The Laryngoscope C 2014 The American Laryngological, V

Rhinological and Otological Society, Inc.

Cytoskeleton of Newborn Vocal Fold Stellate Cells Kiminori Sato, MD; Takashi Kurita, MD; Shun-ichi Chitose, MD; Hirohito Umeno, MD; Tadashi Nakashima, MD Objectives/Hypothesis: Vocal fold stellate cells (VFSCs) in the human maculae flavae located at both ends of the vocal fold mucosa are inferred to be involved in the metabolism of extracellular matrices of the vocal fold mucosa. Tension caused by phonation (vocal fold vibration) likely regulates the behavior of the VFSCs in the human maculae flava. Tensile and compressive strains have direct effects on cell morphology and structure, including changes in cytoskeletal structure and organization. Cytoskeletons play a role as mechanoreceptors for the cells. The microstructure of the intermediate filaments and the expression of their characteristic proteins were investigated regarding the human newborn VFSCs. Study Design: Histopathologic analysis of the human newborn vocal fold. Methods: Three newborn vocal fold mucosae were investigated by immunohistochemistry and electron microscopy. Results: The intermediate filaments in the cytoplasm of the newborn VFSCs were few in number. However, their characteristic proteins (vimentin, desmin, GFAP [Glial fibrillary acidic protein], cytokeratin) had already expressed. Conclusion: The function and fate of VFSCs are regulated by various microenvironmental factors. Not only chemical factors but also mechanical factors could also modulate VFSC behaviors. The cytoskeletal structure of the newborn VFSCs is under development. And the newborn VFSCs have not acquired mechanical regulation. Key Words: Vocal fold stellate cell, cytoskeleton, macula flava, human newborn vocal fold. Level of Evidence: N/A. Laryngoscope, 124:2551–2554, 2014

INTRODUCTION Human adult vocal folds have a layered structure with a vocal ligament.1,2 This structure is based on the differences of extracellular matrix distribution and is essential for the vocal fold vibration and phonation. On the other hand, in human newborn vocal folds, the entire lamina propria appears as a uniform structure with no vocal ligament,2–4 and the layered structure of the human vocal fold matures during adolescence.4 It is generally accepted that tensile and compressive strains have direct effects on cell morphology and structure, including changes in cytoskeletal structure and organization. Cytoskeletons not only provide cytoskeletal structure but also play the role of mechanoreceptor of the cells. Vocal fold stellate cells (VFSCs)5 in the maculae flavae, which are located at both ends of the human vocal fold mucosa, constantly synthesize extracellular matrices

From the Department of Otolaryngology–Head and Neck Surgery, Kurume University School of Medicine, Kurume, Japan. Editor’s Note: This Manuscript was accepted for publication May 20, 2014. This article has been accepted for presentation at the 135th Annual Meeting of the American Laryngological Association, Las Vegas, Nevada, U.S.A, May 14–15, 2014. The authors have no funding, financial relationships, or conflicts of interest to disclose. This investigation was supported by a Grant-in-Aid for Scientific Research (No.24592612) from the Japanese Ministry of Education, Culture, Sports, Science and Technology. Send correspondence to Kiminori Sato, MD, PhD, Department of Otolaryngology–Head and Neck Surgery, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan. E-mail: [email protected] DOI: 10.1002/lary.24776

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that are essential for the viscoelasticity of the human vocal fold mucosa.5–10 The VFSCs in the human maculae flava are inferred to be involved in the metabolism of extracellular matrices essential for the viscoelasticity in the human vocal fold mucosa and form the characteristic layered structure of the human vocal fold mucosa.5–10 The maculae flava containing the VFSCs are considered to be an important structure in the growth, development, and aging of the human vocal fold mucosa.9,10 Our previous studies have supported the hypothesis that the tension caused by phonation (vocal fold vibration) after birth stimulates VFSCs in the anterior and posterior maculae flavae to accelerate production of extracellular matrices and form the vocal ligament, Reinke’s space, and the characteristic layered structure.3,11–14 The tension caused by phonation seems to regulate the behavior of the VFSCs in the maculae flava of the human vocal fold. It is of interest whether the mechanical forces caused by vocal fold vibration from outside the VFSCs in the maculae flava through cell-matrix contacts influence intracellular signaling cascades that ultimately alter many cellular behaviors. In the present study, the microstructure of the intermediate filaments and the expression of their characteristic proteins were investigated regarding VFSCs in the anterior and posterior maculae flavae of the human newborn vocal fold. The roles of vocal fold vibration and phonation (mechanotransduction) after birth were also discussed.

