RESUMO
Tissue regeneration is intricately influenced by the dynamic interplay between the physical attributes of tissue engineering scaffolds and the resulting biological responses. A tunable microporous hydrogel system was engineered using gelatin methacryloyl (GelMA) and polyethylene glycol diacrylate (PEGDA), with polyethylene glycol (PEG) serving as a porogen. Through systematic variation of PEGDA molecular weights, hydrogels with varying mechanical and architectural properties were obtained. The objective of the present study was to elucidate the impact of substrate mechanics and architecture on the immunological and reparative activities of vocal fold tissues. Mechanical characterization of the hydrogels was performed using tensile strength measurements and rheometry. Their morphological properties were investigated using scanning electron microscopy (SEM) and confocal microscopy. A series of biological assays were conducted. Cellular morphology, differentiation, and collagen synthesis of human vocal fold fibroblasts (hVFFs) were evaluated using immunostaining. Fibroblast proliferation was studied using the WST-1 assay, and cell migration was investigated via the Boyden chamber assay. Macrophage polarization and secretions were also examined using immunostaining and ELISA. The results revealed that increasing the molecular weight of PEGDA from 700 Da to 10,000 Da resulted in decreased hydrogel stiffness, from 62.6 to 8.8 kPa, and increased pore dimensions from approximately 64.9 to 137.4 µm. Biological evaluations revealed that hydrogels with a higher stiffness promoted fibroblast proliferation and spreading, albeit with an increased propensity for fibrosis, as indicated by a surge in myofibroblast differentiation and collagen synthesis. In contrast, hydrogels with greater molecular weights had a softer matrix with expanded pores, enhancing cellular migration and promoting an M2 macrophage phenotype conducive to tissue healing. The findings show that the hydrogels formulated with a PEGDA molecular weight of 6000 Da are best among the hydrogels considered for vocal fold repair. The microporous hydrogels could be tuned to serve in other tissue engineering applications.
Assuntos
Proliferação de Células , Fibroblastos , Hidrogéis , Polietilenoglicóis , Prega Vocal , Prega Vocal/citologia , Prega Vocal/metabolismo , Prega Vocal/química , Hidrogéis/química , Humanos , Porosidade , Fibroblastos/citologia , Polietilenoglicóis/química , Elasticidade , Alicerces Teciduais/química , Engenharia Tecidual/métodos , Movimento Celular , Gelatina/química , Diferenciação Celular , Colágeno/química , Colágeno/metabolismoRESUMO
Toward the goal of establishing an engineered model of the vocal fold lamina propria (LP), mesenchymal stem cells (MSCs) are encapsulated in hyaluronic acid (HA)-based hydrogels employing tetrazine ligation with strained alkenes. To mimic matrix stiffening during LP maturation, diffusion-controlled interfacial bioorthogonal crosslinking is carried out on the soft cellular construct using HA modified with a ferocious dienophile, trans-cyclooctene (TCO). Cultures are maintained in MSC growth media for 14 days to afford a model of a newborn LP that is homogeneously soft (nLP), a homogeneously stiffened construct zero (sLP0) or 7 days (sLP7) post cell encapsulation, and a mature LP model (mLP) with a stiff top layer and a soft bottom layer. Installation of additional HA crosslinks restricts cell spreading. Compared to the nLP controls, sLP7 conditions upregulate the expression of fibrous matrix proteins (Col I, DCN, and FN EDA), classic fibroblastic markers (TNC, FAP, and FSP1), and matrix remodeling enzymes (MMP2, TIMP1, and HAS3). Day 7 stiffening also upregulates the catabolic activities, enhances ECM turnover, and promotes YAP expression. Overall, in situ delayed matrix stiffening promotes a fibroblast transition from MSCs and enhances YAP-regulated mechanosensing.
