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Novel immortalized human vocal fold epithelial cell line: In vitro tool for mucosal biology.

ABSTRACT: Study of vocal fold (VF) mucosal biology requires essential human vocal fold epithelial cell (hVFE) lines for use in appropriate model systems. We steadily transfected a retroviral construct containing human telomerase reverse transcriptase (hTERT) into primary normal hVFE to establish a continuously replicating hVFE cell line. Immortalized hVFE across passages have cobblestone morphology, express epithelial markers cytokeratin 4, 13 and 14, induced hTERT gene and protein expression, have similar RNAseq profiling, and can continuously grow for more than 8 months. DNA fingerprinting and karyotype analysis demonstrated that immortalized hVFE were consistent with the presence of a single cell line. Validation of the hVFE, in a three-dimensional in vitro VF mucosal construct revealed a multilayered epithelial structure with VF epithelial cell markers. Wound scratch assay revealed higher migration capability of the immortalized hVFE on the surface of collagen-fibronectin and collagen gel containing human vocal fold fibroblasts (hVFF). Collectively, our report demonstrates the first immortalized hVFE from true VFs providing a novel and invaluable tool for the study of epithelial cell-fibroblast interactions that dictate disease and health of this specialized tissue.

SUBMITTER: Chen X

PROVIDER: S-EPMC7839467 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Novel isolation and biochemical characterization of immortalized fibroblasts for tissue engineering vocal fold lamina propria.

Project description:Tissue regeneration of the vocal fold lamina propria extracellular matrix (ECM) will be facilitated by the use of suitable vocal fold fibroblast (VFF) cell lines in appropriate model systems. Primary human VFFs (hVFFs) were steadily transduced by a retroviral vector containing human telomerase reverse transcriptase (hTERT) gene; immortalized cells grew and divided vigorously for more than 120 days. Biochemical characterization of the six transduced lines included, at different time points, expression of hTERT, telomerase activity, telomere lengths, and transcript levels of ECM constituents. Telomere lengths of the transfected lines were elongated and stable. Gene expression levels of collagen Ialpha1, collagen Ialpha2, collagen VIalpha3, elastin, and fibronectin were measured between the transduced cell clones and the primary hVFFs to verify transcription. Absence of inter- and intraspecies contamination was confirmed with DNA fingerprinting and karyotype analysis. Cell morphology, growth, and transcription expression were examined on 2D scaffolds-collagen, fibronectin, and hyaluronic acid. Immortalized hVFFs demonstrated normal attachment and spread on 2D scaffolds. Collagen Ialpha1, collagen Ialpha2, collagen VIalpha3, elastin, and fibronectin transcript expression was measured from immortalized hVFFs, for all surfaces. This is the first report of immortalization and biochemical characterization of hVFFs, providing a novel and invaluable tool for tissue regeneration applications in the larynx.

| S-EPMC2819707 | biostudies-literature

Exposure to e-cigarette vapor extract induces vocal fold epithelial injury and triggers intense mucosal remodeling.

Project description:Vaping has been reported to cause acute epiglottitis, a life-threatening airway obstruction induced by direct epithelial injury and subsequent inflammatory reaction. Here, we show that we were able to recapitulate this phenomenon in vitro. Exposure of human engineered vocal fold (VF) mucosae to 0.5% and 5% electronic cigarette (e-cigarette) vapor extract (ECVE) for 1 week induced cellular damage of luminal cells, disrupting homeostasis and innate immune responses. Epithelial erosion was likely caused by accumulation of solvents and lipid particles in the cytosol and intercellular spaces, which altered lipid metabolism and plasma membrane properties. Next, we investigated how the mucosal cells responded to the epithelial damage. We withdrew the ECVE from the experimental system and allowed VF mucosae to regenerate for 1, 3 and 7 days, which triggered intense epithelial remodeling. The epithelial changes included expansion of P63 (TP63)-positive basal cells and cytokeratin 14 (KRT14) and laminin subunit α-5 (LAMA5) deposition, which might lead to local basal cell hyperplasia, hyperkeratinization and basement membrane thickening. In summary, vaping presents a threat to VF mucosal health and airway protection, thereby raising further concerns over the safety of e-cigarette use. This article has an associated First Person interview with the first author of the paper.

| S-EPMC9438930 | biostudies-literature

Restricted Water Intake Adversely Affects Rat Vocal Fold Biology.

