Molecular mechanism of corneal neovascularization inhibition by decorin therapy

Abstract

Purpose: Recently we showed that targeted AAV-decorin gene therapy significantly inhibits corneal angiogenesis in vivo in rabbits. This study tested the hypothesis that decorin’s anti-angiogenic effects in the cornea are mediated via the restoration of normal physiologic balance between the pro- and anti-angiogenic cytokines, vascular endothelial growth factor receptor (VEGFR) modulation, and VEGFR-mediated ERK signaling.

Methods: New Zealand White rabbits and decorin deficient mice were used for in vivo studies and were approved by the Institutional Animal Care and Use Committees. Animals were treated in adherence with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Human corneal fibroblast (HCF) cultures were used for in vitro assays. The HCF treated with decorin (250nM) +/- VEGF (50ng/ml) were used for time dependent analysis of VEGFR1 and VEGFR2 phosphorylation using a proteome human receptor tyrosine kinases (RTKs) array. Corneal neovascularization in rabbits and mice was produced with VEGF-implant and alkali burn respectively. Real-time qPCR, immunohistochemistry and fluorescence confocal microscopy analyzed pro-/anti-angiogenic cytokines (VEGF, MCP1, angiopoietin, PEDF etc.), VEGFR mRNA/protein expression, and VEGFR-mediated ERK1/2 signaling in AAV-decorin-transduced, AAV-gfp-transduced neovascularized, and naive rabbit/mouse corneas.

Results: RTK array showed VEGF caused phosphorylation of VEGFR and decorin abrogated VEGF-induced phosphorylation of VEGFR in HCF. Decorin inhibited VEGFR phosphorylation in vivo as revealed by a marked decrease in ERK1/2 phosphorylation in decorin-delivered neovascularized corneal sections. In addition, AAV-decorin delivery rescues normal physiologic balance between pro- and anti-angiogenic factors by down-regulating VEGF, MCP1 (mRNA 3-5 fold p< 0.05 and protein 11-27%, p<0.01) and up-regulating PEDF (mRNA 1.8-2.5 fold p<0.05 and protein 9-13%, p<0.01) in rabbit corneas. Results from in vivo mouse studies are pending.

Conclusions: Decorin inhibits corneal angiogenesis largely by interrupting VEGF signaling, and restoring the pro- and anti-angiogenic factors balance required for corneal transparency.