Faculty Bibliography

OBJECTIVES/OBJECTIVE:Traditionally, disorders of the vocal fold (VF) mucosa and underlying musculature have been regarded as mutually exclusive entities. However, emerging evidence from other organ systems suggests mucosal and muscle compartments engage in reciprocal interactions with functional consequences. We hypothesized that similar crosstalk exists in the VF, whereby fibrotic mucosa influences adjacent muscle. To model this process, we stimulated human VF fibroblasts (HVOX) with TGF-β1, a central mediator of fibrosis, and examined the effects on rat VF myoblasts (rVF-Mbs), as well as reciprocal influences of rVF-Mbs on fibroblasts. METHODS:HVOX fibroblasts were stimulated with 10 ng/mL TGF-β1, and the effects on rVF-Mbs were assessed using conditioned media and co-culture. Myotube formation was evaluated by immunofluorescence, and nuclear localization of Smad2/3 was examined in conditioned media experiments. qRT-PCR quantified transcripts related to myogenic differentiation and Smad2/3 signaling. ALK4/5 inhibition was performed in co-culture to test TGF-β/Smad2/3-signaling pathway involvement. Reciprocal effects were examined by changes in fibrogenic gene expression in HVOX fibroblasts. RESULTS:Both conditioned media and co-culture suppressed myogenic differentiation in rVF-Mbs; increased inhibition was observed in co-culture, as indicated by reduced myotube formation, decreased Myh2 expression, and activation of Smad2/3 signaling. ALK4/5 inhibition abrogated these effects. Differentiating rVF-Mbs attenuated the fibrogenic phenotype of HVOX fibroblasts. CONCLUSIONS:Fibrotic VF mucosal cells can impair myogenic differentiation through TGF-β/Smad2/3-mediated fibroblast-myoblast crosstalk, and myogenic cells may exert reciprocal anti-fibrotic effects. These findings suggest mucosa-muscle interactions may contribute to VF pathology and highlight Smad2/3 as a potential therapeutic target. LEVEL OF EVIDENCE/METHODS:NA STUDY DESIGN: In vitro.

OBJECTIVES/OBJECTIVE:Despite widespread industry knowledge regarding vocal health habits among singers, a paucity of research supports these beliefs, potentially undermining patient counseling. We sought to catalog attitudes of vocal healthcare providers regarding common vocal health practices to compare to vocalists. METHODS:An online survey was administered to fellowship-trained laryngologists and voice-specialized speech-language pathologists (SLPs) to assess attitudes regarding 82 factors frequently associated with vocal health. Respondents rated each item on the following scale: good in the long-term, good in the short-term, bad long-term, bad short-term, or no effect. Results were compared to previously collected data from an analogous survey completed by professional and amateur vocalists. RESULTS:Twenty-four vocal healthcare providers (15 SLPs and nine laryngologists) completed the survey. Consensus among vocal healthcare providers was found on all but six items, but differences between vocalists and providers were observed on 34 items. These items included factors seen as less harmful (eg dairy, whispering) or less beneficial (eg tea, Vitamin B12, jogging on performance day) by healthcare providers than singers. Several items were viewed more cautiously by providers (eg menthol, decongestants, spicy foods, breath-holding, and whiskey shots). CONCLUSIONS:A substantial disconnect between vocal healthcare providers and singers was observed regarding habits conducive to vocal health. These data have significant implications for the counseling of voice users and demonstrate the importance of further study to understand the impact of vocalist habits on vocal health. LEVEL OF EVIDENCE/METHODS:Level 3.

