SMAD3 expression and regulation of fibroplasia in vocal fold injury

Abstract

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.

Quantifying vocal fold wound‐healing biomechanical property changes

“…The current protocol was approved by the New York University School of Medicine Institutional Animal Care and Use Committee. Twelve New Zealand white rabbits (2–3 kg) were anesthetized and underwent laryngoscopy and endoscopic right vocal fold injury using a previously described model by our group . Using ketamine (70 mg/kg) and xylazine (2 mg/kg) for anesthetic induction and isoflurane for maintenance, each rabbit underwent supine laryngoscopy with a size 1 Karl Storz (Flanders, NJ) laryngoscope.…”

Nanoparticle delivery of RNA‐based therapeutics to alter the vocal fold tissue response to injury

“…Under the guidance of 2.7‐mm, 0° or 30° telescope (Karl Storz), the VFs were injured unilaterally by separating the lamina propria from the thyroarytenoid muscle via insertion of a 22‐gauge needle at the lateral portion of right VF, followed by removal of the lamina propria with microscissors and microforceps. This model and the surgical technique have been described previously by our group …”

“…Specifically, Smad3 expression increased 3 days following injury with maximal expression 7 days following VF injury. [4][5][6]11 These data provide some insight into temporal opportunities for intervention targeting Smad3 and serve as the foundation for the timing of treatment in the current study. Globally, we hypothesized that Smad3 is an ideal therapeutic target in vivo, as modulating this pathway can likely redirect wound healing toward a more regenerative, less fibrotic phenotype.…”

“…This model and the surgical technique have been described previously by our group. 11,33 Transfection solutions. Ten microliters of 5 lM Smad3 siRNA was complexed with 10 lL of 210 lM lipitoid diluted in deionized water/13 PBS buffer by adding the siRNA to the lipitoid solution.…”

“…We also described the temporal pattern of Smad3 expression following VF injury in vivo. Specifically, Smad3 expression increased 3 days following injury with maximal expression 7 days following VF injury . These data provide some insight into temporal opportunities for intervention targeting Smad3 and serve as the foundation for the timing of treatment in the current study.…”