PTHrP participates in the bone destruction of middle ear cholesteatoma via promoting macrophage differentiation into osteoclasts induced by RANKL. / PTHrP促进RANKLè¯±å¯¼å·¨å™¬ç»†èƒžåˆ†åŒ–ä¸ºç ´éª¨ç»†èƒžå‚ä¸Žä¸­è€³èƒ†è„‚ç˜¤éª¨ç ´å.

OBJECTIVES: Progressive bone resorption and destruction is one of the most critical clinical features of middle ear cholesteatoma, potentially leading to various intracranial and extracranial complications. However, the mechanisms underlying bone destruction in middle ear cholesteatoma remain unclear. This study aims to explore the role of parathyroid hormone-related protein (PTHrP) in bone destruction associated with middle ear cholesteatoma. METHODS: A total of 25 cholesteatoma specimens and 13 normal external auditory canal skin specimens were collected from patients with acquired middle ear cholesteatoma. Immunohistochemical staining was used to detect the expressions of PTHrP, receptor activator for nuclear factor-kappa B ligand (RANKL), and osteoprotegerin (OPG) in cholesteatoma and normal tissues. Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the presence of TRAP positive multi-nucleated macrophages in cholesteatoma and normal tissues. Mono-nuclear macrophage RAW264.7 cells were subjected to interventions, divided into a RANKL intervention group and a PTHrP+ RANKL co-intervention group. TRAP staining was used to detect osteoclast formation in the 2 groups. The mRNA expression levels of osteoclast-related genes, including TRAP, cathepsin K (CTSK), and nuclear factor of activated T cell cytoplasmic 1 (NFATc1), were measured using real-time polymerase chain reaction (real-time PCR) after the interventions. Bone resorption function of osteoclasts was assessed using a bone resorption pit analysis. RESULTS: Immunohistochemical staining showed significantly increased expression of PTHrP and RANKL and decreased expression of OPG in cholesteatoma tissues (all P<0.05). PTHrP expression was significantly positively correlated with RANKL, the RANKL/OPG ratio, and negatively correlated with OPG expression (r=0.385, r=0.417, r=-0.316, all P<0.05). Additionally, the expression levels of PTHrP and RANKL were significantly positively correlated with the degree of bone destruction in cholesteatoma (r=0.413, r=0.505, both P<0.05). TRAP staining revealed a large number of TRAP-positive cells, including multi-nucleated osteoclasts with three or more nuclei, in the stroma surrounding the cholesteatoma epithelium. After 5 days of RANKL or PTHrP+RANKL co-intervention, the number of osteoclasts was significantly greater in the PTHrP+RANKL co-intervention group than that in the RANKL group (P<0.05), with increased mRNA expression levels of TRAP, CTSK, and NFATc1 (all P<0.05). Scanning electron microscopy of bone resorption pits showed that the number (P<0.05) and size of bone resorption pits on bone slices were significantly greater in the PTHrP+RANKL co-intervention group compared with the RANKL group. CONCLUSIONS: PTHrP may promote the differentiation of macrophages in the surrounding stroma of cholesteatoma into osteoclasts through RANKL induction, contributing to bone destruction in middle ear cholesteatoma.

m6A modification of lncRNA in middle ear cholesteatoma. / 中耳胆脂瘤中lncRNA的m6A修饰.

