Faculty Bibliography

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.

OBJECTIVES/HYPOTHESIS/OBJECTIVE:Glucocorticoids (GC)s are commonly employed to treat vocal fold (VF) pathologies. However, VF atrophy has been associated with intracordal GC injections. Dexamethasone-induced skeletal muscle atrophy is well-documented in other tissues and believed to be mediated by increased muscle proteolysis via upregulation of Muscle Ring Finger (MuRF)-1 and Atrogin-1. Mechanisms of dexamethasone-mediated VF atrophy have not been described. This pilot study employed in vitro and in vivo models to investigate the effects of dexamethasone on VF epithelium, thyroarytenoid (TA) muscle, and TA-derived myoblasts. We hypothesized that dexamethasone will increase atrophy-associated gene expression in TA muscle and myoblasts and decrease TA muscle fiber size and epithelial thickness. STUDY DESIGN/METHODS:In vitro, pre-clinical. METHODS:TA myoblasts were isolated from a female Sprague-Dawley rat and treated with 1 μM dexamethasone for 24-h. In vivo, 15 New Zealand white rabbits were randomly assigned to three treatment groups: (1) bilateral intracordal injection of 40 μL dexamethasone (10 mg/ml; n = 5), (2) volume-matched saline (n = 5), and (3) untreated controls (n = 5). Larynges were harvested 7-days post-injection. Across in vivo and in vitro experimentation, MuRF-1 and Atrogin-1 mRNA expression were measured via RT-qPCR. TA muscle fiber cross-sectional area (CSA) and epithelial thickness were also quantified in vivo. RESULTS:Dexamethasone increased MuRF-1 gene expression in TA myoblasts. Dexamethasone injection, however, did not alter atrophy-associated gene expression, TA CSA, or epithelial thickness in vivo. CONCLUSION/CONCLUSIONS:Dexamethasone increased atrogene expression in TA myoblasts, providing foundational insight into GC induced atrophic gene transcription. Repeated dexamethasone injections may be required to elicit atrophy in vivo. LEVEL OF EVIDENCE/METHODS:N/A Laryngoscope, 2022.

OBJECTIVES/OBJECTIVE:Effective treatments for vocal fold fibrosis remain elusive. Tamoxifen (TAM) is a selective estrogen receptor modulator and was recently reported to have antifibrotic actions. We hypothesized that TAM inhibits vocal fold fibrosis via altered transforming growth factor beta 1 (TGF-β1) signaling. Both in vitro and in vivo approaches were employed to address this hypothesis. METHODS: M) ± TGF-β1 (10 ng/ml) to quantify cell proliferation. The effects of TAM on genes related to fibrosis were quantified via quantitative real-time polymerase chain reaction. In vivo, rat vocal folds were unilaterally injured, and TAM was administered by oral gavage from pre-injury day 5 to post-injury day 7. The rats were randomized into two groups: 0 mg/kg/day (sham) and 50 mg/kg/day (TAM). Histological changes were examined on day 56 to assess tissue architecture. RESULTS: M) + TGF-β1, however, significantly increased Smad7 and Has3 expression and decreased Col1a1 and Acta2 expression compared to TGF-β1 alone. In vivo, TAM significantly increased lamina propria area, hyaluronic acid concentration, and reduced collagen deposition compared to sham treatment. CONCLUSIONS:TAM has antifibrotic potential via the regulation of TGF-β1/Smad signaling in vocal fold injury. These findings provide foundational data to develop innovative therapeutic options for vocal fold fibrosis. LEVEL OF EVIDENCE/METHODS:NA Laryngoscope, 2022.

OBJECTIVES/HYPOTHESIS/UNASSIGNED:Systemic glucocorticoids (GC)s are employed to treat various voice disorders. However, GCs have varying pharmacodynamic properties with adverse effects ranging from changes in epithelial integrity, skeletal muscle catabolism, and altered body weight. We sought to characterize the acute temporal effects of systemic dexamethasone and methylprednisolone on vocal fold (VF) epithelial glucocorticoid receptor (GR) nuclear translocation, epithelial tight junction (ZO-1) expression, thyroarytenoid (TA) muscle fiber morphology, and body weight using an established pre-clinical model. We hypothesized dexamethasone and methylprednisolone will elicit changes in VF epithelial GR nuclear translocation, epithelial ZO-1 expression, TA muscle morphology, and body weight compared to placebo-treated controls. METHODS/UNASSIGNED: = 15) into the iliocostalis/longissimus muscle for 6 consecutive days. Vocal folds from 5 rabbits from each treatment group were harvested at 1-, 3-, or 7 days following the final injection and subjected to immunohistochemistry for ZO-1 and GR as well as TA muscle fiber cross-sectional area (CSA) measures. RESULTS/UNASSIGNED: = .004). CONCLUSIONS/UNASSIGNED:Systemic dexamethasone may more efficiently activate GR in the VF epithelium with a lower risk of body weight loss, suggesting a role for more refined approaches to GC selection for laryngeal pathology.

