title: Growth factor regulation of corneal keratocyte mechanical phenotypes in 3-D collagen matrices.
abstract: OBJECTIVE
  To assess how wound healing cytokines and the extracellular matrix (ECM) environment regulate the keratocyte mechanical phenotype.
  
  
  METHODS
  Rabbit corneal keratocytes were plated within standard bovine or rat tail type I collagen matrices (2.5 mg/mL), compressed collagen matrices (approximately 100 mg/mL), or on collagen-coated dishes and cultured for up to 7 days in serum-free media, platelet derived growth factor BB (PDGF BB), insulin-like growth factor (IGF), TGFβ1, TGFβ2, or FGF2. F-actin, α-smooth muscle actin (α-SMA) and collagen fibrils were imaged using confocal microscopy. Cell morphology, local matrix reorganization, and global matrix contraction were quantified digitally.
  
  
  RESULTS
  IGF and PDGF BB stimulated elongation of keratocytes and extension of dendritic processes within 3-D matrices, without inducing stress fiber formation or collagen reorganization. In contrast, treatment with TGFβ1 and TGFβ2 increased keratocyte contractility, as indicated by stress fiber formation and matrix compaction and alignment. This transformation was enhanced at higher cell densities within standard 3-D matrices, in which α-SMA was incorporated into stress fibers. In contrast, α-SMA was expressed within compressed 3-D matrices even at low cell density. FGF2 did not produce significant cytoskeletal or matrix reorganization in standard 3-D matrices; however, stress fibers were consistently expressed within compressed collagen matrices and on rigid two-dimensional substrates. Inhibiting Rho kinase blocked both TGFβ- and FGF2-induced stress fiber formation.
  
  
  CONCLUSIONS
  Keratocytes cultured in IGF or PDGF BB maintain a quiescent mechanical phenotype over a range of matrix environments. In contrast, the mechanical phenotypes induced by FGF and TGFβ vary in response to the structural and/or mechanical properties of the ECM.
authors.affiliation: Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9057, USA.
authors.forename: Neema
authors.initials: N
authors.lastname: Lakshman
authors.forename: W Matthew
authors.initials: WM
authors.lastname: Petroll
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002452:Cell Count; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D024042:Collagen Type I / Q000378:metabolism; D060527:Corneal Keratocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology*; D018613:Microscopy, Confocal; D010641:Phenotype; D011817:Rabbits; D014945:Wound Healing / Q000502:physiology
chemical_list: D000199:Actins; D024042:Collagen Type I; D036341:Intercellular Signaling Peptides and Proteins; D003094:Collagen