BACKGROUND/OBJECTIVES/OBJECTIVE:Vocal fold (VF) muscle atrophy, often associated with neuromuscular disorders and aging, can lead to voice-related disability. Myostatin is well-known to mediate skeletal muscle atrophy via Smad2/3 signaling, whereas TGF-β1, a potent inducer of Smad2/3 signaling, is upregulated following VF injury. However, the impact of Smad2/3 signaling on laryngeal muscles remains unclear. This study provides foundational insight regarding Smad2/3-dependent atrophic responses of VF skeletal muscle cells, to ultimately develop novel therapeutic strategies for VF muscle atrophy. STUDY DESIGN/METHODS:In vitro. METHODS:Myoblasts isolated from the rat thyroarytenoid muscle were differentiated into myotubes in myogenic differentiation medium ±500 ng/mL myostatin or 10 ng/mL TGF-β1, in the presence or absence of an ALK4/5 inhibitor or siRNA targeting Smad2 and Smad3. Myotube formation and activation of Smad2/3 (nuclear localization of Smad2/3) were assessed via immunofluorescence. Transcription related to myotube differentiation and Smad2/3 signaling was quantified by qRT-PCR. RESULTS:Both myostatin and TGF-β1 suppressed myogenic differentiation, increased Smad2/3 nuclear intensity, downregulated Myh2, and upregulated downstream targets of Smad2/3 (Ccn2 and Serpine1) and Fbox32, an atrophy-related gene. These effects were more pronounced with TGF-β1 than with myostatin and were reversed by inhibition of ALK4/5. Furthermore, Smad2/3 knockdown via siRNA promoted myogenic differentiation, further supporting the role of Smad2/3 signaling in the atrophic response in VF myoblasts. CONCLUSIONS:Smad2/3 signaling mediates differentiation of VF myoblasts and TGF-β1, a potent mediator of fibrosis, elicited a more pronounced atrophic response than myostatin. Smad2/3 may be an attractive therapeutic target for VF muscle atrophy. LEVEL OF EVIDENCE/METHODS:NA.

Rapid and precise activation of the laryngeal musculature, particularly the thyroarytenoid (TA), is required for the production of rat ultrasonic vocalizations (USVs). Although the role of the TA in USV production has been established through denervation and excised larynx studies, how TA muscle dysfunction affects USV acoustics remains unknown. This study examined how iatrogenic, transmuscular vocal fold injury influences USV production by comparing acoustic parameters between rats receiving bilateral transmuscular vocal fold injury (including the TA muscle) versus sham surgery. Twenty adult male rats were randomly assigned to injury or sham groups and assessed at 30 or 60 days post-surgery. USVs were recorded at each timepoint (pre- and post-surgery) and analyzed for changes in principal frequency, frequency complexity (standard deviation and sinuosity), power (intensity of the USV), tonality, and duration. Injury significantly decreased USV frequency complexity and reduced the likelihood of producing frequency-modulated vocalizations in both post-surgical time groups. These findings demonstrate that while TA muscle integrity is crucial for frequency modulation, it is not necessary for basic USV production, offering new insights into the biomechanical role of the TA muscle in rat vocalization and laying the groundwork for investigating therapeutic interventions targeting laryngeal muscle function.

OBJECTIVE:Surgical treatment of recurrent respiratory papillomatosis (RRP) has been shown to aerosolize human papillomavirus (HPV), putting healthcare workers at risk for exposure, infection, and disease. Knowledge of HPV infection risk among otolaryngologists who treat HPV-related diseases is limited. We sought to characterize the prevalence of oral HPV infection in otolaryngologists treating RRP. METHODS:This observational cohort study enrolled otolaryngologists at a national meeting. Participants completed a survey concerning HPV vaccination, disease history, and practice techniques for patients with RRP. An oral rinse was collected from participants; DNA was extracted and analyzed using commercially available kits. RESULTS:laser (N = 78), microdebrider (N = 80), and cold steel (N = 21). Oral rinses from three participants (2.2%) tested positive for HPV, including Subtypes 6 (N = 2) and 16 (N = 1). All three were male with no history of HPV vaccination. CONCLUSION/CONCLUSIONS:Otolaryngologists treating HPV-related diseases do not seem to be at a higher risk of HPV infection compared to the general adult population. Vaccination, the use of N95 masks, and minimizing aerosol-generating techniques are likely protective for healthcare workers dealing with HPV-related conditions.

