https://orcid.org/0000-0001-5219-2428
Figures
Abstract
Studies have shown that certain vocal fold pathologies are more common in one sex than the other. This is often explained by differences in the composition of the lamina propria and anatomical differences between female and male vocal folds, resulting in e.g. different fundamental frequencies. Here, we investigated a potential sex-specific voice frequency effect in an in vitro setting using vocal fold fibroblasts from one male and one female donor with and without cigarette smoke extract (CSE) addition. After exposure to either male or female vibration frequency with or without CSE, cells and supernatants were harvested. Gene and protein analysis were performed by means of qPCR, western blot, ELISA and Luminex. We found that exposure of cells to both male and female vibration pattern did not elicit significant changes in the expression of extracellular matrix-, inflammation-, and fibrosis-related genes, compared to control cells. The addition of CSE to vibration downregulated the gene expression of COL1A1 in cells exposed to the female vibration pattern, as well as induced MMP1 and PTGS2 in cells exposed to both female and male vibration pattern. The protein expression of MMP1 and COX2 was found to be significantly upregulated only in cells exposed to CSE and female vibration pattern. To conclude, different vibration patterns alone did not cause different responses of the cells. However, the female vibration pattern in combination with CSE had a tendency to elicit/maintain more pro-inflammatory responses in cells than the male vibration pattern.
Citation: Kirsch A, Grossmann T, Steffan B, Groselj-Strele A, Gerstenberger C, Gugatschka M (2024) Vocal fold fibroblasts and exposure to vibration in vitro: Does sex matter? PLoS ONE 19(2): e0297168. https://doi.org/10.1371/journal.pone.0297168
Editor: Andre van Wijnen, University of Vermont College of Medicine, UNITED STATES
Received: June 20, 2023; Accepted: December 30, 2023; Published: February 9, 2024
Copyright: © 2024 Kirsch et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Voice disorders affect almost 30% of the general population during their lifetime [1]. Several studies have shown that vocal fold (VF) pathologies occur more often in women than men, with some pathologies being more common in one gender [2, 3]. Likewise, a disproportionately higher rate of benign VF lesions is observed in women compared to men [4]. This susceptibility to phonotraumatic injury in women is often explained by structural differences in male and female laryngeal anatomy. Female larynges have shorter VF, producing voice at a higher fundamental frequency. As a consequence, there is less tissue mass to absorb a larger amount of vibratory force [1]. Additionally, female VF contain less hyaluronic acid (HA) in the superficial layer of the lamina propria, which would make the VF more prone to injury, as HA plays an important role in wound healing [5]. Vocal fold fibroblasts (VFF) are the most abundant cell type within the extracellular matrix (ECM) of the VF lamina propria and are key regulators of the ECM homeostasis, ensuring normal tissue architecture and function. Moreover, VFF are involved in wound healing upon VF injury, and play an important role in various VF pathologies [6–8]. It has been shown that vibrational biomechanical strain can increase VFF proliferation [9] and reduce inflammation after injury [10, 11]. However, a potential sex-specific voice frequency effect has not been investigated so far. As differences between male and female patients are clinically evident, we sought to address this question on the cellular basis.
Using a phonomimetic device [12], we exposed immortalized human VFF to vibration with or without a common noxious stimulus (cigarette smoke extract (CSE)). We applied either a typical male (100–135 Hz), or a typical female (200–250 Hz) frequency range/pattern, to determine whether the difference in stimulation frequency itself elicits different responses in the VFF in terms of ECM component production and inflammatory mediators. Since primary human VFF are difficult to obtain, two immortalized VFF cell lines, of female and male origin, were used for the study.
Materials and methods
Bioreactor settings
Two bioreactors [12] were used in parallel for the experiments, both using the same vibration pattern per experiment. The frequency ranges of the generated patterns were designed to include the fundamental frequencies of the male and female voice [13] (100–135 Hz and 200–250 Hz, respectively). The vibration pattern sound files were created using Audacity software (version 2.2.2, audacityteam.org, registered trademark of Dominic Mazzoni). Spectrums of membrane displacements of BioFlex® plates (Flexcell International Corporation, Burlington, NC, US) were measured using laser Doppler vibrometry as previously described [12].
