On-tissue amidation of sialic acid with aniline for sensitive imaging of sialylated N-glycans from FFPE tissue sections via MALDI mass spectrometry.

Spatial visualization of glycans within clinical tissue samples is critical for discovery of disease-relevant glycan dysregulations. Herein, we develop an on-tissue derivatization strategy for sensitive spatial visualization of N-glycans from formalin-fixed paraffin-embedded (FFPE) tissue sections, based on amidation of sialic acid residues with aniline. The sialylated N-glycans were stabilized and given enhanced signal intensity owing to selective capping of a phenyl group to the sialic acid residue after aniline labeling. Proof-of-concept experiments, including determinations of sialylglycopeptide and N-glycans enzymatically released from glycoproteins, were performed. Further, mass spectrometry (MS) imaging of N-glycans on human laryngeal cancer FFPE tissue sections was conducted via matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), based on our strategy for on-tissue amidation of sialylated N-glycans. We obtained higher sialylated N-glycan coverages for both the glycoproteins and cancer tissue samples, demonstrating that the detection sensitivity for sialylated N-glycans is notably improved by amidation derivatization. We also characterized N-glycan heterogeneity across the human laryngeal cancer tissue section, showing N-glycan dysregulation in the tumor region.

High-resolution magnetic resonance and mass spectrometry imaging of the human larynx.

High-resolution, noninvasive and nondestructive imaging of the subepithelial structures of the larynx would enhance microanatomic tissue assessment and clinical decision making; similarly, in situ molecular profiling of laryngeal tissue would enhance biomarker discovery and pathology readout. Towards these goals, we assessed the capabilities of high-resolution magnetic resonance imaging (MRI) and matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) imaging of rarely reported paediatric and adult cadaveric larynges that contained pathologies. The donors were a 13-month-old male, a 10-year-old female with an infraglottic mucus retention cyst and a 74-year-old female with advanced polypoid degeneration and a mucus retention cyst. MR and molecular imaging data were corroborated using whole-organ histology. Our MR protocols imaged the larynges at 45-117 µm2 in-plane resolution and capably resolved microanatomic structures that have not been previously reported radiographically-such as the vocal fold superficial lamina propria, vocal ligament and macula flavae; age-related tissue features-such as intramuscular fat deposition and cartilage ossification; and the lesions. Diffusion tensor imaging characterised differences in water diffusivity, primary tissue fibre orientation, and fractional anisotropy between the intrinsic laryngeal muscles, mucosae and lesions. MALDI-MS imaging revealed peptide signatures and putative protein assignments for the polypoid degeneration lesion and the N-glycan constituents of one mucus retention cyst. These imaging approaches have immediate application in experimental research and, with ongoing technology development, potential for future clinical application.

On-Tissue Derivatization with Girard's Reagent P Enhances N-Glycan Signals for Formalin-Fixed Paraffin-Embedded Tissue Sections in MALDI Mass Spectrometry Imaging.

Glycosylation is a major protein post-translational modification whose dysregulation has been associated with many diseases. Herein, an on-tissue chemical derivatization strategy based on positively charged hydrazine reagent (Girard's reagent P) coupled with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was developed for analysis of N-glycans from FFPE treated tissue sections. The performance of the proposed approach was evaluated by analysis of monosaccharides, oligosaccharides, N-glycans released from glycoproteins, as well as MS imaging of N-glycans from human cancer tissue sections. The results demonstrated that the signal-to-noise ratios for target saccharides were notably improved after chemical derivatization, in which signals were enhanced by 230-fold for glucose and over 28-fold for maltooctaose. Improved glycome coverage was obtained for N-glycans derived from glycoproteins and tissue samples after chemical derivatization. Furthermore, on-tissue derivatization was applied for MALDI-MSI of N-glycans from human laryngeal cancer and ovarian cancer tissues. Differentially expressed N-glycans among the tumor region, adjacent normal tissue region, and tumor proximal collagen stroma region were imaged, revealing that high-mannose type N-glycans were predominantly expressed in the tumor region. Overall, our results indicate that the on-tissue labeling strategy coupled with MALDI-MSI shows great potential to spatially characterize N-glycan expression within heterogeneous tissue samples with enhanced sensitivity. This study provides a promising approach to better understand the pathogenesis of cancer related aberrant glycosylation, which is beneficial to the design of improved clinical diagnosis and therapeutic strategies.

Modeling fibrosis using fibroblasts isolated from scarred rat vocal folds.

