Concentration-dependent effects of transforming growth factor β1 on corneal wound healing

Purpose: There is an unmet challenge to promote wound healing in non-healing wounds such as in the post-LASIK (laserassisted in situ keratomileusis) cornea. Using human corneal fibroblasts (HCFs) in cell culture, we investigated the concentration dependence of the growth factor transforming growth factor beta 1 (TGF beta 1) on wound closure. Although high concentrations of TGF beta 1 leads to scarring, we asked whether low concentrations of TGF beta 1 could promote wound healing without generating a large fibrotic response. Methods: HCFs were cultured in supplemented serum-free media (SSFM). Cell migration was assessed by scratchwounding. SMAD 2/3 and p38 mitogen-activated protein kinase (p38MAPK) localization and a-smooth muscle actin (a-SMA) organization were evaluated by immunocytochemistry. Active TGF beta was quantified using a luciferase bio-assay. Results: We found that neutralizing antibody to TGF beta 1 reduced cell migration by 73%, compared to immunoglobulin G (IgG) control, establishing that endogenous TGF beta 1 (determined to be 0.01 ng/ml) is necessary to promote cell migration. To evaluate the concentration-dependent effects of TGF beta 1 on wound closure, HCF migration was quantified to determine the impact of increasing concentrations of TGF beta 1 (0.01-1.0 ng/ml). Compared to control (cells in SSFM), the higher concentrations (0.1 and 1.0 ng/ml TGF beta 1) significantly decreased cell migration (63%-86%), induced myofibroblast differentiation (83%-88%), increased SMAD 2/3 localization into the nucleus (72%-79%) and inhibited the activation of p38MAPK (51%-63%). In contrast, addition of the lower concentration of TGF beta 1 (0.01 ng/ml TGF beta 1) promoted a cell migration rate that was similar to endogenous TGF beta, reduced SMAD 2/3 nuclear localization, and stimulated p38MAPK activation. A TGF beta 1 blocking antibody and the p38MAPK inhibitor, SB202192, was used to demonstrate that p38MAPK activation is necessary for TGF beta 1-induced cell migration. Conclusions: Together, our data demonstrate that low concentrations of TGF beta 1 promote p38MAPK activation that is a key to HCF migration, suggesting that a low concentration of TGF beta may be useful in treating non-healing corneal wounds.