Genetic variants and altered expression of SERPINF1 confer disease susceptibility in patients with otosclerosis.

Otosclerosis (OTSC) is a focal and diffuse bone disorder of the human middle ear characterized by abnormal bone growth and deposition at the stapes' footplate. This hinders the transmission of acoustic waves to the inner ear leading to subsequent conductive hearing loss. The plausible convections for the disease are genetic and environmental factors with yet an unraveled root cause. Recently, exome sequencing of European individuals with OTSC revealed rare pathogenic variants in the Serpin Peptidase Inhibitor, Clade F (SERPINF1) gene. Here, we sought to investigate the causal variants of SERPINF1 in the Indian population. The gene and protein expression was also evaluated in otosclerotic stapes to ameliorate our understanding of the potential effect of this gene in OTSC. A total of 230 OTSC patients and 230 healthy controls were genotyped by single-strand conformational polymorphism and Sanger sequencing methods. By comparing the case controls, we identified five rare variants (c.72 C > T, c.151 G > A, c.242 C > G, c.823 A > T, and c.826 T > A) only in patients. Four variants c.390 T > C (p = 0.048), c.440-39 C > T (p = 0.007), c.643 + 9 G > A (p = 0.035), and c.643 + 82 T > C (p = 0.005) were found to be significantly associated with the disease. Down-regulation of SERPINF1 transcript level in otosclerotic stapes was quantified by qRT-PCR, ddPCR and further validated by in situ hybridization. Similarly, reduced protein expression was observed by immunohistochemistry and immunofluorescence in otosclerotic stapes that corroborate with immunoblotting of patients' plasma samples. Our findings identified that SERPINF1 variants are associated with the disease. Furthermore, reduced expression of SERPINF1 in otosclerotic stapes might contribute to OTSC pathophysiology.

Region-specific endodermal signals direct neural crest cells to form the three middle ear ossicles.

Defects in the middle ear ossicles - malleus, incus and stapes - can lead to conductive hearing loss. During development, neural crest cells (NCCs) migrate from the dorsal hindbrain to specific locations in pharyngeal arch (PA) 1 and 2, to form the malleus-incus and stapes, respectively. It is unclear how migratory NCCs reach their proper destination in the PA and initiate mesenchymal condensation to form specific ossicles. We show that secreted molecules sonic hedgehog (SHH) and bone morphogenetic protein 4 (BMP4) emanating from the pharyngeal endoderm are important in instructing region-specific NCC condensation to form malleus-incus and stapes, respectively, in mouse. Tissue-specific knockout of Shh in the pharyngeal endoderm or Smo (a transducer of SHH signaling) in NCCs causes the loss of malleus-incus condensation in PA1 but only affects the maintenance of stapes condensation in PA2. By contrast, knockout of Bmp4 in the pharyngeal endoderm or Smad4 (a transducer of TGFß/BMP signaling) in the NCCs disrupts NCC migration into the stapes region in PA2, affecting stapes formation. These results indicate that region-specific endodermal signals direct formation of specific middle ear ossicles.

A Biochemical Analysis of the Stapes.

BACKGROUND Otosclerosis is a primary disease of the bony labyrinth. In the course of otosclerosis, abnormal resorption and recalcification of the endochondral layer of the temporal bone is observed. The otosclerotic process most commonly develops in the anterior part of the oval window. MATERIAL AND METHODS We analyzed stapes superstructures from 4 patients undergoing surgery for otosclerosis. The first step involved tissue assessment under a scanning electron microscope. The resulting images were analyzed in terms of morphological changes. The stapes superstructure was then divided into small "ossicles", including fragments from the closest vicinity of the stapes footplate and a fragment of the head of the stapes. This material was examined using a scanning electron microscope with a unit for chemical analysis in microareas. RESULTS Chemical analysis confirms the appearance of considerable quantities of the following elements: carbon, oxygen, potassium, and calcium, and the appearance of small quantities of sodium and magnesium. Based on a detailed analysis of the chemical composition, these fragments could represent a calcium phosphate compound from the following system: CaO-P2O5-H2O. Fragments of the superstructure from the region closest to the base of the stapes demonstrated a considerably larger presence of carbon, oxygen, and nitrogen, which most likely suggests an increased metabolic process in this region. CONCLUSIONS Our analysis revealed an increased metabolic activity in the closest vicinity of the otosclerotic focus, the fissula ante fenestram. The increased metabolism correlated with the bone tissue changes seen on scanning electron microscopy.

