High-dose methylprednisolone mediates YAP/TAZ-TEAD in vocal fold fibroblasts with macrophages.

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 +/-0.3-3000nM methylprednisolone +/- 3µM 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.

Concentration Effects of Methylprednisolone in Human Vocal Fold Fibroblast-Macrophage Co-Culture.

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: In vitro. METHODS: THP-1 monocyte-derived macrophages were stimulated with interferon-γ (IFN-γ), lipopolysaccharide (LPS), or transforming growth factor-ß (TGF-ß) 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: 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: N/A Laryngoscope, 133:3116-3122, 2023.

Dose-Dependent Glucocorticoid Regulation of Transcription Factors in Vocal Fold Fibroblasts and Macrophages.

OBJECTIVE: Variable outcomes of glucocorticoid (GC) therapy for laryngeal disease are putatively due to diverse interactions of the GC receptor (GR) with cell signaling pathways, limited consideration regarding concentration-dependent effects, and inconsistent selection of GCs. In the current study, we evaluated the concentration-dependent effects of three frequently administered GCs on transcription factors with an emphasis on the phosphorylation of GR at Ser203 and Ser211 regulating the nuclear translocation of GR. This study provides foundational data regarding the diverse functions of GCs to optimize therapeutic approaches. STUDY DESIGN: In vitro. METHODS: Human vocal fold fibroblasts and THP1-derived macrophages were treated with different concentrations of dexamethasone, methylprednisolone, and triamcinolone in combination with IFN-γ, TNF-α, or IL4. Phosphorylated STAT1, NF-κB family molecules, and phosphorylated STAT6 were analyzed by Western blotting. Ser211-phosphorylated GR (S211-pGR) levels relative to GAPDH and Ser203-phosphorylated GR (S203-pGR) were also analyzed. RESULTS: GCs differentially altered phosphorylated STAT1 and NF-κB family molecules in different cell types under IFN-γ and TNF-α stimuli. GCs did not alter phosphorylated STAT6 in IL4-treated macrophages. The three GCs were nearly equivalent. A lower concentration of dexamethasone increased S211-pGR/GAPDH ratios relative to increased S211-pGR/S203-pGR ratios regardless of cell type and treatment. CONCLUSION: The three GCs employed in two cell lines had nearly equivalent effects on transcription factor regulation. Relatively high levels of Ser203-phosphorylation at low GC concentrations may be related to concentration-dependent differential effects of GCs in the two cell lines. LEVEL OF EVIDENCE: NA Laryngoscope, 133:2704-2711, 2023.

Glucocorticoid Dose Dependency on Gene Expression in Vocal Fold Fibroblasts and Macrophages.

OBJECTIVE: Glucocorticoids (GCs) modulate multiple cellular activities including inflammatory and fibrotic responses. Outcomes of GC treatment for laryngeal disease vary, affording opportunity to optimize treatment. In the current study, three clinically employed GCs were evaluated to identify optimal in vitro concentrations at which GCs mediate favorable anti-inflammatory and fibrotic effects in multiple cell types. We hypothesize a therapeutic window will emerge as a foundation for optimized therapeutic strategies for patients with laryngeal disease. STUDY DESIGN: In vitro. METHODS: Human vocal fold fibroblasts and human macrophages derived from THP-1 monocytes were treated with 0.03-1000 nM dexamethasone, 0.3-10,000 nM methylprednisolone, and 0.3-10,000 nM triamcinolone in combination with interferon-γ, tumor necrosis factor-α, or interleukin-4. Real-time polymerase chain reaction was performed to analyze inflammatory (CXCL10, CXCl11, PTGS2, TNF, IL1B) and fibrotic (CCN2, LOX, TGM2) genes, and TSC22D3, a target gene of GC signaling. EC50 and IC50 to alter inflammatory and fibrotic gene expression was calculated. RESULTS: Interferon-γ and tumor necrosis factor-α increased inflammatory gene expression in both cell types; this response was reduced by GCs. Interleukin-4 increased LOX and TGM2 expression in macrophages; this response was also reduced by GCs. GCs induced TSC22D3 and CCN2 expression independent of cytokine treatment. EC50 for each GC to upregulate CCN2 was higher than the IC50 to downregulate other genes. CONCLUSION: Lower concentrations of GCs repressed inflammatory gene expression and only moderately induced genes involved in fibrosis. These data warrant consideration as a foundation for optimized clinical care paradigms to reduce inflammation and mitigate fibrosis. LEVEL OF EVIDENCE: NA Laryngoscope, 133:1169-1175, 2023.

