Faculty Bibliography

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

Extracellular matrix (ECM) is a complex structure composed of bioactive molecules representative of the specific local tissue microenvironment. Decellularized ECM biomaterials harness these biomolecules for regenerative medicine applications. One potential therapeutic application is the use of vocal fold (VF) specific ECM to restore the VFs after injury. ECM scaffolds are derived through a process of decellularization, which aims to remove unwanted immunogenic biomolecules (e.g., DNA) while preserving the composition of the ECM. The effectiveness of the decellularization is typically assessed at the end by quantifying ECM attributes such as final dsDNA content. However, batch-to-batch variability in ECM manufacturing remains a significant challenge for the process standardization, cost-effectiveness, and scale-up. The limited number of tools available for in-process control heavily restricts the uncovering of the correlations between decellularization process parameters and ECM attributes. In this study, we developed a technique applicable to both the classical batch method and semi-continuous decellularization system to trace the decellularization of two laryngeal tissues in real-time. We hypothesize that monitoring the bioreactor's effluent absorbance at 260 nm as a function of time will provide a representative DNA release profile from the tissue and thus allowing for process optimization. The DNA release profiles were obtained for laryngeal tissues and were successfully used to optimize the derivation of VF lamina propria-ECM (auVF-ECM) hydrogels. This hydrogel had comparable rheological properties to commonly used biomaterials to treat VF injuries. Also, the auVF-ECM hydrogel promoted the down-regulation of CCR7 by THP-1 macrophages upon lipopolysaccharide stimulation in vitro suggesting some anti-inflammatory properties. The results show that absorbance profiles are a good representation of DNA removal during the decellularization process thus providing an important tool to optimize future protocols.

OBJECTIVES/HYPOTHESIS/OBJECTIVE:Vocal fold fibrosis remains a significant clinical challenge. Estrogens, steroid hormones predominantly responsible for secondary sexual characteristics in women, have been shown to alter wound healing and limit fibrosis, but the effects on vocal fold fibrosis are unknown. We sought to elucidate the expression of estrogen receptors and the effects of estrogens on TGF-β1 signaling in rat vocal fold fibroblasts (VFFs). STUDY DESIGN/METHODS:In vitro. METHODS: M) were employed as antagonists of ERα or GPR30, respectively. qPCR was employed to determine estrogen receptor-mediated effects of E2 on genes related to fibrosis. RESULTS: M) and TGF-β1 significantly increased Smad7 (P = .03) and decreased Col1a1 (P = .04) compared to TGF-β1 alone; this response was negated by the combination of ICI and G36 (P = .009). CONCLUSIONS:E2 regulated TGF-β1/Smad signaling via estrogen receptors in VFFs. These findings provide insight into potential mechanisms of estrogens on vocal fold injury with the goal of enhanced therapeutics for vocal fold fibrosis. LEVEL OF EVIDENCE/METHODS:NA. Laryngoscope, 2020.

OBJECTIVE/UNASSIGNED:Oversimplified clinical dogma suggests that laryngeal diseases fall into two broad, mutually exclusive diagnostic categories-mucosal injury or neuromuscular/functional disorders. Extensive investigation in the lower airway as well as other organ systems suggest complex interactions between tissue types underlying both tissue health and pathological states. To date, no such relationship has been described in the vocal folds, likely the most bioactive organ in the body. We hypothesize interactions between the vocal fold muscle and mucosa likely contribute to aberrant phonatory physiology and warrant further investigation to ultimately develop novel therapeutic strategies. METHODS/UNASSIGNED:Primary culture of myoblasts from rat thyroarytenoid muscle and fibroblasts from the vocal fold mucosa were established. Co-culture and conditioned media experiments were performed to established bidirectional interactions between cell types. Transforming Growth Factor (TGF)-β was employed to stimulate a fibrotic phenotype in culture. In addition to quantitative PCR, standard migration and proliferation assays were performed as well as immunocytochemistry. RESULTS/UNASSIGNED:Bidirectional cell-cell interactions were observed. Without TGF-β stimulation, myoblast conditioned media inhibited fibroblast migration, but enhanced proliferation. Conversely, fibroblast conditioned media increased both myoblast proliferation and migration. Myoblast conditioned media decreased TGF-β-mediated gene expression and of particular interest, ACTA2 mRNA expression. In both co-culture and in response to fibroblast conditioned media, myosin heavy chain (Myh2) mRNA expression decreased in myoblasts. CONCLUSIONS/UNASSIGNED:These data are the first to describe interactions between cell types within the vocal fold. The implications for these interactions in vivo warrant further investigation to develop and refine optimal treatment strategies.

Vocal fold (VF) fibrosis is a major cause of intractable voice-related disability and reduced quality of life. Excision of fibrotic regions is suboptimal and associated with scar recurrence and/or further iatrogenic damage. Non-surgical interventions are limited, putatively related to limited insight regarding biochemical events underlying fibrosis, and downstream, the lack of therapeutic targets. YAP/TAZ integrates diverse cell signaling events and interacts with signaling pathways related to fibrosis, including the TGF-β/SMAD pathway. We investigated the expression of YAP/TAZ following vocal fold injury in vivo as well as the effects of TGF-β1 on YAP/TAZ activity in human vocal fold fibroblasts, fibroblast-myofibroblast transition, and TGF-β/SMAD signaling. Iatrogenic injury increased nuclear localization of YAP and TAZ in fibrotic rat vocal folds. In vitro, TGF-β1 activated YAP and TAZ in human VF fibroblasts, and inhibition of YAP/TAZ reversed TGF-β1-stimulated fibroplastic gene upregulation. Additionally, TGF-β1 induced localization of YAP and TAZ in close proximity to SMAD2/3, and nuclear accumulation of SMAD2/3 was inhibited by a YAP/TAZ inhibitor. Collectively, YAP and TAZ were synergistically activated with the TGF-β/SMAD pathway, and likely essential for the fibroplastic phenotypic shift in VF fibroblasts. Based on these data, YAP/TAZ may evolve as an attractive therapeutic target for VF fibrosis.