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Paola Bargagna-Mohan, Ling Lei, Anthony Shelley, Eric C Mohan, Cindy Caruk, Daniel L Lau, Royce Mohan; Role of Vimentin in Regulation of Cell Spreading during Early Stages of Corneal Fibrosis. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4703.
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© ARVO (1962-2015); The Authors (2016-present)
Soluble vimentin (sVim) along with its cytoskeletal filamentous form (fVim) are expressed during corneal fibrosis. Vimentin is known to promote cell adhesion and spreading through interactions with filamin A (FLNa), an actin cross-linking protein involved in tissue remodeling and wound healing. Here we have investigated withaferin A (WFA) as a chemical probe of sVim during early events of cell adhesion and spreading.
We exploited a cell culture model of cell spreading using primary rabbit corneal stromal fibroblast cells (RbCFs). Cells between 2nd and 8th passage were serum starved, trypsinized and allowed to re-attach in presence or absence of WFA in culture medium containing 10% serum. Cell behavior was analyzed by a computer software algorithm. Expression of sVim, phosphorylated (Ser38)-sVim, and FLNa were analyzed by immunohistochemistry and western blot at different time points.
WFA binding to sVim affected cell spreading within 60 minutes of treatment. Interestingly, we found that cell passage was a key feature in driving cell attachment; RbCF2 cells had a 3-fold and RbCF5 a 2-fold higher replating rate than RbCF8 cells within 1 hour-post-adhesion. WFA treatment (1 μΜ) did not significantly alter the rate of adhesion in each cell group, suggesting a non-toxic effect of the drug. Also, cell spreading and cell shape changes were clearly influenced by cell passage number. Majority of RbCF2 and RbCF5 cells spread with an anisotropic shape and displayed a complete assembly of fVim within 1 h post-plating, whereas RbCF8 cells maintained an isotropic shape and showed a less extensive vimentin staining pattern. Soluble protein extracts analyzed by western blots showed no significant changes in both sVim and p-Ser38vim expression in RbCF2 and RbCF5 cells after WFA treatment; however, WFA-treated RbCF8 cells displayed a 5-fold decrease in vimentin expression with appearance of high molecular weight p-Ser38Vim isoforms. RbCF8 cells displayed a 2-fold decrease in soluble FLNa after WFA treatment compared to RbCF2 cells.
We show that modulation of sVim by WFA regulates its interactions with FLNa in a manner dependent on cell passage. As corneal fibroblasts are known to advance into a myofibroblastic phenotype with cell passage, our findings underscore a novel role for sVim as a target for differentially controlling myofibroblastic behavior during fibrosis.
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