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HYPOTHESIS: To evaluate the effectiveness of the menin-MLL inhibitor, MI503, as a conservative treatment of middle ear cholesteatoma (cholesteatoma) in a mouse model and to confirm its safety profile regarding auditory function in vivo. BACKGROUND: Cholesteatoma is a mass formed by the keratinizing squamous epithelium in the tympanic cavity and/or mastoid and subepithelial connective tissue and by the progressive accumulation of keratin debris with/without a surrounding inflammatory reaction. Although the main treatment is surgical therapy, the techniques to prevent recurrence remain a critical area of research. Recently, the use of MI503 in experiments resulted in the inhibition of the growth of cholesteatoma in vivo under histone modification. METHODS: After cholesteatoma was induced in ICR mice (n = 7) by keratinocyte growth factor expression vector transfection, MI503 (50 µM) or phosphate-buffered saline was topically injected for 14 days. The effects of MI503 against cholesteatoma were analyzed by micro-computed tomography images. For the in vivo ototoxicity study, a single intratympanic injection of MI503 (50 or 500 µM) or phosphate-buffered saline (n = 4 each) was done in the ICR mice. An auditory brainstem response was performed at days 0, 1, and 14. For morphological analysis, immunostaining for Phalloidin/F-actin and Myo7a was performed. RESULTS: MI503 reduced keratinocyte growth factor-induced cholesteatoma in vivo (4 of 4 [100%]). No difference was found in the mean variation of the average of the auditory brainstem response thresholds between the three groups in the in vivo ototoxicity study, thus confirming its safety profile regarding auditory function. MI503 does not demonstrate any deleterious effects on murine hair cells when assessed by immunostaining. CONCLUSION: These findings demonstrate an encouraging safety profile for the use of menin-MLL inhibitor for the conservative treatment of cholesteatoma.
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Colesteatoma da Orelha Média , Colesteatoma , Ototoxicidade , Animais , Camundongos , Colesteatoma da Orelha Média/tratamento farmacológico , Fator 7 de Crescimento de Fibroblastos , Epigênese Genética , Camundongos Endogâmicos ICR , Microtomografia por Raio-X , Colesteatoma/cirurgia , Orelha Média , FosfatosRESUMO
During development, cranial neural crest (NC) cells display a striking transition from collective to single-cell migration and undergo a mesenchymal-to-epithelial transformation to form a part of the middle ear epithelial cells (MEECs). While MEECs derived from NC are known to control homeostasis of the epithelium and repair from otitis media, paracrine action of keratinocyte growth factor (KGF) promotes the growth of MEECs and induces middle ear cholesteatoma (cholesteatoma). The animal model of cholesteatoma was previously established by transfecting a human KGF-expression vector. Herein, KGF-inducing cholesteatoma was studied in Wnt1-Cre/Floxed-enhanced green fluorescent protein (EGFP) mice that conditionally express EGFP in the NC lineages. The cytokeratin 14-positive NC lineage expanded into the middle ear and formed cholesteatoma. Moreover, the green fluorescent protein-positive NC lineages comprising the cholesteatoma tissue expressed p75, an NC marker, with high proliferative activity. Similarly, a large number of p75-positive cells were observed in human cholesteatoma tissues. Injections of the immunotoxin murine p75-saporin induced depletion of the p75-positive NC lineages, resulting in the reduction of cholesteatoma in vivo. The p75 knockout in the MEECs had low proliferative activity with or without KGF protein in vitro. Controlling p75 signaling may reduce the proliferation of NC lineages and may represent a new therapeutic target for cholesteatoma.
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BACKGROUND: External auditory canal (EAC) squamous cell carcinoma (SCC) is a rare disease, and the survival rate is low in the advanced stages. It has been reported that miR-34a expression is low in many cancers and acts as a tumor suppressor, but its function in EACSCC has not yet been reported. AIMS: To analyze the miR-34a expression levels in EACSCC specimens using in situ hybridization (ISH). MATERIAL AND METHODS: We performed microRNA ISH for miR-34a detection and immunohistochemical analysis of p53 and Ki67 in the EACSCC and otitis externa (OE) specimens. RESULTS: miR-34a was expressed in the basal and suprabasal layers in the OE epidermis. The pronounced expression of miR-34a was observed in the two cases of T2 (Stage II). In the one case of T3 (Stage III), it was almost the same as that of the OE. On the other hand, the expression levels of miR-34a in the one case of T3 (Stage IV) and two cases of T4 (Stage IV) were apparently reduced. CONCLUSION: We demonstrated that the expression level of miR-34a was higher in early-stage EACSCC and lower in advanced-stage EACSCC. SIGNIFICANCE: The expression level of miR-34a may predict a prognosis in patients with advanced-stage EACSCC.