OBJECTIVES: Middle ear cholesteatoma is a non-tumorous condition that typically leads to hearing loss, bone destruction, and other severe complications. Despite surgery being the primary treatment, the recurrence rate remains high. Therefore, exploring the molecular mechanisms underlying cholesteatoma is crucial for discovering new therapeutic approaches. This study aims to explore the involvement of N6-methyladenosine (m6A) methylation in long non-coding RNAs (lncRNAs) in the biological functions and related pathways of middle ear cholesteatoma. METHODS: The m6A modification patterns of lncRNA in middle ear cholesteatoma tissues (n=5) and normal post-auricular skin tissues (n=5) were analyzed using an lncRNA m6A transcriptome microarray. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted to identify potential biological functions and signaling pathways involved in the pathogenesis of middle ear cholesteatoma. Methylated RNA immunoprecipitation (MeRIP)-PCR was used to validate the m6A modifications in cholesteatoma and normal skin tissues. RESULTS: Compared with normal skin tissues, 1 525 lncRNAs were differentially methylated in middle ear cholesteatoma tissues, with 1 048 showing hypermethylation and 477 showing hypomethylation [fold change (FC)≥3 or <1/3, P<0.05]. GO enrichment analysis indicated that hypermethylated lncRNAs were involved in protein phosphatase inhibitor activity, neuron-neuron synapse, and regulation of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor activity. Hypomethylated lncRNAs were associated with mRNA methyltransferase activity, secretory granule membrane, and mRNA methylation. KEGG analysis revealed that hypermethylated lncRNAs were mainly associated with 5 pathways: the Hedgehog signaling pathway, viral protein interaction with cytokines and cytokine receptors, mitogen-activated protein kinase (MAPK) signaling pathway, cytokine-cytokine receptor interaction, and adrenergic signaling in cardiomyocytes. Hypomethylated lncRNAs were mainly involved in 4 pathways: Renal cell carcinoma, tumor necrosis factor signaling pathway, transcriptional misregulation in cancer, and cytokine-cytokine receptor interaction. Additionally, MeRIP-PCR confirmed the changes in m6A methylation levels in NR_033339, NR_122111, NR_130744, and NR_026800, consistent with microarray analysis. Real-time PCR also confirmed the significant upregulation of MAPK1 and NF-κB, key genes in the MAPK signaling pathway. CONCLUSIONS: This study reveals the m6A modification patterns of lncRNAs in middle ear cholesteatoma, suggests a direction for further research into the role of lncRNA m6A modification in the etiology of cholesteatoma. The findings provide potential therapeutic targets for the treatment of middle ear cholesteatoma.

Osteopontin-driven partial epithelial-mesenchymal transition governs the development of middle ear cholesteatoma.

Cholesteatoma is a common disease of the middle ear. Currently, surgical removal is the only treatment option and patients face a high risk of relapse. The molecular basis of cholesteatoma remains largely unknown. Here, we show that Osteopontin (OPN), a predominantly secreted protein, plays a crucial role in the development of middle ear cholesteatoma. Global transcriptome analysis revealed the loss of epithelial features and an enhanced immune response in human cholesteatoma tissues. Quantitative RT-PCR and immunohistochemical staining of middle ear cholesteatoma validated the reduced expression of epithelial markers, as well as the elevated expression of mesenchymal markers including Vimentin and Fibronectin, but not N-Cadherin, α-smooth muscle actin (α-SMA) or ferroptosis suppressor protein 1 (FSP1), indicating a partial epithelial-mesenchymal transition (EMT) state. Besides, the expression of OPN was significantly elevated in human cholesteatoma tissues. Treatment with OPN promoted cell proliferation, survival and migration and led to a partial EMT in immortalized human keratinocyte cells. Importantly, blockade of OPN signaling could remarkably improve the cholesteatoma-like symptoms in SD rats. Our mechanistic study demonstrated that the AKT-zinc finger E-box binding homeobox 2 (ZEB2) axis mediated the effects of OPN. Overall, these findings suggest that targeting the OPN signaling represents a promising strategy for the treatment of middle ear cholesteatoma.

Expression of MMP-14 and its role in bone destruction in middle ear cholesteatoma: A prospective observational study.