MATERIALS AND METHODS Three human newborn vocal fold mucosae obtained from autopsy cases (Department of Pathology, Kurume University,

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Fig. 1. Transmission electron microscopy of the newborn vocal fold stellate cell in the macula flava. (A, B) Tannic acid stain. (C) Uranyl acetate and lead citrate stain. AM 5 amorphous material; CF 5 collagenous fibers; CP 5 cytoplasmic process; EF 5 elastic fibers; FR 5 free ribosome; GJ 5 gap junction; V 5 vesicle.

Japan) were investigated by light and electron microscopy. Any diseases that could possibly affect the tissue of the vocal fold were not observed. For light microscopy, specimens were fixed in 10% formalin, dehydrated in graded concentrations of ethanol, and embedded in paraffin. Hematoxylin-Eosin stain was used for each section, and immunohistochemical staining was carried out. Cytokeratin, vimentin, glial fibrillary acidic protein (GFAP), and desmin were detected histologically in formalin-fixed and paraffin-embedded tissue by immunohistochemistry, for which a universal immuno-enzyme polymer method staining kit (Histofine Simple Stain MAX-PO; Nichirei, Tokyo, Japan) was used. All specimens were sectioned to a thickness of 5 mm to 6 mm and mounted on glass slides. Deparaffinized and hydrated sections were rinsed with 0.01-mol/L phosphate buffered saline (PBS) at pH 7.4. The specimens were covered with 3% hydrogen peroxide for 10 minutes and rinsed with 0.01-mol/L PBS, followed by treatment with normal mouse serum. The specimens were then incubated with the primary antibody overnight at 4 C. A 1:100 diluted monoclonal antibody against cytokeratin, a 1:100 diluted monoclonal antibody against vimentin, a 1:50 diluted monoclonal antibody against GFAP, and a 1:100 diluted monoclonal antibody against desmin (Abcam plc. Cambridge, UK) were used. After rinsing with PBS and labeling with the universal immuno-enzyme polymer method staining kit, a color reaction was developed with 3,30 -diaminobenzidine for 2 to 30 minutes at room temperature. Immunoreactivity was examined by light microscopy. For transmission electron microscopy, the specimens were fixed in 2.5% glutaraldehyde at 4 C for 2 hours, rinsed with cacodylate buffer solution, and postfixed in 2% osmium tetroxide with cacodylate buffer solution at 4 C for 2 hours. After rinsing with cacodylate buffer solution, the specimens were dehydrated in graded concentrations of ethanol and embedded in epoxy resin. Semi-thin sections were prepared with an ultramicrotome, stained with 1% toluidine blue, and examined with a light microscope. Thin sections were made with an ultramicrotome and stained with uranyl acetate and lead citrate and tannic acid to make the elastin apparent. Observation was conducted with a H-7650 transmission electron microscope (Hitachi High-Technologies Corporation, Tokyo, Japan).

RESULTS Many VFSCs were distributed in the anterior and posterior macula flavae in the human newborn vocal fold (Fig. 1; Fig. 2). Laryngoscope 124: November 2014

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Electron Microscopic Findings of the Vocal Fold Stellate Cells in the Newborn Maculae Flava The VFSCs in the anterior and posterior macula flavae were stellate or oval in shape and possessed cytoplasmic processes (Fig. 1A), and some VFSCs formed gap junctions with each other (Fig. 1B). A few lipid droplets were present in the cytoplasm. The nucleus in the newborn VFSCs was oval. The mucleus-cytoplasm ration was high, and intracellular organelles such as endoplasmic reticulum and Golgi apparatus were not very well developed (Fig. 1A). There were few mitochondria and they were small. Free ribosomes were well developed in the cytoplasm (Fig. 1C). The intermediate filaments in the cytoplasm were few in number (Fig. 1C). Along the periphery of the cytoplasm of the newborn VFSCs that had developed intracellular organelles, vesicles were present (Fig. 1C). Newly released amorphous materials from the vesicles were present on the surface of the newborn VFSCs (Fig. 1C). These electron microscopic findings indicate that the VFSCs in the newborn maculae flava were immature and possessed some features of mesenchymal cells.

Light Microscopic Findings of the Vocal Fold Stellate Cells in the Newborn Maculae Flava The intermediate filaments in the cytoplasm of the newborn VFSCs (in the anterior and posterior macula flavae) were few in number. However, their characteristic proteins (vimentin, desmin, GFAP, cytokeratin) had already expressed (Fig. 2).