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Carcinoma de Células Escamosas , MicroRNAs , Carcinoma de Células Escamosas/patologia , Meato Acústico Externo/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , PrognósticoRESUMO
Objectives: The epidermal growth factor receptor (EGFR) is related to the invasion and metastasis of external auditory canal (EAC) squamous cell carcinoma (SCC). The phosphoinositide-dependent protein kinase-1 (PDPK1) accelerates tumor cell growth through anti-apoptotic signaling under the influence of downstream EGFR-mediated signaling pathways. In this study, we investigated the EGFR/PDPK1 axis in the EAC under EGF stimulation. Methods: We confirmed EGFR and PDPK1 expression in human EACSCC specimens immunohistochemically. We next transfected the EGF expression vector in the mouse EAC and then conducted a PDPK1 inhibitory experiment. Immunohistochemical analysis was performed in the mouse EAC, using anti-EGF, anti-EGFR, anti-PDPK1, and anti-Ki67 antibodies. Immunohistochemical analysis of cleaved caspase-3 and terminal deoxy(d)-UTP nick end labeling (TUNEL) detection assays were also performed for the assessment of apoptosis in the inhibitory experiment. Results: Immunohistochemical analysis revealed overexpression and colocalization of EGFR and PDPK1 in human EACSCC specimens. The growth of a protuberant tumor was observed in the mouse EAC in which EGF expression vector was transfected, and EGF, EGFR, PDPK1, and Ki67 labeling indexes (LIs) were significantly increased. PDPK1 inhibition then induced normal epithelial appearance in the EAC. Moreover, EGF, EGFR, PDPK1, and Ki67 LIs were decreased, and cleaved caspase-3 and TUNEL LIs were increased in the EAC. Conclusion: We demonstrated the possibility that PDPK1 plays an important role in EACSCC.Level of Evidence: NA.
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Distinct histone modifications regulate gene expression in certain diseases, but little is known about histone epigenetics in middle ear cholesteatoma. It is known that histone acetylation destabilizes the nucleosome and chromatin structure and induces gene activation. The association of histone acetylation with chronic inflammatory diseases has been indicated in recent studies. In this study, we examined the localization of variously modified histone H3 acetylation at lysine 9, 14, 18, 23, and 27 in paraffin-embedded sections of human middle ear cholesteatoma (cholesteatoma) tissues and the temporal bones of an animal model of cholesteatoma immunohistochemically. As a result, we found that there was a significant increase of the expression levels of H3K27ac both in human cholesteatoma tissues and the animal model. In genetics, super-enhancers are clusters of enhancers that drive the transcription of genes involved in cell identity. Super-enhancers were originally defined using the H3K27ac signal, and then we used H3K27ac chromatin immunoprecipitation followed by sequencing to map the active cis-regulatory landscape in human cholesteatoma. Based on the results, we identified increased H3K27ac signals as super-enhancers of the FOXC2 loci, as well as increased protein of FOXC2 in cholesteatoma. Recent studies have indicated that menin-MLL inhibitor could suppress tumor growth through the control of histone H3 modification. In this study, we demonstrated that the expression of FOXC2 was inhibited by menin-MLL inhibitor in vivo. These findings indicate that FOXC2 expression under histone modifications promoted the pathogenesis of cholesteatoma and suggest that it may be a therapeutic target of cholesteatoma.