Vocal fold (VF) fibrosis, often resulting from phonosurgery, radiation, or trauma, causes irreversible voice dysfunction due to excessive ECM deposition and increased tissue stiffness. No FDA-approved treatments for VF fibrosis exist, highlighting the need for novel antifibrotic therapies. TGF-β1 contributes to fibroblast-to-myofibroblast activation, leading to increased ACTA2 expression and collagen production via SMAD3, YAP1, and integrin signaling pathways. Leveraging the principle that local cells respond to tissue-specific signals, our ECM hydrogel, derived from decellularized vocal fold lamina propria (VFLP-ECM), reduced ACTA2 expression in TGF-β1-stimulated VF fibroblasts, showcasing antifibrotic potential. This study evaluates the therapeutic potential of VFLP-ECM hydrogel in a rabbit VF injury model. VFLP-ECM hydrogel or bovine type I collagen injections were administered 7 days post-injury and evaluated on day 28. We compared two VFLP-ECM formulations: a manual process (VFLP (man)) and an accelerated automated method (VFLP (au)). VFLP (man) modulated fibrosis-associated gene expressions more effectively than controls. Proteomics identified 229 proteins uniquely preserved in VFLP (man), including vitronectin, crucial in TGF-β1 signaling and ECM remodeling. Transcriptomic analysis suggests downregulation of fibrotic markers and inhibition of SMAD3, YAP1, and MRTFA, alongside upregulation of SMAD7, an inhibitor of TGF-β signaling. Notably, VFLP (man) treatment recovered stiffness comparable to uninjured controls (1.84 vs. 1.94 mN), whereas collagen-treated tissues remained stiff (2.7 mN), similar to the injury group (2.6 mN), indicating incomplete mechanical recovery. These in vivo data show that manually decellularized VFLP-ECM hydrogel attenuates fibrosis by disrupting key biochemical and mechanical cues driving myofibroblast activation.

The pro-fibrotic effects of glucocorticoids may lead to a suboptimal therapeutic response for vocal fold (VF) pathology. Targeting macrophage-fibroblast interactions is an interesting therapeutic strategy; macrophages alter their phenotype to mediate both inflammation and fibrosis. In the current study, we investigated concentration-dependent effects of methylprednisolone on the fibrotic response, with an emphasis on YAP/TAZ-TEAD signaling, and inflammatory gene expression in VF fibroblasts in physical contact with macrophages. We sought to provide foundational data to optimize therapeutic strategies for millions of patients with voice/laryngeal disease-related disability. Following induction of inflammatory (M(IFN/LPS)) and fibrotic (M(TGF)) phenotypes, THP-1-derived macrophages were seeded onto HVOX vocal fold fibroblasts. Cells were co-cultured ± 0.3-3000 nM methylprednisolone ± 3 µM verteporfin, a YAP/TAZ inhibitor. Inflammatory (CXCL10, TNF, PTGS2) and fibrotic genes (ACTA2, CCN2, COL1A1) in fibroblasts were analyzed by real-time polymerase chain reaction after cell sorting. Ser211-phosphorylated glucocorticoid receptor (S211-pGR) was assessed by Western blotting. Nuclear localization of S211-pGR and YAP/TAZ was analyzed by immunocytochemistry. Methylprednisolone decreased TNF and PTGS2 in fibroblasts co-cultured with M(IFN/LPS) macrophages and increased ACTA2 and CCN2 in fibroblasts co-cultured with M(IFN/LPS) and M(TGF). Lower concentrations were required to decrease TNF and PTGS2 expression and to increase S211-pGR than to increase ACTA2 and CCN2 expression and nuclear localization of S211-pGR. Methylprednisolone also increased YAP/TAZ nuclear localization. Verteporfin attenuated upregulation of CCN2, but not PTGS2 downregulation. High concentration methylprednisolone induced nuclear localization of S211-pGR and upregulated fibrotic genes mediated by YAP/TAZ activation.