DISCUSSION Mechanotransduction is the term for the ability of living tissues to sense mechanical stress and respond by tissue remodeling. More recently, mechanotransduction expanded to include the sensation of stress, its translation into a biochemical signal, and the sequence of biological responses that it produces. Mechanical stress has become increasingly recognized as one of the primary and essential factors controlling biological functions, ultimately affecting the functions of the cells, tissue, and Sato et al.: Newborn Vocal Fold Stellate Cell Cytoskeleton

Fig. 2. Immunohistochemical staining of VFSCs in the human newborn macula flava. (A) Vimentine. (B) Desmin. (C) Glial fibrillary acidic protein (GFAP). (D) cytokeratin. VFSC 5 vocal fold stellate cell.

organs.15 It is suggested that the mechanical stress caused by phonation (vocal fold vibration) is one of the primary and essential factors controlling biological functions, ultimately affecting the function of the cells in the vocal fold mucosa. The bending stresses on the vocal fold associated with phonation (vocal fold vibration) are greatest in the region of the maculae flavae located at both ends of the vocal fold mucosa.16 However, the role of mechanotransduction in the vibrating vocal fold mucosa remains unclear. Intermediate filaments have a diameter of about 8 nm to 12 nm, which makes them intermediate in size between microtubules and microfilaments.17 They serve as a scaffold to support the entire cytoskeletal framework and play a structural role.17 They are also thought to have a tension-bearing role because they often occur in areas of cells that are subject to mechanical stress.17 The intermediate filaments are distributed in the cytoplasm of the VFSCs.18 The VFSCs are present in maculae flava subject to a mechanical stress from vocal fold vibration. Therefore, the intermediate filaments of the VFSCs may have a tension-bearing role. Because of the tissue specificity of intermediate filaments, cells from different tissues can be distinguished on the basis of the intermediate filament protein present.17 Our previous study revealed that the intermediate filaments, including vimentin, desmin and GFAP, are distributed in the cytoplasm of the adult VFSCs.18 And Laryngoscope 124: November 2014

cytokeratin is also expressed in the cytoplasm of the adult VFSCs (unpublished data). Vimentin is the major subunit protein of the intermediate filaments of mesenchymal cells. Desmin belongs to the intermediate filament protein family. It is characteristic of myogenic crest cells and is found in muscle cells. GFAP is a member of the intermediate filament protein family and characteristic of neural crest cells. It is heavily and specifically expressed in astrocytes and certain other astroglia in the central nervous system. In addition, the neural stem cells strongly expressed GFAP on a frequent basis. Cytokeratin is the protein of the intermediate filaments of epithelial cells. Therefore, the VFSCs express proteins of all three germ layers. Consequently, it is suggested that the VFSCs are undifferentiated cells and have the ability of multipotency. As a result of this heterogeneity, the VFSCs are not yet as fully differentiated as conventional fibroblasts, and it is uncertain whether the VFSCs derive from the same embryonic source as conventional fibroblasts in the human vocal fold mucosa. The VFSCs show the morphologic features of the hepatic stellate cells, which are desmin-positive cells with perinuclear vitamin A droplets.18 And GFAP and vimentin seen in the hepatic stellate cells are also identified in the VFSCs.18 Consequently, the VFSCs in the human maculae flava are extrahepatic stellate cells and a member of the proposed diffuse stellate cell system.19 There is growing evidence that the VFSCs in the human maculae flava are somatic (mesenchymal) stem Sato et al.: Newborn Vocal Fold Stellate Cell Cytoskeleton

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cells or progenitor cells in the human vocal fold mucosa.20 Human maculae flavae, located at both ends of the vocal fold mucosa can be a candidate for a stem cell niche, which is a microenvironment nurturing a pool of stem cells that are VFSCs.20 It is readily apparent that tensile and compressive strains can have direct effects on cell morphology and structure, including changes in the cell membrane, shape, and volume as well as cytoskeletal structure and organization.21 These physical changes can be converted into changes in cell signaling and transcriptional activities in the nucleus to cause alterations in cellular differentiation, proliferation, and migration.21 The function and fate of stem cells are regulated by various microenvironmental factors.21 In addition to chemical factors, mechanical factors can also modulate stem cell survival, organization, migration, proliferation, and differentiation.21 Stem cells are potentially one of the main players in the phenotype determination of a tissue in response to mechanical loading.21 In the present investigation, the microstructure of the intermediate filaments and the expression of their characteristic proteins were investigated regarding the VFSCs in the maculae flava of the human newborn vocal fold mucosa. Present investigation revealed that the intermediate filaments in the cytoplasm of the newborn VFSCs were few in number. However, their characteristic proteins (vimentin, desmin, GFAP, cytokeratin) had already expressed. It is suggested that vocal fold vibration has effects on cell morphology and structure, including cytoskeletal structure and organization. Consequently, these physical changes can be converted into changes in VFSC signaling and transcriptional activities in the nucleus to cause alterations in the differentiation, proliferation, and migration of the VFSCs. Current scientific findings suggest that the magnitude and frequency of tensile strain are particularly important in determining the type of mechanically induced differentiation that stem cells will undergo.21 The VFSCs reside in the macula flava, which is the microenvironment where the magnitude and frequency of tensile strain are greatest during vocal fold vibration. Mechanotransduction of the VFSCs in the maculae flava caused by vocal fold vibration could possibly be an important factor in regulating the function and fate of the VFSCs. The function and fate of the VFSCs are regulated by various microenvironmental factors. In addition to chemical factors, mechanical factors could also modulate the behavior of the VFSCs.