Macrophages alter inflammatory and fibrotic gene expression in human vocal fold fibroblasts.

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-ß1 (TGF-ß), 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.

The Patient Experience: The Relationship Between Vocal Handicap, Congruency, Perceived Present Control, and Mood Across Four Voice Disorders.

The potential for negative sequalae in psychosocial well-being presents clinical importance to the assessment of voice disorders. Despite the impairment voice disorders cause in the psychosocial domain, the clinical assessment of these disorders relies heavily on visual perceptual judgments of the larynx, audio-perceptual, as well as acoustic and aerodynamic measures. While these measures aid in accurate diagnosis and are necessary for standard of care, they present little insight into the patient experience of having a voice disorder. DESIGN: Retrospective between-subject, non-experimental design. METHODS: Data from 335 patients from the University of Pittsburgh Voice Center were collected from scores of the Voice Handicap Index-10 (VHI-10) and two recent questionnaires, the Voice Present Perceived Control scale (VPPC), and the Vocal Congruency Scale (VCS). Examining how these voice-specific scales related to three mental health screeners for stress (Perceived Stress Scale-4), anxiety (Generalized Anxiety Disorder-7) and depression (Patient Health Questionnaire-9) were also examined. Patient diagnoses included primary muscle tension dysphonia (pMTD), unilateral vocal fold paralysis (UVFP), vocal fold atrophy, and mid membranous vocal fold lesions. RESULTS: There were significant differences in scores from the voice-specific scales between diagnostic groups with UVFP being the highest (worst) in VHI-10 and UVFP being the lowest (worst) in VCS compared to healthy controls. There was no significant difference in VPPC scores between diagnostic groups. Results showed statistically significant inverse relationships between the VHI-10 and the VPPC and between the VHI-10 and VCS for all diagnostic groups. A significant direct relationship was found between the VPPC and the VCS for patients diagnosed with MTD, UVFP and Lesions. In sum, patients with UVFP presented with the most frequent and sometimes strongest relationships between voice and mental health measures. DISCUSSION: This study marks an initial investigation into the nuanced patient experience of having a voice disorder. Three theoretically unrelated voice constructs: handicap, perceived control, and sense of self, were measured via self-report. Results from this study describe the patient experience correlating to these constructs with weak correlations to stress, anxiety, and depression. Findings also clearly suggest that patient experience varies among diagnostic groups, as well as varying constructs. Measures of multiple constructs of patient perception provide valuable insight into a patient's experience of their voice disorder, guidance on the direction of voice treatment, and justification for such treatments.

Different Vibratory Conditions Elicit Different Structural and Biological Vocal Fold Changes in an In-Vivo Rabbit Model of Phonation.

OBJECTIVE: Vibration of the vocal folds can disrupt the tissue and induce structural, functional, and molecular changes; the presence or absence of contact between the vocal folds during vibration can affect the type and extent of these changes. The purpose of this study was to characterize vocal fold changes following 2 hours of contact phonation or phonation without vibratory contact. METHODS: Six New Zealand white breeder rabbits underwent 120 minutes of phonation with or without vibratory contact, and four served as nonphonated controls. The larynx was exposed and current was applied to the cricothyroids bilaterally to achieve vocal fold adduction while humidified airflow was delivered to induce vocal fold vibration. Laryngeal position, airflow, and stimulation levels were adjusted to obtain phonation with or without contact, and phonation was elicited for 120 minutes. Following excision, larynges were stained using Hematoxylin & Eosin, Elastica van Gieson, and Grocott's Methenamine Silver, or labeled with immunofluorescent markers for E-cadherin, CD31, CD11b, and Vimentin. All images were captured using a Nikon 90i microscope and analyzed using ImageJ. RESULTS: Differences between vibratory conditions and control samples were observed. There was more extensive epithelial thinning, reduced epithelial integrity and increased vascularity in the contact phonation group, while both phonatory groups demonstrated a decreased presence of mucous on the luminal surface and a decrease in elastin band thickness and lamina propria depth. Neither condition showed differences in inflammatory cell presence compared to control tissue. CONCLUSIONS: By showing that these two vibratory conditions result in structural changes of different types and magnitude, we have provided the first empirical evidence that vocal fold tissue is sensitive to differences in forces, and that changes in vibratory pattern can elicit different downstream biological changes within the tissue. The differences described herein are an important step toward understanding the vocal folds' potential for differential response to phonotraumatic damage following different vibratory behaviors.

