Human Vocal Fold Fibroblasts Research Articles

The Antiproliferative and Antifibrotic Effects of Cisplatin on Primary Human Vocal Fold Fibroblasts

Objective: Vocal fold scarring and laryngeal stenosis are major clinical challenges. Current drugs do not efficiently reduce scarring. We examined the antiproliferative and antifibrotic effects of cisplatin on primary human vocal fold fibroblasts (HVFFs). Methods: HVFFs were cultured in vitro and identified by immunocytochemistry. The relative viability of HVFFs was analyzed by Cell Counting Kit-8 assays (CCK-8). The fibrogenic phenotype was induced by transforming growth factor-β₁ (TGF-β₁) and reversed by cisplatin as shown by immunocytochemistry. Real-time PCR and Western blotting assessed collagen III and I. Western blotting for Smad2, p-Smad2, Smad-3, p-Smad3 and caspase-3 were performed. Results: CCK-8 results showed that cisplatin decreased the relative viability of HVFFs, and Western blots revealed elevation of the apoptosis-related protein caspase-3 in HVFFs. Cisplatin treatment reduced α-smooth muscle actin staining intensity in the presence of TGF-β₁. Real-time PCR revealed the downregulation of collagen III and I in cisplatin-treated HVFFs. The TGF-β₁-induced increased fibrogenic protein levels were decreased by cisplatin. Reduced levels were detected at late time points. Conclusions: Cisplatin induces antiproliferative and antifibrotic alterations in HVFFs. Cisplatin may prevent postoperative vocal fold scarring and laryngeal stenosis in patients treated with CO₂ laser microsurgery and undergoing delayed wound healing.

Proteomic Analysis of Vocal Fold Fibroblasts Exposed to Cigarette Smoke Extract: Exploring the Pathophysiology of Reinke's Edema*[S

Reinke's edema is a smoking-associated, benign, mostly bilateral lesion of the vocal folds leading to difficulties in breathing and voice problems. Pronounced histological changes such as damaged microvessels or immune cell infiltration have been described in the vocal fold connective tissue, the lamina propria Thus, vocal fold fibroblasts, the main cell type of the lamina propria, have been postulated to play a critical role in disease mediation. Yet information about the pathophysiology is still scarce and treatment is only surgical, i.e. symptomatic. To explore the pathophysiology of Reinke's edema, we exposed near-primary human vocal fold fibroblasts to medium conditioned with cigarette smoke extract for 24 h as well as 4 days followed by quantitative mass spectrometry.Proteomic analyses after 24 h revealed that cigarette smoke increased proteins previously described to be involved in oxidative stress responses in other contexts. Correspondingly, gene sets linked to metabolism of xenobiotics and reactive oxygen species were significantly enriched among cigarette smoke-induced proteins. Among the proteins most downregulated by cigarette smoke, we identified fibrillar collagens COL1A1 and COL1A2; this reduction was validated by complementary methods. Further, we found a significant increase of UDP-glucose 6-dehydrogenase, generating a building block for biosynthesis of hyaluronan, another crucial component of the vocal fold lamina propria In line with this result, hyaluronan levels were significantly increased because of cigarette smoke exposure. Long term treatment of 4 days did not lead to significant changes.The current findings corroborate previous studies but also reveal new insights in possible disease mechanisms of Reinke's edema. We postulate that changes in the composition of the vocal folds' extracellular matrix -reduction of collagen fibrils, increase of hyaluronan- may lead to the clinical findings. This might ease the identification of better, disease-specific treatment options.

Carbon nanotube composite hydrogels for vocal fold tissue engineering: Biocompatibility, rheology, and porosity

Porous composite hydrogels were prepared using glycol chitosan as the matrix, glyoxal as the chemical crosslinker, and carbon nanotubes (CNTs) as the fibers. Both carboxylic and hydroxylic functionalized CNTs were used. The homogeneity of CNTs dispersion was evaluated using scanning electron microscopy. Human vocal fold fibroblasts were cultured and encapsulated in the composite hydrogels with different CNT concentrations to quantify cell viability. Rheological tests were performed to determine the gelation time and the storage modulus as a function of CNT concentration. The gelation time tended to decrease for low concentrations and increase at higher concentrations, reaching a local minimum value. The storage modulus obeyed different trends depending on the functional group. The porosity of the hydrogels was found to increase by 120% when higher concentrations of carboxylic CNTs were used. A high porosity may promote cell adhesion, migration, and recruitment from the surrounding native tissue, which will be investigated in a future work aiming at applying this injectable biomaterial for vocal fold tissue regeneration.