[Otosclerosis. 1st part: pathogenesis]. / Otosclerosis. 1. rész: patogenezis.

Otosclerosis can be found exclusively in the human otic capsule of the temporal bone. Its etiology is still unknown. In the past decades, several potential etiopathogenetic factors have been revealed, however, most studies were based on otosclerotic patients diagnosed by clinical symptoms only. The current experience indicates that one third of this group suffer from non-otosclerotic stapes fixation. In our experimental series, we have diagnosed and classified otosclerotic patients based on histologic examination, and analyzed also the pathogenetic factors. Recent data demonstrate that measles virus and rs1800472 SNP of transforming growth factor beta 1 (TGFß1) gene are marked obvious etiologic factors, which have no therapeutic consequences so far. Furthermore, we summarize the genetic and environmental factors to be found in the literature, which may play a fundamental role in the pathogenesis of otosclerosis. Orv Hetil. 2018; 159(30): 1215-1220.

The origin of the stapes and relationship to the otic capsule and oval window.

BACKGROUND: The stapes, an ossicle found within the middle ear, is involved in transmitting sound waves to the inner ear by means of the oval window. There are several developmental problems associated with this ossicle and the oval window, which cause hearing loss. The developmental origin of these tissues has not been fully elucidated. RESULTS: Using transgenic reporter mice, we have shown that the stapes is of dual origin with the stapedial footplate being composed of cells of both neural crest and mesodermal origin. Wnt1cre/Dicer mice fail to develop neural crest-derived cartilages, therefore, have no middle ear ossicles. We have shown in these mice the mesodermal stapedial footplate fails to form and the oval window is induced but underdeveloped. CONCLUSIONS: If the neural crest part of the stapes fails to form the mesodermal part does not develop, indicating that the two parts are interdependent. The stapes develops tightly associated with the otic capsule, however, it is not essential for the positioning of the oval window, suggesting that other tissues, perhaps within the inner ear are needed for oval window placement.

Cellular voids in the pathogenesis of otosclerosis.

BACKGROUND: Otosclerosis is a common ear disease that causes fixation of the stapes and conductive hearing impairment. However, the pathogenesis of otosclerosis is still unknown. Otosclerosis could be associated with the unique bony environment found in the otic capsule. Normal bone remodelling is almost completely absent around the inner ear after birth allowing degenerative changes and dead osteocytes to accumulate. High levels of inner ear anti resorptive osteoprotegerin (OPG) is most likely responsible for this capsular configuration. Studies have demonstrated how osteocyte lifespan variation creates occasional clusters of dead osteocytes, so-called cellular voids, at otosclerotic predilection sites in the human otic capsule. These cellular voids have been suggested as possible starting points of otosclerosis. AIM: To describe the cellular viability in otosclerotic lesions and compare it to that of cellular voids. MATERIALS AND METHODS: The study was based on unbiased stereological quantifications in undecalcified human temporal bones with otosclerosis. RESULTS: Osteocyte viability was found to vary within the otosclerotic lesions. Furthermore, the results presented here illustrate that inactive otosclerotic lesions consist of mainly dead interstitial bone, much like cellular voids. CONCLUSIONS AND SIGNIFICANCE: Focal degeneration in the otic capsule may play an important role in the pathogenesis of otosclerosis.

Effect of angiotensin II on inflammation pathways in human primary bone cell cultures in otosclerosis.

INTRODUCTION: The aim of this study was to assess the expression and production of inflammation mediators in basal condition and after angiotensin II (AngII) in otosclerosis. MATERIALS AND METHODS: Human stapedial cell cultures (6 otosclerosis and 6 controls) were incubated with AngII (10(-7)M, 24 h) or vehicle. Cytokines and their mRNA expression were assessed by antibody and cDNA arrays. RESULTS: In basal conditions, otosclerotic cultures produced higher amounts of interleukin (IL)-1ß and interferon-inducible protein 10, and smaller amounts of tissue inhibitor of metalloproteinase 2. AngII promoted inflammation by increasing interferon γ and IL-10, and by decreasing macrophage inflammatory protein 1α and soluble tumor necrosis factor receptor II. CONCLUSIONS: Otosclerotic cultures produced higher proinflammatory cytokines in basal condition. AngII appeared to promote inflammation via these mediators in otosclerosis.

