Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Filters applied. Clear all
. 2010 May;119(5):350-7.
doi: 10.1177/000348941011900513.

Inhibitory Effects of Hepatocyte Growth Factor and interleukin-6 on Transforming Growth factor-beta1 Mediated Vocal Fold Fibroblast-Myofibroblast Differentiation

Affiliations
Free PMC article

Inhibitory Effects of Hepatocyte Growth Factor and interleukin-6 on Transforming Growth factor-beta1 Mediated Vocal Fold Fibroblast-Myofibroblast Differentiation

Bimal Vyas et al. Ann Otol Rhinol Laryngol. .
Free PMC article

Abstract

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-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.

Figures

Figure 1
Figure 1
Human vocal fold fibroblasts (hVFF) express myofibroblast marker α-smooth muscle actin (α-SMA) when treated with TGF-β1. VFF were plated on glass slides and treated with 20 ng/ml of TGF-β1 for five days. Cells were fixed with 4% paraformaldehyde and immunostained for α-SMA or vimentin. The top and middle panelsare representative images captured at 20× magnification and the lower panel were captured at 100×. The top panel represents hVFF treated with or without TGF-β1. Cells are larger in size. The middle panel represents hVFF treated with or without TGF-β1. Vimentin (red) is used as control. The lower panel represents hVFF treated with or without TGF-β1. α-SMA (green) is expressed in TGF-β1 treated hVFF.
Figure 2
Figure 2
Dose and time response for human vocal fold fibroblasts (hVFF) as measured by α-SMA when treated with TGF-β1. (A.) Western blot analysis of hVFF incubated with TGF-β1 (0, 5 or 10 ng/mL) for 7 days. Blots were analyzed by densitometry and α-SMA activity was normalized to GAPDH. The histogram is representative of three independent experiments. (B). Western blot analysis of hVFF incubated with TGF-β1 (10 ng/mL) for 1, 3, 5 and 7 days. Blots were analyzed by densitometry and α-SMA activity was normalized to GAPDH. The western blot is representative of three independent experiments.
Figure 3
Figure 3
Time response for rabbit vocal folds treated with TGF-β1 and saline for 1, 3, 5 and 7 days. Western blots were analyzed by densitometry and α-SMA activity was normalized to GAPDH. The histogram is representative of three independent experiments.
Figure 4
Figure 4
HGF decreased TGF-β1 induced α-SMA protein in human vocal fold fibroblasts (hVFF). HVFF were incubated with 10 ng/ml TGF-β1 with and without HGF (5, 10, 20 and 40 ng/ml). After 24 hours, cells were lysed and samples containing equal amounts of total protein were resolved by western blots against α-SMA and GAPDH. Relative expression of α-SMA was evaluated by densitometric analysis by calculating α-SMA to GAPDH ratios. Results shown are representative of 3 independent experiments.
Figure 5
Figure 5
IL-6 decreased TGF-β1 induced α-SMA protein in human vocal fold fibroblasts (hVFF). HVFF were incubated with 10 ng/ml TGF-β1 with and without IL-6 (5, 10, 20 and 40 ng/ml). After 24 hours, cells were lysed and samples containing equal amounts of total protein were resolved by western blot against α-SMA and GAPDH. Relative expression of α-SMA was evaluated by densitometric analysis by calculating α-SMA to GAPDH ratios. Results shown are representative of 3 independent experiments.

Similar articles

See all similar articles

Cited by 21 articles

See all "Cited by" articles

Publication types

Feedback