Assuntos
Colesteatoma da Orelha Média/metabolismo , Epigênese Genética/genética , Fatores de Transcrição Forkhead , Histonas/metabolismo , Animais , Epigenômica , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji , Camundongos Endogâmicos BALB CRESUMO
OBJECTIVE: Cholesteatoma is an epithelial lesion that expands into the middle ear, resulting in bone destruction. The acceleration of the proliferative activity of epithelial stem/progenitor cells is involved in the pathogenesis of cholesteatoma. Recently, the use of a menin-mixed lineage leukemia 1 (MLL1) inhibitor, MI503, in experiments has resulted in inhibition of the growth of tumors under histone modification. In this study, we investigated the effects of the menin-MLL inhibitor against cholesteatoma growth in an in vivo model. METHODS: We first correlated the expression level of histone H3 trimethylation at lysine 4 (H3K4me3) among cholesteatoma cases, chronic otitis media cases and normal skin tissues. Based on the role of keratinocyte growth factor (KGF) in the development of cholesteatoma, KGF-expression vector was transfected into the ear and we analyzed the expression level of H3K4me3. After cholesteatoma was induced, MI503 was administered daily into the ear for 14 days. RESULTS: We detected the highest labeling index of H3K4me3 in the cholesteatoma specimens. After KGF-expression vector transfection in the mouse ear, a high expression level of H3K4me3 was observed in the epithelial layers. The use of MI503 reduced cholesteatoma in the in vivo model and decreased the proliferation of epithelial stem/progenitor cells in a dose-dependent manner. CONCLUSION: We demonstrated that inhibition of the menin-MLL interaction may be a potentially useful strategy in the conservative treatment of cholesteatoma.
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Colesteatoma da Orelha Média , Animais , Orelha Média , Camundongos , Otite Média , Células-Tronco , TransfecçãoRESUMO
PURPOSE OF REVIEW: Middle ear cholesteatoma is an epithelial lesion that expands into the middle ear, resulting in bone destruction. However, the pathogenesis of this has been unknown. The purpose of this review is to understand the role of keratinocyte growth factor (KGF) during epithelial stem and/or progenitor cell proliferation in middle ear cholesteatoma. RECENT FINDINGS: Many researchers have investigated the molecular mechanism of middle ear cholesteatoma to establish a conservative treatment. Recently, some studies have focused on the stem cells of middle ear cholesteatoma and their detection, but the key molecules for stem cell formation were not shown. SUMMARY: We established an animal model for middle ear cholesteatoma and are showing the results of our studies. KGF expression accelerates the proliferation of stem/progenitor cells through the induction of transcription factor p63 expression in the epithelium of the tympanic membrane and mucosal epithelium overlying the promontory of the cochlea and within the attic. This is typical in middle ear cholesteatoma. Moreover, the partial epithelial-mesenchymal transition under the p63 signaling pathway plays an essential role in epithelial cell growth in middle ear cholesteatoma formation. Understanding p63 expression following KGF expression and associated signaling events can improve therapeutic outcomes in patients with middle ear cholesteatoma.
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Colesteatoma da Orelha Média/metabolismo , Colesteatoma da Orelha Média/patologia , Fator 7 de Crescimento de Fibroblastos/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proliferação de Células , Colesteatoma da Orelha Média/etiologia , Transição Epitelial-Mesenquimal , Humanos , Transdução de SinaisRESUMO
Middle-ear cholesteatoma (cholesteatoma) is a chronic otitis media with an enhanced proliferation of epithelial cells. Negative pressure in the middle ear is thought to be important for the etiology of cholesteatoma. However, the mechanism of cholesteatoma formation remains unclear. Integrin-linked protein kinase (ILK), an important modulator of actin cytoskeletal dynamics, interacts with extracellular matrix and results in the up-regulation of mechanotransduction effector Yes-associated protein (YAP). The L1 cell adhesion molecule (L1CAM) has recently been reported as an activator of the mechanotransduction effectors related to cell proliferation and migration. In this study, we demonstrated a stretch assay for middle-ear cultured cells and performed immunohistochemistry using antibodies against Ilk, Yap, and L1cam. The tympanic membrane was also analyzed within a new middle-ear negative-pressure animal model and human cholesteatoma tissues, using immunohistochemistry with antibodies against ILK, YAP, Ki-67, and L1CAM. The expression of cytoplasmic ILK and nuclear shift of YAP increased in the thickened epithelium of the tympanic membrane under a negative-pressure load and the cholesteatoma. The expression of L1CAM was detected in the stromal cells, which enhanced epithelial cell proliferation depending on ILK signaling events. In conclusion, we demonstrated the possibility that the stromal L1CAM and epithelial ILK-YAP signaling played an important role in epithelial growth under mechanotransduction in cholesteatoma formation.