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Holly B. Hindman, Jennifer N. Swanton, Richard P. Phipps, Patricia J. Sime, Krystel R. Huxlin; Differences in the TGF-β1–Induced Profibrotic Response of Anterior and Posterior Corneal Keratocytes In Vitro. Invest. Ophthalmol. Vis. Sci. 2010;51(4):1935-1942. doi: 10.1167/iovs.09-3823.
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To characterize phenotypic differences between anterior and posterior corneal keratocytes after stimulation with the profibrotic agent transforming growth factor-beta1 (TGF-β1) in vitro.
Sixteen corneas from healthy felines were obtained immediately after death. Lamellar dissection was performed to separate the anterior and posterior stroma at approximately 50% depth either manually (n = 2) or with a Moria microkeratome (300-μm head; n = 14). Cells from the anterior and posterior stroma were cultured separately but under identical conditions. Using immunohistochemistry and Western blot techniques, Ki-67 staining and relative expression of Thy-1, alpha smooth muscle actin (α-SMA), and fibronectin were assessed after stimulation with different TGF-β1 concentrations. In addition, anterior and posterior cells cultured in different concentrations of TGF-β1 were wounded with a razor blade, and the wound area and time to closure were determined.
Stimulation by all concentrations of TGF-β1 increased the proportion of Ki-67–positive cells in anterior and posterior cell cultures, but this increase was noted earlier in posterior cells than in anterior cells. Increasing TGF-β1 concentration also increased the relative expression of Thy-1, α-SMA, and fibronectin in anterior and posterior fibroblasts. However, anterior cells expressed these fibrotic markers at lower TGF-β1 concentrations than did posterior keratocytes. After mechanical wounding, posterior cells closed the wound area faster than did anterior cells at all concentrations of TGF-β1.
The present experiments show that anterior and posterior corneal keratocytes exhibit different sensitivities to the profibrotic growth factor TGF-β1. This heterogeneity of keratocyte response may impact wound closure after mechanical wounding.
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