Differences in the TGF-{beta}1-induced profibrotic response of anterior and posterior corneal keratocytes in vitro

Abstract

Purpose. To characterize phenotypic differences between anterior and posterior corneal keratocytes after stimulation with the profibrotic agent transforming growth factor-beta1 (TGF-beta1) 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-mum 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-beta1 concentrations. In addition, anterior and posterior cells cultured in different concentrations of TGF-beta1 were wounded with a razor blade, and the wound area and time to closure were determined. Results. Stimulation by all concentrations of TGF-beta1 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-beta1 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-beta1 concentrations than did posterior keratocytes. After mechanical wounding, posterior cells closed the wound area faster than did anterior cells at all concentrations of TGF-beta1. Conclusions. The present experiments show that anterior and posterior corneal keratocytes exhibit different sensitivities to the profibrotic growth factor TGF-beta1. This heterogeneity of keratocyte response may impact wound closure after mechanical wounding.

Figure 1.

Percentage of Ki-67–positive cells at 0 and 2 ng/mL TGF-β1. At 0 ng/mL TGF-β1, there are small and similar percentages of anterior and posterior cells entering the cell cycle. At 2 ng/mL TGF-β1, there is a greater percentage of posterior cells staining positive for Ki-67 at earlier time points (<7 days). At this same concentration, at later time points (greater than 7 days), there is a greater percentage of anterior cells staining positive for Ki-67.

Figure 2.

Isolated anterior and posterior keratocytes cultured in different concentrations of TGF-β1. (A) Immunofluorescence staining of DAPI (blue), Thy-1 (green), and α-SMA (red). (B) Western blot demonstrating posterior cells required higher concentrations of TGF-β1 than anterior cells for Thy-1 expression (1 vs. 0.1 ng/mL TGF-β1, respectively). (C) Western blot demonstrating posterior cells required higher concentrations of TGF-β1 than anterior cells for α-SMA expression (1 vs. 0.1 ng/mL TGF-β1, respectively).

Figure 3.

Isolated anterior and posterior keratocytes cultured in various concentrations of TGF-β1. (A) Immunofluorescence staining of DAPI (blue), α-SMA (red), and fibronectin (green). (B) Western blot analysis demonstrating that posterior cells required higher concentrations of TGF-β1 than anterior cells for fibronectin expression (1 vs. 0.1 ng/mL TGF-β1, respectively). (C) Western blot demonstrating posterior cells required higher concentrations of TGF-β1 than anterior cells for α-SMA expression (1 vs. 0.1 ng/mL TGF-β1, respectively).

Figure 4.

Expression of Thy-1 (A), α-SMA (B), and fibronectin (C) relative to GAPDH in cat corneal keratocytes cultured for 72 hours with different concentrations of TGF-β1. Significantly greater relative expression of all three proteins was observed in anterior cells at 0.1 ng/mL TGF-β1 compared with posterior cells (Thy-1, P < 0.0001; α-SMA, P = 0.004; fibronectin, P < 0.0001). With 1 ng/mL TGF-β1, this difference begins to resolve, whereas exposure to 2 ng/mL TGF-β1 induces near maximal relative expression of all three proteins in both anterior and posterior cells.

Figure 5.

Closure of a mechanically induced wound in cultured anterior and posterior cells. (A) At TGF-β1 concentration of 1 ng/mL, posterior cells demonstrate faster wound closure than anterior cells over a 12-hour period. (B) Photomicrographs of α-SMA–stained wounded cell cultures exposed to 1 ng/mL TGF-β1. Note the consistently larger wound area exhibited by anterior cells relative to their posterior counterparts at any given postscratch time point. (C) At a TGF-β1 concentration of 2 ng/mL, posterior cells again demonstrate faster wound closure than anterior cells. (D) Photomicrographs of α-SMA–stained wounded cell cultures exposed to 2 ng/mL TGF-β1. Note the consistently larger wound area exhibited by anterior cells relative to their posterior counterparts at each time point after wounding.