Faculty Bibliography

Due to their anatomical location, vocal folds are highly susceptible to injury from external and internal stressors that can lead to irreversible damage and changes in function. As the structure and composition of the vocal folds are heavily linked to their unique function, we hypothesize that a vocal fold-derived extracellular matrix (ECM) would be the ideal scaffold in a regenerative approach to vocal fold repair. Our group has previously described a porcine-derived vocal fold lamina propria ECM (VFLP-ECM)¹. In order to optimize the delivery modality of the VFLP-ECM, we have developed an injectable hydrogel form of the ECM scaffold and have studied the effects of tissue specificity using human vocal fold fibroblasts (hVFF) and human peripheral blood-derived macrophages (hPB-Macrophages). Both cell types play unique roles during the inflammatory and wound healing response at the site of vocal fold injury. In the present study, we compare VFLP-ECM with other ECM hydrogels (such as collagen, heart, bladder) in their ability to activate and modify gene expression of hVFFs and hPB-macrophages. This information will help us tailor the VFLP-ECM hydrogel to mod-ulate the environment present during vocal fold injury

Importance/UNASSIGNED:Compromised cough effectiveness is correlated with dysphagia and aspiration. Glottic insufficiency likely yields decreased cough strength and effectiveness. Although vocal fold augmentation favorably affects voice and likely improves cough strength, few data exist to support this hypothesis. Objective/UNASSIGNED:To assess whether vocal fold augmentation improves peak airflow measurements during maximal-effort cough following augmentation. Design, Setting, and Participants/UNASSIGNED:This case series study was conducted in a tertiary, academic laryngology clinic. Participants included 14 consecutive individuals with glottic insufficiency due to vocal fold paralysis, which was diagnosed via videostrobolaryngoscopy as a component of routine clinical examination. All participants who chose to proceed with augmentation were considered for the study whether office-based or operative augmentation was planned. Postaugmentation data were collected only at the first follow-up visit, which was targeted for 14 days after augmentation but varied on the basis of participant availability. Data were collected from June 5, 2014, to October 1, 2015. Data analysis took place between October 2, 2015, and March 3, 2017. Main Outcomes and Measures/UNASSIGNED:Peak airflow during maximal volitional cough was quantified before and after vocal fold augmentation. Participants performed maximal coughs, and peak expiratory flow during the maximal cough was captured according to American Thoracic Society guidelines. Results/UNASSIGNED:Among the 14 participants (7 men and 7 women), the mean (SD) age was 62 (18) years. Three types of injectable material were used for vocal fold augmentation: carboxymethylcellulose in 5 patients, hyaluronic acid in 5, and calcium hydroxylapatite in 4. Following augmentation, cough strength increased in 11 participants and decreased cough strength was observed in 3. Peak airflow measurements during maximal cough varied from a decrease of 40 L/min to an increase of 150 L/min following augmentation. When preaugmentation and postaugmentation peak airflow measurements were compared, the median improvement was 50 L/min (95% CI, 10-75 L/min; P = .01). Immediate peak airflow measurements during cough collected within 30 minutes of augmentation varied when compared with measurements collected at follow-up (103-380 vs 160-390 L/min). Conclusions and Relevance/UNASSIGNED:Peak airflow during maximal cough may improve with vocal fold augmentation. Additional assessment and measurements are needed to further delineate which patients will benefit most regarding their cough from vocal fold augmentation.