Cell culture
Immortalized human VFF [14] were kindly provided by Prof. Susan Thibeault, University of Wisconsin-Madison. Cells from both male (A8, age 21, RRID:CVCL_A4GG) and female (E7, age 59, RRID:CVCL_A4GJ) donors were used for the experiments. Cells were cultured at 37˚C with 5% CO2 in high glucose Dulbecco’s modified Eagle’s medium (DMEM, Sigma Aldrich, Vienna, AT) supplemented with 10% fetal bovine serum (FBS, Biowest, Nuaillé, FR) and 100 μg/ml Normocin (Invivogen, San Diego, CA, USA). Cells were trypsinized using 0.25% Trypsin/EDTA (Sigma Aldrich) and seeded on the flexible culture plates coated with pronectin at a density of 144 000 cells/well (A8) and 150 000 cells/well (E7), each in 3 wells/plate. E7 cells have a slightly different morphology, compared to A8 cells, therefore different cell numbers were used to achieve a similar cell confluency. Cigarette smoke extract (CSE) and air bubbled control (ABC) were prepared as previously described [15]. Cells were allowed to attach under static conditions for 24 hours, after which the medium was changed to DMEM containing 1% FBS and either 5% CSE or 5% ABC. Cells were then transferred to the vibration bioreactor for 72 hours. Cells were exposed to the vibration pattern as followed: 8 hours without vibration (rest) followed by 16 hours composed of 30 seconds vibration (either male or female vibration pattern) and 90 seconds rest (total of 4 hours vibration per day). Non-vibrational control cells were cultivated in parallel in a separate incubator. After 72 hours, cells were harvested and medium was collected for subsequent analysis. Six experiments were performed for each vibration pattern.
Analyses
LDH assay.
Quantification of cytotoxicity was performed with cell culture supernatants using the Pierce LDH Cytotoxicity Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) as previously described [12], according to the manufacturer’s instructions. LDH activity is expressed as percentage of maximal LDH activity.
RNA isolation and RT-qPCR.
RNA isolation was performed as previously described [12] using QIAZOL Lysis Reagent and miRNeasy Mini Kit (both from Qiagen, Hilden, Germany). Purified RNA was eluated in RNase-free water and concentration was determined using the NanoDrop 2000c spectrophotometer (Thermo Scientific). Reverse transcription (RT) was performed as described previously [6]. RT quantitative PCR (RT-qPCR) was performed using the Bio-Rad CFX 384 real-time PCR Detection System (Bio-Rad, Hercules, CA, USA) and quantified on CFX Maestro 1.1 Software (version 4.1.2433.1219, Bio-Rad). Self-designed primers were validated and melting curves were generated for all qPCR runs. For detection of the cDNA of one gene of interest, all samples were run on the same qPCR plate. Primer sequences are provided in Table 1. Cq values of technical triplicates were averaged and relative quantification of all mRNAs of interest was performed based on the 2-ΔΔCq method [16] with a minor modification, the geometric mean of the Cq values of B2M and UXT reference RNAs was used as an internal normalization factor.
Western blot.
Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in RIPA buffer (Cell Biolabs, San Diego, CA, USA) supplemented with 1x Halt Protease and Phosphatase Inhibitor Cocktail and 5 mM EDTA (both Thermo Fisher Scientific). Protein content was determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Fifteen μg of total protein was mixed with appropriate amounts of 4x Laemmli Buffer (Bio- Rad, Hercules, CA, USA) and dithiothreitol (DTT) and boiled for 5 minutes at 95 ˚C. SDS-PAGE was performed using 4–20% Mini PROTEAN TGX gels (Bio-Rad), after which the proteins were blotted onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad). The blots were blocked in 5% milk for 2 hours, followed by incubation with the primary antibody over night at 4 ˚C (COL1A1: Nordic Bio Site, AB_2892675; COX2: Thermo Fisher Scientific; AB_2533224; GAPDH: Cell Signaling Technology, AB_561053; MMP1: Proteintech, AB_2297741). After washing, the blots were incubated with the appropriate HRP-conjugated secondary antibody (Agilent Dako, AB_2617137 and AB_2617138). Bands were detected after the addition of SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). Blot images were acquired with the ChemiDoc Touch system (Bio-Rad) and densitometric analysis was performed using ImageLab software (Bio-Rad). After detection, the blots were stripped for 10 minutes using Restore PLUS western blot stripping buffer (Thermo Fisher Scientific), and detection of further proteins was performed as indicated above.
Magnetic Luminex® Assay.
Proteins of interest were determined in supernatants using custom Human High Sensitivity Cytokine A Premixed Magnetic Luminex Performance Assay (Biotechne, R&D Biosystems) for analysis of VEGFA according to manufacturer’s instructions. Standard curves were generated from provided analyte standards. Sample dilution, determined from previous experiments, was 1:4. The assay was measured on the Bio-Plex 200 assay reader and concentrations were calculated using the Bio-Plex Manager Software, version 6.2 (both Bio-Rad).