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/fisiologia , Colesteatoma da Orelha Média/metabolismo , Células Epiteliais/metabolismo , Mecanotransdução Celular/fisiologia , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Células Cultivadas , Colesteatoma da Orelha Média/patologia , Modelos Animais de Doenças , Células Epiteliais/patologia , Camundongos , Membrana Timpânica/metabolismo , Membrana Timpânica/patologia , Proteínas de Sinalização YAPRESUMO
DNA methylation is an important epigenetic mechanism for cellular maintenance. However, the methylation pattern and the key molecule regulated epigenetically in oral mucosal regeneration is unclear. In this study, we generated a rat oral ulcer model and investigated the cell proliferative activities and DNA methylation patterns immunohistochemically. We also performed immunohistochemical analysis of a regulator of epithelial stem/progenitor cell differentiation in the rat model. We demonstrated immunohistochemistry using antibodies for the molecules as follows: Ki-67, a marker of cellular proliferation; 5-methylcytosine (5-mC), a marker of DNA methylation; 5-hydroxymethylcytosine (5-hmC), a marker of DNA demethylation; Dnmt1, a maintenance DNA methyltransferase; Dnmt3a and Dnmt3b, de novo DNA methyltransferases; and Wnt5a, a regulator of stem/progenitor cell differentiation. In this model, re-epithelialization was completed at Day 4 after ulceration. Regenerating mucosal hypertrophy reached a peak at Day 5 and appeared normal at Day 14. Ki-67-positive cells increased at Day 2 and returned to normal at Day 6 after ulceration. The ratio of the expression level of 5-mC to 5-hmC declined at Day 5 and returned to normal at Day 6. The expression level of Dnmt1 had not changed compared to the normal control at every time point. On the other hand, the expression levels of Dnmt3a and Dnmt3b had decreased significantly at Day 5 and returned to normal at Day 6. Moreover, Wnt5a-positive cells increased at Day 5. In conclusion, oral mucosal regeneration was strictly regulated by DNA methylation. Moreover, Wnt5a might play a critical role in oral mucosal regeneration.
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Metilação de DNA , Regulação da Expressão Gênica , Mucosa Bucal/crescimento & desenvolvimento , Úlceras Orais/patologia , Regeneração , 5-Metilcitosina/análogos & derivados , Animais , Proliferação de Células , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Imuno-Histoquímica , Ligantes , Masculino , Ratos , Ratos Wistar , Proteína Wnt-5a/metabolismoRESUMO
OBJECTIVE: The middle ear epithelium is derived from the neural crest and endoderm, which line distinct regions of the middle ear cavity. In this study, we investigated the localization of stem/progenitor cells in the middle ear mucosa of adult mice and the effects of keratinocyte growth factor (KGF) on the cell kinetics of stem/progenitor cells in vivo. METHODS: In this study, after KGF-expression vector was transfected in the ear, two kinds of thymidine analogues, BrdU and EdU, were transferred at different time points. BrdU was detected by immunohistochemistry and EdU was detected by click chemistry. We also performed immunohistochemistry using anti-Keratin14 (K14) antibody (an undifferentiated epithelial cell marker), anti-p63 antibody (a stem/progenitor cell marker) and anti-acetylated α-tubulin antibody (a ciliated epithelial cell marker). RESULTS: A large number of EdU-positive cells were detected in the thickened mucosal epithelium of the pars flaccida and attic region at Day 1 after KGF transfection. Interestingly, in the mucosal epithelium overlying the promontory of the cochlea, many EdU-positive cells were detected. These cells were also positive for K14 and p63. The acetylated α-tubulin positive cells were reduced in the attic region at Day 1 after KGF transfection. CONCLUSION: These findings indicate that KGF over-expression may increase stem/progenitor cell proliferation in the mucosal epithelium not only within the attic which is typical in middle ear cholesteatoma, but also overlying the promontory of the cochlea.
