SMAD3 expression and regulation of fibroplasia in vocal fold injury

Abstract

Objective: 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 profibrotic, TGF-β signaling, and extracellular matrix metabolism genes at 6 and 24 hours following TGF-β1 stimulation.

Conclusion: 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.

Level of evidence: NA. Laryngoscope, 127:E308-E316, 2017.

Keywords: PCR array; SMAD3; Vocal fold; fibrosis; siRNA; transforming growth factor-β.

Figure 1

SMAD3 and SMAD7 mRNA expression changes following rabbit vocal fold injury. SMAD3 gene expression of injured right vocal fold increased at 1 hour post-injury and peaked at day 7; significant differences were observed at days 3 and 7 compared to control (A) (both p<0.001). SMAD7 expression peaked on day 7; significant differences were observed at days 3, 7, and 14 after injury (B) (p=0.02, p<0.001, and p<0.001, respectively). Data presented as mean ± SEM (n=4 per group; *p<0.05, **p<0.01, and ***p<0.001).

Figure 2

Clustergram of eight representative genes. mRNA expression was examined 6 and 24 hours following TGF-β1 stimulation +/− siRNA for SMAD3; the average of each condition is displayed. TGF-β1 = transforming growth factor-β1.