The role of <scp>S</scp>mad3 in the fibrotic phenotype in human vocal fold fibroblasts

Branski, Ryan C.; Bing, Renjie; Kraja, Iv; Amin, Milan R.

doi:10.1002/lary.25673

Cited by 28 publications

(64 citation statements)

References 23 publications

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“…Lee et al recently demonstrated that suppression of Smad3 using a topical siRNA gel conferred resistance to radiation‐induced cutaneous fibrosis in a murine model . Our laboratory also has recently demonstrated that SMAD3 siRNA reduced the fibrotic phenotype in vocal fold fibroblasts in vitro . Most recently, our laboratory described successful knockdown of SMAD3 via local siRNA injection in a leporine model of vocal fold injury.…”

Section: Discussionsupporting

confidence: 91%

“…Our laboratory and others have identified SMAD3 as a principle mediator of fibrotic VF wound healing due to its role in TGF‐β1 signaling, which in turn regulates numerous fibroblast activities including migration, proliferation, and production of extracellular matrix proteins . SMAD7 is a homolog of SMAD3 and serves as an antifibrotic regulator opposing the actions of SMAD3 through competitive inhibition .…”

Section: Discussionmentioning

confidence: 99%

“…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 . Cumulatively, these data suggest that gene therapy‐mediated knockdown of SMAD3 may have the potential to be an efficacious therapeutic to alter wound‐healing following vocal fold injury.…”

Section: Introductionmentioning

confidence: 99%

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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: Discussionsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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SMAD3 expression and regulation of fibroplasia in vocal fold injury

et al. 2017

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“…Previous reports suggested that GR inhibited TGF‐β signaling by directly targeting the transcriptional activation function of SMAD3, which is consistent with our data. Our laboratory and others identified SMAD3 as a principle mediator of fibrotic VF wound healing due to its role in TGF‐β1 signaling, which in turn regulates numerous fibroblast activities including migration, proliferation, and production of extracellular matrix . Clearly, these data suggest that DM has antifibrotic effects.…”

Section: Discussionmentioning

confidence: 84%

“…Conversely, SMAD7 is a competitive inhibitor of SMAD activation, and therefore is thought to be antifibrotic. Activated Smads have been implicated in a variety of fibrotic processes, suggesting that Smad activation plays a central role in fibrosis . Ideally, therapies for vocal fold fibrosis should likely alter the fibroblast phenotype, and emerging data suggest that GCs attenuate TGF‐β/SMAD signaling in human fetal lung fibroblasts .…”

Section: Introductionmentioning

confidence: 99%

Phosphorylation of the glucocorticoid receptor alters SMAD signaling in vocal fold fibroblasts

Mukudai¹,

Hiwatashi²,

Bing³

et al. 2018

The Laryngoscope

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Objectives/Hypothesis Direct glucocorticoid (GC) injection for vocal fold (VF) scarring has evolved as a therapeutic strategy, but the mechanisms underlying the antifibrotic effects remain unclear. GCs act via the glucocorticoid receptor (GR), which is phosphorylated at multiple serine residues in a hormone‐dependent manner to affect bioactivity. We hypothesize that GCs regulate SMAD signaling via GR phosphorylation in vocal fold fibroblasts (VFFs). Study Design In vitro. Methods Human VFFs were treated with dexamethasone (DM; 10−5–10−7M) ± transforming growth factor (TGF)‐β1 (10 ng/mL). RU486 (10−6M) was employed to isolate the regulatory effects of GR. Total GR, Ser211, and Ser203 phosphorylation was examined via sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and immunocytochemistry. Quantitative polymerase chain reaction was employed to determine GR‐mediated effects of DM on genes related to fibrosis. Results Total GR and Ser211 phosphorylation was observed predominantly in the nucleus 1 hour after DM administration. DM decreased total GR expression, but Ser203 and Ser211 phosphorylation increased. RU486 limited the effects of DM. SMAD3 and SMAD7 mRNA expression significantly decreased 4 hours after DM administration (P < 0.05); this response was negated by RU486. COL1A1 remained unchanged, and ACTA2 significantly increased following 24 hours of DM treatment (P < 0.05). Conclusion DM regulated TGF‐β1 signaling via altered SMAD3 and SMAD7 expression. This response was associated with altered GR phosphorylation. These findings provide insight into the mechanisms of steroidal effects on vocal fold repair; ultimately, we seek to enhance therapeutic strategies for these challenging patients. Level of Evidence NA Laryngoscope, 129:E187–E193, 2019

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Preliminary study of a novel transfection modality for in vivo siRNA delivery to vocal fold fibroblasts

et al. 2016

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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

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

Cited by 28 publications

References 23 publications

SMAD3 expression and regulation of fibroplasia in vocal fold injury

SMAD3 expression and regulation of fibroplasia in vocal fold injury

Phosphorylation of the glucocorticoid receptor alters SMAD signaling in vocal fold fibroblasts

Preliminary study of a novel transfection modality for in vivo siRNA delivery to vocal fold fibroblasts

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