Inhibitory effects of hepatocyte growth factor and interleukin-6 on transforming growth factor-β1 mediated vocal fold fibroblast-myofibroblast differentiation

Objectives: 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-β1 (TGF-β1). Cultures were analyzed by immunofluorescence and Western blotting, with an α-smooth muscle actin (α-SMA) antibody used as a myofibroblast marker. Normal rabbit vocal folds were treated with 10 ng/mL of TGF-β1 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 α-SMA labeling in cells stimulated by 10 and 20 ng/mL TGF-β1, indicating that hVFFs were capable of differentiation to myofibroblasts. Transforming growth factor-β1 induced the largest increase in α-SMA at 10 ng/mL on day 5 of treatment. Both HGF and IL-6 suppressed the expression of TGF-β1-induced α-SMA. Conclusions: Our work characterizes a useful in vitro model of TGF-β1-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-β1 response.