OBJECTIVES/HYPOTHESIS: NR4A1 was recently identified as an endogenous inhibitor of transforming growth factor (TGF)-beta-induced fibrosis, and the role of this nuclear receptor has not been elucidated in tissue health or the response to injury in the vocal folds. Given the clinical implications of vocal fold fibrosis, we investigated NR4A1 expression during vocal fold wound healing in vivo and the regulatory roles of NR4A1 on vocal fold fibroblasts (VFFs) in vitro with the ultimate goal of developing targeted therapies for this challenging patient population. STUDY DESIGN: In vivo and in vitro. METHODS: In vivo, the temporal pattern of NR4A1 mRNA expression was quantified following rat vocal fold injury. In vitro, the role of NR4A1 on TGF-beta1-mediated transcription of genes underlying fibrosis as well as myofibroblast differentiation and collagen gel contraction was quantified in our human VFF line. Small interfering RNA was employed to alter NR4A1 expression to further elucidate this complex system. RESULTS: Nr4a1 mRNA increased 1 day after injury and peaked at 7 days. Knockdown of NR4A1 resulted in upregulation of COL1A1 and TGF-beta1, with TGF-beta1 stimulation (both P < .001) in VFFs. NR4A1 knockdown also resulted in increased alpha-smooth muscle actin-positive cells (P = .013) and contraction (P = .002) in response to TGF-beta1. CONCLUSIONS: NR4A1 has not been described in vocal fold health or disease. Upregulation of TGF-beta following vocal fold injury was concurrent with increased NR4A1 expression. These data provide a foundation for the development of therapeutic strategies given persistent TGF-beta signaling in vocal fold fibrosis. LEVEL OF EVIDENCE: N/A Laryngoscope, 2017.

OBJECTIVE: Recent reports highlight the efficacy of small interfering RNA (siRNA) targeting SMAD3 to regulate transforming growth factor beta (TGF-beta)-mediated fibroplasia in vocal fold fibroblasts. The current study sought to investigate SMAD3 expression during wound healing in vivo and quantify the downstream transcriptional events associated with SMAD3 knockdown in vitro. STUDY DESIGN: In vivo and in vitro. METHODS: Unilateral vocal fold injury was created in a rabbit model. SMAD3 and SMAD7 mRNA expression was quantified at 1 hour and 1, 3, 7, 14, 30, 60, and 90 days following injury. In vitro, multi-gene analysis technology was employed in our immortalized human vocal-fold fibroblast cell line following TGF-beta1 stimulation +/- SMAD3 knockdown across time points. RESULTS: SMAD3 mRNA expression increased following injury; upregulation was significant at 3 and 7 days compared to control (both P < 0.001). SMAD7 mRNA was also upregulated at 3, 7, and 14 days (P = 0.02, P < 0.001, and P < 0.001, respectively). In vitro, SMAD3 knockdown reduced the expression of multiple profibrotic, TGF-beta signaling, and extracellular matrix metabolism genes at 6 and 24 hours following TGF-beta1 stimulation. CONCLUSION: Cumulatively, these data support SMAD3 as a potential master regulator of TGF-beta-mediated fibrosis. SMAD3 transcription peaked 7 days following injury. Multi-gene analysis indicated that the therapeutic effectiveness of SMAD3 knockdown may be related to regulation of downstream mediators of fibroplasia and altered TGF-beta signaling. LEVEL OF EVIDENCE: NA. Laryngoscope, 2017.

OBJECTIVES: Interactions between mesenchymal stem cells (MSCs) and native vocal fold fibroblasts (VFFs) have not been described in spite of promising preliminary data regarding the effects of MSCs on vocal fold repair in vivo. The current study employed a conditioned media (CM) model to investigate the paracrine effects of bone marrow-derived mesenchymal stem cells (BMSCs) on VFFs. METHODS: Human VFFs were treated with transforming growth factor-beta1 (TGF-beta1; 10 ng/mL), CM from human BMSCs following 48 hours of TGF-beta1 stimulation, or CM+TGF-beta1. Proliferation, immunocytochemistry for alpha smooth muscle actin (alphaSMA), migration, and collagen gel contraction were quantified as well as transcription of components of the TGF-beta signaling pathway. RESULTS: Transforming growth factor-beta1 accelerated proliferation and induced alphaSMA in VFFs; these effects were suppressed with CM ( P = .009, P < .001, respectively). The CM+TGF-beta1 condition increased cell migration ( P = .02) and decreased gel contraction; CM+TGF-beta1 also inhibited TGF-beta signaling via significant upregulation of NR4A1 as well as downregulation of S MAD3 and TGF-beta1 relative to TGF-beta1 stimulation in the absence of CM ( P = .002, P < .001, and P = .005, respectively). CONCLUSIONS: Conditioned media affected many profibrotic cell activities in TGF-beta1-stimulated VFFs, likely related to altered TGF-beta signaling. These data provide preliminary insight regarding the antifibrotic effects of MSCs and further support their progression to clinical utility.