Following injury, pathologically activated vocal fold fibroblasts (VFFs) can engage in disordered extracellular matrix (ECM) remodeling, leading to VF fibrosis and impaired voice function. Given the importance of scar VFFs to phenotypically appropriate in vitro modeling of VF fibrosis, we pursued detailed characterization of scar VFFs obtained from surgically injured rat VF mucosae, compared with those obtained from experimentally naïve, age-matched tissue. Scar VFFs initially exhibited a myofibroblast phenotype characterized by increased proliferation, increased Col1a1 transcription and collagen, type I synthesis, increased Acta2 transcription and α-smooth muscle actin synthesis, and enhanced contractile function. These features were most distinct at passage 1 (P1); we observed a coalescence of the scar and naïve VFF phenotypes at later passages. An empirical Bayes statistical analysis of the P1 cell transcriptome identified 421 genes that were differentially expressed by scar, compared with naïve, VFFs. These genes were primarily associated with the wound response, ECM regulation, and cell proliferation. Follow-up comparison of P1 scar VFFs and their in vivo tissue source showed substantial transcriptomic differences. Finally, P1 scar VFFs responded to treatment with hepatocyte growth factor and transforming growth factor-ß3, two biologics with reported therapeutic value. Despite the practical limitations inherent to working with early passage cells, this experimental model is easily implemented in any suitably equipped laboratory and has the potential to improve the applicability of preclinical VF fibrosis research.

Interspecies comparison of stellate cell-containing macula flavae and vitamin A storage in vocal fold mucosa.

The macula flavae (MF), populated by vitamin A-storing stellate cells (SCs), are believed to play a fundamental role in development, maintenance and repair of the vocal fold (VF) mucosa; however, to date, they have mostly been examined in observational human cadaver studies. Here, we conducted an interspecies comparison of MF and SC phenotype, as well as vitamin A quantification and localization, in human, pig, dog, rabbit and rat VF mucosae. MF containing vitamin A-positive SCs were only identified in human and rat specimens. Pig, dog and rabbit VF mucosae contained no discernable MF, but rather exhibited preferential vitamin A localization to mucous (pig), serous (dog) or mixed (rabbit) glands. This glandular vitamin A storage corresponded to exceedingly high concentrations of retinol in pig and dog mucosae, and retinyl ester in dog mucosa. These findings have significant implications for the presumed role of the MF and SCs in VF biology, the nature of vitamin A storage within the VF mucosa, and the selection of an appropriate animal model for future experimental studies.

Single-cell lipidomics enabled by dual-polarity ionization and ion mobility-mass spectrometry imaging.

Single-cell (SC) analysis provides unique insight into individual cell dynamics and cell-to-cell heterogeneity. Here, we utilize trapped ion mobility separation coupled with dual-polarity ionization mass spectrometry imaging (MSI) to enable high-throughput in situ profiling of the SC lipidome. Multimodal SC imaging, in which dual-polarity-mode MSI is used to perform serial data acquisition runs on individual cells, significantly enhanced SC lipidome coverage. High-spatial resolution SC-MSI identifies both inter- and intracellular lipid heterogeneity; this heterogeneity is further explicated by Uniform Manifold Approximation and Projection and machine learning-driven classifications. We characterize SC lipidome alteration in response to stearoyl-CoA desaturase 1 inhibition and, additionally, identify cell-layer specific lipid distribution patterns in mouse cerebellar cortex. This integrated multimodal SC-MSI technology enables high-resolution spatial mapping of intercellular and cell-to-cell lipidome heterogeneity, SC lipidome remodeling induced by pharmacological intervention, and region-specific lipid diversity within tissue.

E-cadherin and transglutaminase-1 epithelial barrier restoration precedes type IV collagen basement membrane reconstruction following vocal fold mucosal injury.

The vocal fold epithelium is critical to upper airway immunologic defense and water/ion transport; therefore, any form of physical trauma or insult increases the vulnerability of this structure to functional impairment and pathogen invasion/infection. In this study, we examined the reestablishment of epithelial and basement membrane barrier structures in a well-established rat model of vocal fold mucosal injury. We observed active cell recruitment culminating in peak hyperplasia at 3 days postinjury, the establishment of robust E-cadherin+ and transglutaminase-1+ biochemical barrier signals along the epithelial surface by 3 days postinjury, and the persistent absence of a type IV collagen+ basement membrane at 7 days postinjury. The distinct spatial and temporal immunoactivity of these molecules is consistent with a programmed repair process driving the restoration of vocal fold mucosal integrity and permeability. These data may inform future efforts to optimize functional mucosal recovery postinjury and avoid undesirable events such as barrier compromise or epithelial metaplasia.

