Purchase this article with an account.
R. Mohan, P. Bargagna–Mohan; Towards a Chemical Biology Approach to Probe Corneal Tissue Stasis . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4493.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: We hypothesize that perturbation of the ubiquitin proteasome pathway (UPP) may block or reverse alkali–induced corneal angiogenesis and promote regenerative healing. In this study, we investigated the molecular mode of action of withaferin A (WA), a newly identified small molecule UPP–targeting agent and angiogenesis inhibitor, in the corneal inflammatory–driven model of angiogenesis. Methods: We employed C57BL/6 mice in which corneal angiogenesis was induced in one eye by application of 0.15 M sodium hydroxide followed by complete debridement of corneal and limbal epithelia. Injured mice in groups of 4 each were treated with vehicle (DMSO) or WA by daily i. p. injection. Mouse corneas were analyzed with the aid of a dissecting microscope and angiogenesis assessed by digital photography with quantification using NIH ImageJ software. In one experiment, mice were treated daily for 6 days with vehicle or WA at 2.5 mg/kg/d. In a second experiment, mice were treated with vehicle or WA at doses of 5 mg/kg/d, 500 µg/kg/d or 50 µg/kg/d for first 4 days and subsequently sacrificed on day 7 for tissue analysis. Corneal buttons were obtained from non injured eyes and 7–day–old healing mouse eyes. Tissue extracts using equal amounts of protein pooled from 4 mice per group were then subjected to western blot analysis and blots were probed with antibodies against IΚB–α, heme oxygenase–1 (HO–1), ubiquitin and ß–actin. Results: WA dose–dependently inhibits corneal angiogenesis to greater than 70%, with reduction in corneal opacity also being observed at the higher doses of WA. In vehicle–treated mice, corneal expression of native form of IΚB–α was high, but the phosphorylated form was low. Both IΚB–α forms were dose–dependently reduced in WA–treated mice to levels found in uninjured corneas. The oxidative stress–response protein HO–1 that was also highly elevated in vehicle–treated corneas was reduced by WA treatment to barely detectable levels found in uninjured corneas. The profile of polyubiquitinated proteins that was highly elevated in vehicle–treated mouse corneas was minimal in uninjured corneas and also diminished in mice treated with WA. Conclusions: WA treatment downregulates expression of key stress– and inflammation–response proteins and potently inhibits corneal angiogenesis. These findings suggest that the UPP–targeting function of WA has pharmacological relevance to many angio–inflammatory ocular diseases. The use of WA as a pharmacological agent for corneal disease treatment and chemical probe of corneal tissue stasis warrants further investigation.
This PDF is available to Subscribers Only