Assuntos
Hidrogéis , Células-Tronco Mesenquimais , Humanos , Recém-Nascido , Hidrogéis/metabolismo , Prega Vocal/metabolismo , Fibroblastos , Ácido Hialurônico/metabolismoRESUMO
OBJECTIVE: To investigate the role of H+ /K+ ATPase in the proliferation of pepsin-induced vocal cord leukoplakia (VCL) cells. STUDY DESIGN: Translation research. SETTING: Affiliated Hospital of University. METHODS: Immunohistochemistry was used to detect pepsin, H+ /K+ ATPase (ATP4A and ATP4B subunits) in VCL cells with varying degrees of dysplasia. After primary cultures of VCL cells had been established, the effects of acidified pepsin on the proliferation, autophagy, and H+ /K+ -ATPase distribution of VCL cells were investigated. RESULTS: The levels of pepsin, ATP4A, and ATP4B were significantly higher in VCL tissue with moderate-to-severe dysplasia than in normal tissue (p < .05); these levels gradually increased according to dysplasia severity. The expression levels of ATP4A and ATP4B were significantly correlated with the amount of pepsin in VCL cells (p < .01). Acidified pepsin enhanced the levels of proliferation and autophagy in human VCL epithelial cells. The cloning- and autophagy-promoting effects of acidified pepsin on VCL cells were partially reversed by pantoprazole; these effects were completely blocked by the autophagy inhibitor chloroquine. Finally, acidified pepsin promoted the colocalization of H+ /K+ -ATPase and lysosomes in VCL cells; it also mediated lysosome acidification. CONCLUSION: Pepsin and H+ /K+ -ATPase may contribute to the progression of VCL. Specifically, acidified pepsin may regulate lysosome acidification by promoting lysosomal localization of H+ /K+ -ATPase.
Assuntos
Doenças da Laringe , Pepsina A , Humanos , Prega Vocal/metabolismo , Autofagia , Células Epiteliais/metabolismo , Adenosina Trifosfatases , Proliferação de Células , Leucoplasia/metabolismoRESUMO
Suppressor of cytokine signaling 3 (SOCS3) is a negative regulatory protein that has been identified as a key inhibitory regulator of JAK/STAT signaling pathway. However, the mutual regulatory relationship between SOCS3 and JAK2/STAT3 signaling pathway after vocal fold injury remains unclear. In this study, we used small interfering RNA (siRNA) to investigate the mechanism of SOCS3 regulating of fibroblasts through JAK2/STAT3 signaling pathway after vocal fold injury. Our data shows that SOCS3 silencing promotes the transformation of normal vocal fold fibroblasts (VFFs) into an fibrotic phenotype and activates the JAK2/STAT3 signaling pathway. JAK2 silencing significantly inhibits the increase in type I collagen and α-smooth muscle actin (α-SMA) secretion in VFFs induced by TGF-ß but has no significant effect on normal VFFs. The silencing of SOCS3 and JAK2 reverses the fibrotic phenotype of VFFs induced by SOCS3 silencing. Therefore, we suggest that SOCS3 can affect the activation of vocal fold fibroblasts by regulating the JAK2/STAT3 signaling pathway after vocal fold injury. It provides a new insight for promoting the repair of vocal fold injury and preventing the formation of fibrosis.
Assuntos
Proteínas Supressoras da Sinalização de Citocina , Prega Vocal , Fibroblastos/metabolismo , Janus Quinase 2/metabolismo , RNA Interferente Pequeno/farmacologia , Transdução de Sinais , Fator de Transcrição STAT3/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/genética , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Prega Vocal/metabolismo , Animais , RatosRESUMO
Long-term voice abuse or sudden vocal fold microvascular disruption may lead to injury and subsequent repair/remodeling in the vocal fold mucosa. Periostin is known to be involved in airway remodeling and also in various otolaryngological diseases. D-ß-aspartic acid is the major isomer of D-aspartic acid found in elderly tissue. In this study we investigated the expression and the role of D-ß-aspartic acid and periostin in the formation of vocal fold polyps. The expression patterns of D-ß-aspartic acid and periostin in 36 surgical specimens of vocal fold polyps from 36 patients were investigated immunohistochemically. In the epithelium of vocal polyps, D-ß-aspartic acid was expressed in all cases. Expression of D-ß-aspartic acid was detected in 25 samples obtained from patients with vocal fold polyps stroma. Expression of periostin was detected in 28 samples obtained from patients with vocal fold polyps. Two patterns of D-ß-aspartic acid expression were observed in vocal fold polyps stroma: positive type and negative type. The following four patterns of periostin expression were observed in vocal fold polyps: negative type, superficial type, infiltrative type, and diffuse type. An association was observed between D-ß-aspartic acid expression patterns and periostin expression patterns. From these findings we speculate that periostin and D-ß-aspartic acid participate in certain pathological changes in vocal fold polyps, such as extracellular matrix accumulation, local fibrosis, and the formation and development of vocal fold polyps.