Investigation of surgical adhesives for vocal fold wound closure.

Surgical adhesives are increasingly used for vocal fold microsurgery to assist wound closure and reduce the risks of scar formation. Currently used vocal fold adhesives such as fibrin glue, however, have thus far not been found to promote wound closure or reduce scarring. The objectives of this study were to investigate the mechanical strength and the cytotoxicity of three commercially available adhesives (Glubran 2, GEM, Viareggio, Italy; BioGlue, CryoLife, Kennesaw, GA; and Tisseel, Baxter Healthcare, Deerfield, IL) for vocal fold wound closure. Shear and tension tests were performed on 150 porcine larynges. The cytotoxicity of the adhesives to immortalized human vocal fold fibroblasts was investigated using neutral red uptake assays. The average shear adhesive strength for Tisseel, BioGlue, and Glubran 2 was 13.86 ± 5.03 kilopascal (kPa), 40.92 ± 17.94 kPa, and 68.79 ± 13.29 kPa, respectively. The tensile adhesive strength for Tisseel, BioGlue, and Glubran 2 was 10.70 ± 6.42 kPa, 34.27 ± 12.59 kPa, and 46.67 ± 12.13 kPa, respectively. The vocal fold cell viabilities in extracts of Tisseel, BioGlue, and Glubran 2 were 99.27%, 43.05%, and 1.79%, respectively. There was a clear tradeoff between adhesive strength and toxicity. The maximum failure strength in shear or tension of the three surgical adhesives ranked from strongest to the weakest was: 1) Glubran 2, 2) BioGlue, and 3) Tisseel. Tisseel was found to be the least toxic of the three adhesives, whereas Glubran 2 was the most toxic. NA Laryngoscope, 129:2139-2146, 2019.

Development and validation of a novel phonomimetic bioreactor.

Vocal fold fibroblasts (VFF) constitute the main cell type of the vocal fold’s lamina propria, produce the extracellular matrix and thereby determine the tissue characteristics. To study VFF behavior under in vitro conditions it is important to mimic the dynamic environment of the in vivo state. The aim of our study was to develop and validate a novel phonomimetic bioreactor system mainly based on commercially available components. The use of cell culture dishes with flexible silicone bottoms in combination with a suitable loudspeaker made it possible to expose the cells to various kinds of phonatory stimuli. The fundamental vibration characteristics of silicone membranes were investigated with and without cell culture medium by laser Doppler vibrometry. Human VFF were seeded in flexible-bottomed plates and placed in a custom-made housing containing a loudspeaker. After the cells were exposed to a predefined audio stimulation protocol, cell viability was assessed and gene as well as protein expression levels were compared to static controls. Laser Doppler vibrometry revealed that addition of cell culture medium changed the resonance frequencies of vibrating membranes. Gene expression of hyaluronan synthase 2, collagen III, fibronectin and TGFβ-1 was significantly upregulated in VFF exposed to vibration, compared to static control. Vibration also significantly upregulated collagen I gene and protein expression. We present a new type of phonomimetic bioreactor. Compared to previous models, our device is easy to assemble and cost-effective, yet can provide a wide spectrum of phonatory stimuli based on the entire dynamic range of the human voice. Gene expression data of VFF cultured in our phonomimetic bioreactor show a significant effect of vibration on ECM metabolism, which illustrates the efficacy of our device.

The role of pepsin in epithelia-mesenchymal transition in idiopathic subglottic stenosis.

