Abstract
Purpose: :
Injury to the cornea leads to formation of distinct classes of mediators that initiate and amplify inflammatory and neovascular responses including the CYP4B1–derived 12–HETrE, a potent inflammatory and angiogenic eicosanoid acting via induction of VEGF. We used small interfering RNA (siRNA) targeting CYP4B1 in a rabbit model of corneal neovascularization to substantiate the cause/effect relationship between CYP4B1 expression, 12–HETrE production and neovascularization.
Methods: :
SiRNA sequences were derived from the coding sequence of the rabbit CYP4B1. The efficacy of each siRNA duplex to inhibit CYP4B1 catalytic activity, i.e., 12–HETrE production, was assayed using RCE cells expressing the CYP4B1. Corneal neovascularization was induced by placing a single 7.0 silk suture at midstromal depth approximately 2 mm from the limbus in anesthetized rabbits. CYP4B1 or scrambled siRNAs (20 µM, 20µl) were administered by subconjunctival injection adjacent to the suture location. Injections of siRNA were repeated at day 2 and 4 after suture placement. Corneal neovascularization was examined by slit lamp microscopy and quantified by image analysis. Corneas were dissected and processed for measurement of CYP4B1 and VEGF expression by real time PCR and for determination of 12–HETrE levels by GC/MS.
Results: :
12–HETrE production in CYP4B1–expressing RCE cells was markedly inhibited (81 and 85%) by CYP4B1–specific siRNAs but was unaltered by CYP4B1–scrambled siRNA, Corneal neovascularization was markedly reduced in eyes treated with the CYP4B1–specific siRNA as compared to eyes treated with the control siRNA. Inhibition of neovascularization by CYP4B1–specific siRNA was 50 and 60% at day 4 and 7 after suture placement, respectively. Endogenous levels of 12–HETrE were barely detected in corneal homogenates from eyes injected with CYP4B1–specific siRNA Relative expression of VEGF mRNA measured by real time PCR (ΔCP ratio VEGF/28S) in suture–containing corneal limbal wedges was 0.12±0.08 indicating inhibition of VEGF expression in CYP4B1 siRNA–treated eyes.
Conclusions: :
The results further substantiate the role of CYP4B1 as an angiogenic pathway in the cornea. CYP4B1 through its catalytic activity produces the angiogenic eicosanoid 12–HETrE, which contributes to corneal neovascularization by mechanisms that include VEGF induction. Inhibition of CYP4B1 may constitute part of therapeutic strategies that target angiogenic factors such as VEGF.
Keywords: neovascularization • eicosanoids • inflammation