Cholesteatoma is a noncancerous cystic lesion caused by an abnormal growth of keratinizing squamous epithelium which is invasive and capable of destroying structures. A prospective study on the expression of membrane type1-matrix metalloproteinases (MMP-14) and its related influencing factors in middle ear cholesteatoma was conducted to fully understand the pathogenesis of cholesteatoma in the molecular level. We examined the expression of MMP-14 by immunohistochemical staining 39 middle ear cholesteatoma specimens and 10 external auditory meatus epithelial cell specimens. The cholesteatoma specimens were divided into 4 groups according to the degree of destruction of the ossicles during surgery. The associated factors affecting MMP-14 expression were analyzed using statistical methods; The positive expression of MMP-14 in the epithelium of the external auditory canal was significantly different between middle ear cholesteatoma and normal patients (P < .05); Gender, age, and the degree of hearing loss had no statistically significant effect on MMP-14 expression (P > .05); The expression of MMP-14 was positively correlated with the severity of bone destruction (R = 0.535, P < .05); MMP-14 plays an important role in the pathological development of the epithelium of cholesteatoma; MMP-14 expression in middle ear cholesteatoma tissue was not strongly correlated with the level of hearing loss, age or gender, but was positively correlated with the degree of middle ear bone destruction.

The Hyperproliferation Mechanism of Cholesteatoma Based on Proteomics: SNCA Promotes Autophagy-Mediated Cell Proliferation Through the PI3K/AKT/CyclinD1 Signaling Pathway.

Cholesteatoma is a chronic inflammatory ear disease with abnormal keratinized epithelium proliferation and tissue damage. However, the mechanism of keratinized epithelium hyperproliferation in cholesteatoma remains unknown. Hence, our study sought to shed light on mechanisms affecting the pathology and development of cholesteatoma, which could help develop adjunctive treatments. To investigate molecular changes in cholesteatoma pathogenesis, we analyzed clinical cholesteatoma specimens and paired ear canal skin with mass spectrometry-based proteomics and bioinformatics. From our screen, alpha-synuclein (SNCA) was overexpressed in middle ear cholesteatoma and might be a key hub protein associated with inflammation, proliferation, and autophagy in cholesteatoma. SNCA was more sensitive to lipopolysaccharide-induced inflammation, and autophagy marker increase was accompanied by autophagy activation in middle ear cholesteatoma tissues. Overexpression of SNCA activated autophagy and promoted cell proliferation and migration, especially under lipopolysaccharide inflammatory stimulation. Moreover, inhibiting autophagy impaired SNCA-mediated keratinocyte proliferation and corresponded with inhibition of the PI3K/AKT/CyclinD1 pathways. Also, 740Y-P, a PI3K activator reversed the suppression of autophagy and PI3K signaling by siATG5 in SNCA-overexpressing cells, which restored proliferative activity. Besides, knockdown of SNCA in RHEK-1 and HaCaT cells or knockdown of PI3K in RHEK-1 and HaCaT cells overexpressing SNCA both resulted in attenuated cell proliferation. Our studies indicated that SNCA overexpression in cholesteatoma might maintain the proliferative ability of cholesteatoma keratinocytes by promoting autophagy under inflammatory conditions. This suggests that dual inhibition of SNCA and autophagy may be a promising new target for treating cholesteatoma.

Morphopathogenesis of Adult Acquired Cholesteatoma.

Background and Objectives. The aim of this study was to compare the distribution of proliferation markers (Ki-67, NF-κß), tissue-remodeling factors (MMP-2, MMP-9, TIMP-2, TIMP-4), vascular endothelial growth factor (VEGF), interleukins (IL-1 and IL-10), human beta defensins (HßD-2 and HßD-4) and Sonic hedgehog gene protein in cholesteatoma and control skin. Methods. Nineteen patient cholesteatoma tissues and seven control skin materials from cadavers were included in the study and stained immunohistochemically. Results. Statistically discernible differences were found between the following: the Ki-67 in the matrix and the Ki-67 in the skin epithelium (p = 0.000); the Ki-67 in the perimatrix and the Ki-67 in the connective tissue (p = 0.010); the NF-κß in the cholesteatoma matrix and the NF-κß in the epithelium (p = 0.001); the MMP-9 in the matrix and the MMP-9 in the epithelium (p = 0.008); the HßD-2 in the perimatrix and the HßD-2 in the connective tissue (p = 0.004); and the Shh in the cholesteatoma's perimatrix and the Shh in the skin's connective tissue (p = 0.000). Conclusion. The elevation of Ki-67 and NF-κß suggests the induction of cellular proliferation in the cholesteatoma. Intercorrelations between VEGF, NF-κß and TIMP-2 induce neo-angiogenesis in adult cholesteatoma. The similarity in the expression of IL-1 and IL-10 suggests the dysregulation of the local immune status in cholesteatoma. The overexpression of the Sonic hedgehog gene protein in the cholesteatoma proves the selective local stimulation of perimatrix development.