Therapeutic monocolonal antibodies (MAbs) are a new, rapidly growing class of medications that frequently have poorly characterized side-effect profiles. We present a patient who developed inflammatory lesions of the vocal folds in temporal relation to the initiation of alirocumab. Lesions of the vocal folds represent a previously unreported adverse effect of alirocumab therapy, making it the second MAb documented with such a side effect. The potential laryngeal effects of alirocumab specifically, and of MAbs more broadly, warrant investigation. Laryngoscope, 2016.

OBJECTIVE: An obstacle to clinical use of RNA-based gene suppression is instability and inefficiency of current delivery modalities. Nanoparticle delivery likely holds great promise, but the kinetics and transfection conditions must be optimized prior to in vivo utility. We investigated a RNA nanoparticle complex incorporating a lipitoid transfection reagent in comparison to a commercially available reagent. STUDY DESIGN: In vitro. METHODS: We investigated which variables influence transfection efficiency of lipitoid oligomers and a commercially available reagent across species, in vitro. These variables included duration, dose, and number of administrations, as well as serum and media conditions. The target gene was Smad3, a signaling protein in the transforming growth factor-beta cascade implicated in fibroplasia in the vocal folds and other tissues. RESULTS: The two reagents suppressed Smad3 mRNA for up to 96 hours; lipitoid performed favorably and comparably. Both compounds yielded 60% to 80% mRNA knockdown in rat, rabbit, and human vocal fold fibroblasts (P < 0.05 relative to control). Dose and number of administrations played a significant role in gene suppression (P < 0.05). Suppression was more dose-sensitive with lipitoid. At a constant siRNA concentration, a 50% decrease in gene expression was observed in response to a five-fold increase in lipitoid concentration. Increased number of administrations enhanced gene suppression, approximately 45% decrease between one and four administrations. Neither serum nor media type altered efficiency. CONCLUSION: Lipitoid effectively knocked down Smad3 expression across multiple transfection conditions. These preliminary data are encouraging, and lipitoid warrants further investigation with the goal of clinical utility. LEVEL OF EVIDENCE: NA. Laryngoscope, 2016.

OBJECTIVES/HYPOTHESIS: Quantification of clinical outcomes after vocal fold (VF) interventions is challenging with current technology. High-speed digital imaging and optical coherence tomography (OCT) of excised larynges assess intact laryngeal function, but do not provide critical biomechanical information. We developed a protocol to quantify tissue properties in intact, excised VFs using dynamic nanomechanical analysis (nano-DMA) to obtain precise biomechanical properties in the micrometer scale. STUDY DESIGN: Experimental animal study. METHODS: Three pig larynges were bisected in the sagittal plane, maintaining an intact anterior commissure, and subjected to nano-DMA at nine locations with a 250-mum flat-tip punch and frequency sweep load profile (10-105 Hz, 1,000 muN peak force) across the free edge of the VF and inferiorly along the conus elasticus. RESULTS: Storage, loss, and complex moduli increased inferiorly from the free edge. Storage moduli increased from a mean of 32.3 kPa (range, 6.5-55.38 kPa) at the free edge to 46.3kPa (range, 7.4-71.6) 5 mm below the free edge, and 71.4 kPa (range, 33.7-112 kPa) 1 cm below the free edge. Comparable values were 11.6 kPa (range, 5.0-20.0 kPa), 16.7 kPa (range, 5.7-26.8 kPa), and 22.6 kPa (range, 9.7-38.0 kPa) for loss modulus, and 35.7 kPa (range, 14.4-56.4 kPa), 50.1 kPa (range, 18.7-72.8 kPa), and 75.4 kPa (range, 42.0-116.0 kPa) for complex modulus. Another larynx repeatedly frozen and thawed during technique development had similarly increased storage, loss, and complex modulus trends across locations. CONCLUSIONS: Nano-DMA of the intact hemilarynx provides a platform for quantification of biomechanical responses to a myriad of therapeutic interventions to complement data from high-speed imaging and OCT. LEVEL OF EVIDENCE: NA Laryngoscope, 2016.