Assuntos
Doenças da Laringe , Pólipos , Humanos , Idoso , Prega Vocal/metabolismo , Prega Vocal/patologia , Prega Vocal/cirurgia , Ácido Isoaspártico , Doenças da Laringe/metabolismo , Doenças da Laringe/patologia , Doenças da Laringe/cirurgia , Pólipos/metabolismo , Pólipos/patologia , Pólipos/cirurgiaRESUMO
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.
Assuntos
Glucocorticoides , NF-kappa B , Humanos , Glucocorticoides/farmacologia , Fator de Necrose Tumoral alfa , Prega Vocal/metabolismo , Interleucina-4 , Receptores de Glucocorticoides/metabolismo , Dexametasona/farmacologia , Fibroblastos/metabolismoRESUMO
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: In vitro. METHODS: Human vocal fold fibroblasts and human macrophages derived from THP-1 monocytes were treated with 0.03-1000 nM dexamethasone, 0.3-10,000 nM methylprednisolone, and 0.3-10,000 nM triamcinolone in combination with interferon-γ, tumor necrosis factor-α, or interleukin-4. Real-time polymerase chain reaction was performed to analyze inflammatory (CXCL10, CXCl11, PTGS2, TNF, IL1B) and fibrotic (CCN2, LOX, TGM2) genes, and TSC22D3, a target gene of GC signaling. EC50 and IC50 to alter inflammatory and fibrotic gene expression was calculated. RESULTS: Interferon-γ and tumor necrosis factor-α increased inflammatory gene expression in both cell types; this response was reduced by GCs. Interleukin-4 increased LOX and TGM2 expression in macrophages; this response was also reduced by GCs. GCs induced TSC22D3 and CCN2 expression independent of cytokine treatment. EC50 for each GC to upregulate CCN2 was higher than the IC50 to downregulate other genes. CONCLUSION: 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: NA Laryngoscope, 133:1169-1175, 2023.
Assuntos
Glucocorticoides , Interleucina-4 , Humanos , Glucocorticoides/farmacologia , Interleucina-4/farmacologia , Interleucina-4/metabolismo , Dexametasona/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Interferon gama/metabolismo , Interferon gama/farmacologia , Prega Vocal/metabolismo , Receptores de Glucocorticoides/metabolismo , Macrófagos/metabolismo , Expressão Gênica , Fibroblastos/metabolismo , FibroseRESUMO
PURPOSE: Vocal fold scarring is abnormal scar tissue in the lamina propria layer of the vocal fold. To facilitate investigation of vocal fold scarring, we established and characterized immortalized human vocal fold fibroblast (iHVFF) cell lines. METHODS: Human vocal fold fibroblasts were immortalized by introducing Simian virus 40 large T antigen (SV40TAg) by transfection. Successfully transfected fibroblasts were sorted using flow cytometry. Immunofluorescence cytochemistry and western blot were applied to analyze the expression of fibronectin, vimentin, alpha-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP). Cell proliferation rate was measured by CCK-8 assay. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the mRNA expression level. RESULTS: The iHVFFs continued to proliferate for more than 30 generations and appeared spindle-shaped. The expression of Vimentin and α-SMA were detected in both iHVFFs and primary fibroblasts, and enhanced expression of FAP was observed in iHVFFs. Furthermore, iHVFFs exhibited an increased proliferative capability compared with the primary fibroblasts. RT-qPCR results suggested that collagen type III alpha 1 chain (COL3A1), interleukin-6, cyclooxygenase 2 (COX2), hyaluronan synthase 2 (HAS2), hepatocyte growth factor (HGF) in the iHVFFs significantly increased, whereas transforming growth factor-ß1 (TGF-ß1), elastin and matrix metallopeptidase-1 (MMP-1) expression significantly downregulated. No differences in mRNA expression of α-SMA, fibronectin and collagen type I alpha 2 chain (COL1A2) were noted between iHVFFs and primary fibroblasts. CONCLUSION: iHVFFs can be used as a novel tool cell for future researches on the mechanisms of pathogenesis and treatment of vocal fold scarring.