Idiopathic subglottic stenosis (iSGS) is commonly characterized by laryngeal fibrosis thought to arise by epithelia-mesenchymal transition (EMT) induced by chronic inflammation. Pepsin is a potent inducer of inflammation in the airways during chronic laryngopharyngeal reflux and has been observed in the subglottic mucosa of patients with iSGS, absent in normal mucosa. The aim of this study was to examine the effect of pepsin on mechanisms of EMT in laryngeal cells with implications for iSGS. In vitro translational research study. Human laryngeal epithelial cell cultures were exposed to 0.1 mg/mL or 1.0 mg/mL pepsin at pH7 for 24 and 48 hours, or media pH5 ± 0.1 mg/mL pepsin for 10 minutes and harvested after 24 and 48 hours. EMT marker expression was measured by qPCR and enzyme-linked immunosorbent assays. Wound-healing scratch assay was performed on immortalized human vocal fold fibroblasts pretreated with media pH5 ± 0.1 mg/mL pepsin (10 minutes) or continuously treated with media pH7 ± 0.1 to 1 mg/mL pepsin for 24 hours. Pepsin yielded no effect on MMP1, MMP9, FN1, COL1A1, HAS2, or CDH1 gene expression or matrix metalloproteinase-9 or fibronectin protein expression, either alone or in the presence of weak acid. Pepsin and/or acid produced no effect on fibroblast migration. Whereas pepsin has been shown to be present in the subglottic mucosa of patients with iSGS, this in vitro acute exposure investigation does not provide evidence of a direct causal role for development of fibrosis in subglottic epithelial cell cultures. NA. Laryngoscope, 130:154-158, 2020.

Incorporation of types I and III collagen in tunable hyaluronan hydrogels for vocal fold tissue engineering

Vocal fold scarring is the fibrotic manifestation of a variety of voice disorders, and is difficult to treat. Tissue engineering therapies provide a potential strategy to regenerate the native tissue microenvironment in order to restore vocal fold functionality. However, major challenges remain in capturing the complexity of the native tissue and sustaining regeneration. We hypothesized that hydrogels with tunable viscoelastic properties that present relevant biological cues to cells might be better suited as therapeutics. Herein, we characterized the response of human vocal fold fibroblasts to four different biomimetic hydrogels: thiolated hyaluronan (HA) crosslinked with poly(ethylene glycol) diacrylate (PEGDA), HA-PEGDA with type I collagen (HA-Col I), HA-PEGDA with type III collagen (HA-Col III) and HA-PEGDA with type I and III collagen (HA-Col I-Col III). Collagen incorporation allowed for interpenetrating fibrils of collagen within the non-fibrillar HA network, which increased the mechanical properties of the hydrogels. The addition of collagen fibrils also reduced hyaluronidase degradation of HA and hydrogel swelling ratio. Fibroblasts encapsulated in the HA-Col gels adopted a spindle shaped fibroblastic morphology by day 7 and exhibited extensive cytoskeletal networks by day 21, suggesting that the incorporation of collagen was essential for cell adhesion and spreading. Cells remained viable and synthesized new DNA throughout 21 days of culture. Gene expression levels significantly differed between the cells encapsulated in the different hydrogels. Relative fold changes in gene expression of MMP1, COL1A1, fibronectin and decorin suggest higher degrees of remodeling in HA-Col I-Col III gels in comparison to HA-Col I or HA-Col III hydrogels, suggesting that the former may better serve as a natural biomimetic hydrogel for tissue engineering applications. Statement of SignificanceVoice disorders affect about 1/3rd of the US population and significantly reduce quality of life. Patients with vocal fold fibrosis have few treatment options. Tissue engineering therapies provide a potential strategy to regenerate the native tissue microenvironment in order to restore vocal fold functionality. Various studies have used collagen or thiolated hyaluronan (HA) with gelatin as potential tissue engineering therapies. However, there is room for improvement in providing cells with more relevant biological cues that mimic the native tissue microenvironment and sustain regeneration. The present study introduces the use of type I collagen and type III collagen along with thiolated HA as a natural biomimetic hydrogel for vocal fold tissue engineering applications.

