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James V. Jester, Jiying Huang, Stephen Fisher, Jennifer Spiekerman, Jin Ho Chang, Woodring E. Wright, Jerry W. Shay; Myofibroblast Differentiation of Normal Human Keratocytes and hTERT, Extended-Life Human Corneal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2003;44(5):1850-1858. doi: 10.1167/iovs.02-0973.
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purpose. The purpose of this study was to determine whether TGFβ 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 TGFβ 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 α-smooth muscle actin (α-SMA).
results. Treatment of cultured keratocytes with TGFβ (1 ng/mL) induced cell spreading, assembly of actin filaments, formation of focal adhesions, and expression of α-SMA, which was blocked by the addition of RGD-containing peptides (100 μM). A similar response was identified in hTERT-expressing human corneal fibroblast cell lines, showing a 69-fold increase in α-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 TGFβ 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.
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