Hyaluronan ELISA.
Hyaluronan (HA) content was measured in supernatants (1:40 dilution) using the Quantikine ELISA (R&D Systems, Abingdon, UK), according to the manufacturer’s instructions.
Statistical analysis.
Shapiro-Wilk test was used to assess the normal distribution of the data. Due to the experimental setup, the statistical analysis was performed in two steps. First, for each vibration pattern (male or female) and cell line, a repeated measures (RM) one-way ANOVA with Šídák multiple comparison test or Friedman test with Dunn’s multiple comparisons test was used to test for differences between vibration, combination of CSE and vibration, and static control. In a second step, the data was normalized as fold change compared to control, and a two-way ANOVA followed by Tukey’s multiple comparison test was used to compare the effect of different vibration patterns and cell lines. P values below 0.05 were considered statistically significant. All statistical analyses were performed using GraphPad Prism version 9.0 (San Diego, CA, USA).
Results
The male and female vibration pattern induced membrane displacements of 60–80 μm and 20–130 μm, as shown in Fig 1A and 1B, respectively. The LDH activity assay revealed that exposure of cells to vibration alone, or in combination with CSE was not cytotoxic (Fig 1C).
Spectrum of membrane displacement in the Bioflex 6-well culture plates when stimulated in the fundamental frequency range of the male (A) and female (B) voice. LDH activity assay shows no cytotoxicity induced by the treatment (C). Membrane displacement data are shown as median from 6 wells of a plate; measured at the center of the membranes. LDH assay data are shown as mean with SD of six independent experiments (N = 6).
Gene expression analysis of ECM-related genes showed no statistically significant effect of vibration on the expression of collagen (COL1A1, COL1A2 and COL3A1), fibronectin (FN1) and matrix metallopeptidase 1 (MMP1) expression (Fig 2A–2E, respectively). However, the addition of CSE significantly reduced the gene expression of COL1A in both cell lines exposed to the female pattern. The gene expression of MMP1 was significantly upregulated with CSE addition in all conditions compared to control. The protein expression of COL1α1 tended to be downregulated with the addition of CSE to vibration, but was significantly downregulated only in E7 cells exposed to male vibration and CSE (Fig 2F and 2H). MMP1 protein expression was significantly upregulated in E7 cell line samples exposed to the female pattern and CSE, compared to control, although a trend toward upregulation was seen for all cell lines and vibration patterns in combination with CSE (Fig 2G and 2H). Protein levels of COL1α2, Col3α1 and FN1 were not significantly altered (S1 Fig).
Gene expression levels of ECM-related genes COL1A1 (A), COL1A2 (B), COL3A1 (C), FN1 (D) and MMP1 (E) were analyzed by RT-qPCR and protein levels of COL1α1 (F) and MMP1 (G) were analyzed by Western blot. Representative blots of COL1α1 and MMP1 are shown in (H). All data are shown as mean and SD of fold changes compared to static control from six independent experiments (N = 6). Differences between the treatments within one vibration pattern and cell line were tested using RM one-way ANOVA with Šídák multiple comparison test or Friedman test with Dunn’s multiple comparisons test, depending on the result of the Shapiro-Wilk test of normality. Two-way ANOVA followed by Tukey’s multiple comparison test was used to compare the effect of different vibration patterns and cell lines. Unless otherwise indicated, asterisks show difference to static control, * P < 0.05, ** P < 0.01. f = female vibration pattern, m = male vibration pattern.
Gene expression of HA metabolism genes was not affected by vibration (Fig 3A–3D). However, the combination of CSE and female vibration pattern significantly reduced the gene expression of HA synthase 2 (HAS2) in the E7 cell line, compared to the control. HA levels measured by ELISA were slightly but significantly elevated in A8 cells exposed to CSE and female vibration pattern (Fig 3E).
Gene expression levels of HA metabolism-related genes HAS2 (A), HAS3 (B), HYAL1 (C) and HYAL2 (D) were analyzed by RT-PCR. Total HA content in supernatants was analyzed with ELISA (E). All data are shown as mean and SD of fold changes compared to static control from six independent experiments (N = 6). Differences between the treatments within one vibration pattern and cell line were tested using RM one-way ANOVA with Šídák multiple comparison test or Friedman test with Dunn’s multiple comparisons test, depending on the result of the Shapiro-Wilk test of normality. Two-way ANOVA followed by Tukey’s multiple comparison test was used to compare the effect of different vibration patterns and cell lines. Unless otherwise indicated, asterisks show difference to static control, * P < 0.05.
