title: Concentration-dependent effects of transforming growth factor β1 on corneal wound healing.
abstract: OBJECTIVE
  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 β1 (TGFβ1) on wound closure. Although high concentrations of TGFβ1 leads to scarring, we asked whether low concentrations of TGFβ1 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 TGFβ was quantified using a luciferase bio-assay.
  
  
  RESULTS
  We found that neutralizing antibody to TGFβ1 reduced cell migration by 73%, compared to immunoglobulin G (IgG) control, establishing that endogenous TGFβ1 (determined to be 0.01 ng/ml) is necessary to promote cell migration. To evaluate the concentration-dependent effects of TGFβ1 on wound closure, HCF migration was quantified to determine the impact of increasing concentrations of TGFβ1 (0.01-1.0 ng/ml). Compared to control (cells in SSFM), the higher concentrations (0.1 and 1.0 ng/ml TGFβ1) 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 TGFβ1 (0.01 ng/ml TGFβ1) promoted a cell migration rate that was similar to endogenous TGFβ, reduced SMAD 2/3 nuclear localization, and stimulated p38MAPK activation. A TGFβ1 blocking antibody and the p38MAPK inhibitor, SB202192, was used to demonstrate that p38MAPK activation is necessary for TGFβ1-induced cell migration.
  
  
  CONCLUSIONS
  Together, our data demonstrate that low concentrations of TGFβ1 promote p38MAPK activation that is a key to HCF migration, suggesting that a low concentration of TGFβ may be useful in treating non-healing corneal wounds.
authors: Mount Sinai School of Medicine, Dept of Ophthalmology, New York, NY 10029, USA.
authors: Lingyan
authors: L
authors: Wang
authors: Chun-Ying
authors: CY
authors: Ko
authors: Erin E
authors: EE
authors: Meyers
authors: Benjamin S
authors: BS
authors: Pedroja
authors: Nadia
authors: N
authors: Pelaez
authors: Audrey M
authors: AM
authors: Bernstein
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D019138:Antibodies, Blocking / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects*; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000601:surgery; D016895:Culture Media, Serum-Free; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D051785:Smad Proteins / Q000235:genetics / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D014945:Wound Healing / Q000187:drug effects*; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; D019138:Antibodies, Blocking; D016895:Culture Media, Serum-Free; D047428:Protein Kinase Inhibitors; D051785:Smad Proteins; D053773:Transforming Growth Factor beta1; D048051:p38 Mitogen-Activated Protein Kinases