4B1 transfected cells. C) VEGF mRNA levels in RCE cells treated with 12-HETrE (0.1–100 nM) or subjected to hypoxia (2% O₂). Results are expressed as relative expression normalized to 28S levels and are mean±SE; n=3; *p<0.05 from control.