Project description:ObjectivesA holistic understanding of the many ways that systemic dehydration affects vocal fold biology is still evolving. There are also myriad physiologically relevant methodologies to induce systemic dehydration. To untangle the effects of systemic dehydration on vocal fold biology, we need to utilize realistic, clinically translatable paradigms of systemic dehydration in lab animals. Restricted access to water accommodates clinical translation. We investigated whether systemic dehydration via reduced water intake would negatively affect vocal fold biology.Study designProspective, in vivo study design.MethodsMale Sprague Dawley rats (N = 13) were provided 4 mL/100 g of water/day for 5 days, whereas male control rats (N = 8) were given ad lib access to water. Following euthanasia, tissues were processed for histological staining, gene expression, and protein assays.ResultsRenin gene expression level in kidneys increased significantly (P ≤ .05), validating dehydration. Dehydration induced by restricted water access downregulated the gene expression of interleukin-1α and desmoglein-1 (P ≤ .05). Hyaluronidase-2 gene expression increased after dehydration (P ≤ .05). The protein level of desmoglein-1 decreased after dehydration (P ≤ .05). Histological analyses suggested decreased hyaluronan (P ≤ .05) in the water-restricted rat vocal fold.ConclusionReduced daily water intake for just 5 days impairs vocal fold biology by disrupting inflammatory cytokine release, reducing plasma membrane integrity, and disrupting the hyaluronan network. This is the first study investigating the dehydrating effects of restricted water intake on vocal fold tissue in an in vivo model.Level of evidenceNA (prospective animal study). Laryngoscope, 131:839-845, 2021.

| S-EPMC8404141 | biostudies-literature

Expression data from rat vocal fold at different periods after vocal fold injury

Project description:Vocal cord healing is a dynamic process, and many genes and proteins are involved, which play varying roles at different regeneration stages after injury. Previous studies have shown that inflammatory responses occur at the early stage of vocal cord injury, where the fibroblasts proliferate exuberantly with intensive secretion and deposition of ECM. These activities reach the peak at 3-7 days and their intensity begins to decline 15 days later. A study based on the dermal system has shown that ECM remodeling during the repair of injury can last for several months. However, few studies have been conducted as to the dynamic changes of gene expressions and signaling pathway during the healing process of vocal cord injury. Plotting these changes will facilitate the understanding about the physiological changes during healing and the identification of key time points and target genes in fibrosis formation.

2019-10-26 | GSE139383 | GEO

Practice variations in voice treatment selection following vocal fold mucosal resection.

Project description:Objectives/hypothesisTo characterize initial voice treatment selection following vocal fold mucosal resection in a Medicare population.Study designRetrospective analysis of a large, nationally representative Medicare claims database.MethodsPatients with > 12 months of continuous Medicare coverage who underwent a leukoplakia- or cancer-related vocal fold mucosal resection (index) procedure during calendar years 2004 to 2009 were studied. The primary outcome of interest was receipt of initial voice treatment (thyroplasty, vocal fold injection, or speech therapy) following the index procedure. We evaluated the cumulative incidence of each postindex treatment type, treating the other treatment types as competing risks, and further evaluated postindex treatment utilization using the proportional hazards model for the subdistribution of a competing risk. Patient age, sex, and Medicaid eligibility were used as predictors.ResultsA total of 2,041 patients underwent 2,427 index procedures during the study period. In 14% of cases, an initial voice treatment event was identified. Women were significantly less likely to receive surgical or behavioral treatment compared to men. From age 65 to 75 years, the likelihood of undergoing surgical treatment increased significantly with each 5-year age increase; after age 75 years, the likelihood of undergoing either surgical or behavioral treatment decreased significantly every 5 years. Patients with low socioeconomic status were significantly less likely to undergo speech therapy.ConclusionThe majority of Medicare patients do not undergo voice treatment following vocal fold mucosal resection. Further, the treatments analyzed here appear disproportionally utilized based on patient sex, age, and socioeconomic status. Additional research is needed to determine whether these observations reflect clinically explainable differences or disparities in care.Level of evidence2c. Laryngoscope, 126:2505-2512, 2016.