Chronic rhinosinusitis with nasal polyps: a proteomic analysis.

OBJECTIVES: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a severe subtype of chronic rhinosinusitis that can affect patients despite medical and surgical interventions. The purpose of this study was to utilize the techniques of proteomics to investigate differences in protein abundance within the sinonasal mucosa of patients with CRSwNP compared to healthy controls. METHODS: In a case-control study at a tertiary-care academic medical center, sinonasal mucosa was harvested from 3 patients with CRSwNP and 3 control patients undergoing transsphenoidal excision of pituitary tumors. Two-dimensional gel electrophoresis was used to identify proteins with elevated or reduced abundance in CRSwNP patients compared to controls. The proteins showing the greatest abundance differences were characterized by mass spectrometry. RESULTS: More than 300 differentially abundant proteins (p < or = 0.05) were identified. Many of these protein species were involved in the host inflammatory response. Proteins up-regulated in CRSwNP patients included eosinophil lysophospholipase by a ratio (R) of 18.13, RHO-GDP dissociation inhibitor 2 (R = 2.80), and apolipoprotein A-1 (R = 1.73). Down-regulated proteins in CRSwNP patients included catalase (R = -5.87), annexin A1 (R = -6.27), and keratin II-8 (R = -6.73). A detailed analysis of additional protein species is outlined. CONCLUSIONS: The proteomic approach allows detection of significant differences in protein abundance in CRSwNP and provides unique insight into the pathophysiology of this common disease.

An atlas of protein turnover rates in mouse tissues.

Protein turnover is critical to cellular physiology as well as to the growth and maintenance of tissues. The unique synthesis and degradation rates of each protein help to define tissue phenotype, and knowledge of tissue- and protein-specific half-lives is directly relevant to protein-related drug development as well as the administration of medical therapies. Using stable isotope labeling and mass spectrometry, we determine the in vivo turnover rates of thousands of proteins-including those of the extracellular matrix-in a set of biologically important mouse tissues. We additionally develop a data visualization platform, named ApplE Turnover, that enables facile searching for any protein of interest in a tissue of interest and then displays its half-life, confidence interval, and supporting measurements. This extensive dataset and the corresponding visualization software provide a reference to guide future studies of mammalian protein turnover in response to physiologic perturbation, disease, or therapeutic intervention.

Proteomic and Genomic Methylation Signatures of Idiopathic Subglottic Stenosis.

OBJECTIVE: Idiopathic subglottic stenosis (iSGS) is a chronic inflammatory condition that causes dyspnea and affects middle-aged women of White race and non-Latino or Hispanic ethnicity. To better characterize its phenotype and pathogenesis, we assessed the proteomic and genomic methylation signatures of subglottic tissue collected from iSGS patients compared to controls. STUDY DESIGN: Molecular analysis of clinical biospecimens. METHODS: We collected subglottic tissue biopsies from 12 patients during direct laryngoscopy, immediately prior to surgical treatment of iSGS; as well as from 4 age-, sex-, and race/ethnicity-matched control patients undergoing other direct laryngoscopic procedures. We isolated protein and genomic DNA, acquired proteomic data using label-free quantitative mass spectrometry techniques, and acquired genome-wide methylation data using bisulfite conversion and a microarray platform. We compared molecular profiles across the iSGS and control groups, and with respect to clinical course in the iSGS group. Eight of the 12 iSGS patients underwent subsequent blood collection and plasma isolation for further assessment. RESULTS: Proteomic analysis revealed 42 differentially abundant proteins in the iSGS biopsies compared to controls, inferring enrichment of biological pathways associated with early wound healing, innate immunity, matrix remodeling, and metabolism. Proteome-based hierarchical clustering organized patients into two iSGS and one control subgroups. Methylation analysis revealed five hypermethylated genes in the iSGS biopsies compared to controls, including the biotin recycling enzyme biotinidase (BTD). Follow-up analysis showed elevated plasma BTD activity in iSGS patients compared to both controls and published normative data. CONCLUSION: iSGS exhibits distinct proteomic and genomic methylation signatures. These signatures expand current understanding of the iSGS phenotype, support the possibility of disease subgroups, and should inform the direction of future experimental studies. LEVEL OF EVIDENCE: Not applicable Laryngoscope, 131:E540-E546, 2021.

Quantification and molecular imaging of fatty acid isomers from complex biological samples by mass spectrometry.

