Inhibitory effects of hepatocyte growth factor and interleukin-6 on transforming growth factor-beta1 mediated vocal fold fibroblast-myofibroblast differentiation.
OBJECTIVE
The role of myofibroblasts in vocal fold scarring has not been extensively studied, partly because of the lack of a robust in vitro model. The objective of this investigation was to develop and characterize a myofibroblast in vitro model that could be utilized to investigate the molecular mechanism of myofibroblast differentiation and function in injured vocal fold tissue.


METHODS
Differentiation of human primary vocal fold fibroblasts (hVFFs) to myofibroblasts was stimulated with 5, 10, or 20 ng/mL of recombinant transforming growth factor-beta1 (TGF-beta1). Cultures were analyzed by immunofluorescence and Western blotting, with an alpha-smooth muscle actin (alpha-SMA) antibody used as a myofibroblast marker. Normal rabbit vocal folds were treated with 10 ng/mL of TGF-beta1 for 7 days for in vivo corroboration. The effects of interleukin-6 (IL-6) and hepatocyte growth factor (HGF) on myofibroblast differentiation were studied with Western blots.


RESULTS
The hVFFs demonstrated positive alpha-SMA labeling in cells stimulated by 10 and 20 ng/mL TGF-beta1, indicating that hVFFs were capable of differentiation to myofibroblasts. Transforming growth factor-beta1 induced the largest increase in alpha-SMA at 10 ng/mL on day 5 of treatment. Both HGF and IL-6 suppressed the expression of TGF-beta1-induced alpha-SMA.


CONCLUSIONS
Our work characterizes a useful in vitro model of TGF-beta1-mediated vocal fold fibroblast-myofibroblast differentiation. The extent of differentiation appears to be attenuated by HGF, suggesting a potential mechanism to support prior work indicating that HGF plays a protective role in reducing scar formation in vocal fold injuries. Paradoxically, IL-6, which has been shown to play a profibrotic role in dermal studies, also attenuated the TGF-beta1 response.
Division of Otolaryngology-Head and Neck Surgery, Department of Surgery, University of Wisconsin-Madison, Madison, Wisconsin 53705-2275, USA.
Bimal
B
Vyas
Keiko
K
Ishikawa
Suzy
S
Duflo
Xia
X
Chen
Susan L
SL
Thibeault
D000199:Actins / Q000276:immunology; D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects*; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology*; D005455:Fluorescent Antibody Technique; D017228:Hepatocyte Growth Factor / Q000494:pharmacology*; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology*; D009130:Muscle, Smooth / Q000166:cytology*; D011817:Rabbits; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D014827:Vocal Cords / Q000166:cytology*
D000199:Actins; D000906:Antibodies; D015850:Interleukin-6; D053773:Transforming Growth Factor beta1; C544013:alpha-smooth muscle actin, mouse; D017228:Hepatocyte Growth Factor