Assuntos
Cicatriz , Fibronectinas , Humanos , Fibronectinas/metabolismo , Cicatriz/metabolismo , Cicatriz/patologia , Vimentina/metabolismo , Prega Vocal/metabolismo , Prega Vocal/patologia , Linhagem Celular , Fator de Crescimento Transformador beta1/metabolismo , Fibroblastos/metabolismo , Células CultivadasRESUMO
BACKGROUND: A considerable body of clinical evidence suggests that systemic dehydration can negatively affect voice production, leading to the common recommendation to rehydrate. Evidence for the corrective benefits of rehydration, however, is limited with mixed conclusions, and biological data on the underlying tissue changes with rehydration is lacking. In this study, we used a rabbit model (n = 24) of acute (5 days) water restriction-induced systemic dehydration with subsequent rehydration (3 days) to explore the protein-level changes underlying the molecular transition from euhydration to dehydration and following rehydration using LC-MS/MS protein quantification in the vocal folds. We show that 5-day water restriction led to an average 4.3% decrease in body weight with relative increases in anion gap, Cl-, creatinine, Na+, and relative decreases in BUN, iCa2+, K+, and tCO2 compared to control (euhydrated) animals. A total of 309 differentially regulated (p < 0.05) proteins were identified between the Control and Dehydration groups. We observed a noteworthy similarity between the Dehydration and Rehydration groups, both well differentiated from the Control group, highlighting the distinct timelines of resolution of the clinical symptoms of systemic dehydration and the underlying molecular changes. SIGNIFICANCE: Voice disorders are a ubiquitous problem with considerable economic and psychological impact. Maintenance of proper hydration is commonly prescribed as a general vocal hygiene practice. There is evidence that dehydration negatively impacts phonation, but our understanding of the state of vocal folds in the context of systemic dehydration are limited, particular from a molecular perspective. Further, ours is a novel molecular study of the short-term impact of rehydration on the tissue. Given the relatively minimal difference in vocal fold proteomic profiles between the Dehydration and Rehydration groups, our data demonstrate a complex physiological response to acute systemic dehydration, and highlight the importance of considering persistent underlying molecular pathology despite the rapid resolution of clinical measures. This study sets a foundation for future research to confirm the nature of potential beneficial outcomes of clinical recommendations related to hydration.
Assuntos
Desidratação , Prega Vocal , Animais , Coelhos , Prega Vocal/metabolismo , Desidratação/metabolismo , Proteômica , Cromatografia Líquida , Espectrometria de Massas em Tandem , Hidratação , Água/metabolismoRESUMO
OBJECTIVES: There is growing evidence that the cells in the maculae flavae (MFe) are candidates for tissue stem cells of the vocal fold mucosa and the MFe are a stem cell niche. Distribution of label-retaining cells and their properties in the postnatal vocal fold mucosa were investigated. METHODS: Oral administration of bromodeoxyuridine (BrdU) was given to pregnant Sprague-Dawley rats and the label-retaining cells in the postnatal vocal fold mucosa were observed by immunohistochemistry. Immunoreactivity to antibodies directed to Ki-67 was studied to investigate the cell cycle. RESULTS: At day 1 after birth, BrdU positive cells were identified in the MFe (60.1 ± 1.7%), epithelium (58.7 ± 10.6%) and lamina propria (52.4 ± 7.8%) of the vocal fold mucosa. At day 56 after birth, the number of BrdU positive cells in the epithelium (4.8 ± 2.2%) and lamina propria (32.3 ± 16.5%) were significantly lower compared to day 1 after birth (P < 0.05). However, the number of BrdU positive cells remaining in the MFe was still high (56.2 ± 2.5%). The label-retaining cells were distributed throughout the MFe. Few Ki-67 positive cells were identified in the MFe indicating they were resting cells. CONCLUSIONS: The results of this study are consistent with the hypothesis that the cells in the postnatal MFe are candidates for tissue stem cells. At birth, these cells are already present in the MFe of the newborn vocal fold and they are likely ready to start the growth and development of the vocal fold mucosa.
