Differences in the TGF-{beta}1-induced profibrotic response of anterior and posterior corneal keratocytes in vitro.
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.
Hindman
Holly B
HB
Flaum Eye Institute, University of Rochester Medical Center, Rochester, New York, USA. holly_hindman@urmc.rochester.edu
Swanton
Jennifer N
JN
Phipps
Richard P
RP
Sime
Patricia J
PJ
Huxlin
Krystel R
KR
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*
D000199:Actins; D005353:Fibronectins; D019394:Ki-67 Antigen; D018800:Thy-1 Antigens; D053773:Transforming Growth Factor beta1