Faculty Bibliography

Macrophage phenotypes are simplistically classified as pro-inflammatory (M1) or anti-inflammatory/pro-fibrotic (M2). Phenotypically different macrophages are putatively involved in vocal fold (VF) fibrosis. The current study investigated interactions between macrophages and VF fibroblasts. THP-1 monocyte-derived macrophages were treated with interferon-gamma (IFN-γ), lipopolysaccharide (LPS)/IFN-γ, interleukin-10 (IL10), transforming growth factor-β1 (TGF-β), or interleukin-4 (IL4) for 24 h (M(IFN), M(IFN/LPS), M(IL10), M(TGF), and M(IL4), respectively; M(-) denotes untreated macrophages). Differentially activated macrophages and human VF fibroblasts were co-cultured ± direct contact. Expression of CXCL10, CCN2, ACTA2, FN1, TGM2, and LOX was quantified by real-time polymerase chain reaction. Type I collagen and smooth muscle actin (SMA) were observed by immunofluorescence. CXCL10 and PTGS2 were upregulated in fibroblasts indirectly co-cultured with M(IFN) and M(IFN/LPS). M(TGF) stimulated CCN2, ACTA2, and FN1 in fibroblasts. Enzymes involved in extracellular matrix crosslinking (TGM2, LOX) were increased in monocultured M(IL4) compared to M(-). Direct co-culture with all macrophages increased type I collagen and SMA in fibroblasts. Macrophage phenotypic shift was consistent with stimulation and had downstream differential effects on VF fibroblasts. Direct contact with macrophages, regardless of phenotype, stimulated a pro-fibrotic response in VF fibroblasts. Collectively, these data suggest meaningful interactions between macrophages and fibroblasts mediate fibrosis.

Introduction: The inner lining of the upper airway includes ciliated epithelium and lamina propria essential for barrier function. This layer is disrupted upon injury and results in inflammation and fibrotic scarring[1]. We developed a model to study the impact of basement architectural cues on the epithelial-fibroblast interaction at air-liquid interface by using randomly-oriented and aligned polycaprolactone (PCL) fibers.
Material(s) and Method(s): Plasma treated randomly oriented and aligned PCL electrospun fibers were placed in transwell chambers and were seeded with human tracheal fibroblasts (HTFs) for 7 days and then human bronchial epithelial cells (HBEs) were introduced above the HTF layer. An air-liquid interface was established on day 14 to promote HBE differentiation. Permeability, cell proliferation, and expression of fibroblast (fibronectin and S100A4) and epithelial (MUC5A) markers were evaluated using ELISA and immunofluorescence (IHC) imaging (n = 6). Quantitative data were compared using one-way Analysis of Variance (ANOVA) followed by Tukey's test for post hoc determination of significant differences at p < 0.05.Results and Discussion: Fiber alignment resulted in higher expression of fibroblast markers during the first 7 days while randomly oriented fibers generally caused higher (27%) cell proliferation over time. In addition, IHC images revealed homogenous HBE growth above the HTFs layer with significant laminin- rich matrix deposited at the interface and dispersed spheroidal epithelial clusters observed in both groups. Larger epithelial spheres were observed in coculture on randomly oriented fibers with rudimentary ciliated structures.
Conclusion(s): A successful epithelial-fibroblast coculture system with pro-fibrotic behavior was achieved by controlling architectural cues introduced during initial fibroblastepithelial interactions

Extracellular matrix (ECM) is a complex structure composed of bioactive molecules representative of the specific local tissue microenvironment. Decellularized ECM biomaterials harness these biomolecules for regenerative medicine applications. One potential therapeutic application is the use of vocal fold (VF) specific ECM to restore the VFs after injury. ECM scaffolds are derived through a process of decellularization, which aims to remove unwanted immunogenic biomolecules (e.g., DNA) while preserving the composition of the ECM. The effectiveness of the decellularization is typically assessed at the end by quantifying ECM attributes such as final dsDNA content. However, batch-to-batch variability in ECM manufacturing remains a significant challenge for the process standardization, cost-effectiveness, and scale-up. The limited number of tools available for in-process control heavily restricts the uncovering of the correlations between decellularization process parameters and ECM attributes. In this study, we developed a technique applicable to both the classical batch method and semi-continuous decellularization system to trace the decellularization of two laryngeal tissues in real-time. We hypothesize that monitoring the bioreactor's effluent absorbance at 260 nm as a function of time will provide a representative DNA release profile from the tissue and thus allowing for process optimization. The DNA release profiles were obtained for laryngeal tissues and were successfully used to optimize the derivation of VF lamina propria-ECM (auVF-ECM) hydrogels. This hydrogel had comparable rheological properties to commonly used biomaterials to treat VF injuries. Also, the auVF-ECM hydrogel promoted the down-regulation of CCR7 by THP-1 macrophages upon lipopolysaccharide stimulation in vitro suggesting some anti-inflammatory properties. The results show that absorbance profiles are a good representation of DNA removal during the decellularization process thus providing an important tool to optimize future protocols.

