Faculty Bibliography

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.

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.

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.

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.

OBJECTIVE:Vocal tremor (VT) poses treatment challenges due to uncertain pathophysiology. VT is typically classified into two phenotypes: isolated vocal tremor (iVT) and essential tremor-related voice tremor (ETvt). The impact of phenotypes on upper aerodigestive tract physiology during swallowing remains unclear. Qualitative and quantitative measures were employed to characterize tremor phenotypes and investigate the effects on swallowing physiology. METHODS:Eleven ETvt participants (1 Male, 10 Female; x̄ age = 74) and 8 iVT participants (1 Male, 7 Female; x̄ age = 71) swallowed 20 mL boluses in cued and uncued conditions under standardized fluoroscopic visualization. Sustained/a/productions were captured to assess the rate and extent of fundamental frequency (F0) modulation. Penetration and Aspiration Scale (PAS) scores were obtained and swallowing biomechanics were captured using Swallowtail™ software. Participants also completed the Swallowing Quality of Life (SWAL-QOL) questionnaire. RESULTS:Hypopharyngeal transit was faster in both VT phenotypes compared with Swallowtail™ normative reference data. Total pharyngeal transit times, however, were only faster in patients with iVT, relative to reference data. No significant differences were observed on the SWAL-QOL or PAS between tremor phenotypes. SWAL-QOL scores revealed that these patients rarely reported dysphagia symptoms. CONCLUSIONS:Subtle differences in swallowing patterns were observed across VT phenotypes, possibly related to adaptive mechanisms resulting in quicker pharyngeal bolus transit. Most patients did not report swallowing issues or dysphagia symptoms. This study is foundational for larger studies on this challenging population. LEVEL OF EVIDENCE/METHODS:4 Laryngoscope, 134:4599-4603, 2024.

Endotracheal Tubes (ETTs) maintain and secure a patent airway; however, prolonged intubation often results in unintended injury to the mucosal epithelium and inflammatory sequelae which complicate recovery. ETT design and materials used have yet to adapt to address intubation associated complications. In this study, a composite coating of electrospun polycaprolactone (PCL) fibers embedded in a four-arm polyethylene glycol acrylate matrix (4APEGA) is developed to transform the ETT from a mechanical device to a dual-purpose device capable of delivering multiple therapeutics while preserving coating integrity. Further, the composite coating system (PCL-4APEGA) is capable of sustained delivery of dexamethasone from the PCL phase and small interfering RNA (siRNA) containing polyplexes from the 4APEGA phase. The siRNA is released rapidly and targets smad3 for immediate reduction in pro-fibrotic transforming growth factor-beta 1 (TGFϐ1) signaling in the upper airway mucosa as well as suppressing long-term sequelae in inflammation from prolonged intubation. A bioreactor was used to study mucosal adhesion to the composite PCL-4APEGA coated ETTs and investigate continued mucus secretory function in ex vivo epithelial samples. The addition of the 4APEGA coating and siRNA delivery to the dexamethasone delivery was then evaluated in a swine model of intubation injury and observed to restore mechanical function of the vocal folds and maintain epithelial thickness when observed over 14 days of intubation. This study demonstrated that increase in surface lubrication paired with surface stiffness reduction significantly decreased fibrotic behavior while reducing epithelial adhesion and abrasion.

OBJECTIVE:The diversity of glucocorticoid (GC) properties may underlie variability of clinical efficacy for vocal fold (VF) disease. Optimized therapeutic approaches must account for tissue complexity as well as interactions between cell types. We previously reported that reduced GC concentrations inhibited inflammation without eliciting fibrosis in mono-cultured VF fibroblasts and macrophages. These data suggested that a refined approach to GC concentration may improve outcomes. In the current study, co-culture of VF fibroblasts and macrophages was employed to investigate the effects of different concentrations of methylprednisolone on fibrotic and inflammatory response genes in VF fibroblasts to optimize management paradigms. STUDY DESIGN/METHODS:In vitro. METHODS:THP-1 monocyte-derived macrophages were stimulated with interferon-γ (IFN-γ), lipopolysaccharide (LPS), or transforming growth factor-β (TGF-β) to induce inflammatory (M(IFN/LPS)) and fibrotic (M(TGF)) phenotypes. Macrophages were then co-cultured with a human VF fibroblast cell line using a 0.4 μm pore membrane with or without 0.1-3000 nM methylprednisolone. Inflammatory (CXCL10, TNF, and PTGS2) and fibrotic (ACTA2, CCN2, and COL1A1) gene expression was quantified in fibroblasts. RESULTS:Incubating VF fibroblasts with M(IFN/LPS) macrophages increased expression of TNF and PTGS2, and this effect was inhibited by methylprednisolone. Incubation of VF fibroblasts with M(TGF) macrophages increased expression of ACTA2, CCN2, and COL1A1, and this effect was enhanced by methylprednisolone. The concentration of methylprednisolone required to downregulate inflammatory genes (TNF and PTGS2) was lower than that to upregulate fibrotic genes (ACTA2, CCN2, and COL1A1). CONCLUSION/CONCLUSIONS:Reduced concentration of methylprednisolone effectively suppressed inflammatory genes without enhancing fibrotic genes, suggesting that a refined approach to GC concentration may improve clinical outcomes. LEVEL OF EVIDENCE/METHODS:N/A Laryngoscope, 2023.

OBJECTIVES/HYPOTHESIS/OBJECTIVE:Myofiber culture has been employed to investigate muscle physiology in vitro and is well-established in the rodent hind limb. Thyroarytenoid (TA) myofiber culture has not been described, providing an opportunity to employ this method to investigate distinct TA myofiber functions. The purpose of this study was to assess the feasibility of a TA myofiber culture model. STUDY DESIGN/METHODS:In vitro. METHODS:for 2 h. Myofiber specificity was determined via immunolabeling for desmin and myosin heavy chain (MHC). Myofibers viability was assessed over 7 days via esterase assay. Additional myofibers were immunolabeled for satellite cell marker Pax-7. Glucocorticoid (GC) receptor (GR) was immunolabeled following GC treatment. RESULTS:The harvest technique yielded ~120 myofibers per larynx. By day 7, ~60% of the fibers remained attached and were calcein AM-positive/ethidium homodimer-negative, indicating viability. Myofibers were positive for desmin and MHC, indicating muscle specificity. Cells surrounding myofibers were positive for Pax-7, indicating the presence of myogenic satellite cells. Myofibers also responded to GC treatment as determined by GR nuclear translocation. CONCLUSION/CONCLUSIONS:TA myofibers remained viable in culture for at least 7 days with a predictable response to exogenous stimuli. This technique provides novel investigative opportunities regarding TA structure and function. LEVEL OF EVIDENCE/METHODS:N/A Laryngoscope, 2023.