Purchase this article with an account.
Royce Mohan, Sunil Deokule, Kyle Thompson, John Wizeman, Paola Bargagna-Mohan; Potent Anti-Fibrotic Activity of Withaferin A in Ocular Injury Models. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2512.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To determine the efficacy of withaferin A (WFA) on fibrosis in different injury models and validate vimentin as a target for fibrosis treatment.
We employed a mouse corneal injury model (0.15 N NaOH application for 1 min with epithelial cell debridement) to induce corneal fibrosis. 129 Svev wild type and vimentin-deficient mice were treated with vehicle (DMSO) or WFA (2 mg/kg/d) solubilized in DMSO by i.p. injection for a period of 2 weeks or 1 month. Corneas were photographed and digital images were subjected to analysis for opacity. Immunohistochemistry (IHC) and western blot (WB) analysis was also performed. A surgical model of trabeculectomy was employed in six New Zealand White rabbits to cause injury in both eyes. Three of the study eyes received high dose of WFA and three received low dose of WFA by subconjuctival injection on days 0, 4, 7, 14 and 21, while control eyes received saline injection at the same time points. Eyes were monitored for intraocular pressure, bleb survival and adverse events over 28 days. Rabbit eyes were also subjected to IHC and WB. Tissue sections and blots were probed with antibodies to α-SMA, vimentin, desmin and Skp2.
Mice subjected to corneal injury in vimentin deficiency recovered significant corneal clarity by 2 weeks, which was further improved with WFA treatment. These mice treated with WFA for 1 month and then left untreated for 2.5 months maintained corneal clarity. Western blot analysis and IHC confirmed that corneal clarity restored to wild-type mice by WFA treatment was due to potent downregulation of α-SMA, vimentin and desmin in wild type mice, and prolonged downregulation of α-SMA and desmin in vimentin-deficient mice leads to recovery of corneal transparency. In the rabbit study, three blebs from study eyes (2 high-dose and 1 low-dose) survived for 3 weeks and 4 blebs survived for 2 weeks. All blebs in control eyes failed by 1 week. High dose WFA treatment reduced fibrotic overexpression of vimentin in wound fibroblasts surrounding the surgical site as demonstrated by IHC. By WB analysis, uninjured tenon sample showed basal vimentin expression that was increased in study eye tenon samples by 5.8-fold, but potently reduced to basal levels in study eye tenon samples by both doses of WFA. Consequently, both WFA doses also caused potent inhibition of injury-induced α-SMA and Skp2 expression, reducing their respective expression to background levels.
Vimentin genetic and pharmacological deficiency promotes regenerative healing in the cornea. WFA also elicits anti-fibrotic activity in the rabbit trabeculectomy model. Our findings highlight the efficacy of WFA as a lead for drug development towards the treatment of eye diseases and injuries impacted by fibrosis.
This PDF is available to Subscribers Only