Assuntos
Mucosa , Prega Vocal , Ratos , Animais , Prega Vocal/metabolismo , Bromodesoxiuridina/metabolismo , Antígeno Ki-67/metabolismo , Ratos Sprague-DawleyRESUMO
Mechanoreceptors are implicated as functional afferents within mucosa of the airways and the recent discovery of mechanosensitive channels Piezo1 and Piezo2 has proved essential for cells of various mechanically sensitive tissues. However, the role for Piezo1/2 in vocal fold (VF) mucosal epithelia, a cell that withstands excessive biomechanical insult, remains unknown. The purpose of this study was to test the hypothesis that Piezo1 is required for VF mucosal repair pathways of epithelial cell injury. Utilizing a sonic hedgehog (shh) Cre line for epithelial-specific ablation of Piezo1/2 mechanoreceptors, we investigated 6wk adult VF mucosa following naphthalene exposure for repair strategies at 1, 3, 7 and 14 days post-injury (dpi). PIEZO1 localized to differentiated apical epithelia and was paramount for epithelial remodeling events. Injury to wildtype epithelium was most appreciated at 3 dpi. Shhcre/+; Piezo1loxP/loxP, Piezo2 loxP/+ mutant epithelium exhibited severe cell/nuclear defects compared to injured controls. Conditional ablation of Piezo1 and/or Piezo2 to uninjured VF epithelium did not result in abnormal phenotypes across P0, P15 and 6wk postnatal stages compared to heterozygote and control tissue. Results demonstrate a role for Piezo1-expressing VF epithelia in regulating self-renewal via effects on p63 transcription and YAP subcellular translocation-altering cytokeratin differentiation.
Assuntos
Proteínas Hedgehog , Queratinas , Proteínas Hedgehog/metabolismo , Queratinas/genética , Prega Vocal/metabolismo , Células Epiteliais/metabolismo , Epitélio/metabolismoRESUMO
Repeated mechanical and chemical insults cause an irreversible alteration of extracellular matrix (ECM) composition and properties, giving rise to vocal fold scarring that is refractory to treatment. Although it is well known that fibroblast activation to myofibroblast is the key to the development of the pathology, the lack of a physiologically relevant in vitro model of vocal folds impedes mechanistic investigations on how ECM cues promote myofibroblast differentiation. Herein, we describe a bio-orthogonally cross-linked hydrogel platform that recapitulates the alteration of matrix adhesiveness due to enhanced fibronectin deposition when vocal fold wound healing is initiated. The synthetic ECM (sECM) was established via the cycloaddition reaction of tetrazine (Tz) with slow (norbornene, Nb)- and fast (trans-cyclooctene, TCO)-reacting dienophiles. The relatively slow Tz-Nb ligation allowed the establishment of the covalent hydrogel network for 3D cell encapsulation, while the rapid and efficient Tz-TCO reaction enabled precise conjugation of the cell-adhesive RGDSP peptide in the hydrogel network. To mimic the dynamic changes of ECM composition during wound healing, RGDSP was conjugated to cell-laden hydrogel constructs via a diffusion-controlled bioorthognal ligation method 3 days post encapsulation. At a low RGDSP concentration (0.2 mM), fibroblasts residing in the hydrogel remained quiescent when maintained in transforming growth factor beta 1 (TGF-ß1)-conditioned media. However, at a high concentration (2 mM), RGDSP potentiated TGF-ß1-induced myofibroblast differentiation, as evidenced by the formation of an actin cytoskeleton network, including F-actin and alpha-smooth muscle actin. The RGDSP-driven fibroblast activation to myofibroblast was accompanied with an increase in the expression of wound healing-related genes, the secretion of profibrotic cytokines, and matrix contraction required for tissue remodeling. This work represents the first step toward the establishment of a 3D hydrogel-based cellular model for studying myofibroblast differentiation in a defined niche associated with vocal fold scarring.