Dual growth factor-immobilized bioactive injection material for enhanced treatment of glottal insufficiency

With increasing demand for treatment of glottal insufficiency, several injection materials have been examined. However, biological resorption, degradation of injected materials, and the subsequent need to perform multiple injections still remain major clinical problems. In this study, we fabricated two different growth factor (GF) [single basic fibroblast growth factor (bFGF), single hepatocyte growth factor (HGF), or dual bFGF/HGF]-immobilized polycaprolactone (PCL)/Pluronic F127 microspheres. These materials were investigated for their potential use as bioactive injection laryngoplasty agents. HGF was found to be continuously released over 20 days and the bFGF was found to be continuously released over 25 days, as demonstrated by ELISA assay. Human vocal fold fibroblasts (hVFFs) showed significantly higher proliferative ability on dual GF-immobilized microspheres. GF-immobilized microspheres (bFGF, HGF, and dual GF) were injected into paralyzed vocal folds of New Zealand white rabbits. Through endoscopic observation and H&E staining, we identified that the microspheres remained localized at the injection site, resulting in constant volume augmentation of the paralyzed vocal fold without significant loss of the initial volume after 4 weeks. The expression of genes related to the extracellular matrix (ECM) in the vocal fold was upregulated by dual GF-immobilized microspheres. Furthermore, dual GF-immobilized microspheres inhibited muscle degeneration and upregulation of myogenic-related genes. In conclusion, dual GF-immobilized microspheres passively augmented the volume of the paralyzed vocal fold while actively inducing ECM synthesis at the injected vocal fold and preserving muscle tissue. Dual GF-immobilized microspheres could be a new and promising injection material for paralyzed vocal folds. Statement of SignificanceLimitation of prolonged augmentation of vocal fold and degeneration of vocal fold tissue still remain as major clinical problems in the treatment of vocal fold paralysis. Herein, we fabricated the polycaprolactone (PCL)/Pluronic F127 microspheres to augment volume of paralyzed vocal folds. On top of that, we additionally immobilized the growth factors (bFGF, HGF, or dual bFGF/HGF) on the surface of these microspheres. We highlight the efficacy of the dual GF-immobilized microspheres which augmented the volume of the paralyzed vocal fold passively, induced ECM synthesis actively at the injected vocal fold and preserved laryngeal muscle tissue. Our results suggest that the dual GF-immobilized microsphere could be a new promising injection material for injection laryngoplasty to treat paralyzed vocal fold.

The effects of cytosporone-B, a novel antifibrotic agent, on vocal fold fibroblasts.

Our laboratory recently described NR4A1 as an endogenous inhibitor of TGF-β-induced vocal fold (VF) fibrosis. Our prior report described the temporal expression of NR4A1 during VF healing in vivo and the effects of NR4A1 knockdown on fibroplastic cell activities in vitro. Based on these findings, we hypothesized that cytosporone-B (Csn-B), an NR4A1 agonist, may hold significant therapeutic potential. In vitro. Human VF fibroblasts were exposed to TGF-β1+/-Csn-B. Expression of genes related to fibrosis were quantified. In addition, contraction was assayed as a surrogate for the fibrotic phenotype in our cell line. TGF-B1 stimulated COL1A1 and ACTA2, as expected. Csn-B significantly downregulated TGF-β1-mediated upregulation of these genes (P = .009, P = .03, respectively). Csn-B had no effect on genes related to TGF-β/Smad signaling. Csn-B also decreased the TGF-β1-mediated contractile phenotype in our cells (P = .004). NR4A1 is an endogenous inhibitor of fibrosis in the vocal folds and Csn-B, as an NR4A1 agonist, may evolve as an ideal, therapeutic candidate for this challenging condition. NA Laryngoscope, 128:E425-E428, 2018.

Phosphorylation of the glucocorticoid receptor alters SMAD signaling in vocal fold fibroblasts.

