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R. Mohan, P. Bargagna–Mohan; Identification of a Potential Novel Anti–Angiogenic Mechanism in the Injury–Healing Cornea . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3906.
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© ARVO (1962-2015); The Authors (2016-present)
Withaferin A (WA) is a potent small molecule inhibitor of angiogenesis and targets the ubiquitin proteasome pathway (UPP). In this study, we investigated the abundant corneal protein transketolase (TKT) as a potential downstream mediator of WA’s anti–angiogenic mechanism.
Corneal angiogenesis was initiated in C57BL/6 mice in one eye by application of 0.15M sodium hydroxide followed by complete debridement of corneal and limbal epithelia. Injured mice in groups of 4 were treated daily with vehicle (DMSO) or WA by intraperiteoneal injection and, time–course and dose–response experiments were performed. Equal amounts of protein pooled from samples in each group were subjected to immunoblotting or Coomassie blue staining after fractionation on 4–20% SDS– polyacrylamide gels. Blots were probed with antibodies against ubiquitin, TKT and ß–actin. In other such experiments, whole eyes were isolated after 4 and 8 days post–injury. Frozen tissue sections were subjected to immunohistochemical analysis using anti–TKT antibody and detected with FITC–conjugated secondary antibodies. Whole corneal buttons were also isolated and stained with FITC–conjugated anti–CD–31 antibodies. Images were captured on a Leica confocal microscope.
We observed an increase in global ubiquitinated proteins in the injured corneas compared to control uninjured fellow eyes. WA treatment downregulates the levels of injury–induced ubiquitinated proteins in healing corneas. Intriguingly, the abundant 70 kDa TKT band was also found to be ubiquitinated and it becomes the most predominant ubiquitinated species in epithelium of vascularized corneas. This pattern of TKT ubiquitination increases in levels over time post–injury. The striking upregulation of TKT ubiquitination was downregulated by WA treatment in association with the inhibition of angiogenesis.
WA exerts a novel mechanism of anti–angiogenesis by downregulating expression of ubiquitinated TKT in injured corneas. The characterization of TKT ubiquitinated isoform(s) and their role in corneal angiogenesis is currently under investigation.
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