title: 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.
authors.affiliation: Flaum Eye Institute, University of Rochester Medical Center, Rochester, New York, USA. holly_hindman@urmc.rochester.edu
authors.forename: Holly B
authors.initials: HB
authors.lastname: Hindman
authors.forename: Jennifer N
authors.initials: JN
authors.lastname: Swanton
authors.forename: Richard P
authors.initials: RP
authors.lastname: Phipps
authors.forename: Patricia J
authors.initials: PJ
authors.lastname: Sime
authors.forename: Krystel R
authors.initials: KR
authors.lastname: Huxlin
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002415:Cats; D002478:Cells, Cultured; D065306:Corneal Injuries; D003319:Corneal Stroma / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D007150:Immunohistochemistry; D019394:Ki-67 Antigen / Q000378:metabolism; D010641:Phenotype; D018800:Thy-1 Antigens / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
chemical_list: D000199:Actins; D005353:Fibronectins; D019394:Ki-67 Antigen; D018800:Thy-1 Antigens; D053773:Transforming Growth Factor beta1