Controversies in RELN/reelin expression in otosclerosis.

Several studies have reported a potential genetic association between disease-specific single nucleotide polymorphism (SNPs) of RELN and otosclerosis and confirmed RELN expression in human stapes footplates. These are conflicting results, since RELN expression has been attributed exclusively to neural tissues and to odontoblasts. Otosclerosis is a disease of complex bone remodeling disorder, which is limited to the human otic capsule. Genetic predisposition has long been suspected, however, the pathogenesis remained unclear. Ankylotic stapes footplates (n = 85), cortical bone fragments (n = 4), hearing ossicles (n = 2) and human brain tissue specimens (n = 4) were processed to RELN-specific RT-PCR and reelin-specific immunofluorescent assay (IFA). The first group of ankylotic stapes footplates (n = 22) showed a consistent positive reaction against reelin by IFA; however, RELN-specific mRNA could not be detected in the second, RT-PCR group (n = 63). Brain specimens were characterized by robust expression of reelin (n = 2) and RELN-specific mRNA (n = 2). In case of bone-specific controls (n = 6), reelin/RELN expression was excluded obviously. Concerning current observations, RELN gene does not show active expression in adult stapes footplates. Since, the otic capsule surrounds a special neural structure (membranous labyrinth), reelin might play a coordinative role in the early embryonic stage of development. As being a part of the otic capsule, stapes footplate might be characterized by persisting reelin detectability without mRNA expression. Between these conditions, the etiologic role of RELN is questionable in the pathogenesis of otosclerosis.

No evidence for disturbed COL1A1 and A2 expression in otosclerosis.

Otosclerosis is a complex bone remodeling disorder of the human otic capsule that might be associated with various mutations of A1 and A2 alleles of type-I collagen. The study herein presented, investigates the possibilty of the genetic involvement of type-I collagen in the pathogenesis of histologically confirmed otosclerosis. A total of 55 ankylotic stapes footplates were analyzed. Cortical bone fragments (n = 30), incus (n = 3) and malleus (n = 2) specimens were employed as negative controls. Specimens were divided into two groups. The first group was processed using conventional H.E. hematoxylin-eosin (H.E.) staining and type-I collagen-specific immunofluorescent assay (IFA), while the second group was examined by COL1A1 and A2-specific RT-PCR. Otosclerotic- (n = 31) and non-otosclerotic stapes footplates (n = 9) as well as cortical bones (n = 20), incus (n = 2) and malleus specimens (n = 1) showed normal and quite similar A1 and A2 allele expression confirmed by IFA. RT-PCR analysis revealed normal and consistent mRNA expression of both alleles in each specimen. Expression levels and patterns of COL1A1/A2 alleles did not show significant correlation with the histological diagnosis of otosclerosis. Type-I collagen is a highly conserved structure protein, which plays a fundamental role in the integritiy of various connective tissues. Mutations of A1 and A2 alleles result in serious systemic disorders of the skeleton, tendons and skin. Since otosclerosis is an organ-specific disease, it is difficult to explain its genetic association with type-I collagen. In conclusion, we found no evidence supporting the putative link of COL1A1 and COL1A2 alleles with otosclerosis.

Restriction analysis of otosclerosis-associated CD46 splicing variants.

Otosclerosis is a primary bone remodeling disorder of the human otic capsule and is associated with persistent measles virus infection. The human cellular receptor of measles virus is the membrane cofactor protein (MCP, CD46), which has 14 well-described splicing variants. Unique CD46 expression pattern of the otic capsule and the stapes footplate may determine the susceptibility for persistent measles virus infection. A total of 51 surgically removed ankylotic stapes footplates were analyzed by histopathological and molecular biological methods, respectively. Nucleic acids were extracted. Measles virus sequences were detected by nucleoprotein RNA-specific reverse transcriptase polymerase chain reaction (RT-PCR). Alternatively spliced RNA of CD46 isoforms was amplified by RT-PCR; cDNA amplimers were separated by SDS poly-acrylamide gel electrophoresis and were purified from the gel. Complementary DNA of CD46 isoforms was restricted by endonuclease enzymes having CD46-specific recognition sites. The presence of viral RNA was associated exclusively with the histopathological diagnosis of otosclerosis; the stapes specimens with negative measles virus belonged to non-otosclerotic stapes fixations. All specimens (N = 51) were characterized by the consecutive expression of five CD46 variants (c, d, e, f and one shorter unidentified isoform). Histologically confirmed ostosclerotic specimens (N = 21) were characterized by increased expression levels of variant "f" and the unknown isoform. Increased expression levels of these isoforms and special CD46 expression pattern of the human otic capsule might produce modified or pathological intracellular signalization that could create the possibility of persistent measles virus infection.