Direct glucocorticoid (GC) injection for vocal fold (VF) scarring has evolved as a therapeutic strategy, but the mechanisms underlying the antifibrotic effects remain unclear. GCs act via the glucocorticoid receptor (GR), which is phosphorylated at multiple serine residues in a hormone-dependent manner to affect bioactivity. We hypothesize that GCs regulate SMAD signaling via GR phosphorylation in vocal fold fibroblasts (VFFs). In vitro. Human VFFs were treated with dexamethasone (DM; 10-5 -10-7 M) ± transforming growth factor (TGF)-β1 (10 ng/mL). RU486 (10-6 M) was employed to isolate the regulatory effects of GR. Total GR, Ser211 , and Ser203 phosphorylation was examined via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunocytochemistry. Quantitative polymerase chain reaction was employed to determine GR-mediated effects of DM on genes related to fibrosis. Total GR and Ser211 phosphorylation was observed predominantly in the nucleus 1 hour after DM administration. DM decreased total GR expression, but Ser203 and Ser211 phosphorylation increased. RU486 limited the effects of DM. SMAD3 and SMAD7 mRNA expression significantly decreased 4 hours after DM administration (P < 0.05); this response was negated by RU486. COL1A1 remained unchanged, and ACTA2 significantly increased following 24 hours of DM treatment (P < 0.05). DM regulated TGF-β1 signaling via altered SMAD3 and SMAD7 expression. This response was associated with altered GR phosphorylation. These findings provide insight into the mechanisms of steroidal effects on vocal fold repair; ultimately, we seek to enhance therapeutic strategies for these challenging patients. NA Laryngoscope, 129:E187-E193, 2019.

Cryotherapy has antifibrotic and regenerative effects on human vocal fold fibroblasts.

Vocal fold scarring remains a major treatment challenge, and scar prevention without residual lesions remains a dilemma. Cryotherapy has shown cosmetic outcomes on skin lesions with minimal scarring. The aim of this study was to clarify the beneficial effects of cryotherapy for the prevention and the treatment of vocal fold scarring. In vitro. Primary cultures of human vocal fold fibroblasts (VFFs) were used in this study. Myofibroblast differentiation was stimulated by transforming growth factor β1 (TGF-β1). We mimicked the cryotherapy effect on vocal fold healing in vivo by freezing VFFs ± TGF-β1 in vitro. The influence of freezing on cell viability, proliferation, migration, and contractile properties were analyzed. The expression of collagen I, collagen III, fibronectin, TGF-β1, matrix metallopeptidase 1 (MMP1), hyaluronan synthase 1 (HAS1) were investigated by real-time polymerase chain reaction (RT-PCR), and the expression of alpha smooth muscle actin (α-SMA) and decorin were investigated by RT-PCR and Western blot. Freezing was found to modify extracellular matrix (ECM) synthesis and differentiation of VFFs. Expression of collagen I, collagen III, fibronectin, α-SMA, and TGF-β1 was downregulated, and MMP1 was upregulated in VFFs + TGF-β1 (myofibroblast) by freezing. HAS1 and decorin were upregulated in both VFFs ± TGF-β1 by freezing. Freezing VFFs + TGF-β1 (myofibroblast) with fast thawing had a lower expression of α-SMA when compared with slow thawing. Freezing reduced the migration and collagen contraction of VFFs + TGF-β1 (myofibroblast). Cryotherapy induces antifibrotic and regenerative ECM alterations in VFFs. These data provide insight into the prevention and the treatment of vocal fold scarring with cryotherapy in phonomicrosurgery. NA Laryngoscope, 129:E143-E150, 2019.

Photodynamic therapy induces antifibrotic alterations in primary human vocal fold fibroblasts.

Photodynamic therapy (PDT) is a promising treatment modality for laryngeal dysplasia, early-stage carcinoma, and papilloma, and was reported to have the ability to preserve laryngeal function and voice quality without clinical fibrotic response. We aimed to investigate the mechanism behind the antifibrotic effects of PDT on primary human vocal fold fibroblasts (VFFs) in vitro. In vitro analysis from one human donor. Cell viability of VFFs in response to varying doses of PDT was investigated by the Cell Counting Kit-8 method. Sublethal-dose PDT (SL-PDT) was used for the following experiments. Expression of genes related to vocal fold extracellular matrix formation was analyzed by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blotting. Effects of PDT on cell migration, collagen contraction, and transforming growth factor β-1 (TGF-β1)-induced myofibroblast differentiation were also analyzed. PDT affects the viability of VFFs in a dose-dependent manner. SL-PDT significantly changed the expression profile of VFFs with antifibrotic effects. It also inhibited cell migration, reduced collagen contraction, and reversed the fibroblast-myofibroblast differentiation induced by TGF-β1. SL-PDT induces antifibrotic alterations in VFFs. This could explain the low incidence of vocal fold scar associated with PDT. Moreover, PDT may be useful in treating existing vocal fold scars. Further studies should focus on the in vivo effect of PDT on vocal fold wound healing and scar remodeling. NA Laryngoscope, 128:E323-E331, 2018.

