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GA Abrams, AI Teixeira, JD Foley, PF Nealey, PJ Bertics, CJ Murphy; The Interaction Of Rho And Substratum Topography In Orientation Of Human Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1647.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To test the concept that signaling mechanisms involving the G-protein Rho are associated with cell orientation in response to surfaces with well-defined nanostructured features. Methods: Cell culture substrates consisted of silicon wafers with etched patterns of grooves and ridges with periods between 400 nm and 4µm and smooth surfaces, coated with silicon oxide to be chemically equivalent. Primary human corneal epithelial cells were plated at a density of 10,000 cells/cm2 in media containing 10% fetal bovine serum or serum free media. A population of cells was treated with lysophosphatidic acid (LPA), a known activator of Rho, or exoenzyme C3 from Clostrudium botulinum, a known inhibitor of Rho. Fluorescent labeling with phalloidin was used to visualize the actin cytoskeleton. Cells were considered aligned with the grooves when the angle between the major axis of the cell and the grooves was less than 10 degrees. 3T3 fibroblasts, that demonstrate a very high degree of orientation (approx. 90%) on anisotropic patterns, were used as controls throughout. Results: Approximately 30% of untreated epithelial cells orient parallel to substratum grooves and ridges. Treatment of cells with LPA more than doubled the percentage of aligned cells on all patterned surfaces. In addition, cells treated with LPA resulted in a more highly elongated morphology than non-treated cells or cells on smooth surfaces. F-actin polymerization was more prominent in the highly elongated cells. Cells treated with exoenzyme C3 resulted in a rounded morphology with complete loss of orientation and F-actin polymerization on all surfaces. Conclusion: LPA stimulation increases the percentage of aligned epithelial cells and exoenzyme C3 treatment results in a loss of cell orientation. These observations suggest that downstream effectors of LPA and exoenzyme C3 substrates, such as Rho, appear critical in cellular responses to topopgraphy.
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