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.

Selectively targeting the E-type prostanoid receptor 4 (EP4) might be a new therapeutic option in the treatment of glomerulonephritis (GN), since the EP4 receptor is expressed on different immune cells, resident kidney cells, and endothelial cells, which are all involved in the pathogenesis of immune-complex GN. This study aimed to evaluate the therapeutic potential and to understand the mode of action of EP4 agonist in immune-complex GN using the murine model of nephrotoxic serum nephritis (NTS). In vivo, NTS mice were treated two times daily with two different doses of an EP4 agonist ONO AE1-329 or vehicle for 14 days total. The effect of PGE2 and EP4 agonism and antagonism was tested on murine distal convoluted tubular epithelial cells (DCT) in vitro. In vivo, the higher dose of the EP4 agonist led to an improved NTS phenotype, including a reduced tubular injury score and reduced neutrophil gelatinase-associated lipocalin (NGAL) and blood urea nitrogen (BUN) levels. EP4 agonist treatment caused decreased CD4⁺ T cell infiltration into the kidney and increased proliferative capacity of tubular cells. Injection of the EP4 agonist resulted in dose-dependent vasodilation and hypotensive episodes. The low-dose EP4 agonist treatment resulted in less pronounced episodes of hypotension. In vitro, EP4 agonism resulted in cAMP production and increased distal convoluted tubular (DCT) proliferation. Taken together, EP4 agonism improved the NTS phenotype by various mechanisms, including reduced blood pressure, decreased CD4⁺ T cell infiltration, and a direct effect on tubular cells leading to increased proliferation probably by increasing cAMP levels.

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.

Objectives: The aim of the study was to increase muscle volume and improve phonation characteristics of the aged ovine larynx by functional electrical stimulation (FES) using a minimally invasive surgical procedure. Methods: Stimulation electrodes were placed bilaterally near the terminal adduction branch of the recurrent laryngeal nerves (RLN). The electrodes were connected to battery powered pulse generators implanted subcutaneously at the neck region. Training patterns were programmed by an external programmer using a bidirectional radio frequency link. Training sessions were repeated automatically by the implant every other day for 1 week followed by every day for 8 weeks in the awake animal. Another group of animals were used as sham, with electrodes positioned but not connected to an implant. Outcome parameters included gene expression analysis, histological assessment of muscle fiber size, functional analysis, and volumetric measurements based on three-dimensional reconstructions of the entire thyroarytenoid muscle (TAM). Results: Increase in minimal muscle fiber diameter and an improvement in vocal efficiency were observed following FES, compared with sham animals. Conclusion: This is the first study to demonstrate beneficial effects in the TAM of FES at molecular, histological, and functional levels. FES of the terminal branches of the RLN reversed the effects of age-related changes and improved vocal efficiency.

The vast majority of voice disorders is associated with changes of the unique, but delicate, human vocal fold mucosa. The ability to develop new effective treatment methods is significantly limited by the physical inaccessibility and the extremely rare occasions under which healthy tissue biopsies can be obtained. Therefore, the interest in laryngological research has shifted to human oral (buccal) mucosa, a similar and more easily available tissue. The harvesting process is less invasive and accompanied with faster healing and less scarring, compared to vocal fold mucosa. Here we report a descriptive proteomic comparison of paired human buccal and vocal fold mucosa by high‐resolution mass spectrometry (CID‐MS/MS). Our study identified a total of 1575 proteins detected within both tissues that are highly consistent in several crucial biological processes, cellular components, and molecular functions. Hence, our proteomic analysis will provide a fundamental resource for the laryngological research community.