Clinical Effect of Ear Endoscopic Intervention on CMEC Patients and Analysis of the Relationship between ROS, P-Akt, and HIF-1α Expression and the Degree of Bone Destruction.

The clinical efficacy of ear endoscopic intervention in patients with congenital middle ear cholesteatoma (CMEC) is explored, and the relationship between the expression of reactive oxygen species (ROS), phosphorylated protein kinase B (P-Akt), hypoxia-inducible factor-1 α (HIF-1α) and the degree of bone damage are analyzed. A total of 72 CMEC patients admitted to the otolaryngology department of our hospital from 2019 to January 2021 for surgical treatment are selected. According to the different intervention methods, the microscope group and the otolaryngology intervention group are established, respectively, with 36 patients in each group. The patients in the microscope group are treated with a microscope for middle ear cholesteatoma surgery, and the patients in the otoscope intervention group are treated with an otoscope for middle ear cholesteatoma surgery. The experimental results show that ear endoscopic intervention has better clinical efficacy for CMEC patients, which can effectively shorten the operation time, reduce the incidence of postoperative complications, and effectively improve the hearing of patients.

Review of potential medical treatments for middle ear cholesteatoma.

Middle ear cholesteatoma (MEC), is a destructive, and locally invasive lesion in the middle ear driven by inflammation with an annual incidence of 10 per 100,000. Surgical extraction/excision remains the only treatment strategy available and recurrence is high (up to 40%), therefore developing the first pharmaceutical treatments for MEC is desperately required. This review was targeted at connecting the dysregulated inflammatory network of MEC to pathogenesis and identification of pharmaceutical targets. We summarized the numerous basic research endeavors undertaken over the last 30+ years to identify the key targets in the dysregulated inflammatory pathways and judged the level of evidence for a given target if it was generated by in vitro, in vivo or clinical experiments. MEC pathogenesis was found to be connected to cytokines characteristic for Th1, Th17 and M1 cells. In addition, we found that the inflammation created damage associated molecular patterns (DAMPs), which further promoted inflammation. Similar positive feedback loops have already been described for other Th1/Th17 driven inflammatory diseases (arthritis, Crohn's disease or multiple sclerosis). A wide-ranging search for molecular targeted therapies (MTT) led to the discovery of over a hundred clinically approved drugs already applied in precision medicine. Based on exclusion criteria designed to enable fast translation as well as efficacy, we condensed the numerous MTTs down to 13 top drugs. The review should serve as groundwork for the primary goal, which is to provide potential pharmaceutical therapies to MEC patients for the first time in history. Video Abstract.

Middle ear cholesteatoma (MEC) is a destructive and locally invasive ulcerated lesion in the middle ear driven by inflammation which occurs in 10 out of 100,000 people annually. Surgical extraction/excision is the only treatment strategy available and recurrence is high (up to 40% after ten years), therefore developing the first pharmaceutical treatments for MEC is desperately required. This review is focused on the connections between inflammation and MEC pathogenesis. These connections can be used as attack points for pharmaceuticals. For this we summarized the results of research undertaken over the last 30 + years. MEC pathogenesis can be described by specific inflammatory dysregulation already known from arthritis, Crohn's disease or multiple sclerosis. A hallmark of this dysregulation are positive feedback loops of the inflammation further amplifying itself in a vicious circle-like manner. We have identified over one hundred drugs which are already used in clinic to treat other inflammatory diseases, and could potentially be repurposed to treat MEC. To improve and expedite clinical success rates, we applied certain criteria based on our literature searches and condensed these drugs down to the 13 top drugs. We hope the review will serve as groundwork for the primary goal, which is to provide potential pharmaceutical therapies to MEC patients for the first time in history.