OBJECTIVES/HYPOTHESIS/OBJECTIVE:Vocal fold fibrosis remains a significant clinical challenge. Estrogens, steroid hormones predominantly responsible for secondary sexual characteristics in women, have been shown to alter wound healing and limit fibrosis, but the effects on vocal fold fibrosis are unknown. We sought to elucidate the expression of estrogen receptors and the effects of estrogens on TGF-β1 signaling in rat vocal fold fibroblasts (VFFs). STUDY DESIGN/METHODS:In vitro. METHODS: M) were employed as antagonists of ERα or GPR30, respectively. qPCR was employed to determine estrogen receptor-mediated effects of E2 on genes related to fibrosis. RESULTS: M) and TGF-β1 significantly increased Smad7 (P = .03) and decreased Col1a1 (P = .04) compared to TGF-β1 alone; this response was negated by the combination of ICI and G36 (P = .009). CONCLUSIONS:E2 regulated TGF-β1/Smad signaling via estrogen receptors in VFFs. These findings provide insight into potential mechanisms of estrogens on vocal fold injury with the goal of enhanced therapeutics for vocal fold fibrosis. LEVEL OF EVIDENCE/METHODS:NA. Laryngoscope, 2020.

OBJECTIVE/UNASSIGNED:Oversimplified clinical dogma suggests that laryngeal diseases fall into two broad, mutually exclusive diagnostic categories-mucosal injury or neuromuscular/functional disorders. Extensive investigation in the lower airway as well as other organ systems suggest complex interactions between tissue types underlying both tissue health and pathological states. To date, no such relationship has been described in the vocal folds, likely the most bioactive organ in the body. We hypothesize interactions between the vocal fold muscle and mucosa likely contribute to aberrant phonatory physiology and warrant further investigation to ultimately develop novel therapeutic strategies. METHODS/UNASSIGNED:Primary culture of myoblasts from rat thyroarytenoid muscle and fibroblasts from the vocal fold mucosa were established. Co-culture and conditioned media experiments were performed to established bidirectional interactions between cell types. Transforming Growth Factor (TGF)-β was employed to stimulate a fibrotic phenotype in culture. In addition to quantitative PCR, standard migration and proliferation assays were performed as well as immunocytochemistry. RESULTS/UNASSIGNED:Bidirectional cell-cell interactions were observed. Without TGF-β stimulation, myoblast conditioned media inhibited fibroblast migration, but enhanced proliferation. Conversely, fibroblast conditioned media increased both myoblast proliferation and migration. Myoblast conditioned media decreased TGF-β-mediated gene expression and of particular interest, ACTA2 mRNA expression. In both co-culture and in response to fibroblast conditioned media, myosin heavy chain (Myh2) mRNA expression decreased in myoblasts. CONCLUSIONS/UNASSIGNED:These data are the first to describe interactions between cell types within the vocal fold. The implications for these interactions in vivo warrant further investigation to develop and refine optimal treatment strategies.

Vocal fold (VF) fibrosis is a major cause of intractable voice-related disability and reduced quality of life. Excision of fibrotic regions is suboptimal and associated with scar recurrence and/or further iatrogenic damage. Non-surgical interventions are limited, putatively related to limited insight regarding biochemical events underlying fibrosis, and downstream, the lack of therapeutic targets. YAP/TAZ integrates diverse cell signaling events and interacts with signaling pathways related to fibrosis, including the TGF-β/SMAD pathway. We investigated the expression of YAP/TAZ following vocal fold injury in vivo as well as the effects of TGF-β1 on YAP/TAZ activity in human vocal fold fibroblasts, fibroblast-myofibroblast transition, and TGF-β/SMAD signaling. Iatrogenic injury increased nuclear localization of YAP and TAZ in fibrotic rat vocal folds. In vitro, TGF-β1 activated YAP and TAZ in human VF fibroblasts, and inhibition of YAP/TAZ reversed TGF-β1-stimulated fibroplastic gene upregulation. Additionally, TGF-β1 induced localization of YAP and TAZ in close proximity to SMAD2/3, and nuclear accumulation of SMAD2/3 was inhibited by a YAP/TAZ inhibitor. Collectively, YAP and TAZ were synergistically activated with the TGF-β/SMAD pathway, and likely essential for the fibroplastic phenotypic shift in VF fibroblasts. Based on these data, YAP/TAZ may evolve as an attractive therapeutic target for VF fibrosis.

Fibrosis of the vocal folds poses a substantive clinical challenge potentially underlying the rapid proliferation of direct steroid injections into the upper airway. The variable clinical response to glucocorticoids (GCs) in the vocal folds is likely related to diversity inherent to GCs and both patient-specific, and upstream, cell-specific responses to GCs. Broadly, we hypothesize the disparity in clinical outcomes are due to undesirable effects of GCs on resident fibroblasts. Transcriptome analysis identified significant GC-mediated modulation of Hippo signaling, a known regulator of fibrotic gene expression. Subsequent analysis confirmed GC-mediated YAP activation, a transcriptional co-factor in the Hippo signaling pathway. YAP inhibition attenuated ACTA2 expression in GC-treated human vocal fold fibroblasts. Nuclear localization and phosphorylation at Ser211, however, was not affected by YAP inhibition, suggesting nuclear translocation of YAP is indirectly driven by GR. RNA-seq analysis confirmed the influence of GCs on Wnt signaling, and canonical Wnt signaling target genes were upregulated by GCs. These data implicate YAP and its downstream targets as putative mediators of a pro-fibrotic response to GCs. Therapeutic YAP inhibition may ultimately be clinically relevant and warrants further consideration.

