RESUMO
PURPOSE: There is an unmet challenge to promote wound healing in non-healing wounds such as in the post-LASIK (laser-assisted in situ keratomileusis) cornea. Using human corneal fibroblasts (HCFs) in cell culture, we investigated the concentration dependence of the growth factor transforming growth factor ß1 (TGFß1) on wound closure. Although high concentrations of TGFß1 leads to scarring, we asked whether low concentrations of TGFß1 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 scratch-wounding. SMAD 2/3 and p38 mitogen-activated protein kinase (p38MAPK) localization and α-smooth muscle actin (α-SMA) organization were evaluated by immunocytochemistry. Active TGFß was quantified using a luciferase bio-assay. RESULTS: We found that neutralizing antibody to TGFß1 reduced cell migration by 73%, compared to immunoglobulin G (IgG) control, establishing that endogenous TGFß1 (determined to be 0.01 ng/ml) is necessary to promote cell migration. To evaluate the concentration-dependent effects of TGFß1 on wound closure, HCF migration was quantified to determine the impact of increasing concentrations of TGFß1 (0.01-1.0 ng/ml). Compared to control (cells in SSFM), the higher concentrations (0.1 and 1.0 ng/ml TGFß1) 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 TGFß1 (0.01 ng/ml TGFß1) promoted a cell migration rate that was similar to endogenous TGFß, reduced SMAD 2/3 nuclear localization, and stimulated p38MAPK activation. A TGFß1 blocking antibody and the p38MAPK inhibitor, SB202192, was used to demonstrate that p38MAPK activation is necessary for TGFß1-induced cell migration. CONCLUSIONS: Together, our data demonstrate that low concentrations of TGFß1 promote p38MAPK activation that is a key to HCF migration, suggesting that a low concentration of TGFß may be useful in treating non-healing corneal wounds.