Smad3: An emerging target for vocal fold fibrosis

Paul, Benjamin C.; Rafii, Benjamin; Gandonu, Sonate; Bing, Renjie; Born, Hayley; Amin, Milan R.; Branski, Ryan C.

doi:10.1002/lary.24723

Cited by 24 publications

(36 citation statements)

References 23 publications

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“…For example, a topical siRNA gel effectively attenuated Smad3 expression in a mouse model, leading to resistance to radiation‐induced cutaneous fibrosis . Regarding the vocal fold, our laboratory first described increased SMAD3 mRNA expression following vocal fold injury, and recently achieved local VF knockdown of SMAD3 expression in vivo (data under review) . In vitro, our laboratory also confirmed that siRNA targeting SMAD3 suppressed collagen gene expression in human vocal fold fibroblasts under stimulation with exogenous TGF‐β1, and knockdown of SMAD3 limited TGF‐β‐induced contraction .…”

Section: Introductionsupporting

confidence: 56%

SMAD3 expression and regulation of fibroplasia in vocal fold injury

et al. 2017

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Objectives/Hypothesis Recent reports highlight the efficacy of small interfering RNA (siRNA) targeting SMAD3 to regulate transforming growth factor β (TGF-β)-mediated fibroplasia in vocal fold fibroblasts. The current study sought to investigate SMAD3 expression during wound healing in vivo and quantify the downstream transcriptional events associated with SMAD3 knockdown in vitro. Study Design In vivo and in vitro Methods Unilateral vocal fold injury was created in a rabbit model. SMAD3 and SMAD7 mRNA expression was quantified at 1 hour and 1, 3, 7, 14, 30, 60, and 90 days following injury. In vitro, multi-gene analysis technology was employed in our immortalized human vocal fold fibroblast cell line following TGF-β1 stimulation +/− SMAD3 knockdown across time points. Results SMAD3 mRNA expression increased following injury; upregulation was significant at 3 and 7 days compared to control (both p<0.001). SMAD7 mRNA was also upregulated at 3, 7, and 14 days (p=0.02, p<0.001, and p<0.001, respectively). In vitro, SMAD3 knockdown reduced the expression of multiple pro-fibrotic, TGF-β signaling, and extracellular matrix metabolism genes at 6 and 24 hours following TGF-β1 stimulation. Conclusions Cumulatively, these data support SMAD3 as a potential master regulator of TGF-β-mediated fibrosis. SMAD3 transcription peaked 7 days following injury. Multi-gene analysis indicated that the therapeutic effectiveness of SMAD3 knockdown may be related to regulation of downstream mediators of fibroplasia and altered TGF-β signaling.

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Section: Introductionsupporting

confidence: 56%

SMAD3 expression and regulation of fibroplasia in vocal fold injury

et al. 2017

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“…Activated Smads have been implicated in a variety of fibrotic processes, suggesting that Smad activation plays a central role in both collagen production and accumulation in these diseases. Preliminary investigation by our laboratory confirmed upregulation of Smad3 messenger (m)RNA following acute vocal fold injury . Globally, we hypothesize that Smad3 is a master regulator of fibrosis, and manipulation of Smad3 via RNA‐based therapies holds significant clinical promise.…”

Section: Introductionmentioning

confidence: 70%

The role of Smad3 in the fibrotic phenotype in human vocal fold fibroblasts

Branski¹,

Bing²,

Kraja³

et al. 2015

The Laryngoscope

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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.

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“…The development of physiologically relevant therapeutics for fibrosis in general, and more specifically, VF scar requires increased insight into the biochemical mechanisms underlying these aberrant processes. Our group previously reported TGF‐β signaling was Smad3‐dependant in VF fibroblasts, and RNA‐based therapy to alter Smad3 expression may evolve into the clinical milieu to limit the TGF‐β1–mediated profibrotic phenotype . However, an alternative approach has evolved.…”

Section: Introductionmentioning

confidence: 99%

NR4A1 is an endogenous inhibitor of vocal fold fibrosis

et al. 2017

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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

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Smad3: An emerging target for vocal fold fibrosis

Abstract: These data provide a foundation for further investigation regarding the development of novel RNA-based therapeutics for the VF, specifically locally delivered siRNA for challenging fibrotic conditions of the VF.

Cited by 24 publications

References 23 publications

SMAD3 expression and regulation of fibroplasia in vocal fold injury

SMAD3 expression and regulation of fibroplasia in vocal fold injury

The role of Smad3 in the fibrotic phenotype in human vocal fold fibroblasts

NR4A1 is an endogenous inhibitor of vocal fold fibrosis

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