Investigation of EGF, IL1-α and IL-6 levels and selected hematological parameters (NLR, MPV) in patients with the chronic cholesteatomatous otitis media.

In this study we aimed to investigate epidermal growth factor (EGF), interleukin (IL1)-α and IL-6 levels, and hematological parameters in the serum samples of patients with chronic cholesteatomatous otitis media (CCOM). This prospective included 40 patients who underwent surgery due to CCOM between June 2020 and May 2021. The stage of middle ear cholesteatoma was determined on each chart using the EAONO/JOS system. The control group comprised of 30 adults who were scheduled for septoplasty over the same period in our hospital, had no otological complaints, and had normal otological findings. The demographic, clinical, and laboratory data of the patients were obtained from the electronic medical record system of our hospital. The serum EGF, IL1-α and IL-6 levels, and hematological parameters (neutrophil-lymphocyte ratio (NLR) and mean platelet volume (MPV)) were compared between the CCOM and control groups. Seven patients had Stage 1 and 33 patients had Stage 2 middle ear cholestatoma. There was no statistically significant difference between the CCOM and control groups in terms of age and gender (p=0.092 and p=0.616, respectively). The serum EGF and IL1-α levels of the CCOM group were statistically significantly higher than those of the control group (p=0.047 and p=0.013, respectively). No statistically significant difference was observed in the serum IL-6 levels of the CCOM and control groups (p=0.675). There was also no significant difference between the CCOM and control groups in terms of the mean NLR and MPV values ​​(p=0.887 and p=0.164, respectively). There was no significant difference between the Stage 1 and Stage 2 cholesteatoma subgroups in terms of the mean EGF, IL1-α, IL-6 levels (p=0.204, p=0.557 and p=0.613, respectively), and the mean NLR and MPV values (p=0.487, p=0.439, respectively). Increased serum EGF and IL1-α levels in patients with CCOM suggest that these cytokines may play a role in cholesteatomatous epithelial hyperproliferation.

The Effect of Zbxz23ir-21 NANO(nanomaterials) Delivery Vector on Apoptosis and PTEN(phosphatase and tensin homolog deleted on chromosome ten)/PI3K(Intracellular phosphatidylinositol kinase)/AKT(related to the A and C kinase) in Children with CHOLESTEATOMA in Middle Ear.

Cholesteatoma of the middle ear is a kind of cystic disease with clear boundary formed by the abnormal growth of keratosquamous epithelium in temporal bone. Cholesteatoma otitis caused by it is a common disease in otorhinolaryngology. The EPR effect promotes the selective distribution of macromolecular substances in tumor tissues, which can increase drug efficacy. The purpose of this paper is to prepare and deliver the mir34a small molecule regulator, rubine, by nanotechnology, and to deliver it to the cells successfully. It can passively target tumor tissue through EPR effect, and play its regulatory role on miR-34a, thus inhibiting the growth of cholesteatoma cells. The effects of nano delivery on apoptosis and PIEN/P13K/AKt of children with middle ear choledochoma were tested in this paper. The experimental results were conducted on cholesteatoma cells as cell lines and balb/c nude mice as experimental objects. The expression of PTEN/PI3K/AKT in experimental group and control group was detected by immunohistochemistry. Apoptosis was discussed by cell activity detection. The physical and chemical properties, encapsulation efficiency, drug release ability in vitro and antitumor activity of nanoparticles in vitro and in vivo were studied. The results of cell level experiments in vitro showed that free RUBINE caused about 15% apoptosis, which was not different from RC NPs. The results showed that the nanoparticles could improve the expression of miR-34 in the cells, and then regulate the expression of Bcl-2, Cdk6 and CyclinD1, and play the inhibitory effect of miR-34a on the proliferation and migration of tumor cells.

Osteoclastic activity in chronic otitis media with cholesteatoma-related bone destruction.