Both vibration alone and the combination of CSE and vibration did not affect the fibrosis-related genes actin alpha 2 (ACTA2) and transforming growth factor beta 1 (TGFB1) (Fig 4A and 4B, respectively). Vascular endothelial growth factor A (VEGFA), a factor that promotes angiogenesis was not significantly altered at the gene expression level (Fig 4C). However, a small, but significant upregulation at the protein level was measured in the supernatant of cells exposed to CSE in combination with vibration (Fig 4D). The expression of cyclooxygenase 2 (PTGS2, COX2, Fig 4E), an inflammation associated gene, was significantly upregulated only with the addition of CSE to vibration in E7 cells exposed to the female vibration patterns, compared to control, while a significant difference between vibration only and vibration and CSE was observed with the male vibration pattern for both cell lines. On the protein level, COX2 was significantly upregulated only in the E7 cell line exposed to the female vibration pattern and CSE, although a trend was seen for all cell lines and vibration patterns combined with CSE exposure (Fig 4F and 4G). The gene expression of interleukin 6 (IL6), was not significantly upregulated with vibration or the combination of CSE and vibration, compared to control. However, a significant difference was observed in the E7 cell line exposed to the female pattern when comparing cells exposed to the vibration alone and the combination of vibration and CSE (Fig 4H).
Gene expression levels of fibrosis-related genes ACTA2 (A) and TGFB (B) and angiogenesis-associated genes VEGFA (C) were analyzed by RT-qPCR. Protein expression of VEGF (D) was measured by Western blot. Gene expression levels of inflammation-associated PTGS2 (COX2) and IL6 were analyzed by RT-qPCR (E and H, respectively) and protein levels of. COX2 were analyzed by Western blot (F). A representative COX2 blot is shown in (G). All data are shown as mean and SD of fold changes compared to static control from six independent experiments (N = 6). Differences between the treatments within one vibration pattern and cell line were tested using RM one-way ANOVA with Šídák multiple comparison test or Friedman test with Dunn’s multiple comparisons test, depending on the result of the Shapiro-Wilk test of normality. Two-way ANOVA followed by Tukey’s multiple comparison test was used to compare the effect of different vibration patterns and cell lines. Unless otherwise indicated, asterisks show difference to static control, * P < 0.05, *** P < 0.001. f = female vibration pattern, m = male vibration pattern.
Discussion
Sex-specific differences in voice frequency are biologically and anatomically defined, as males have larger larynges and longer VF, compared to females [17], but the cellular effect of different frequency ranges cannot be studied in vivo, which is why we used an in vitro setting. The VFs are physiologically exposed to high frequency forces, thus the exposure to vibrational force in VF research is important in order to mimic those conditions in vitro. However, different studies used different vibration frequencies, ranging from 50–300 Hz, with most using a typical male frequency of 100 Hz [9, 12, 18–23]. As sex specific-differences in research, including basic research, have become more relevant, we wanted to answer the question if simply exposing VFF to sex specific vibration frequencies would evoke a different response of the cells. A positive answer might indicate that sex specific findings at the level of the VF (e.g. prevalence of VF polyps) have their origin starting at the cellular level. As a consequence, vibration frequency would need to be taken into consideration when conducting in vitro VF research.
The used female and male frequency patterns caused different membrane deflection ranges of the BioFlex® cell culture dish wells, however, a similar average deflection of approximately 60μm was achieved. We showed that a moderate exposure to vibration alone (male or female pattern, 4 hours per day) did not statistically alter the expression of ECM-, inflammation-, and fibrosis-related genes, compared to control cells. This is in line with a previous publication in which cells were exposed to the same amount of vibration per day, with the difference being the vibration frequency pattern, using a range of 50–250 Hz [11]. The addition of CSE to vibration, however, induced changes in the expression of several genes and proteins. A previous publication by Gugatschka et al. showed that CSE alone can cause a downregulation of COL1A1 gene expression in VFF [15]. Here we observed that the combination of CSE and vibration downregulated COL1A1 by 20–27% in cells exposed to the male vibration pattern, and by 35–40% in cells exposed to the female vibration pattern, compared to control. Only the downregulation in cells exposed to the female vibration pattern was statistically significant. The downregulation of Col1α1 was also observed on the protein level, although the combination of CSE and male pattern had the greatest effect. Therefore, we could not observe a clear impact of a specific vibration pattern. MMP1 gene expression was upregulated by CSE and vibration, which was not surprising since MMP1 has been reported to contain a promotor region responsive to CSE [24]. Different vibration patterns did not modulate the effect of CSE on MMP1 expression. The upregulation of MMP1 protein compared to control, was also seen, and although similar mean values were observed