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R. Mohan, E. Alimova, B. Trucchi, P. Bargagna-Mohan; Pharmaological Control of Angiofibrosis by Withaferin A. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1487.
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We previously showed that the angiogenesis inhibitor withaferin A (WFA) downregulates injury-induced protein polyubiquitination in the inflammatory model of corneal neovascularization. We also discovered that WFA’s anti-angiogenic activity is mediated by direct binding to the intermediate filament (IF) protein vimentin (Bargagna-Mohan et al., Chemistry & Biology, 2007). Here, we have investigated the vimentin-targeting activity of WFA on the ubiquitin proteasome pathway in this model.
Corneal neovascularization was induced by application of 0.15 M sodium hydroxide to mouse corneas followed by deepithelialization of epithelium from limbus to central cornea. 129/Svev vimentin-deficient (vim-/-) and wild-type mice subjected to this injury were treated with vehicle or WFA (2 mg/kg/d). At different times post injury, mice were killed and total proteins from corneal tissues was isolated and subjected to western blotting. Additionally, cryo-sections of eyes were stained with antibodies to ubiquitin, vimentin, desmin and α-smooth muscle (α-sm) actin.
WFA downregulates the injury-induced expression of polyubiquitinated proteins in a time- and dose-dependent manner. Desmin expression in injured corneas was upregulated and localized to invasive blood vessels and WFA treatment downregulated its expression. In vim-/- mice compared to wild-type mice, desmin expression was surprisingly reduced and correlated also with the low expression of α-sm actin. The injury-induced high expression of polyubiquitinated proteins was comparable between vim-/- and wild-type mice, however WFA treatment did not reduce injury-induced expression of polyubiquitinated proteins in vim-/- mice.
WFA exerts its anti-angiogenic activity through vimentin and causes downregulation of expression of polyubiquitinated proteins and fibrotic markers such as α-sm actin and desmin. WFA’s pharmacological control of this angiofibrotic axis in vimentin deficiency is severely attenuated. These findings unveil vimentin-targeting as a novel mechanism to control the diseased upregulation of protein ubiquitination. WFA's potent in vivo activities suggest that this withanolide could be exploited as a drug lead in development of therapeutics to control angiofibrosis.
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