BACKGROUND: Cholesteatoma-related bone destruction is the cause of many complications due to chronic otitis media. This study aimed to evaluate osteoclastic activity in cholesteatoma-related bone destruction using tartrate-resistant acid phosphatase 5b, an enzyme specific to osteoclastic activity. METHOD: Seventy-two patients diagnosed with chronic otitis media were included in this study and were divided into two groups: with and without bone destruction. The blood serum and tissue tartrate-resistant acid phosphatase 5b levels from both groups were compared. RESULTS: There were no significant differences in the level of serum enzymes between both groups. However, in tissue samples, tartrate-resistant acid phosphatase 5b levels were significantly lower in the bone destruction group than the group without bone destruction. CONCLUSION: This study determined that the level of tartrate-resistant acid phosphatase 5b, a specific enzyme for osteoclastic activity in cholesteatoma-related bone destruction, is locally decreased. This data suggests that osteoclastic activity may decrease in cholesteatoma-related bone destruction. However, further experimental and clinical studies are required to clarify this highly complex mechanism.

Super-enhancer Acquisition Drives FOXC2 Expression in Middle Ear Cholesteatoma.

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.

Expression of endoplasmic reticulum stress mRNAs in otitis media.

BACKGROUND: Endoplasmic reticulum (ER) stress is a cellular defense mechanism that occurs when ER function is impaired. OBJECTIVE: This study was designed to evaluate the expression of major mRNAs of ER stress in patients with otitis media with effusion (OME), chronic otitis media (COM), and COM with cholesteatoma (CholeOM). MATERIAL AND METHODS: Specimens were collected during surgery from patients with OME, COM, and CholeOM, and the levels of ER stress mRNAs measured by real-time polymerase chain reaction. Levels of ER stress mRNAs were compared in the three groups and correlated with clinical findings and pus culture results. RESULTS: The level of CHOP mRNA was higher, and the levels of sXBP1 and ATF6 mRNAs lower, in the OME than in the other two groups (p < .05 each). Evaluation of bacterial pus culture negative patients showed that the level of ATF6 mRNA was higher in the CholeOM than in the other two groups (p < .05), whereas evaluation of bacterial pus culture positive patients showed that the level of CHOP mRNA was higher in the OME than in the other groups (p < .05). CONCLUSIONS AND SIGNIFICANCE: ER stress may be involved in the pathophysiology of OM and the levels of ER stress mRNAs were expressed differently in each type of otitis media according to bacterial culture test results.

Chronic inflammation of middle ear cholesteatoma promotes its recurrence via a paracrine mechanism.

BACKGROUND: Cholesteatoma disease is an expanding lesion in the middle ear. Hearing loss and facial paralysis alongside with other intracranial complications are found. No pharmaceutical treatment is available today and recurrence after surgical extraction occurs. We investigated possible TLR4-based mechanisms promoting recurrence and explore possible treatments strategies. METHODS: We isolated fibroblasts and epidermal stem cells from cholesteatoma tissue and healthy auditory canal skin. Subsequently, their expression under standard culture conditions and after stimulation with LPS was investigated by RT-qPCR. Cell metabolism and proliferation were analysed upon LPS treatment, with and without TLR4 antagonist. An indirect co-culture of fibroblasts and epidermal stem cells isolated from cholesteatoma tissue was utilized to monitor epidermal differentiation upon LPS treatment by RT-qPCR and immunocytochemistry. RESULTS: Under standard culture conditions, we detected a tissue-independent higher expression of IL-1ß and IL-8 in stem cells, an upregulation of KGF and IGF-2 in both cell types derived from cholesteatoma and higher expression of TLR4 in stem cells derived from cholesteatoma tissue. Upon LPS challenge, we could detect a significantly higher expression of IL-1α, IL-1ß, IL-6 and IL-8 in stem cells and of TNF-a, GM-CSF and CXCL-5 in stem cells and fibroblasts derived from cholesteatoma. The expression of the growth factors KGF, EGF, EREG, IGF-2 and HGF was significantly higher in fibroblasts, particularly when derived from cholesteatoma. Upon treatment with LPS the metabolism was elevated in stem cells and fibroblasts, proliferation was only enhanced in fibroblasts derived from cholesteatoma. This could be reversed by the treatment with a TLR4 antagonist. The cholesteatoma fibroblasts could be triggered by LPS to promote the epidermal differentiation of the stem cells, while no LPS treatment or LPS treatment without the presence of fibroblasts did not result in such a differentiation. CONCLUSION: We propose that cholesteatoma recurrence is based on TLR4 signalling imprinted in the cholesteatoma cells. It induces excessive inflammation of stem cells and fibroblasts, proliferation of perimatrix fibroblasts and the generation of epidermal cells from stem cells thru paracrine signalling by fibroblasts. Treatment of the operation site with a TLR4 antagonist might reduce the chance of cholesteatoma recurrence. Video Abstract.