| S-EPMC5018919 | biostudies-literature

Expression data from rat vocal fold at different periods after vocal fold injury (miRNA)

Project description:Vocal cord healing is a dynamic process, and many genes and proteins are involved, which play varying roles at different regeneration stages after injury. Previous studies have shown that inflammatory responses occur at the early stage of vocal cord injury, where the fibroblasts proliferate exuberantly with intensive secretion and deposition of ECM. These activities reach the peak at 3-7 days and their intensity begins to decline 15 days later. A study based on the dermal system has shown that ECM remodeling during the repair of injury can last for several months. However, few studies have been conducted as to the dynamic changes of gene and microRNA expressions during the healing process of vocal cord injury. Plotting these changes will facilitate the understanding about the physiological changes during healing and the identification of key time points and target genes and microRNAs in fibrosis formation.

2020-03-25 | GSE147473 | GEO

Transcription in wounded rat vocal folds and vocal fold fibroblasts

Project description:We used microarrays to characterize transcriptome profiles of rat vocal fold tissue following surgical injury (vs. naive tissue); rat vocal fold fibroblasts harvested from scar tissue at the 60 d time point (vs. naive fibroblasts); rat vocal fold scar fibroblasts treated with siRNA against the collagen chaperone protein rat gp46 (vs. scramble siRNA). Adult Fischer 344 rat vocal fold tissue was harvested at 3, 14, and 60 days following surgical injury (control = age-matched naive tissue); rat vocal fold scar fibroblasts were obtained via explant culture of tissue obtained 60 days following surgical injury and harvested at 80% confluence during passage 1 (control = age-matched naive rat vocal fold fibroblasts); rat vocal fold scar fibroblasts were treated for 1 h with 50 nM liposome-delivered siRNA against rat gp46 when 80% confluent at passage 1, cultured for an additional 24 h in fresh media, then harvested (control = rat vocal fold scar fibroblasts treated with 50 nM liposome-delivered scramble siRNA).

2014-10-09 | E-GEOD-62204 | biostudies-arrayexpress

Biocompatibility of a synthetic extracellular matrix on immortalized vocal fold fibroblasts in 3-D culture.

Project description:In order to promote wound repair and induce tissue regeneration, an engineered hyaluronan (HA) hydrogel - Carbylan GSX, which contains di(thiopropionyl) bishydrazide-modified hyaluronic acid, di(thiopropionyl) bishydrazide-modified gelatin and polyethylene glycol diacrylate - has been developed for extracellular matrix (ECM) defects of the superficial and middle layers of the lamina propria. The purpose of this study was to evaluate the biocompatibility of Carbylan GSX in a previously established immortalized human vocal fold fibroblast (hVFF) cell line prior to human clinical trials. Immortalized hVFF proliferation, viability, apoptosis and transcript analysis for both ECM constituents and inflammatory markers were measured for two-dimensional and three-dimensional (3-D) culture conditions. There were no significant differences in morphology, cell marker protein expression, proliferation, viability and apoptosis of hVFF cultured with Carbylan GSX compared to Matrigel, a commercial 3-D control, after 1 week. Gene expression levels for fibromodulin, transforming growth factor-beta1 and tumor necrosis factor-alpha were similar between Carbylan GSX and Matrigel. Fibronectin, hyaluronidase 1 and cyclooxygenase II expression levels were induced by Carbylan GSX, whereas interleukins 6 and 8, Col I and hyaluronic acid synthase 3 expression levels were decreased by Carbylan GSX. This investigation demonstrates that Carbylan GSX may serve as a natural biomaterial for tissue-engineering of human vocal folds.

| S-EPMC2883010 | biostudies-literature

Transcription in wounded rat vocal folds and vocal fold fibroblasts

2014-10-09 | GSE62204 | GEO

Protein turnover during in vitro human vocal fold mucosa remodeling

Project description:We present here the first study evaluating the dynamic proteome changes in the remodeling process of decellularized tissue by reseeded cells using vocal fold mucosa as the model system.

2016-01-06 | PXD002734 | Pride

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