Characteristics and Responses of Human Vocal Fold Cells in a Vibrational Culture Model.

This study was conducted to provide a vibrational culture model to investigate the effects of mechanical environments on cellular functions, and elucidate physiological characteristics of two different types of cells in vocal folds under static and vibrational conditions. In vitro study of human vocal fold fibroblasts (hVFFs) and human macula flava stellate cells (hMF-SCs). hVFFs and hMF-SCs were exposed to a 2-second-on/2-second-off, 205 Hz vibration regime for 4 hours by using a vibrational culture model. We compared cell morphology, cell viability, and gene expression in extracellular matrix-related components, growth factors, and differentiation markers under static and vibratory conditions. hVFFs and hMF-SCs differed in their morphologies and gene expression levels under static condition. The applied vibration did not induce changes in morphology and viability of either cell type. However, gene expression levels changed in both cell types in response to vibration; in particular, hMF-SCs exhibited a more sensitive response to vibration than that shown by hVFFs. The vibrational culture model developed in this study enabled us to investigate the effects of the applied vibration on two types of vocal fold resident cells. As a result, we could demonstrate that hVFFs and hMF-SCs exhibited distinctively different characteristics under vibrational conditions. NA. Laryngoscope, 128:E258-E264, 2018.

Stem Cell-Mediated Paracrine Signaling Alters Fibroplasia in Human Vocal Fold Fibroblasts in Vitro.

Interactions between mesenchymal stem cells (MSCs) and native vocal fold fibroblasts (VFFs) have not been described in spite of promising preliminary data regarding the effects of MSCs on vocal fold repair in vivo. The current study employed a conditioned media (CM) model to investigate the paracrine effects of bone marrow-derived mesenchymal stem cells (BMSCs) on VFFs. Human VFFs were treated with transforming growth factor-β1 (TGF-β1; 10 ng/mL), CM from human BMSCs following 48 hours of TGF-β1 stimulation, or CM+TGF-β1. Proliferation, immunocytochemistry for alpha smooth muscle actin (αSMA), migration, and collagen gel contraction were quantified as well as transcription of components of the TGF-β signaling pathway. Transforming growth factor-β1 accelerated proliferation and induced αSMA in VFFs; these effects were suppressed with CM ( P = .009, P < .001, respectively). The CM+TGF-β1 condition increased cell migration ( P = .02) and decreased gel contraction; CM+TGF-β1 also inhibited TGF-β signaling via significant upregulation of NR4A1 as well as downregulation of S MAD3 and TGF-β1 relative to TGF-β1 stimulation in the absence of CM ( P = .002, P < .001, and P = .005, respectively). Conditioned media affected many profibrotic cell activities in TGF-β1-stimulated VFFs, likely related to altered TGF-β signaling. These data provide preliminary insight regarding the antifibrotic effects of MSCs and further support their progression to clinical utility.