Elucidating the isomeric structure of free fatty acids (FAs) in biological samples is essential to comprehend their biological functions in various physiological and pathological processes. Herein, we report a novel approach of using peracetic acid (PAA) induced epoxidation coupled with mass spectrometry (MS) for localization of the C[double bond, length as m-dash]C bond in unsaturated FAs, which enables both quantification and spatial visualization of FA isomers from biological samples. Abundant diagnostic fragment ions indicative of the C[double bond, length as m-dash]C positions were produced upon fragmentation of the FA epoxides derived from either in-solution or on-tissue PAA epoxidation of free FAs. The performance of the proposed approach was evaluated by analysis of FAs in human cell lines as well as mapping the FA isomers from cancer tissue samples with MALDI-TOF/TOF-MS. Merits of the newly developed method include high sensitivity, simplicity, high reaction efficiency, and capability of spatial characterization of FA isomers in tissue samples.

Microarray-driven validation of reference genes for quantitative real-time polymerase chain reaction in a rat vocal fold model of mucosal injury.

Relative quantification by normalization against a stably expressed reference gene is a widely used data analysis method in microarray and quantitative real-time polymerase chain reaction (qRT-PCR) platforms; however, recent evidence suggests that many commonly utilized reference genes are unstable in certain experimental systems and situations. The primary aim of this study, therefore, was to screen and identify stably expressed reference genes in a well-established rat model of vocal fold mucosal injury. We selected and evaluated the expression stability of nine candidate reference genes. Ablim1, Sptbn1, and Wrnip1 were identified as stably expressed in a model-specific microarray dataset and were further validated as suitable reference genes in an independent qRT-PCR experiment using 2(-DeltaCT) and pairwise comparison-based (geNorm) analyses. Parallel analysis of six commonly used reference genes identified Sdha as the only stably expressed candidate in this group. Sdha, Sptbn1, and the geometric mean of Sdha and Sptbn1 each provided accurate normalization of target gene Tgfb1; Gapdh, the least stable candidate gene in our dataset, provided inaccurate normalization and an invalid experimental result. The stable reference genes identified here are suitable for accurate normalization of target gene expression in vocal fold mucosal injury experiments.

Morphological and extracellular matrix changes following vocal fold injury in mice.

Mouse experimental models are commonly utilized tools in biomedical research but remain underrepresented in vocal fold biology, presumably due to the small size of the larynx and limited description of the anatomical, cellular and extracellular composition of the vocal folds. In this study, we provide a whole-mount serial section-based histological description of vocal fold morphology of wild-type FVB strain mice, alongside a histological and immunohistochemical (IHC)-based quantitative analysis of extracellular matrix (ECM) alteration 1, 7, 14, 28, 42 and 56 days following unilateral vocal fold injury. IHC was specific for procollagen type I, collagen type I, collagen type III, collagen type IV, elastin, decorin, fibronectin and hyaluronic acid binding protein 2. The histological description confirmed the presence of a laryngeal alar structural complex in the mouse, which appears to be a morphological feature unique to rodents. The lamina propria appeared uniform without evidence of a distinct layer structure as has been reported in larger animals and humans. Time-dependent alterations in vocal fold morphology, ECM organization and ECM protein/glycoconjugate abundance were observed in injured vocal folds compared to control. The presence of a mature scar was observed between 28 and 42 days postinjury. Morphological and ECM changes following vocal fold injury in the mouse were generally consistent with those reported in other animal models, particularly the rat, although wound repair in the mouse appears to occur at a faster rate.

Reactive response of fibrocytes to vocal fold mucosal injury in rat.

Fibrocytes hold a prominent role in inflammatory and tissue repair processes in various organ systems. In this study, we identified and quantified a reactive fibrocyte population in the vocal fold mucosa postinjury using immunohistochemistry and stereological analysis. These cells, which expressed CD11b on their surface and prolyl-4-hydroxylase ß (P4H-ß) intracellularly, were largely restricted to the lamina propria, and were morphologically and immunochemically distinguishable from newly recruited epithelial cells. We validated our immunohistochemistry findings using flow cytometry, and additionally characterized a reactive fibrocyte population in circulating peripheral blood using a novel detection panel (CD16(-) CD11b(+) P4H-ß(+) ). Fibrocyte recruitment peaked at 3 days postinjury in peripheral blood, and 5 days postinjury in the vocal fold mucosa. These findings suggest that circulating fibrocytes are recruited to sites of tissue injury in the vocal fold mucosa, and may play an important role in vocal fold tissue repair. The results of this study are consistent with published data from other organ systems and strongly suggest the importance of fibrocytes as therapeutic targets. Our newly reported antigen panel facilitating the direct characterization of fibrocytes via flow cytometry is a useful tool with the potential to facilitate improved study of this cell population.