Importance/UNASSIGNED:Human papillomavirus (HPV) vaccination is recommended for children and younger adults but not older adults or those with prior HPV exposure, leaving a large portion of the population at risk for HPV-mediated disease. Emerging data suggest a possible role for vaccination as an adjuvant treatment for individuals with HPV-related clinical disease. Objective/UNASSIGNED:To systematically review the literature regarding HPV vaccination for secondary disease prevention after treatment of active clinical disease across disease sites to serve as a platform for the management of HPV-related disease of the head and neck. Evidence Review/UNASSIGNED:A systematic search from August 3 to 21, 2015, of the PubMed, MEDLINE, EMBASE, CINAHL, Cochrane Library, Web of Science, Biosis Citation Index, Current Contents Connect, Scientific Library Online, and Global Health databases used PRISMA guidelines to identify 326 relevant articles related to adjuvant use of HPV vaccination. Primary search terms were (HPV vaccine OR human papillomavirus vaccine OR papillomarvirus vaccines OR alphapapillomavirus vaccine) AND (HPV OR human papillomavirus OR alphapapillomavirus OR papillomaviridae OR virus warts OR wart virus) AND (recurrence OR relapse OR reoccurrence OR recurrences OR relapses OR relapsing). Forty-five full texts in English were reviewed, with 19 articles included in the final review. In some studies, subpopulations of individuals with HPV DNA positivity and/or seropositivity were extracted for inclusion. Included studies were assessed for bias and separated based on the presence of active clinical disease or HPV DNA positivity or seropositivity. Findings/UNASSIGNED:Nineteen studies with 22 474 unique patients were included in the review. When HPV vaccination was used as an adjuvant treatment for active clinical disease, 9 of 12 studies reported decreased disease recurrence, decreased disease burden, or increased intersurgical interval. In contrast, none of the 7 studies of vaccination in individuals with HPV DNA positivity and/or seropositivity without clinical disease reported improved outcomes. Conclusions and Relevance/UNASSIGNED:Differences between adjuvant vaccination in HPV-mediated clinical disease and vaccination in HPV DNA-positive and/or HPV-seropositive populations posit underlying differences in disease and immune processes. These data suggest that additional evaluation of adjuvant HPV vaccination in individuals with active clinical disease is warranted.

The potential for the misinterpretation of positron-emission tomography (PET) scans in the context of a possible malignancy has been confirmed in a case report showing increased 18F-fluorodeoxyglucose (FDG) uptake after unilateral vocal fold augmentation medialization. We sought to expand these findings by investigating FDG uptake in a larger cohort of patients via a retrospective chart review. We examined the records of 15 adults-8 men and 7 women-who had undergone vocal fold augmentation for unilateral vocal fold paralysis and at least one subsequent PET scan. The differences in PET standard uptake value (SUV) between the injected and noninjected vocal folds were assessed via the Wilcoxon signed-rank test. A Spearman rank correlation coefficient was then used to estimate the relationship between differences in PET uptake and the length of time between the injection and the follow-up PET scan. The mean SUV of the injected vocal folds was 3.70, and the mean in the noninjected folds was 2.97. The difference did not achieve statistical significance (p = 0.34). In addition, the rank correlation coefficient with regard to the association between the difference in PET uptake and the duration between injection and PET was -0.24, suggesting an inverse relationship. However, the correlation coefficient did not differ significantly from zero (p = 0.34). We conclude that PET uptake after vocal fold augmentation medialization is variable and that it can increase substantially. This information should be considered in the context of the diagnostic accuracy of malignancy on PET.