TFIIB-related factor 2 regulates glucose-regulated protein 78 expression in acquired middle ear cholesteatoma.

Acquired middle ear cholesteatoma leads to hearing loss, ear discharge, ear pain, and more serious intracranial complications. However, there is still no effective treatment other than surgery. TFIIB-related factor 2 (BRF2) acted as a redox sensor overexpressing in oxidative stress which linked endoplasmic reticulum (ER) stress, while glucose-regulated protein 78 (GRP78) was a biomarker of ER stress in cancer, atherosclerosis and inflammation. In our study, we investigated the roles of BRF2 and GRP78 in acquired middle ear cholesteatoma. Our results revealed that the expression of BRF2 was significant increased in acquired middle ear cholesteatoma, and which was positively correlated with the expression of GRP78. In addition, BRF2 and GRP78 showed colocalization in epithelium of acquired middle ear cholesteatomas and HaCaT cells. Prolongation of LPS stimulation in HaCaT cells escalated the expression of BRF2 and GRP78. To confirm the role of BRF2 and GRP78, we transfected si-BRF2 into HaCaT cells. All results indicated that BRF2 expression positively regulates the expression of GRP78 and may participate in the pathogenesis of acquire middle ear cholesteatoma.

Keratinocyte growth factor signaling promotes stem/progenitor cell proliferation under p63 expression during middle ear cholesteatoma formation.

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.

Serum sclerostin levels in chronic otitis media with and without cholesteatoma.

OBJECTIVE Sclerostin is a glycoprotein that plays a catabolic role in bone and is involved in the regulation of bone metabolism by increasing the osteoclastic bone resorption. In this study, serum sclerostin levels were measured in chronic otitis media (COM) with and without cholesteatoma, assuming that it might have a role in the aetiopathogenesis of bone resorption. METHODS A total of 44 patients with cholesteatomatous COM (cCOM) (n = 22) and non-cholesteatomatous COM (ncCOM) (n = 22) were included in this study, and 26 healthy volunteers without any chronic ear disease problem(s) constituted the control group (n = 26). RESULTS No significant difference was not found in terms of serum iPTH, ALP, and vitamin D levels between ncCOM, cCOM, and the control groups. A significant difference was found in terms of serum sclerostin, Ca, and P levels between ncCOM, cCOM, and the control groups (p<0.05). Serum sclerostin levels in the study groups were significantly higher but their serum Ca and P levels were significantly lower compared to the control group. CONCLUSION We think that serum sclerostin concentrations, which were significantly higher in patients with cCOM and ncCOM compared to healthy controls are associated with bone erosion. There is a need for further studies with larger samples in order to determine the relationship between sclerostin and bone erosion in cholesteatoma to help in establishing preventive measures against cholesteatoma and set new targets for the development of non-surgical treatments.

L1CAM-ILK-YAP Mechanotransduction Drives Proliferative Activity of Epithelial Cells in Middle Ear Cholesteatoma.

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.

Expression of PTHrP and RANKL in acquired middle ear cholesteatoma epithelium.

