Differences in the TGF-β1–Induced Profibrotic Response of Anterior and Posterior Corneal Keratocytes In Vitro

Abstract

Purpose To characterize phenotypic differences between anterior and posterior corneal keratocytes following stimulation with the pro-fibrotic agent transforming growth factor-beta1 (TGF-β1) in vitro. Methods Sixteen corneas from healthy felines were obtained immediately post-mortem. Lamellar dissection was performed to separate the anterior and posterior stroma at approximately 50% depth either manually (N=2) or using a Moria microkeratome (300μm 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 (α–SMA), and fibronectin were assessed following stimulation with different TGF-β1 concentrations. In addition, anterior and posterior cells cultured in different concentrations of TGF-β1 were wounded with a razor blade and wound area and time to closure determined. Results Stimulation by all concentrations of TGF-β1 increased the proportion of Ki-67 positive cells in anterior and posterior cell cultures, but this increase was noted at earlier time-points in posterior cells as compared to anterior cells. Increasing TGF-β1 concentration also increased the relative expression of Thy-1, α-SMA and fibronectin in anterior and posterior fibroblasts. However, anterior cells expressed these fibrotic markers at lower TGF-β1 concentrations than posterior keratocytes. Following mechanical wounding, posterior cells closed the wound area faster than anterior cells at all concentrations of TGF-β1. Conclusions The present experiments show that anterior and posterior corneal keratocytes exhibit different sensitivities to the pro-fibrotic growth factor, TGF-β1. This heterogeneity of keratocyte response may impact wound closure following mechanical wounding.

Chemical and Topographical Effects on Cell Differentiation and Matrix Elasticity in a Corneal Stromal Layer Model

“…As quiescence or cell morphology alterations are not unique to the keratocyte phenotype,5 we corroborated our mechanical findings with traditional qPCR, to examine the gene expression of characteristic markers, specific to a keratocytic genotype. In previous studies lumican,22, 30 keratocan,22, 31 ALDH 3 ,22, 31, 32 Thy‐1,9, 33, 34 α‐SMA,5, 6, 8, 13, 35 fibronectin,5, 36 CD3435 and vimentin37 have all been examined to help to determine and distinguish the differences in gene and protein expression between corneal keratocytes, fibroblasts and myofibroblast phenotypes in vitro . In this study, we chose to monitor the gene expression profiles of four markers, keratocan, ALDH 3 , Thy‐1 and α‐SMA in response to the chemical and topographical cues.…”

“…Therefore, this gene served as a marker for keratocyte phenotype. Thy‐1 expression can be induced via the transformation of a corneal karatocytes to corneal fibroblasts or myofibroblasts9 and it is regularly used in corneal tissue engineering applications to help distinguish corneal fibroblasts from keratocytes 9, 33, 34. In addition, corneal wound fibroblasts and myofibroblasts express α‐SMA, which is linked to wound contraction 6, 8, 13.…”

Yin Yang 1 Is a Novel Regulator of Pulmonary Fibrosis

“…Hematoxylin and eosin and Masson's trichrome stains were used for analysis of pathologic changes. Immunohistochemical staining for a-SMA and YY1 tissues was performed according to a previous paper (25). The severity of fibrosis was evaluated in stained sections by an individual who was blinded to the genotypes of the mice.…”

“…Cells were supplemented with Dulbecco's modified Eagle medium and 10% fetal bovine serum at 378C for 4 days. Anti-a-SMA antibody (1A4; DAKO Cytomation, Carpinteria, CA), anti-collagen (LF-67; Larry W. Fisher, Ph.D., Matrix Biochemistry Section, Craniofacial and Skeletal Diseases Branch of NIH), and anti-YY1 antibody (H-414; Santa Cruz, Santa Cruz, CA) were stained according to a previous paper (25). The cells were examined with a Zeiss fluorescence microscope.…”

Ocular Fibroblast Diversity: Implications for Inflammation and Ocular Wound Healing

“…11,12 The sensitivity of a tissue to inflammatory stimuli is most likely determined, at least in part, by the regional type of fibroblast. 13,30 In this study, we compared the responses of fibroblasts isolated from the cornea, lacrimal gland, and Tenon's capsule, to the proinflammatory cytokine, IL-1␤. We found that, after IL-1␤ stimulation, corneal fibroblasts exhibit heightened proinflammatory cytokine and PGE 2 production relative to lacrimal gland or Tenon's capsule fibroblasts (Figs.…”