Alteration in cellular morphology, density and distribution in rat vocal fold mucosa following injury.

The vocal fold mucosa plays an important role in voice production. Its cellular composition and density frequently change under various pathological conditions, often contributing to altered extracellular matrix production, tissue viscoelasticity, and voice quality. In this study, cellular changes in the rat mucosa following a unilateral stripping injury were investigated and analyzed semi-quantitatively. Distinctive and sequential changes in cellular morphology, composition, and density were observed in the mucosa post-injury. Cellular recruitment was a major event during the early stage of injury and reached its peak level by day 5 post-injury. Several types of cells, including neutrophil-like cells, epithelial cells, and fibroblast-like cells, were sequentially recruited. The sequential emergence of reactive cell populations following injury and subsequent reconstruction of the mucosa suggests their involvement in vocal fold tissue repair and scar formation processes.

Dynamics of vitamin A uptake, storage, and utilization in vocal fold mucosa.

OBJECTIVE: Extrahepatic vitamin A is housed within organ-specific stellate cells that support local tissue function. These cells have been reported in the vocal fold mucosa (VFM) of the larynx; however, it is unknown how vitamin A reaches and is disseminated among VFM target cells, how VFM storage and utilization vary as a function of total body stores, and how these parameters change in the context of pathology. Therefore, in this study, we investigated fundamental VFM vitamin A uptake and metabolism. METHODS: Using cadaveric tissue and serum from human donors representing the full continuum of clinical vitamin A status, we established a concentration range and analyzed the impact of biologic and clinical covariates on VFM vitamin A. We additionally conducted immunodetection of vitamin A-associated markers and pharmacokinetic profiling of orally dosed α-retinyl ester (a chylomicron tracer) in rats. RESULTS: Serum vitamin A was a significant predictor of human VFM concentrations, suggesting that VFM stores may be rapidly metabolized in situ and replenished from the circulatory pool. On a vitamin A-sufficient background, dosed α-vitamin A was detected in rat VFM in both ester and alcohol forms, showing that, in addition to plasma retinol and local stellate cell stores, VFM can access and process postprandial retinyl esters from circulating chylomicra. Both α forms were rapidly depleted, confirming the high metabolic demand for vitamin A within VFM. CONCLUSION: This thorough physiological analysis validates VFM as an extrahepatic vitamin A repository and characterizes its unique uptake, storage, and utilization phenotype.

Pulsed dye laser-induced inflammatory response and extracellular matrix turnover in rat vocal folds and vocal fold fibroblasts.

BACKGROUND AND OBJECTIVES: Disruption of the vocal fold extracellular matrix (ECM) can induce a profound and refractory dysphonia. Pulsed dye laser (PDL) irradiation has shown early promise as a treatment modality for disordered ECM in patients with chronic vocal fold scar; however, there are limited data addressing the mechanism by which this laser energy might induce cellular and extracellular changes in vocal fold tissues. In this study, we examined the inflammatory and ECM modulating effects of PDL irradiation on normal vocal fold tissues and cultured vocal fold fibroblasts (VFFs). STUDY DESIGN/MATERIALS AND METHODS: We evaluated the effects of 585 nm PDL irradiation on inflammatory cytokine and collagen/collagenase gene transcription in normal rat vocal folds in vivo (3-168 hours following delivery of approximately 39.46 J/cm(2) fluence) and VFFs in vitro (3-72 hours following delivery of 4.82 or 9.64 J/cm(2) fluence). We also examined morphological vocal fold tissue changes 3 hours, 1 week, and 1 month post-irradiation. RESULTS: PDL irradiation altered inflammatory cytokine and procollagen/collagenase expression at the transcript level, both in vitro and in vivo. Additionally, PDL irradiation induced an inflammatory repair process in vivo that was completed by 1 month with preservation of normal tissue morphology. CONCLUSIONS: PDL irradiation can modulate ECM turnover in phenotypically normal vocal folds. Additional work is required to determine if these findings extend to disordered ECM, such as is seen in vocal fold scar. Lasers Surg. Med. 41:585-594, 2009. (c) 2009 Wiley-Liss, Inc.

Surgical method to create vocal fold injuries in mice.