Introduction.In vitro experimentation is intentionally contrived to isolate specific phenomena in the context of profound biological complexity. Mycoplasmas in the upper airway likely contribute to this complexity and play a largely unknown role in both health and disease. Similarly, the presence and role of mycoplasma in in vitro investigation are largely unknown.Hypothesis. We hypothesize mycoplasma in human vocal fold fibroblasts (VFF) will affect both basal gene-expression patterns as well as the cell response to exogenous stimuli.Aim. We sought to determine mycoplasma presence across vocal fold fibroblast cultures, basal transcriptional changes as a function of mycoplasma, and responsiveness to exogenous glucocorticoids in mycoplasma-positive and -negative VFF.Methodology. PCR-based mycoplasma detection was performed in an immortalized human VFF line as well as rat and rabbit primary VFF cultures and extracted rat laryngeal tissue. RNA sequencing was performed in mycoplasma-positive and -negative human cells at baseline and in response to dexamethasone.Results. Mycoplasma was identified in the human cell line as well as primary culture from rabbits. Mycoplasma was not detected in tissue or primary culture from rat vocal folds. Basal mRNA expression in human VFF differed significantly following mycoplasma treatment. In addition, differential responses to dexamethasone were observed across multiple pathways as a function of mycoplasma presence in these cells. Pathways including apoptosis, DNA damage repair, and G1 to S cell cycle signalling were significantly enriched in mycoplasma-positive cells.Conclusion. Variability of mycoplasma presence across culture conditions and differential responses to exogenous stimuli as a function of mycoplasma presence are potentially problematic for the translation of in vitro experimentation in the upper aerodigestive tract. It remains unclear if these findings represent contamination or the baseline state of this specialized tissue.

Similar to the hypertrophic scar and keloids, the efficacy of glucorticoids (GC) for vocal fold injury is highly variable. We previously reported dexamethasone enhanced the pro-fibrotic effects of transforming growth factor (TGF)-β as a potential mechanism for inconsistent clinical outcomes. In the current study, we sought to determine the mechanism(s) whereby GCs influence the fibrotic response and mechanisms underlying these effects with an emphasis on TGF-β and nuclear receptor subfamily 4 group A member 1 (NR4A1) signaling. Human VF fibroblasts (HVOX) were treated with three commonly-employed GCs+ /-TGF-β1. Phosphorylation of the glucocorticoid receptor (GR:NR3C1) and activation of NR4A1 was analyzed by western blotting. Genes involved in the fibrotic response, including ACTA2, TGFBR1, and TGFBR2 were analyzed by qPCR. RNA-seq was performed to identify global changes in gene expression induced by dexamethasone. GCs enhanced phosphorylation of GR at Ser211 and TGF-β-induced ACTA2 expression. Dexamethasone upregulated TGFBR1, and TGFBR2 in the presence of TGF-β1 and increased active NR4A1. RNA-seq results confirmed numerous pathways, including TGF-β signaling, affected by dexamethasone. Synergistic pro-fibrotic effects of TGF-β were observed across GCs and appeared to be mediated, at least partially, via upregulation of TGF-β receptors. Dexamethasone exhibited diverse regulation of gene expression including NR4A1 upregulation consistent with the anti-fibrotic potential of GCs.

OBJECTIVES/HYPOTHESIS/OBJECTIVE:To assess the safety and efficacy of autologous cultured fibroblasts (ACFs) to treat dysphonia related to vocal fold scar and age-related vocal atrophy (ARVA). STUDY DESIGN/METHODS:Randomized, double-blinded, placebo-controlled, multi-institutional, phase II trial. METHODS:cells or placebo saline was performed at 4-week intervals for each vocal fold. Follow-up was performed at 4, 8, and 12 months. The primary outcome was improved mucosal waves. Secondary outcomes included Voice Handicap Index (VHI)-30, patient reported voice quality outcomes, and perceptual analysis of voice. RESULTS:Fifteen subjects received ACF and six received saline injections. At 4, 8, and 12 months after ACF treatments, a significant improvement in mucosal wave grade relative to baseline was observed in both vocal scar and ARVA groups. Relative to control group, mucosal waves were significantly improved in the ARVA group at 4 and 8 months. Perceptual analysis significantly improved in the vocal scar group 12 months after ACF treatments compared to controls. Vocal scar group reported significantly improved vocal quality from baseline. VHI and expert rater voice grade improved in both groups, but did not achieve significance. No adverse events related to fibroblast injections were observed. CONCLUSIONS:In this cohort, injection of ACFs into the vocal fold lamina propria (LP) was safe and significantly improved mucosal waves in patients with vocal scar and ARVA. ACF may hold promise to reconstruct the LP. LEVEL OF EVIDENCE/METHODS:1 Laryngoscope, 2019.