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Orelha Média/citologia , Células Epiteliais/metabolismo , Fator 7 de Crescimento de Fibroblastos/metabolismo , Mucosa/citologia , Células-Tronco/fisiologia , Animais , Proliferação de Células , Orelha Média/metabolismo , Fator 7 de Crescimento de Fibroblastos/genética , Queratina-14/metabolismo , Masculino , Camundongos , Mucosa/metabolismo , Transativadores/metabolismo , Transfecção , Tubulina (Proteína)/metabolismoRESUMO
INTRODUCTION: Partial epithelial-mesenchymal transition (p-EMT) is a process by which epithelial cells partially lose their intercellular adhesion and change to obtain migration ability. The transcription factor p63 regulates the expression of cadherin family and induces epithelial cell proliferation. In this study, we hypothesized that p-EMT under p63 expression may be a key factor in epithelial cell growth in middle ear cholesteatoma. METHODS: Specimens were surgically excised from patients with congenital cholesteatoma (CC) (nâ=â48), acquired middle ear cholesteatoma (AC) (nâ=â120), and normal skin tissue (nâ=â34). We analyzed immunohistochemically for the EMT marker (N-cadherin), adherence junction marker (E-cadherin), and tight junction marker (claudin-1, claudin-4, occludin). We also examined the labeling index (LI) of p63 and Proliferating cell nuclear antigen (PCNA) (late S phase marker), and Snail expression as a mobility marker. RESULTS: The expression of p63 (CC 51.0â±â7.4%, AC 50.0â±â5.9%) was significantly higher in the thickened epithelium of CC and AC compared with normal skin tissue (pâ<â0.0001). The loss of E-cadherin was observed (CC 50.0%, AC 55.8%) but the expression patterns in the tight junction were almost normal. N-cadherin was partially detected in the basal and upper layer of epithelium in CC and AC. In contrast to that of normal skin tissue, the LI of PCNA was significantly higher in AC (pâ<â0.0001). The positive rate of Snail was significantly higher in CC (pâ<â0.0001). CONCLUSION: This study indicates that p-EMT via the p63 signaling pathway might plays an essential role in epithelial growth in AC and CC formation, although tight junction formation and terminal differentiation were not affected in those processes.
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Colesteatoma da Orelha Média/patologia , Colesteatoma/congênito , Transição Epitelial-Mesenquimal/fisiologia , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Adulto , Colesteatoma/metabolismo , Colesteatoma/patologia , Colesteatoma da Orelha Média/metabolismo , Epitélio/metabolismo , Epitélio/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
The basal stem/progenitor cell maintains homeostasis of the epidermis. Progressive disturbance of this homeostasis has been implicated as a possible cause in the pathogenesis of epithelial disease, such as middle ear cholesteatoma. In many cases of stem/progenitor cell regulation, the importance of extracellular signals provided by the surrounding cells is well-recognized. Keratinocyte growth factor (KGF) is a mesenchymal-cell-derived paracrine growth factor that specifically participates in skin homeostasis; however, the overexpression of KGF induces middle ear cholesteatoma. In this study, two kinds of thymidine analogs were transferred at different time points and we investigated the effects of overexpressed KGF on the cell kinetics of stem/progenitor cells in vivo. As a result, BrdU(+)EdU(+) cells (stem/progenitor cells) were detected in the thickened epithelium of KGF-transfected specimens. The use of a high-resolution microscope enabled us to analyze the phosphorylated level of p63 in individual nuclei, and the results clearly demonstrated that BrdU(+)EdU(+) cells are regarded as progenitor cells. In the overexpression of KGF, the stimulation of progenitor cell proliferation was inhibited by SU5402, an inhibitor for tyrosine kinase of KGFR. These findings indicate that KGF overexpression may increase stem/progenitor cell proliferation and block terminal differentiation, resulting in epithelial hyperplasia, which is typical in middle ear cholesteatoma.
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Colesteatoma da Orelha Média/etiologia , Células Epidérmicas/fisiologia , Fator 7 de Crescimento de Fibroblastos/metabolismo , Fosfoproteínas/metabolismo , Células-Tronco/fisiologia , Transativadores/metabolismo , Animais , Proliferação de Células , Colesteatoma da Orelha Média/metabolismo , Fator 7 de Crescimento de Fibroblastos/genética , Técnicas de Transferência de Genes , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos Endogâmicos ICR , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
OBJECTIVES: Mechanotransduction plays an important role in cell-proliferative activities. Negative pressure in the middle ear is thought to be an important factor related to the etiology of acquired middle ear cholesteatoma. However, the correlation between negative pressure in the middle ear and the mechanism of middle ear cholesteatoma formation remains unclear. In this study, we investigated the expression of key molecules for mechanotransduction immunohistochemically. METHODS: An immunohistochemical analysis was performed using anti-Wnt5a (a marker of alternative Wnt signaling), -Yes-associated protein (YAP) (a marker of mechanosensing) and -pYAP (phosphorylated YAP at Ser 127: inactivated YAP) antibody in the tympanic membrane (TM) under a negative pressure load and in human middle ear cholesteatoma tissues. RESULTS: The number of Wnt5a-positive cells had increased and YAP nuclear translocation was observed in epithelial and mesenchymal cells in the pars flaccida (PF) of the TM under a negative-pressure load and in human middle ear cholesteatoma tissues. CONCLUSIONS: We demonstrated that negative pressure in the middle ear might possibly induce cell proliferation PF of TM in response to mechanical force (mechanotransduction) through YAP nuclear translocation mediated by alternative Wnt signaling, thus affecting human middle ear cholesteatoma formation.