OBJECTIVES: The goal of this study was to develop a surgical method for the creation of vocal fold injuries in mice, as a precursor to the use of genetically engineered mouse models in the study of vocal fold wound healing and scar formation. METHODS: Seven FVB strain mice were used in this study. A laryngoscope and 3 micro-instruments were designed and fabricated to facilitate endoscopic vocal fold visualization and the creation of vocal fold surgical injuries. The larynges were harvested 1 and 7 days after surgery, and the vocal fold injury sites were evaluated by routine hematoxylin and eosin staining. Additional immunohistochemical analysis of collagen type I and elastin distribution in the lamina propria was performed for an uninjured control larynx. RESULTS: Endoscopic visualization and vocal fold stripping resulting in thyroarytenoid muscle exposure were successful in all animals. Histologic and immunohistochemical analyses revealed a simple lamina propria structure with relatively even collagen type I and elastin distribution in the control vocal fold, obliteration of vocal fold mucosa 1 day after surgery, and complete reepithelialization by 7 days. CONCLUSIONS: These results demonstrate the feasibility of creating reproducible vocal fold injuries via an endoscopic approach in mice. The observation that the mouse lamina propria may have a relatively simple histologic structure indicates that additional characterization should be performed and caution used in translating findings between this and other model systems.

A rat excised larynx model of vocal fold scar.

PURPOSE: To develop and evaluate a rat excised larynx model for the measurement of acoustic, aerodynamic, and vocal fold vibratory changes resulting from vocal fold scar. METHOD: Twenty-four 4-month-old male Sprague-Dawley rats were assigned to 1 of 4 experimental groups: chronic vocal fold scar, chronic vocal fold scar treated with 100-ng basic fibroblast growth factor (bFGF), chronic vocal fold scar treated with saline (sham treatment), and unscarred untreated control. Following tissue harvest, histological and immunohistochemical data were collected to confirm extracellular matrix alteration in the chronic scar group; acoustic, aerodynamic, and high-speed digital imaging data were collected using an excised larynx setup in all groups. Phonation threshold pressure (P(th)), glottal resistance (R(g)), glottal efficiency (E(g)), vibratory amplitude, and vibratory area were used as dependent variables. RESULTS: Chronically scarred vocal folds were characterized by elevated collagen Types I and III and reduced hyaluronic acid abundance. Phonation was achieved, and data were collected from all control and bFGF-treated larynges; however, phonation was not achieved with 3 of 6 chronically scarred and 1 of 6 saline-treated larynges. Compared with control, the chronic scar group was characterized by elevated P(th), reduced E(g), and intralarynx vibratory amplitude and area asymmetry. The bFGF group was characterized by P(th) below control-group levels, E(g) comparable with control, and vocal fold vibratory amplitude and area symmetry comparable with control. The sham group was characterized by P(th) comparable with control, E(g) superior to control, and vocal fold vibratory amplitude and area symmetry comparable with control. CONCLUSIONS: The excised larynx model reported here demonstrated robust deterioration across phonatory indices under the scar condition and sensitivity to treatment-induced change under the bFGF condition. The improvement observed under the sham condition may reflect unanticipated therapeutic benefit or artifact. This model holds promise as a tool for the functional characterization of biomechanical tissue changes resulting from vocal fold scar and the evaluation of experimental therapies.

Reversal of Vocal Fold Mucosal Fibrosis Using siRNA against the Collagen-Specific Chaperone Serpinh1.

Vocal fold (VF) mucosal fibrosis results in substantial voice impairment and is recalcitrant to current treatments. To reverse this chronic disorder, anti-fibrotic therapies should target the molecular pathology of aberrant collagen accumulation in the extracellular matrix. We investigated the therapeutic potential of siRNA against Serpinh1, a collagen-specific chaperone that enables cotranslational folding and assembly of procollagens in the endoplasmic reticulum. We implemented a previously validated siRNA construct, conducted transfection experiments using in vitro and in vivo rat models, and measured knockdown efficiency, dose responses, delivery strategies, and therapeutic outcomes. Liposome-mediated delivery of Serpinh1-siRNA downregulated collagen production in naive and scar VF fibroblasts as well as naive VF mucosa; moreover, sustained Serpinh1 knockdown in fibrotic VF mucosa reversed scar-associated collagen accumulation within 4 weeks. Analysis of therapeutic effects at the transcriptome level showed evidence of cell cycle upregulation, catabolism, matrix disassembly, and morphogenesis. These findings indicate that Serpinh1-siRNA holds potential as a molecular therapy for chronic VF mucosal fibrosis.