INTRODUCTION/BACKGROUND:Glucocorticoids (GCs) are commonly prescribed for laryngeal indications due to their potent anti-inflammatory properties. However, GCs effect on vocal fold (VF) epithelial morphology and barrier function following injury is overlooked and may be key to efficacy. In this study, the effects of GCs on epithelial morphology and barrier function were quantified in injured VFs. We seek to increase our understanding of biochemical processes underlying GC mechanisms to refine therapeutic strategies. METHODS:Microflap injury was induced in 65 rabbits. Seven days after injury, animals received bilateral 20 μL intracordal injections of saline, dexamethasone, methylprednisolone, or triamcinolone (n = 15 per condition). Five rabbits in each condition were euthanized 1, 7, or 60 days following treatment. An additional five animals served as non-injured/untreated controls. To quantify transepithelial electrical resistance (TEER), 1 mm epithelial biopsies were placed in an Ussing chamber. The contralateral VF was processed for transmission electron microscopy and epithelial depth analysis. RESULTS:At 60 days, GC treatment maintained TEER levels similar to non-injured/untreated controls. However, triamcinolone reduced TEER compared with saline-treated conditions. Acutely, epithelial hyperplasia typically persisted in all injured VFs. At 60 days, only dexamethasone and triamcinolone increased epithelial depth in injured VFs; all GCs increased epithelial depth compared with non-injured/untreated controls. CONCLUSION/CONCLUSIONS:Acutely, GCs did not alter TEER. Additionally, GCs did not alter epithelial depth compared with saline treatment, indicating alignment with natural healing responses. At 60 days, GCs exhibited varying degrees of TEER restoration and epithelial hyperplasia, possibly due to distinct pharmacodynamic profiles. LEVEL OF EVIDENCE/METHODS:NA Laryngoscope, 2024.

BACKGROUND/UNASSIGNED:) and intensity modulation), patient perception of their voice remains unexamined despite its clinical importance. This study aimed to characterize the relationship between patient-reported vocal tremor severity and acoustic voice outcomes at baseline and after botulinum toxin injections. METHOD/UNASSIGNED: RESULTS/UNASSIGNED: DISCUSSION/UNASSIGNED:Participants reporting more severe vocal tremor demonstrated more aberrant acoustic voice outcomes. After botulinum toxin injection, substantial heterogeneity was observed in acoustic voice measures which varied based on patient perception of change. These preliminary, exploratory findings provide a foundation for future investigations to define meaningful change in this population.

OBJECTIVES/OBJECTIVE:Vocal fold scar remains a therapeutic challenge. Vocal fold fibroblasts (VFFs) secrete extracellular matrix (ECM), and transforming growth factor-beta 1 (TGF-β1)-mediated fibroblast to myofibroblast differentiation is central to the development of fibrosis. The transient receptor potential (TRP) channel superfamily is a group of nonselective cation channels, and activation of TRP ankyrin 1 (TRPA1) channel has been shown to have antifibrotic effects through TGF-β1/Smad signaling in various organs. This study aimed to elucidate expression of TRPA1 and the impact of TRPA1 activation on TGF-β1/Smad signaling in VFFs. METHODS: M) for 4 or 24 h. Trpa1, Smad3, Smad7, Col1a1, Acta2, and Has1 mRNA expression were quantified via qPCR. RESULTS:TRPA1 was expressed in cultured VFFs and the lamina propria. TGF-β1 administration significantly increased Trpa1 compared to control. AITC alone did not alter Smad3, Smad7, Acta2, or ECM related genes. However, the combination of AITC and TGF-β1 significantly increased Smad3 and decreased Smad7 and Acta2 compared to TGF-β1 alone; A-967079 significantly reduced this response. CONCLUSIONS:VFFs expressed TRPA1, and the activation of TRPA1 regulated TGF-β1/Smad signaling in VFFs. These findings provide preliminary insights into potential anti-fibrotic mechanisms of TRPA1 activation through TGF-β1/Smad signaling in VFFs. LEVEL OF EVIDENCE/METHODS:NA Laryngoscope, 2024.