The voice disorder Reinke’s edema (RE) is a smoking- and voice-abuse associated benign lesion of the vocal folds, defined by an edema of the Reinke’s space, accompanied by pathological microvasculature changes and immune cell infiltration. Vocal fold fibroblasts (VFF) are the main cell type of the lamina propria and play a key role in the disease progression. Current therapy is restricted to symptomatic treatment. Hence, there is an urgent need for a better understanding of the molecular causes of the disease. In the present study, we investigated differential expression profiles of RE and control VFF by means of RNA sequencing. In addition, fast gene set enrichment analysis (FGSEA) was performed in order to obtain involved biological processes, mRNA and protein levels of targets of interest were further evaluated. We identified 74 differentially regulated genes in total, 19 of which were upregulated and 55 downregulated. Differential expression analysis and FGSEA revealed upregulated genes and pathways involved in extracellular matrix (ECM) remodeling, inflammation and fibrosis. Downregulated genes and pathways were involved in ECM degradation, cell cycle control and proliferation. The current study addressed for the first time a direct comparison of VFF from RE to control and evaluated immediate functional consequences.

To explore the effects of short- and long-term cigarette smoke extract (CSE) stimulation on the expression of extracellular matrix (ECM) components and inflammatory cytokines in an in vitro model for studying Reinke's edema using human vocal fold fibroblasts (hVFF). Experimental pilot study using intervention with CSE in vitro. Immortalized hVFF were pretreated with 5% CSE or control medium over a period of 2 or 8 weeks, followed by a final 3-day incubation time. We evaluated cell proliferation and examined gene and protein expression of control- and CSE-treated cells using quantitative polymerase chain reaction, Western Blot and enzyme linked immunosorbent assay. Cell numbers of CSE-treated hVFF strongly decreased after 8 weeks and limited the overall duration of the experiment. We observed significant upregulations in gene expression and protein levels of inflammatory markers (cyclooxygenase COX1, COX2) and ECM components (decorin, matrix metalloproteinase 1, transglutaminase 2, gremlin 2) induced by CSE after 2 and 8 weeks. Interleukin 1 receptor 1, prostaglandin I2 synthase, collagen- and hyaluronan-related gene expression showed minor upregulations. The majority of the observed genes were similarly regulated at both time points. However, the CSE-induced mRNA level of COX1 was ablated after 8 weeks. Long-term treatment did not yield results significantly different from the short-term protocol. Therefore, we propose that prolonged CSE exposure is not superior to short-term settings, which save both time and materials.

The isolated or combined effects of vibration and smoke extract (SE) from the IQOS™ "heat-not-burn" technology on human vocal fold fibroblasts (hVFF) were evaluated in an in vitro setting in order to elucidate their influence on vocal fold (patho-) physiology. Experimental pilot study using intervention with IQOS™-SE in vitro. Immortalized hVFF were exposed to IQOS™-SE or control medium under static or vibrational conditions. A phonomimetic bioreactor was used to deliver vibrational patterns to hVFF over a period of 5days. Cytotoxicity was quantified by lactate dehydrogenase assay. Effects on extracellular matrix production, inflammation, fibrogenesis, and angiogenesis were assessed by reverse transcription-quantitative polymerase chain reaction, western blot, enzyme-linked immunosorbent assay, and Magnetic Luminex assays. We observed significant changes induced either by IQOS™-SE exposure alone (matrix metalloproteinase 1, fibronectin, cyclooxygenase (COX)1, interleukin-8 gene expression), or by the combination of IQOS™-SE and vibration (hyaluronidase 2, COX2, interleukin-8 protein levels, vascular endothelial growth factor D). Short-term in vitro exposure of hVFF to IQOS™-SE did not result in cytotoxicity and reduced the gene expression of measured inflammation mediators, but had no effect on their protein expression. However, the clinical effects of long-term IQOS™ use are still not known and further research is needed in order to assess, if IQOS™ is in fact less harmful than conventional cigarettes.

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.

This in vitro study investigated the interaction between human vocal fold fibroblasts (hVFF) and macrophages under the influence of cigarette smoke extract (CSE) and vibration as potential regulators of vocal fold (VF) inflammation. Experimental in vitro pilot study. Immortalized hVFF were cultured in flexible-bottomed cell culture plates, treated with CSE, and subjected to static or dynamic conditions in a phonomimetic bioreactor. For coculture, unstimulated or lipopolysaccharide/IFNγ-stimulated THP-1 (human leukemia monocytic cell line) macrophages were added in inserts for a final 24 hours of vibration period. We measured messenger ribonucleic acid (mRNA) (quantitative polymerase chain reaction [qPCR]) and protein levels (Western Blot, ELISA, and LUMINEX®) of hVFF and analyzed the results using two- and three-way ANOVA with post hoc tests. Under inflammatory stimulation, we observed a reduction of collagen (COL) type 1A1, 1A2, and 3A1, and increased gene expression of COL4A1, matrix metallopeptidase 2, and vascular endothelial growth factor A in hVFF. Additionally, the pro-inflammatory markers cyclooxygenase (COX) 1 and 2, interleukin (IL) 1β, IL-6, and IL-8 were upregulated. CSE increased COX1 and COX2 levels, whereas vibration reduced CSE-induced increases of COL4A1 and COX2 in pro-inflammatory stimulated hVFF. This study indicates that vibration may mitigate CSE-induced inflammatory damage in the hVFF, thereby offering new insights into the cellular crosstalk that underlies the pathophysiology of VF inflammation in smoking-related voice disorders.

Research of human vocal fold (VF) biology is hampered by several factors. The sensitive microstructure of the VF mucosa is one of them and limits the in vivo research, as biopsies carry a very high risk of scarring. A laryngeal organotypic model consisting of VF epithelial cells and VF fibroblasts (VFF) may overcome some of these limitations. In contrast to human VFF, which are available in several forms, availability of VF epithelial cells is scarce. Buccal mucosa might be a good alternative source for epithelial cells, as it is easily accessible, and biopsies heal without scarring. For this project, we thus generated alternative constructs consisting of immortalized human VF fibroblasts and primary human buccal epithelial cells. The constructs (n = 3) were compared to native laryngeal mucosa at the histological and proteomic level. The engineered constructs reassembled into a mucosa-like structure after a cultivation period of 35 days. Immunohistochemical staining confirmed a multi-layered stratified epithelium, a collagen type IV positive barrier-like structure resembling the basement membrane, and an underlying layer containing VFF. Proteomic analysis resulted in a total number of 1961 identified and quantified proteins. Of these, 83.8% were detected in both native VF and constructs, with only 53 proteins having significantly different abundance. 15.3% of detected proteins were identified in native VF mucosa only, most likely due to endothelial, immune and muscle cells within the VF samples, while 0.9% were found only in the constructs. Based on easily available cell sources, we demonstrate that our laryngeal mucosa model shares many characteristics with native VF mucosa. It provides an alternative and reproducible in vitro model and offers many research opportunities ranging from the study of VF biology to the testing of interventions (e.g. drug testing).