for all patterns and cell lines, it was statistically significant only in the E7 cell line exposed to the combination of CSE and female vibration pattern. MMP1 digests interstitial collagen [25], therefore a reduction of Col1α1 in cells exposed to CSE and vibration is most likely a combination of gene downregulation and protein degradation. Female VF have less HA in the superficial layer of the lamina propria compared to male ones [26]. Although we observed a significant decrease of HAS2 gene expression in E7 cells exposed to female vibration pattern and CSE, this did not affect the level of HA measured in the supernatant. A trend toward an increase in HAS3 gene expression was observed in cells exposed to CSE and vibration, which, although not significant, may have contributed to HA production, as a small, but significant increase in HA production was observed in A8 cells exposed to the female vibration pattern and CSE. VEGF is involved in angiogenesis, inflammation, wound healing and tissue remodeling in the skin and airway [27–29]. Vibration alone had no effect on both the gene and protein expression, however the addition of CSE caused a slight but significant increase of VEGF protein measured in the supernatant for almost all vibration patterns and cell lines. This is in line with a previous publication by Grossmann et al. where fibroblasts exposed to CSE and vibration showed an increased VEGF protein in the supernatant, compared to control [30]. CSE can induce COX2 [30–33], leading to a pro-inflammatory environment. We found that the gene expression of PTGS2 was significantly upregulated only in the E7 cell line exposed to the female pattern and CSE, although a trend was observed for all cell lines and patterns. This was also confirmed on the protein level. The gene expression of the pro-inflammatory IL6 is also known to be upregulated by CSE [34]. We observed the highest IL6 levels again in the E7 cell line exposed to the female pattern and CSE, compared to control, but missed statistical significance.
It is possible that the female vibration pattern exerted more tensile stress on the cells, due to higher maximum membrane displacements, compared to the male pattern (120 μm vs 80 μm, respectively). This, in combination with CSE, caused a significant change in the expression of the ECM-related genes, as well as MMP1 and COX2 protein, compared to the control. Moreover, the E7 cell line was derived from an older donor compared to the A8 cell line, which could have an impact on the inflammatory response of the E7 cell line. Aging is linked with an increase in inflammation [35, 36]. Thus, the E7 cell line may have epigenetic alterations that contribute to a stronger inflammatory response despite the immortalization, as a study suggests that hTERT immortalization does not prevent epigenetic aging [37].
At the time of immortalization, the karyotypes of used A8 and E7 cell lines contained XY and XX chromosomes [14], respectively. However, one must address the sex of immortalized cells with caution, since losses of Y chromosomes have been seen with higher passages in other cell lines [38]. Therefore, the cells used in this study are only considered as different VFF cell lines, not as biologically female or male cells. Sex hormones play a role in ear, nose, and throat diseases [39]. While there are immunohistochemical studies showing the expression and distribution of estrogen and androgen receptors in human vocal folds [40–42], one study claims that these are unspecific staining patterns and thus false positives [43]. Moreover, to the best of our knowledge, in vitro studies investigating the role of sex hormones on isolated VFF were done only using rat cells [44, 45]. As it is unclear if human VFF express steroid hormone receptors, we decided to omit the additional exposure of cells to different sex hormones.
In conclusion, moderate in vitro exposure to female and male vibration patterns alone had no significant effect on gene and protein expression of immortalized VFF. However, the addition of a noxious stimuli (CSE), did evoke certain differences in the effect of the male and female frequency pattern on the cells and should be investigated further. Therefore, the differences in VF pathologies are most likely not caused by the different vibration frequencies alone, and are rather a consequence of anatomical differences combined with external stimuli.
Supporting information
S1 Fig. Effect of vibration and CSE on ECM-related protein expression.
Protein levels of COL1A2 (A), COL3A1 (B), and FN1 (D) were analyzed by Western blot (A, B) and measured in the supernatant (D). Representative blots of COL1α2 and COL3α1 are shown in (C). All data are shown as mean and SD of fold changes compared to static control from six independent experiments (N = 6). Differences between the treatments within one vibration pattern and cell line were tested using RM one-way ANOVA with Šídák multiple comparison test or Friedman test with Dunn’s multiple comparisons test, depending on the result of the Shapiro-Wilk test of normality. Two-way ANOVA followed by Tukey’s multiple comparison test was used to compare the effect of different vibration patterns and cell lines. f = female vibration pattern, m = male vibration pattern.
https://doi.org/10.1371/journal.pone.0297168.s001
(TIF)
S1 Raw images. Original uncropped and unadjusted Western blot images.
https://doi.org/10.1371/journal.pone.0297168.s002
(PDF)
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