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Proteínas Reguladoras de Apoptose/metabolismo , Colesteatoma da Orelha Média/metabolismo , Mecanotransdução Celular , Membrana Timpânica/metabolismo , Adulto , Idoso , Animais , Humanos , Masculino , Pessoa de Meia-Idade , Ratos Sprague-Dawley , Via de Sinalização Wnt , Proteína Wnt-5a/metabolismo , Proteínas de Sinalização YAPRESUMO
We reported previously that keratinocyte growth factor (KGF), a mesenchymal cell-derived paracrine growth factor, plays an important role in middle ear cholesteatoma formation, which is characterized by marked proliferation of epithelial cells. Here, we investigated whether KGF, the main factor that induces cholesteatoma, overexpression in vivo results in the formation of cholesteatoma. Flag-hKGF cDNA driven by CMV14 promoter was transfected through electroporation into the external auditory canal (EAC) of rats once (short-term model) or five times on every fourth day (long-term model). Ears transfected with empty vector were used as controls. Successful transfection of plasmids into epithelial and stromal cells was confirmed by Flag immunohistochemistry. In the short-term model, the intensity of KGF protein was the strongest in hKGF transfected ear at day 4. KGF expression induced epithelial cell proliferation, reaching a peak level at day 4 and then decreased later, while in the long-term model, KGF expression in the EAC led to middle ear cholesteatoma formation. In conclusion, we described here a new experimental model of human middle ear cholesteatoma, and demonstrated that KGF and KGF receptor paracrine action play an essential role in middle ear cholesteatoma formation in an in vivo model.
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Colesteatoma da Orelha Média/genética , DNA/genética , Fator 7 de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica , Animais , Colesteatoma da Orelha Média/metabolismo , Modelos Animais de Doenças , Fator 7 de Crescimento de Fibroblastos/biossíntese , Humanos , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Histone acetylation is involved in the regulation of chromatin structure and gene function. We reported previously that histone H3 acetylation pattern is subject to dynamic changes and limited to certain stages of germ cell differentiation during murine spermatogenesis, suggesting a crucial role for acetylation in the process. In the present study, we investigated the effects of hyper- and hypo-acetylation on spermatogenesis. Changes in acetylation level were induced by either in vivo administration of sodium phenylbutyrate, a histone deacetylase inhibitor, or by knockdown of histone acetyltransferases using short hairpin RNA plasmids transfection. Administration of sodium phenylbutyrate induced accumulation of acetylated histone H3 at lysine 9 and lysine 18 in round spermatids, together with spermatid morphological abnormalities and induction of apoptosis through a Bax-related pathway. Knockdown of steroid receptor coactivator 1, a member of histone acetyltransferases, but not general control of amino acid synthesis 5 nor elongator protein 3 by in vivo electroporation of shRNA plasmids, reduced acetylated histone H3 at lysine 9 in round spermatids, and induced morphological abnormalities. We concluded that the proper regulation of histone H3 acetylation levels is important for spermatid differentiation and complex chromatin remodeling during spermiogenesis.
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Histonas/metabolismo , Espermátides/patologia , Testículo/fisiopatologia , Acetilação/efeitos dos fármacos , Animais , Apoptose , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Técnicas de Silenciamento de Genes , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histona Desacetilases/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Tamanho do Órgão , Fenilbutiratos/farmacologia , Espermátides/efeitos dos fármacos , Espermátides/enzimologia , Espermátides/ultraestrutura , Espermatogênese/genética , Testículo/efeitos dos fármacos , Testículo/enzimologia , Testículo/ultraestruturaRESUMO
CONCLUSION: We demonstrated that repression of keratinocyte growth factor (KGF) receptor (KGFR) could be a potentially useful strategy in the conservative treatment of middle ear cholesteatoma. OBJECTIVES: Recently, the use of a selective inhibitor of the KGFR, SU5402, in an in vitro experiment resulted in the inhibition of the differentiation and proliferation of epithelial cells through KGF secretion by fibroblasts isolated from the cholesteatoma. In this study, we investigated the effects of the KGFR inhibitor during middle ear cholesteatoma formation in vivo. METHODS: Based on the role of KGF in the development of cholesteatoma, Flag-hKGF cDNA driven by CMV14 promoter was transfected through electroporation into the external auditory canal of rats five times on every fourth day. Ears transfected with empty vector were used as controls. KGFR selective inhibitor (SU5402) or MEK inhibitor (PD0325901) was administered in the right ear of five rats after vector transfection. In the control, 2% DMSO in PBS was administered in the other ears after vector transfection. RESULTS: The use of a selective KGFR inhibitor, SU5402, completely prevented middle ear cholesteatoma formation in the rats.