Assuntos
Miofibroblastos , Fator de Crescimento Transformador beta1 , Humanos , Fator de Crescimento Transformador beta1/farmacologia , Prega Vocal/metabolismo , Hidrogéis/farmacologia , Hidrogéis/metabolismo , Cicatriz/metabolismo , Adesividade , FibroblastosRESUMO
BACKGROUND: Atrophy of the vocal folds and the accompanying glottic insufficiency affect the quality of life. Although growth factors have been used to treat muscle atrophy, their effectiveness is limited by their short half-life. METHODS: In total, 15 rabbits and 24 rats were used for the study. The right recurrent laryngeal nerves of all animals were transected. One month following nerve transection, PBS (PBS group), rHGF (HGF group), or a c-Met agonistic antibody (c-Met group) was injected into the paralyzed vocal folds. The larynges of the rabbits were harvested from each group for histologic examination and subjected to PCR analysis. RESULTS: Cross-sectional areas (CSAs) of thyroarytenoid muscles were evaluated. The c-Met group had increased CSAs compared to the PBS and HGF groups, but there were no significant differences compared to normal controls. The expression levels of myogenesis-related genes were evaluated three weeks after the injection. The expression levels of myosin heavy chain IIa were significantly increased in the PBS group, while the expression levels of MyoD were increased in the c-Met group. CONCLUSIONS: The c-Met agonistic antibody showed promise for promoting muscle regeneration in a vocal fold palsy model.
Assuntos
Paralisia das Pregas Vocais , Prega Vocal , Animais , Músculos Laríngeos , Atrofia Muscular/metabolismo , Qualidade de Vida , Coelhos , Ratos , Paralisia das Pregas Vocais/metabolismo , Paralisia das Pregas Vocais/patologia , Paralisia das Pregas Vocais/terapia , Prega Vocal/metabolismoRESUMO
Long-term voice abuse or sudden vocal fold microvascular disruption may lead to injury and subsequent repair/remodeling in the vocal fold mucosa. Periostin is known to be involved in airway remodeling and also in various otolaryngological diseases. The aim of this article was to investigate the expression and the role of periostin in the formation of vocal fold polyps. The expression patterns of periostin in 59 surgical specimens of vocal fold polyps from 54 patients were investigated immunohistochemically. Normal vocal fold mucosa specimens from 5 patients who had undergone total laryngectomy were used as the control group. Retrospective study with planned data collection was conducted at Tohoku Medical and Pharmaceutical University. Expression of periostin was detected in 43 (72.9%) samples and four patterns of periostin expression were observed in vocal fold polyps: negative type, superficial type, infiltrative type, and diffuse type. An association was observed between periostin expression patterns and the histological subtypes of vocal fold polyps. The infiltrative pattern of periostin expression was significantly dominant in vascular-hyaline types. Expression of transforming growth factor-ß (TGF-ß) was also detected in the vocal fold polyps. Our results confirmed that periostin might be involved in certain pathological changes in vocal fold polyps, such as extracellular matrix accumulation, local fibrosis, and formation and development of vocal fold polyps.
Assuntos
Doenças da Laringe , Pólipos , Humanos , Doenças da Laringe/metabolismo , Doenças da Laringe/patologia , Doenças da Laringe/cirurgia , Pólipos/metabolismo , Pólipos/patologia , Pólipos/cirurgia , Estudos Retrospectivos , Prega Vocal/metabolismo , Prega Vocal/patologia , Prega Vocal/cirurgiaRESUMO
Macrophages aid in wound healing by changing their phenotype and can be a key driver of fibrosis. However, the contribution of macrophage phenotype to fibrosis following vocal fold injury remains unclear. Peroxisome proliferator-activated receptor-γ (PPARγ) is expressed mainly by macrophages during early wound healing and regulates the macrophage phenotype. This study aimed to evaluate the effects of pioglitazone (PIO), a PPARγ agonist, on the macrophage phenotype and fibrosis following vocal fold injury in rats. PIO was injected into the rat vocal folds on days 1, 3, 5, and 7 after injury, and the vocal fold lamina propria was evaluated on days 4 and 56 after injury. Moreover, THP-1-derived macrophages were treated with PIO, and the expression of proinflammatory cytokines under lipopolysaccharide/interferon-γ stimulation was analyzed. PIO reduced the expression of Ccl2 both in vivo and in vitro. Furthermore, PIO decreased the density of inducible nitric oxide synthase+ CD68+ macrophages and inhibited the expression of fibrosis-related factors on day 4 after injury. On day 56 after injury, PIO inhibited fibrosis, tissue contracture, and hyaluronic acid loss in a PPARγ-dependent manner. These results indicate that PPARγ activation could inhibit accumulation of inflammatory macrophages and improve tissue repair. Taken together, these findings imply that inflammatory macrophages play a key role in vocal fold fibrosis.
