Myofibroblast differentiation of normal human keratocytes and hTERT, extended-life human corneal fibroblasts

Abstract

Purpose: The purpose of this study was to determine whether TGFbeta induces myofibroblast differentiation in cultured human keratocytes and in telomerase (hTERT)-immortalized human corneal fibroblast cell lines.

Methods: Normal human corneal keratocytes were isolated from donor corneas of various ages and grown under serum-free (cultured keratocytes) or serum-added (corneal fibroblasts) conditions. Corneal fibroblasts were infected with the MPSV-hTERT retroviral vector, and selected clones were isolated and characterized by chromosomal karyotyping. The responses of normal cultured keratocytes and serum-starved corneal fibroblasts to TGFbeta in the presence or absence of Arg-Gly-Asp (RGD)-containing peptides and neutralizing antibodies to platelet-derived growth factor (PDGF) were characterized by immunocytochemistry, Western blot analysis, and real-time PCR, to identify assembly of actin filaments, formation of focal adhesions, and expression of alpha-smooth muscle actin (alpha-SMA).

Results: Treatment of cultured keratocytes with TGFbeta (1 ng/mL) induced cell spreading, assembly of actin filaments, formation of focal adhesions, and expression of alpha-SMA, which was blocked by the addition of RGD-containing peptides (100 microM). A similar response was identified in hTERT-expressing human corneal fibroblast cell lines, showing a 69-fold increase in alpha-SMA message. Furthermore, treatment of hTERT corneal fibroblasts with RGD or anti-PDGF inhibited myofibroblast differentiation. Karyotype analysis of hTERT corneal fibroblasts identified age-dependent chromosomal aberrations in cells of older donors but not in those of a 10-year-old donor.

Conclusions: Induction of myofibroblast differentiation by TGFbeta in cultured human keratocytes and hTERT corneal fibroblasts occurs through a similar signal transduction pathway to that previously identified in the rabbit, which involves an autocrine PDGF feedback loop.