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Colesteatoma da Orelha Média/prevenção & controle , Pirróis/uso terapêutico , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Animais , Benzamidas , Colesteatoma da Orelha Média/metabolismo , Difenilamina/análogos & derivados , Avaliação Pré-Clínica de Medicamentos , Meato Acústico Externo/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Vetores Genéticos , Masculino , Ratos Sprague-Dawley , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , TransfecçãoRESUMO
OBJECTIVES/HYPOTHESIS: High negative pressure in the middle ear was thought to be closely related to the etiology of retraction-type cholesteatoma. Recently, it has been detected that mechanical forces are important factors in epithelial turnover and affect cytoskeletal remodeling. Continuous negative pressure in the middle ear may possibly accelerate the proliferation and differentiation of epithelial cells of the tympanic membrane. STUDY DESIGN: Animal experimental study. METHODS: Eleven adult male Sprague-Dawley rats were used, and continuous negative pressure was loaded by connecting a catheter from the rat's middle ear to the supply route of an implantable microinfusion pump, iPRECIO. At 7 days after implantation of the device, an otoendoscopic examination and micro-computed tomography (CT) analysis of the temporal bone were performed; the temporal bones were then collected for histological and immunohistochemical analysis. The degree of proliferation and differentiation of epithelial cells of the tympanic membrane was investigated immunohistochemically using the anti-cytokeratin-5 and anti-cytokeratin-10 antibodies. RESULTS: Otoendoscopic examination revealed retraction of the pars flaccida in all of the ears under negative pressure. In the micro-CT analysis, soft tissue density area in the hypotympanum was observed in all ears under negative pressure. Histological analysis revealed thickened epithelium of the pars flaccida. In this region, the thickness of layers with cytokeratin-5-positive cells and cytokeratin-10-positive cells were increased. CONCLUSIONS: Continuous negative pressure in the middle ear can lead to thickening of the epithelium of the pars flaccida, and may accelerate the proliferation and differentiation of epithelial cells.
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Colesteatoma/etiologia , Orelha Média/fisiopatologia , Perfuração da Membrana Timpânica/etiologia , Animais , Proliferação de Células , Colesteatoma/patologia , Modelos Animais de Doenças , Endoscopia , Células Epiteliais/patologia , Masculino , Otoscopia , Pressão/efeitos adversos , Ratos , Ratos Sprague-Dawley , Perfuração da Membrana Timpânica/patologia , Microtomografia por Raio-XRESUMO
Middle-ear mucosa maintains middle-ear pressure. However, the majority of surgical cases exhibit inadequate middle-ear mucosal regeneration, and mucosal transplantation is necessary in such cases. The aim of the present study was to assess the feasibility of transplantation of isolated mucosal cells encapsulated within synthetic self-assembling peptide nanofiber scaffolds using PuraMatrix, which has been successfully used as scaffolding in tissue engineering, for the repair of damaged middle-ear. Middle-ear bullae with mucosa were removed from Sprague Dawley (SD) transgenic rats, transfected with enhanced green fluorescent protein (EGFP) transgene and excised into small pieces, then cultured up to the third passage. After surgical elimination of middle-ear mucosa in SD recipient rats, donor cells were encapsulated within PuraMatrix and transplanted into these immunosuppressed rats. Primary cultured cells were positive for pancytokeratin but not for vimentin, and retained the character of middle-ear epithelial cells. A high proportion of EGFP-expressing cells were found in the recipient middle-ear after transplantation with PuraMatrix, but not without PuraMatrix. These cells retained normal morphology and function, as confirmed by histological examination, immunohistochemistry, and electron microscopy, and multiplied to form new epithelial and subepithelial layers together with basement membrane. The present study demonstrated the feasibility of transplantation of cultured middle-ear mucosal epithelial cells encapsulated within PuraMatrix for regeneration of surgically eliminated mucosa of the middle-ear in SD rats.