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CONCLUSION The cytoskeletal structure of the newborn VFSCs is under development. Consequently, the newborn VFSCs have not acquired mechanical regulation. However, the characteristic proteins of intermediate filament protein (vimentin, desmin, GFAP, cytokeratin) had already expressed in the newborn VFSCs.

BIBLIOGRAPHY 1. Hirano M. Phonosurgery. Basic & clinical investigations. Otologia (Fukuoka) 1975;21(suppl 1):239–260. 2. Hirano M, Sato K. Histological Color Atlas of the Human Larynx. San Diego, CA: Singular Publishing Group Inc.; 1993. 3. Sato K, Hirano M, Nakashima T. Fine structure of the human newborn and infant vocal fold mucosae. Ann Otol Rhinol Laryngol 2001;110:417– 424. 4. Hirano M, Kurita S, Nakashima T. Growth, development and aging of human vocal folds. In: Bless DM, Abbs JH, eds. Vocal Fold Physiology. San Diego, CA: College-Hill Press; 1983: 22–43. 5. Sato K, Hirano M, Nakashima T. Stellate cells in the human vocal fold. Ann Otol Rhinol Laryngol 2001;110:319–325. 6. Sato K, Hirano M, Nakashima T. Vitamin A-storing stellate cells in the human vocal fold. Acta Otolaryngol 2003;123:106–110. 7. Sato K, Hirano M, Nakashima T. Age-related changes in vitamin A-storing stellate cells of human vocal fold. Ann Otol Rhinol Laryngol 2004;113: 108–112. 8. Sato K, Nakashima T. Vitamin A-storing stellate cells in the human newborn vocal fold. Ann Otol Rhinol Laryngol 2005;114:517–524. 9. Sato K, Umeno H, Nakashima T. Functional histology of the macula flava in the human vocal fold. Part 1: its roles in the adult vocal fold. Folia Phoniatr Logop 2010;62:178–184. 10. Sato K, Umeno H, Nakashima T. Functional histology of the macula flava in the human vocal fold. Part 2: its roles in the growth and development of the vocal fold. Folia Phoniatr Logop 2010;62:263–270. 11. Sato K, Hirano M. Histologic investigation of the macula flava of the human newborn vocal fold. Ann Otol Rhinol Laryngol 1995;104:556– 562. 12. Sato K, Nakashima T, Nonaka S, Harabuchi Y. Histopathologic investigations of the unphonated human vocal fold mucosa. Acta Otolaryngol 2008;128:694–701. 13. Sato K, Umeno H, Nakashima T, Nonaka S, Harabuchi Y. Expression and distribution of hyaluronic acid and CD44 in unphonated human vocal fold mucosa. Ann Otol Rhinol Laryngol 2009;118:773–780. 14. Sato K, Umeno H, Nakashima T, Nonaka S, Harabuchi Y. Histopathologic investigations of the unphonated human child vocal fold mucosa. J Voice 2012;26:37–43. 15. Mofrad M, Kamm R. Cellular Mechanotransduction. Diverse Perspectives from Molecules to Tissues. Mofrad M, Kamm R, eds. New York, NY: Cambridge University Press; 2010. 16. Titze IR, Hunter EJ. Normal vibration frequencies of the vocal ligament. J Acoust Soc Am 2004;115:2264–2269. 17. Becker W, Kleinsmith L, Hardin J. Intermediate filament. In: The World of the Cell. 6th ed. San Francisco, CA: Pearson Education, Inc.; 2006: 446–450. 18. Sato K, Umeno T, Nakashima T. Vocal Fold Stellate cells in the human macula flava and the diffuse stellate cell system. Ann Otol Rhinol Laryngol 2012;121:51–56. 19. Zhao L, Burt AD. The diffuse stellate cell system. J Mol Hist 2007;38:53– 64. 20. Sato K, Umeno T, Nakashima T. Vocal fold stem cells and their niche in the human vocal fold. Ann Otol Rhinol Laryngol 2012;121:798–803. 21. Kurpinski K, Janario R, Chien S, Li S. Mechanical regulation of stem cells: implications in tissue remodeling. In: Cellular Mechanotransduction. Diverse Perspectives from Molecules to Tissues. Mofrad M, Kamm R, eds. New York, NY: Cambridge University Press; 2010: 403–416.

Sato et al.: Newborn Vocal Fold Stellate Cell Cytoskeleton

Cytoskeleton of newborn vocal fold stellate cells.

Vocal fold stellate cells (VFSCs) in the human maculae flavae located at both ends of the vocal fold mucosa are inferred to be involved in the metabol...
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