A Comparison of the Localization of Integral Membrane Proteins in Human and Rabbit Vocal Folds.

OBJECTIVES: This study's objective was to identify and compare the localization of Aquaporin (AQP) 1, 4, 7, Na+/K + -ATPase, E-cadherin, zona occludin (ZO)-1, and occludin in human and rabbit vocal folds (VF)s to inform the design of future studies to explore the function of these proteins in the regulation of VF homeostasis. METHODS: Four human larynges and five New Zealand white rabbit larynges were used. Samples were immunolabeled for primary antibodies against AQP1, AQP4, AQP7, the alpha subunit of Na+/K + -ATPase, E-cadherin, and ZO-1 and occludin and then captured digitally using a Nikon Eclipse 90i microscope and Hamamatsu C10600 Camera. Two raters familiar with human and rabbit VF histology identified positive labeling in tissue structures, including the apical epithelium, basal epithelium/basement membrane, and lamina propria (LP). RESULTS: Samples from both species showed positive labeling for AQP1 in the basal epithelium/basement membrane, superficial LP, and deep/intermediate LP. Aquaporin 4, Aquaporin 7, Na+/K + -ATPase, and E-cadherin were primarily localized to the epithelium of both species. Zona occludin-1 was primarily localized apical epithelium and the superficial LP of both species. Occludin was primarily present in the apical epithelium in rabbit samples but not human. CONCLUSION: These data provide evidence of the presence of key ion transport channels and cell adhesion proteins in human and rabbit VFs. Aquaporin 1, 4, 7, Na+/K + -ATPase, E-cadherin, and ZO-1 were similarly localized in both species. These findings will be useful to investigators interested in the exploration of VF homeostasis and barrier integrity in future studies. LEVEL OF EVIDENCE: N/A Laryngoscope, 131:E1265-E1271, 2021.

Continuous Rate Infusion of Ketamine Hydrochloride and Dexmedetomidine for Maintenance of Anesthesia during Laryngotracheal Surgery in New Zealand White Rabbits (Oryctolagus cuniculus).

New Zealand white rabbits (Oryctolagus cuniculus) are an established in vivo model for the study of structural and functional consequences of vocal-fold vibration. Research design requires invasive laryngotracheal procedures, and the presence of laryngospasms or pain responses (or both) hinder phonation-related data collection. Published anesthesia regimens report respiratory depression and muscle tone changes and have been unsuccessful in mitigating autonomic laryngeal responses in our protocol. Infusion of ketamine hydrochloride and dexmedetomidine hydrochloride in pediatric medicine provides effective analgesia and sedation for laryngotracheal procedures including intubation and bronchoscopy; however, data evaluating the use of ketamine-dexmedetomidine infusion in rabbits are unavailable. This study reports a new infusion regimen, which was used in 58 male New Zealand white rabbits that underwent a nonsurvival laryngotracheal procedure to induce phonotraumatic vocal-fold injury. Animals were sedated by using ketamine hydrochloride (20 mg/kg IM) and dexmedetomidine (0.125 mg/kg IM). Maintenance anesthesia was provided by using continuous rate intravenous infusion of ketamine hydrochloride (343 µg/kg/min) and dexmedetomidine (1.60 µg/kg/min). A stable plane of anesthesia with no autonomic laryngeal response (laryngospasm) was achieved in 32 of the 58 rabbits (55%). Laryngospasms occurred in 25 of 58 animals (43%) and were controlled in 20 cases (80%) by providing 0.33 mL 2% topical lidocaine, incremental increase in infusion rate, or both. Continuous rate infusion of ketamine hydrochloride-dexmedetomidine with prophylactic topical lidocaine provides a predictable and adjustable surgical plane of anesthesia, with minimal confounding respiratory and autonomic laryngeal responses, during extended-duration laryngotracheal surgery in rabbits. This regimen should be considered as an alternative to injection maintenance for prolonged, invasive procedures.