In vitro mechanical vibration down-regulates pro-inflammatory and pro-fibrotic signaling in human vocal fold fibroblasts

Abstract

Introduction: Voice rest following phonotrauma or phonosurgery has a considerable clinical impact, but clinical recommendations are inconsistent due to inconclusive data. As biopsies of the vocal folds (VF) for molecular biology studies in humans are unethical, we established a new in vitro model to explore the effects of vibration on human vocal fold fibroblasts (hVFF) in an inflammatory and normal state, which is based on previously published models.

Methods: By using a phonomimetic bioreactor we were able to apply predefined vibrational stress patterns on hVFF cultured under inflammatory or normal conditions. Inflammatory and pro-fibrotic stimuli were induced by interleukin (IL)1β and transforming growth factor (TGF)β1, respectively. Mechanical stimulation was applied four hours daily, over a period of 72 hours. Outcome measurements comprised assessment of extracellular matrix (ECM)-related components, angiogenic factors, and inflammatory and fibrogenic markers on gene expression and protein levels.

Results: Under inflammatory conditions, the inflammatory cytokine IL11, as well as the myofibroblast marker alpha smooth muscle actin (α-SMA) were significantly reduced when additional vibration was applied. The desirable anti-fibrotic ECM component hyaluronic acid was increased following cytokine treatment, but was not diminished following vibration.

Conclusion: Our experiments revealed the effect of vibrational stress on hVFF in an inflammatory state. Elevated levels of certain pro-inflammatory/pro-fibrotic factors could be mitigated by additional vibrational excitation in an in vitro setting. These findings corroborate clinical studies which recommend early voice activation following an acute event.

Fig 1. Experimental design.

Twenty-four hours after seeding the cells, the cell culture medium was changed to a serum-free medium for starvation. The next day, cells were divided into four groups with different conditions (with or without cytokines–static or dynamic). After a further 73 hours, cells were harvested. Two wells of each condition were used for qPCR, Luminex as well as ELISA and one for Western Blot. All samples were taken into account for LDH assay.

Fig 2. Effect of vibration and/or cytokine treatment on cell viability and gene expression.

After 72 hours of exposure to a vibration pattern and/or cytokine treatment and an additional one-hour rest period, supernatants were collected for LDH activity assay. The LDH activity was expressed as percentage of the maximal LDH activity. p<0.05 was considered significant.

Fig 3. Effect of cytokine treatment and/or vibration on HA metabolism.

Supernatants were collected for ELISA to quantify values of HA (a). RT-qPCR analysis of HA-related gene expression (b-e) was performed. Results are represented as mean ± S.D. of four independent experiments (N = 4). Statistical analysis was performed using one-way ANOVA (normally distributed data) or Kruskal-Wallis test (non-parametric data), p<0.05 was considered significant; decimal numbers above the bars represent the statistically significant p-values from post-hoc comparisons. For the p-values not presented here, see S2 File. HA (hyaluronic acid), HAS1 (hyaluronan synthase 1), HAS2 (hyaluronan synthase 2), HAS3 (hyaluronan synthase 3), HYAL2 (hyaluronidase 2).

Fig 4. Effect of cytokine treatment and/or vibration on ECM-related molecules.

mRNA (a-c and f) and protein levels (d and e) of ECM-related molecules were analyzed by RT-qPCR, Western blot and Luminex, respectively. Results are represented as mean ± S.D. of four independent experiments (N = 4). Statistical analysis was performed using one-way ANOVA (normally distributed data) or Kruskal-Wallis test (non-parametric data), p<0.05 was considered significant; decimal numbers above the bars represent the statistically significant p-values from post-hoc comparisons. For the p-values not presented here, see S2 File. COL1A1 (collagen 1 alpha 1), COL1A2 (collagen 1 alpha 2), COL3A1 (collagen 3 alpha 1), MMP1 (matrix metalloproteinase 1).

Fig 5. Effect of cytokine treatment and/or vibration on ECM-related molecules, growth factors and angiogenic factors.

mRNA (a) and protein levels (b-d) of ECM-related molecules as well as growth- and angiogenic factors were analyzed by RT-qPCR and Luminex, respectively. Results are represented as mean ± S.D. of four independent experiments (N = 4). Statistical analysis was performed using one-way ANOVA (normally distributed data) or Kruskal-Wallis test (non-parametric data), p<0.05 was considered significant; decimal numbers above the bars represent the statistically significant p-values from post-hoc comparisons. For the p-values not presented here, see S2 File. FN1 (fibronectin 1), TIMP1 (Tissue inhibitor of metalloproteinase), bFGF (basic fibroblast growth factor), VEGF A (vascular endothelial growth factor A), VEGF C (vascular endothelial growth factor C).

Fig 6. Effect of cytokine treatment and/or vibration on inflammatory and fibrogenic markers.

mRNA (a, b, d, e) and protein levels (c and f) of inflammatory and fibrogenic markers were analyzed by RT-qPCR, Western blot and Luminex, respectively. Results are represented as mean ± S.D. of four independent experiments (N = 4). Statistical analysis was performed using one-way ANOVA (normally distributed data) or Kruskal-Wallis test (non-parametric data), p<0.05 was considered significant; decimal numbers above the bars represent the statistically significant p-values from post-hoc comparisons. For the p-values not presented here, see S2 File. IL1β (interleukin 1β), IL6, IL11, TGFβ1 (transforming growth factor beta 1), ACTA2 (alpha smooth muscle actin), α-SMA (alpha smooth muscle actin).