Assuntos
PPAR gama , Tiazolidinedionas , Animais , Fibrose , Hipoglicemiantes/farmacologia , Ativação de Macrófagos , PPAR gama/genética , Pioglitazona/farmacologia , Ratos , Tiazolidinedionas/farmacologia , Prega Vocal/metabolismoRESUMO
Post-radiation fibrosis of the vocal folds is thought to cause vocal impairment. However, the mechanism by which this occurs has been poorly documented, probably because of the lack of an appropriate experimental animal model. The purpose of this study was to establish a simple and reproducible mouse model of laryngeal radiation to investigate the development of vocal fold fibrosis over time. C57BL/6 mice individually placed in a lead shield were irradiated with a single dose of 20 Gy. At 1, 2, and 6 months after irradiation, larynges were harvested and subjected to histological examination and gene expression analysis. Irradiated vocal folds showed time-dependent tissue contraction and increased collagen deposition, with no significant difference in the changes in hyaluronic acid levels. Transcriptional analysis revealed upregulated expressions of TGF-ß1 and iNOS at 6 months, but downregulated expressions of Acta2, Col1a1, Col3a1, and MMP8. Moreover, elevated TGF-ß1 and reduced downstream gene expression levels indicated the existence of an inhibitory factor over the TGF-ß/Smad pathway. Discrepancies in histological and transcriptional studies of collagen might suggest that radiation-induced vocal fold fibrosis could be caused by the elongated turnover of collagen. Overall, we established a mouse model of radiation-induced vocal fold fibrosis using a simple protocol. Further investigations are warranted to elucidate the pathogenesis of irradiation-induced fibrosis in vocal folds.
Assuntos
Fator de Crescimento Transformador beta1 , Prega Vocal , Animais , Colágeno/genética , Colágeno/metabolismo , Modelos Animais de Doenças , Fibrose , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Transformador beta1/metabolismo , Prega Vocal/metabolismo , Prega Vocal/patologiaRESUMO
Severe dysplasia of vocal cord leukoplakia (VCL) is more likely to occur in laryngeal carcinoma. Alcohol dehydrogenase and acetaldehyde dehydrogenase are both important enzymes in alcohol metabolism. This study aimed to investigate the incidence rate of malignant transformation in patients with VCL and the role of drinking habits and ALDH2 and ADH1B genetic polymorphisms in the malignant transformation of VCL. From January 2007 to January 2017, 136 cases of VCL were included in this retrospective analysis. Information on medical history, alcohol and tobacco consumption habits, ALDH2 and ADH1B genotypes, gastroesophageal reflux, and clinical pathological characteristics of VCL was collected. As a result, patients had a median follow-up of 9.6 years (interquartile range: 7.5-12.5 years). Twenty-three of 136 VCL patients finally developed laryngeal carcinoma, resulting in a cumulative malignant transformation rate of 16.9%. Cox regression analysis demonstrated that the independent risk factors for the malignant transformation of VCL included age over 60 years (hazard ratio [HR]: 13.872, p < 0.001), ALDH2 *2 allele status (HR: 9.694, p < 0.001), alcohol (HR: 10.011, p < 0.001) and tobacco (HR: 8.869, p < 0.001) exposure after operation, and drinking frequency (HR: 2.178, p = 0.016). Therefore, among patients over 60 years old, an ALDH2-inactivating mutation and excessive ethanol and tobacco consumption are potential contributors to the malignant transformation of VCL.
