Objective To investigate the role of Smad3 as a regulator of transforming growth factor (TGF)-β1-mediated cell activities associated with fibrosis in normal human vocal fold fibroblasts. We also sought to confirm the temporal stability of Smad3 knockdown via siRNA. Vocal fold fibroblasts were employed to determine the effects of Smad3 knockdown on TGF-β1-mediated migration and contraction as well as regulation of connective tissue growth factor (CTGF). We hypothesized that Smad3 is an ideal candidate for therapeutic manipulation in vivo based on its role in fibrosis. Study Design In vitro Methods Knockdown of Smad3 via siRNA was performed in our normal human vocal fold cell line. Three-dimensional collagen gel contraction and scratch assays were employed to determine the role of Smad3 on TGF-β1-mediated contraction and migration, respectively. The role Smad3 in the induction of CTGF was characterized via SDS-Page. The effects of Smad3 signaling on Smad7 mRNA and protein were also quantified. Results Smad3 knockdown was temporally stable up to 72 hours (p<0.001). Smad3 knockdown diminished TGF-β1-mediated collagen gel contraction and migration. Smad3 knockdown also blunted induction of CTGF, but had no effect on TGF-β1-mediated Smad7 mRNA or protein induction. Conclusion TGF-β1 stimulated pro-fibrotic cell activities in our cell line and these actions were largely reduced with Smad3 knockdown. These data provide continued support for therapeutic targeting of Smad3 for vocal fold fibrosis as it appears to regulate the fibrotic phenotype.
Objectives/Hypothesis Mesenchymal stem cells (MSCs) hold therapeutic promise for vocal fold scar, yet the precise mechanism(s) underlying tissue level changes remain unclear. We hypothesize that MSCs interact with native fibroblasts to favorably affect healing. Furthermore, we hypothesize these interactions vary based on MSC source. Methods Vocal fold fibroblasts (VFFs), adipose-derived stem cells (ASCs), and bone-marrow derived stem cells (BMSCs) were extracted from Sprague-Dawley rats and a co-culture model was employed culturing VFFs+/-TGF-β1 (10ng/ml) +/− MSCs. Mono-culture MSCs were also prepared as a control. Both extracellular matrix (ECM) and components of the TGF-β signaling pathway were analyzed via polymerase chain reaction and SDS-PAGE. Results Significantly decreased TGF-β1 mRNA and α-smooth muscle actin protein was observed in VFFs in response to TGF-β1 in the co-culture with both MSCs (p<0.05, p<0.01). BMSCs significantly downregulated Collagen I (p<0.05), Collagen III (p<0.05), Smad3 (p<0.01), and TGF-β1 receptor I (p<0.01) mRNA in VFFs. Hyaluronic synthase-1 and 2 increased in co-cultured BMSCs when compared with mono-cultured BMSCs at baseline and in response to TGF-β1 (p<0.01). Conclusions MSCs had a favorable effect on ECM regulation as well as suppression of TGF-β1 signaling in VFF. Bidirectional paracrine signaling was also observed as VFFs altered ECM regulation in MSCs. These data provide insight into the regenerative effects of MSCs and provide a foundation for clinical application.
Objectives/Hypothesis NR4A1 was recently identified as an endogenous inhibitor of TGF-β-induced fibrosis and the role of this nuclear receptor has not been elucidated in tissue health or the response to injury in the vocal folds. Given the clinical implications of vocal fold fibrosis, we investigated NR4A1 expression during vocal fold wound healing in vivo and the regulatory roles of NR4A1 on vocal fold fibroblasts (VFFs) in vitro with the ultimate goal of developing targeted therapies for this challenging patient population. Study Design In vivo and in vitro Methods In vivo, the temporal pattern of NR4A1 mRNA expression was quantified following rat vocal fold injury. In vitro, the role of NR4A1 on TGF-β1-mediated transcription of genes underlying fibrosis as well as myofibroblast differentiation and collagen gel contraction was quantified in our human VFF line. Small interfering RNA was employed to alter NR4A1 expression to further elucidate this complex system. Results Nr4a1 mRNA increased 1 day after injury and peaked at 7 days. Knockdown of NR4A1 resulted in upregulation of COL1A1 and TGF-β1 with TGF-β1 stimulation (both p<0.001) in VFFs. NR4A1 knockdown also resulted in increased alpha smooth muscle actin positive cells (p=0.013) and contraction (p=0.002) in response to TGF-β1. Conclusions NR4A1 has not been described in vocal fold health or disease. Upregulation of TGF-β following vocal fold injury was concurrent with increased NR4A1 expression. These data provide a foundation for the development of therapeutic strategies given persistent TGF-β signaling in vocal fold fibrosis. Level of Evidence N/A
Objective An obstacle to clinical use of RNA-based gene suppression is instability and inefficiency of current delivery modalities. Nanoparticle delivery likely holds great promise, but the kinetics and transfection conditions must be optimized prior to in vivo utility. We investigated a RNA nanoparticle complex incorporating a “lipitoid” transfection reagent in comparison to a commercially-available reagent. Study Design In vitro Methods We investigated which variables influence transfection efficiency of lipitoid oligomers and a commercially-available reagent across species, in vitro. These variables included duration, dose, and number of administrations, as well as serum and media conditions. The target gene was Smad3, a signaling protein in the Transforming Growth Factor (TGF)-β cascade implicated in fibroplasia in the vocal folds and other tissues. Results The two reagents suppressed Smad3 mRNA for up to 96 hours; Lipitoid performed favorably and comparably. Both compounds yielded 60–80% mRNA knockdown in rat, rabbit, and human vocal fold fibroblasts (p< 0.05 relative to control). Dose and number of administrations played a significant role in gene suppression (p<0.05). Suppression was more dose-sensitive with Lipitoid; at a constant siRNA concentration, a 50% decrease in gene expression was observed in response to a 5-fold increase in Lipitoid concentration. Increased number of administrations enhanced gene suppression; ~45% decrease between one and four administrations. Neither serum nor media type altered efficiency. Conclusion Lipitoid effectively knocked down Smad3 expression across multiple transfection conditions. These preliminary data are encouraging and Lipitoid warrants further investigation with the ultimate goal of clinical utility. Level of Evidence N/A
Conditioned media affected many profibrotic cell activities in TGF-β1-stimulated VFFs, likely related to altered TGF-β signaling. These data provide preliminary insight regarding the antifibrotic effects of MSCs and further support their progression to clinical utility.
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