title: Withaferin A effectively targets soluble vimentin in the glaucoma filtration surgical model of fibrosis.
abstract: Withaferin A (WFA) is a natural product that binds to soluble forms of the type III intermediate filament (IF) vimentin. Currently, it is unknown under what pathophysiological contexts vimentin is druggable, as cytoskeltal vimentin-IFs are abundantly expressed. To investigate druggability of vimentin, we exploited rabbit Tenon's capsule fibroblast (RbTCF) cell cultures and the rabbit glaucoma filtration surgical (GFS) model of fibrosis. WFA potently caused G₀/G₁ cell cycle inhibition (IC₅₀ 25 nM) in RbTCFs, downregulating ubiquitin E3 ligase skp2 and inducing p27(Kip1) expression. Transforming growth factor (TGF)-ß-induced myofibroblast transformation caused development of cell spheroids with numerous elongated invadopodia, which WFA blocked potently by downregulating soluble vimentin and α-smooth muscle actin (SMA) expression. In the pilot proof-of-concept study using the GFS model, subconjunctival injections of a low WFA dose reduced skp2 expression in Tenon's capsule and increased p27(Kip1) expression without significant alteration to vimentin-IFs. This treatment maintains significant nanomolar WFA concentrations in anterior segment tissues that correspond to WFA's cell cycle targeting activity. A ten-fold higher WFA dose caused potent downregulation of soluble vimentin and skp2 expression, but as found in cell cultures, no further increase in p27(Kip1) expression was observed. Instead, this high WFA dose potently induced vimentin-IF disruption and downregulated α-SMA expression that mimicked WFA activity in TGF-ß-treated RbTCFs that blocked cell contractile activity at submicromolar concentrations. These findings illuminate that localized WFA injection to ocular tissues exerts pharmacological control over the skp2-p27(Kip1) pathway by targeting of soluble vimentin in a model of surgical fibrosis.
authors.affiliation: Neuroscience, University of Connecticut Health Center, Farmington, Connecticut, United States of America.
authors.forename: Paola
authors.initials: P
authors.lastname: Bargagna-Mohan
authors.forename: Sunil P
authors.initials: SP
authors.lastname: Deokule
authors.forename: Kyle
authors.initials: K
authors.lastname: Thompson
authors.forename: John
authors.initials: J
authors.lastname: Wizeman
authors.forename: Cidambi
authors.initials: C
authors.lastname: Srinivasan
authors.forename: Sunil
authors.initials: S
authors.lastname: Vooturi
authors.forename: Uday B
authors.initials: UB
authors.lastname: Kompella
authors.forename: Royce
authors.initials: R
authors.lastname: Mohan
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects*; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000378:metabolism; D005355:Fibrosis / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects*; D020327:Glaucoma Drainage Implants; D007150:Immunohistochemistry; D011817:Rabbits; D044786:S-Phase Kinase-Associated Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D053719:Tandem Mass Spectrometry; D058475:Tenon Capsule / Q000166:cytology; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism*; D054358:Withanolides / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D044786:S-Phase Kinase-Associated Proteins; D014746:Vimentin; D054358:Withanolides; D044767:Ubiquitin-Protein Ligases; C009684:withaferin A