The leading cause of corneal blindness world wide is from angiogenesis, which is the pathogenic growth of new blood vessels from an existing vasculature. Normal wound healing in the cornea does not involve angiogenesis. Therefore, identifying the molecular mechanisms that are responsible for preventing self- destructive angiogenic processes have important bearing on corneal homeostasis, regeneration, and transplantation. A novel biochemical feature preventing corneal stromal cells from participating in pathogenic fibrotic repair processes has recently been linked to the evolutionary conserved ubiquitin proteasome pathway (UPP). The UPP regulates protein quality control, inflammatory transcription factor NF-kappa B activation, cell cycle, differentiation and antigen presentation. We have investigated the hypothesis that a DPP-mediated genetic program controls the angiogenic phenotype of the cornea. We show that the UPP is upregulated in vascular endothelial cells when activated by angiogenic stimulators and during injury healing in vascularized corneas in vivo. This pattern of UPP activation is attenuated by the potent small molecule angiogenesis inhibitor, withaferin A. To demonstrate that a protein binding target(s) of withaferin A mediates this drug's anti-angiogenic mechanism, we generated a biotinylated analog of withaferin A. We have successfully exploited this affinity reagent and isolated the withaferin binding target. In this R01 proposal, we plan to exploit angiogenic and nonangiogenic models of wound healing to define the UPP-driven healing mechanism(s) of the cornea and validate newly identified molecular targets for anti-angiogenesis. For these investigations, withaferin A will serve both as a pharmacological agent and a cell permeable probe of its binding-protein target's function. Specifically, we will (1) characterize the drug's inhibitory mode of action on targeting the angiogenic activation of the UPP, (2) investigate key components of the UPP as mediators of the drugs activity, and (3) investigate withaferin-protein target-deficient mouse models to validate the requirement for drug-protein interaction in the corneal anti-angiogenic mechanism of WFA. We believe these investigations focused on the UPP and the target of withaferin A will enable us to reach our long-term objectives of discovering new therapeutic approaches to control angiogenesis and promote its homeostasis.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Anterior Eye Disease Study Section (AED)
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Mckie, George Ann
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University of Connecticut
Schools of Medicine
United States
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Wizeman, John W; Mohan, Royce (2017) Expression of peptidylarginine deiminase 4 in an alkali injury model of retinal gliosis. Biochem Biophys Res Commun 487:134-139
Bargagna-Mohan, Paola; Ishii, Akihiro; Lei, Ling et al. (2017) Sustained activation of ERK1/2 MAPK in Schwann cells causes corneal neurofibroma. J Neurosci Res 95:1712-1729
Wizeman, John W; Nicholas, Anthony P; Ishigami, Akihito et al. (2016) Citrullination of glial intermediate filaments is an early response in retinal injury. Mol Vis 22:1137-1155
Ritzel, Rodney M; Pan, Sarah J; Verma, Rajkumar et al. (2016) Early retinal inflammatory biomarkers in the middle cerebral artery occlusion model of ischemic stroke. Mol Vis 22:575-88
Mohan, Royce; Bargagna-Mohan, Paola (2016) The Use of Withaferin A to Study Intermediate Filaments. Methods Enzymol 568:187-218
Bargagna-Mohan, Paola; Lei, Ling; Thompson, Alexis et al. (2015) Vimentin Phosphorylation Underlies Myofibroblast Sensitivity to Withaferin A In Vitro and during Corneal Fibrosis. PLoS One 10:e0133399
Bargagna-Mohan, Paola; Deokule, Sunil P; Thompson, Kyle et al. (2013) Withaferin A effectively targets soluble vimentin in the glaucoma filtration surgical model of fibrosis. PLoS One 8:e63881
Bargagna-Mohan, Paola; Paranthan, Riya R; Hamza, Adel et al. (2012) Corneal antifibrotic switch identified in genetic and pharmacological deficiency of vimentin. J Biol Chem 287:989-1006
Paranthan, Riya R; Bargagna-Mohan, Paola; Lau, Daniel L et al. (2011) A robust model for simultaneously inducing corneal neovascularization and retinal gliosis in the mouse eye. Mol Vis 17:1901-8
Bargagna-Mohan, Paola; Paranthan, Riya R; Hamza, Adel et al. (2010) Withaferin A targets intermediate filaments glial fibrillary acidic protein and vimentin in a model of retinal gliosis. J Biol Chem 285:7657-69

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