Differences in the TGF-β1-Induced Profibrotic Response of Anterior and Posterior Corneal Keratocytes In Vitro

PURPOSE. To characterize phenotypic differences between anterior and posterior corneal keratocytes after stimulation with the profibrotic agent transforming growth factor-beta1 (TGF-beta 1) in vitro. METHODS. 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-mu m 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 (alpha-SMA), and fibronectin were assessed after stimulation with different TGF-beta 1 concentrations. In addition, anterior and posterior cells cultured in different concentrations of TGF-beta 1 were wounded with a razor blade, and the wound area and time to closure were determined. RESULTS. Stimulation by all concentrations of TGF-beta 1 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-beta 1 concentration also increased the relative expression of Thy-1, alpha-SMA, and fibronectin in anterior and posterior fibroblasts. However, anterior cells expressed these fibrotic markers at lower TGF-beta 1 concentrations than did posterior keratocytes. After mechanical wounding, posterior cells closed the wound area faster than did anterior cells at all concentrations of TGF-beta 1. CONCLUSIONS. The present experiments show that anterior and posterior corneal keratocytes exhibit different sensitivities to the profibrotic growth factor TGF-beta 1. This heterogeneity of keratocyte response may impact wound closure after mechanical wounding. (Invest Ophthalmol Vis Sci. 2010; 51:1935-1942) DOI: 10.1167/iovs.09-3823