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/METHODS:In vitro. METHODS:to alter inflammatory and fibrotic gene expression was calculated. RESULTS:to downregulate other genes. CONCLUSION/CONCLUSIONS: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/METHODS:NA Laryngoscope, 133:1169-1175, 2023.

AIM/OBJECTIVE:Because the tongue is a midline structure, studies on the neural correlates of lateralized tongue function are challenging and remain limited. Patients with tongue cancer who undergo unilateral partial glossectomy may be a unique cohort to study tongue-associated cortical activation, particularly regarding brain hemispheric lateralization. This longitudinal functional magnetic resonance imaging (fMRI) study investigated cortical activation changes for three tongue tasks before and after left-sided partial glossectomy in patients with squamous cell carcinoma of the tongue. METHODS:Seven patients with squamous cell carcinoma involving the left tongue who underwent fMRI before and 6 months after unilateral partial glossectomy were studied. Post-surgical changes in laterality index (LI) values for tongue-associated precentral and postcentral gyri fMRI activation were calculated for the dry swallow, tongue press, and saliva sucking tasks. Group analysis fMRI activation maps were generated for each of the three tasks. RESULTS:< 0.05). There was also increased activation in the supplementary motor area following surgery. CONCLUSION/CONCLUSIONS:Post-surgical fMRI changes following left-sided partial glossectomy may suggest task-specific sensitivities to cortical activation changes following unilateral tongue deficits that may reflect the impacts of surgery and adaptive responses to tongue impairment.

OBJECTIVES/OBJECTIVE:Functional outcomes following microflap surgery for vocal fold pathology are favorable. Although the stratified squamous epithelium appears to heal rapidly, persistent physiologic tissue alterations are likely. We sought to elucidate key biochemical processes including recruitment of immune cells, regulation of cellular junction proteins, and long-term alterations to epithelial tissue permeability following microflap with an eye toward enhanced clinical outcomes. METHODS:Forty New Zealand rabbits were assigned to eight groups (n = 5/group): no-injury control or bilateral microflap with survival for 0 h, 12 h, 1 day, 3 days, 7 days, 30 days, and 60 days post-microflap. The epithelium was dissected from one vocal fold and transepithelial resistance was quantified. The contralateral fold was subjected to transmission electron microscopy. Images were evaluated by a blinded rater and paracellular space dilation was quantified using ImageJ. Immune cell infiltration was evaluated and recorded qualitatively. RESULTS:Increased innate immune response was observed 12 h as well as 7 and 30 days after microflap. At 60 days following injury, decreased epithelial resistance was observed. Paracellular spaces were dilated at all time-points following injury. CONCLUSIONS:The vocal fold epithelium was significantly altered at 60 days following microflap. The implications for this tissue phenotype are unclear. However, compromised epithelial barrier function is implicated in various diseases and may increase the risk of subsequent injury. LEVEL OF EVIDENCE/METHODS:Not Applicable Laryngoscope, 2022.

Purpose of Review: This review seeks to illuminate the challenges that arise in the use of steroids in the context of a performing voice, to review pharmacologic principles that can help to guide dosing regimens, to examine emerging science about the mechanistic action of glucocorticoids, and to provide a useful guide for clinicians who treat vocal performers. Recent Findings: Though perceptions and mythologies abound, most saliently (1) the incidence of vocal fold hemorrhage while taking oral steroids is extremely low; (2) appropriate dosing is likely to involve regimens that meet or exceed 30 mg oral Prednisone-equivalent daily to address edema acutely; (3) tapering after short courses may well be unnecessary. Summary: Steroids can be used safely and judiciously to treat vocal performers, guided by physical examination, sound clinical judgment, and a multidisciplinary approach to the individual needs of each unique voice and performer.

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