Acute Nanoparticle Exposure to Vocal Folds: A Laboratory Study

Airway exposure to nanoparticles is common in occupational settings. Inhaled nanoparticles have toxic effects on respiratory tissue. Vocal folds are also at direct risk from inhaled nanoparticles. This study investigated the effects of single-walled carbon nanotubes (SWCNT), a type of nanoparticle, on vocal fold epithelium and fibroblasts. These cell types were selected for study as the epithelium is the outer layer of the vocal folds and fibroblasts are the most common cell type in connective tissue underlying the epithelium. Native porcine vocal fold epithelium and cultured human vocal fold fibroblasts were exposed to SWCNTs (100 ng/mL) and control (no SWCNT) in vitro. Epithelial and fibroblast viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Epithelial barrier integrity was assessed with transepithelial resistance and sodium fluorescein permeability. Epithelial tight junctional protein occludin expression was measured with Western blot. Gene expressions of the fibroblast-specific protein 1 (FSP-1), α-smooth muscle actin (α-SMA), and collagen III (Col-III) were assessed using quantitative polymerase chain reaction. Transcriptional expression of genes encoding FSP-1 and Col-III was increased significantly following SWCNT exposure. There were no significant differences between control and SWCNT groups on any of the other measures. SWCNT exposure induces vocal fold fibroblasts to a fibrotic phenotype. These data help us understand vocal fold defense mechanisms and lay the groundwork for studying the physiological effects of nanoparticle exposure in vivo.

Preliminary study of a novel transfection modality for in vivo siRNA delivery to vocal fold fibroblasts.

An obstacle to clinical use of RNA-based gene suppression is instability and inefficiency of current delivery modalities. Nanoparticle delivery likely holds great promise, but the kinetics and transfection conditions must be optimized prior to in vivo utility. We investigated a RNA nanoparticle complex incorporating a lipitoid transfection reagent in comparison to a commercially available reagent. In vitro. We investigated which variables influence transfection efficiency of lipitoid oligomers and a commercially available reagent across species, in vitro. These variables included duration, dose, and number of administrations, as well as serum and media conditions. The target gene was Smad3, a signaling protein in the transforming growth factor-β cascade implicated in fibroplasia in the vocal folds and other tissues. The two reagents suppressed Smad3 mRNA for up to 96 hours; lipitoid performed favorably and comparably. Both compounds yielded 60% to 80% mRNA knockdown in rat, rabbit, and human vocal fold fibroblasts (P < 0.05 relative to control). Dose and number of administrations played a significant role in gene suppression (P < 0.05). Suppression was more dose-sensitive with lipitoid. At a constant siRNA concentration, a 50% decrease in gene expression was observed in response to a five-fold increase in lipitoid concentration. Increased number of administrations enhanced gene suppression, ∼45% decrease between one and four administrations. Neither serum nor media type altered efficiency. Lipitoid effectively knocked down Smad3 expression across multiple transfection conditions. These preliminary data are encouraging, and lipitoid warrants further investigation with the goal of clinical utility. NA. Laryngoscope, 127:E231-E237, 2017.

An in vitro scaffold-free epithelial-fibroblast coculture model for the larynx.

Physiologically relevant, well-characterized in vitro vocal fold coculture models are needed to test the effects of various challenges and therapeutics on vocal fold physiology. We characterize a healthy state coculture model, created by using bronchial/tracheal epithelial cells and immortalized vocal fold fibroblasts. We also demonstrate that this model can be induced into a fibroplastic state to overexpress stress fibers using TGFβ1. In vitro. Cell metabolic activity of immortalized human vocal fold fibroblasts incubated in different medium combinations was confirmed with an MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay. Fibroblasts were grown to confluence, and primary bronchial/tracheal epithelial cells suspended in coculture medium were seeded directly over the base layer of the fibroblasts. Cells were treated with transforming growth factor β1 (TGFβ1) to induce myofibroblast formation. Cell shape and position were confirmed by live cell tracking, fibrosis was confirmed by probing for α smooth muscle actin (αSMA), and phenotype was confirmed by immunostaining for vimentin and E-cadherin. Fibroblasts retain metabolic activity in coculture epithelial medium. Live cell imaging revealed a layer of epithelial cells atop fibroblasts. αSMA expression was enhanced in TGFβ1-treated cells, confirming that both cell types maintained a healthy phenotype in coculture, and can be induced into overexpressing stress fibers. Vimentin and E-cadherin immunostaining show that cells retain phenotype in coculture. These data lay effective groundwork for a functional coculture model that retains the reproducibility necessary to serve as a viable diagnostic and therapeutic screening platform. NA Laryngoscope, 127:E185-E192, 2017.