Background: Regarded as the most important clinical characteristic of middle ear cholesteatoma, the exact mechanism of bone resorption in cholesteatoma still remains unknown.Objectives: To investigate protein expression of PTHrP and RANKL in acquired middle ear cholesteatoma epithelium and analyze their functional roles in the etiopathogenesis of bone resorption in middle ear cholesteatoma.Material and methods: A total of 22 patients who underwent surgical treatment for middle ear cholesteatoma were recruited in the study. Protein expression of PTHrP and RANKL in middle ear cholesteatoma and normal postauricular skin was investigated by immunohistochemical staining. Correlations between bone resorption degree and expression of PTHrP and RANKL were also analyzed.Results: Protein expression of PTHrP and RANKL in cholesteatoma epithelium significantly increased when compared with normal postauricular skin epithelium. In cholesteatoma epithelium, a significantly positive association was observed between PTHrP and RANKL expression. Meanwhile, obviously positive correlations between protein expression of PTHrP and RANKL and bone resorption degree were discovered.Conclusions and significance: The increased protein expression of PTHrP and RANKL in cholesteatoma epithelium, and their associations with the degree of bone resorption, revealing that PTHrP might promote bone resorption process in middle ear cholesteatoma through RANKL signaling pathway.

Serum sclerostin levels in chronic otitis media with and without cholesteatoma

SUMMARY OBJECTIVE Sclerostin is a glycoprotein that plays a catabolic role in bone and is involved in the regulation of bone metabolism by increasing the osteoclastic bone resorption. In this study, serum sclerostin levels were measured in chronic otitis media (COM) with and without cholesteatoma, assuming that it might have a role in the aetiopathogenesis of bone resorption. METHODS A total of 44 patients with cholesteatomatous COM (cCOM) (n = 22) and non-cholesteatomatous COM (ncCOM) (n = 22) were included in this study, and 26 healthy volunteers without any chronic ear disease problem(s) constituted the control group (n = 26). RESULTS No significant difference was not found in terms of serum iPTH, ALP, and vitamin D levels between ncCOM, cCOM, and the control groups. A significant difference was found in terms of serum sclerostin, Ca, and P levels between ncCOM, cCOM, and the control groups (p<0.05). Serum sclerostin levels in the study groups were significantly higher but their serum Ca and P levels were significantly lower compared to the control group. CONCLUSION We think that serum sclerostin concentrations, which were significantly higher in patients with cCOM and ncCOM compared to healthy controls are associated with bone erosion. There is a need for further studies with larger samples in order to determine the relationship between sclerostin and bone erosion in cholesteatoma to help in establishing preventive measures against cholesteatoma and set new targets for the development of non-surgical treatments.

RESUMO OBJETIVO A esclerostina é uma glicoproteína que desempenha um papel catabólico no osso e também envolve a regulação do metabolismo ósseo, aumentando a reabsorção óssea osteoclástica. Neste estudo, os níveis séricos de esclerostina foram medidos em otite média crônica (OMC) com e sem colesteatoma, e presumiu-se se que ela poderia ter um papel na etiopatogênese da reabsorção óssea. MÉTODOS Um total de 44 pacientes com otite média crônica colesteatomatosa (OMCc) (n=22), não colesteatomatosa (OMCnc)(n=22) foram incluídos neste estudo, e 26 voluntários saudáveis e sem doenças crônicas do ouvido constituíram o grupo de controle (n=26). RESULTADOS Não foi encontrada diferença significativa em termos de níveis séricos de iPTH, ALP e vitamina D entre OMCnc, OMCc e o grupo de controle. Foi encontrada uma diferença significativa em termos de níveis séricos de esclerostina, Ca e P entre OMCnc, OMCc e o grupo de controle (p<0,05). Os níveis séricos de esclerostina nos grupos de estudo foram significativamente mais altos, mas os níveis séricos de Ca e P foram significativamente mais baixos em comparação com o grupo de controle. CONCLUSÃO Acreditamos que as concentrações séricas de esclerostina, significativamente maiores em pacientes com OMCc e OMCnc em relação aos controles saudáveis, estão associadas à erosão óssea. Há necessidade de mais estudos com amostras maiores para determinar a relação entre esclerostina e erosão óssea no colesteatoma, já que essas pesquisas podem ajudar a estabelecer medidas preventivas contra o colesteatoma e novas metas para o desenvolvimento de tratamentos não cirúrgicos.