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Tamoxifen Alters TGF-β1/Smad Signaling in Vocal Fold Injury

Matsushita, Hiroki; Mukudai, Shigeyuki; Ozawa, Satomi; Kinoshita, Shota; Hashimoto, Keiko; Kaneko, Mami; Sugiyama, Yoichiro; Branski, Ryan C; Hirano, Shigeru
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.
PMID: 36250536
ISSN: 1531-4995
CID: 5352332

Acute In Vitro and In Vivo Effects of Dexamethasone in the Vocal Folds: a Pilot Study

Gartling, Gary; Nakamura, Ryosuke; Sayce, Lea; Zimmerman, Zachary; Slater, Alysha; Wilson, Azure; Bing, Renjie; Branski, Ryan C; Rousseau, Bernard
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.
PMID: 36317801
ISSN: 1531-4995
CID: 5358512

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

Nakamura, Ryosuke; Bing, Renjie; Gartling, Gary J; Garabedian, Michael J; Branski, Ryan C
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.
PMID: 37246727
ISSN: 1531-4995
CID: 5543132

A Novel Method for Thyroarytenoid Myofiber Culture

Gartling, Gary; Nakamura, Ryosuke; Bing, Renjie; Branski, Ryan C
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.
PMID: 37227163
ISSN: 1531-4995
CID: 5543832

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

Nakamura, Ryosuke; Bing, Renjie; Gartling, Gary J; Garabedian, Michael J; Branski, Ryan C
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.
PMID: 36779842
ISSN: 1531-4995
CID: 5466622

Functional MRI during tongue strength tasks before and after partial glossectomy: Insights into the cortical activation of tongue motor function

Peck, Kyung K; Cho, Nicholas S; Pasquini, Luca; Jenabi, Mehrnaz; Branski, Ryan C; Lazarus, Cathy L; Kraus, Dennis H; Holodny, Andrei I
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.
PMID: 37118651
ISSN: 2385-1996
CID: 5465692

Epithelial response to vocal fold microflap injury in a preclinical model

Sayce, Lea; Zimmerman, Zachary; Gartling, Gary; Rousseau, Bernard; Branski, Ryan C
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.
PMID: 35538915
ISSN: 1531-4995
CID: 5214392

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

Nakamura, Ryosuke; Bing, Renjie; Gartling, Gary J.; Garabedian, Michael J.; Branski, Ryan C.
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, 2023.
ISSN: 0023-852x
CID: 5424902

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

Nakamura, Ryosuke; Bing, Renjie; Gartling, Gary J; Branski, Ryan C
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.
PMID: 35931141
ISSN: 1090-2422
CID: 5288372


Miar, S; Dion, G; Branski, R; Ong, J; Bizios, R; Guda, T
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
ISSN: 1937-335x
CID: 5252472