Assuntos
Álcool Desidrogenase , Carcinoma , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Consumo de Bebidas Alcoólicas/efeitos adversos , Consumo de Bebidas Alcoólicas/genética , Aldeído Desidrogenase/metabolismo , Aldeído-Desidrogenase Mitocondrial/genética , Aldeído Oxirredutases , China , Etanol , Genótipo , Humanos , Leucoplasia/genética , Pessoa de Meia-Idade , Polimorfismo Genético , Estudos Retrospectivos , Prega Vocal/metabolismoRESUMO
Whole-body dehydration (i.e., systemic dehydration) leads to vocal fold tissue dehydration. Furosemide, a common diuretic prescribed to treat hypertension and edema-associated conditions, induces systemic dehydration. Furosemide also causes voice changes in human speakers, making this method of systemic dehydration particularly interesting for vocal fold dehydration studies. Our objective was to obtain a comprehensive proteome of vocal folds following furosemide-induced systemic dehydration. New Zealand White rabbits were used as the animal model and randomly assigned to euhydrated (control) or furosemide-dehydrated groups. Systemic dehydration, induced by injectable furosemide, was verified by an average body weight loss of -5.5% and significant percentage changes in blood analytes in the dehydrated rabbits compared to controls. Vocal fold specimens, including mucosa and muscle, were processed for proteomic analysis using label-free quantitation LC-MS/MS. Over 1600 proteins were successfully identified across all vocal fold samples; and associated with a variety of cellular components and ubiquitous cell functions. Protein levels were compared between groups showing 32 proteins differentially regulated (p ≤ 0.05) in the dehydrated vocal folds. These are mainly involved with mitochondrial translation and metabolism. The downregulation of proteins involved in mitochondrial metabolism in the vocal folds suggests a mechanism to prevent oxidative stress associated with systemic dehydration. SIGNIFICANCE: Voice disorders affect different population demographics worldwide with one in 13 adults in the United States reporting voice problems annually. Vocal fold systemic hydration is clinically recognized for preventing and treating voice problems and depends on optimal body hydration primarily achieved by water intake. Herein, we use the rabbit as a translatable animal model, and furosemide as a translatable method of systemic dehydration, to reveal a comprehensive proteomic profile of vocal fold mucosa and muscle in response to systemic dehydration. The significant subset of proteins differentially regulated due to furosemide-induced dehydration offer novel insights into the molecular mechanisms of systemic dehydration in the vocal folds. These findings also deepen our understanding of changes to tissue biology after diuretic administration.
Assuntos
Proteoma , Prega Vocal , Animais , Cromatografia Líquida , Desidratação/induzido quimicamente , Desidratação/metabolismo , Furosemida/metabolismo , Furosemida/farmacologia , Proteoma/metabolismo , Proteômica , Coelhos , Espectrometria de Massas em Tandem , Prega Vocal/metabolismoRESUMO
The vast majority of voice disorders is associated with changes of the unique, but delicate, human vocal fold mucosa. The ability to develop new effective treatment methods is significantly limited by the physical inaccessibility and the extremely rare occasions under which healthy tissue biopsies can be obtained. Therefore, the interest in laryngological research has shifted to human oral (buccal) mucosa, a similar and more easily available tissue. The harvesting process is less invasive and accompanied with faster healing and less scarring, compared to vocal fold mucosa. Here we report a descriptive proteomic comparison of paired human buccal and vocal fold mucosa by high-resolution mass spectrometry (CID-MS/MS). Our study identified a total of 1575 proteins detected within both tissues that are highly consistent in several crucial biological processes, cellular components, and molecular functions. Hence, our proteomic analysis will provide a fundamental resource for the laryngological research community.
Assuntos
Proteômica , Prega Vocal , Cicatriz/metabolismo , Cicatriz/patologia , Humanos , Mucosa Bucal/metabolismo , Mucosa Bucal/patologia , Espectrometria de Massas em Tandem , Prega Vocal/metabolismo , Prega Vocal/patologiaRESUMO
OBJECTIVE: The aim of the present study was to investigate hyaluronic acid (HA) concentrations in vocal folds among patients with Reinke's edema. STUDY DESIGN: Prospective and experimental study. SETTING: Single tertiary center. METHODS: An HA binding protein isolated from bovine nasal cartilage was used to identify and isolate the HA from samples. Plates coated with biotin-conjugated binding protein and streptavidin-europium conjugate were sequentially incubated with 18 Reinke's edema samples and 11 female vocal fold cover samples from cadavers (the superficial layer of the lamina propria; control group). After the release of europium from streptavidin in enhancement solution, final fluorescence was measured in a fluorometer. RESULTS: The mean HA concentration in Reinke's edema vocal folds was significantly higher than that in the control vocal folds (9.2 × 103 vs 0.9 × 103µg/g). CONCLUSION: Vocal fold covers affected by Reinke's edema present a higher concentration of HA than do vocal fold covers with no edema.