The Protective Role of Autophagy in Human Vocal Fold Fibroblasts under Cigarette Smoke Extract Exposure: A New Insight into the Study of Reinke's Edema

Background/Aims: Reinke's edema (RE), one of the most common benign lesions of the human vocal folds, has been associated with cigarette smoking. We have observed an abundant number of autophagosomes in the cytoplasm of fibroblasts in the lamina propria of human vocal folds under transmission electron microscopy in RE tissue. Thus, we conducted a study about autophagy induced by cigarette smoke extract (CSE) exposure in cultured human vocal fold fibroblasts (hVFFs). Methods: Sampled tissues were cultured and exposed to varying levels of CSE. Immunochemistry staining and Western blot analyses were then performed on the cultures. Results: We demonstrated that CSE significantly promoted intracellular reactive oxygen species accumulation and reduced the viability of hVFFs in a concentration-dependent manner. Both immunocytochemistry and Western blot analysis showed increased expression of the autophagy marker type II-membrane-associated light chain 3 in fibroblasts following 24 h of CSE exposure, indicating the upregulation of autophagy activity. Furthermore, the results demonstrate that the induction of autophagy played a protective role in hVFFs exposed to CSE. Conclusion: Autophagy can be induced by CSE in hVFFs, and it is a protective response of hVFFs to the exposure of CSE. This study provides a new insight into the process involved in the development of RE.

Early Cellular Response to Radiation in Human Vocal Fold Fibroblasts.

Radiation therapy is a common treatment strategy for laryngeal carcinoma. However, radiation is not without adverse side effects, especially toward healthy vocal fold tissue, which can lead to long-term impairments in vocal function. The objective of this preliminary study was to investigate early responses of healthy human vocal fold fibroblasts (VFF) to radiation. VFF were exposed to a single or fractionated dose radiation scheme. Nonradiated VFF served as controls. Morphology of radiated and control VFF was subjectively examined. Quantitative polymerase chain reaction was used to evaluate the effect of radiation on extracellular matrix and inflammatory-related genes. VFF viability was investigated using a LIVE/DEAD and clonogenic assay. Single or fractioned dose radiated VFF were morphologically indistinguishable from control VFF. No significant differences in gene expression were observed following either radiation scheme and as compared to controls. Clonogenic assay revealed reduced VFF viability following the fractionated but not single dose scheme. No changes in viability were detected using the LIVE/DEAD assay. We present one of the first investigations to evaluate early responses of healthy VFF to radiation. Findings will contribute to a growing body of literature seeking to elucidate the biological mechanisms underlying voice changes following radiation therapy for laryngeal carcinoma.

The role of Smad3 in the fibrotic phenotype in human vocal fold fibroblasts.

To investigate the role of Smad3 as a regulator of transforming growth factor (TGF)-β1-mediated cell activities associated with fibrosis in normal human vocal fold fibroblasts. We also sought to confirm the temporal stability of Smad3 knockdown via small inhibitor ribonucleic acid (siRNA). Vocal fold fibroblasts were employed to determine the effects of Smad3 knockdown on TGF-β1-mediated migration and contraction, as well as regulation of connective tissue growth factor (CTGF). We hypothesized that Smad3 is an ideal candidate for therapeutic manipulation in vivo based on its role in fibrosis. In vitro. Knockdown of Smad3 via siRNA was performed in our normal human vocal fold cell line. Three-dimensional collagen gel contraction and scratch assays were employed to determine the role of Smad3 on TGF-β1-mediated contraction and migration, respectively. The role Smad3 in the induction of CTGF was characterized via sodium dodecyl sulfate polyacrylamide gel electrophoresis. The effects of Smad3 signaling on Smad7 messenger (m)RNA and protein were also quantified. Smad3 knockdown was temporally-stable up to 72 hours (P < 0.001), diminished TGF-β1-mediated collagen gel contraction and migration, and blunted induction of CTGF, but it had no effect on TGF-β1-mediated Smad7 mRNA or protein induction. Transforming growth factor-β1 stimulated profibrotic cell activities in our cell line and these actions were largely reduced with Smad3 knockdown. These data provide continued support for therapeutic targeting of Smad3 for vocal fold fibrosis because it appears to regulate the fibrotic phenotype. N/A. Laryngoscope, 126:1151-1156, 2016.