Intrathecal Application of a Fluorescent Dye for the Identification of Cerebrospinal Fluid Leaks in Cochlear Malformation.

In cases of cerebrospinal fluid (CSF) leaks, reliable detection of their origins is needed to seal the leak sufficiently and prevent complications, such as meningitis. A method is presented here using intrathecal administered fluorescein in a clinical case of bilateral congenital ear malformation. A fluorescent dye is administered intrathecally to achieve intraoperative visualization of CSF leaks. The dye is applied 20 min before surgery, and concentration of 5% is used. Per every 10 kg of body weight, 0.1 mL of the fluid is applied intrathecally. The fluorescein is visualized using a fully digital microscope. The origin of the fluid leak is identified in the stapes footplate. During primary surgery, it is sealed, and cochlea implantation is performed for hearing restoration. In this specific case, 6 weeks later, the implant was explanted due to acute meningitis, and the electrode array was left as a spacer. Postoperatively, in the aural smear, ß-transferrin was detected. During a revision mastoidectomy, dislocated coverage of the leak was found. The stapes was removed and oval window sealed. Five days after revision surgery, no ß-transferrin was detected in the aural smear. During the revision of cochlea implantation 6 months later, intact coverage of the oval niche was observed. Thus, intrathecal fluorescein application proves to be a reliable tool for the detection of CSF leaks. It facilitates the orientation in malformations and complicated or unknown surgical situs. In the literature, its use is described for CSF fistulas in endonasal surgery but is rarely described in skull base and mastoid surgeries. The method has been used successfully in several cases with CSF leaks, and the results confirm the feasibility of safely accessing the origin of the leak.

A New Subtype of Multiple Synostoses Syndrome Is Caused by a Mutation in GDF6 That Decreases Its Sensitivity to Noggin and Enhances Its Potency as a BMP Signal.

Growth and differentiation factors (GDFs) are secreted signaling molecules within the BMP family that have critical roles in joint morphogenesis during skeletal development in mice and humans. Using genetic data obtained from a six-generation Chinese family, we identified a missense variant in GDF6 (NP_001001557.1; p.Y444N) that fully segregates with a novel autosomal dominant synostoses (SYNS) phenotype, which we designate as SYNS4. Affected individuals display bilateral wrist and ankle deformities at birth and progressive conductive deafness after age 40 years. We find that the Y444N variant affects a highly conserved residue of GDF6 in a region critical for binding of GDF6 to its receptor(s) and to the BMP antagonist NOG, and show that this mutant GDF6 is a more potent stimulator of the canonical BMP signaling pathway compared with wild-type GDF6. Further, we determine that the enhanced BMP activity exhibited by mutant GDF6 is attributable to resistance to NOG-mediated antagonism. Collectively, our findings indicate that increased BMP signaling owing to a GDF6 gain-of-function mutation is responsible for loss of joint formation and profound functional impairment in patients with SYNS4. More broadly, our study highlights the delicate balance of BMP signaling required for proper joint morphogenesis and reinforces the critical role of BMP signaling in skeletal development.

Codetection of measles virus and tumor necrosis factor-alpha mRNA in otosclerotic stapes footplates.

HYPOTHESIS: Otosclerosis is a disease of unknown etiology causing conductive or sensorineural hearing loss. Persistent measles virus infection of the otic capsule is considered to be one of the etiologic factors in otosclerosis. BACKGROUND: Determinants of measles virus infection and reactive inflammation were studied in otosclerosis. The presence of measles virus was shown in otosclerotic patients by reverse-transcription polymerase chain reaction (RT-PCR) amplification of the viral RNA. No report is available, however, on the types and features of paracrine cytokines in otosclerosis. METHODS: Nucleic acid was extracted from stapes footplate samples of clinically otosclerotic patients. Measles virus nucleoprotein RNA was amplified by seminested RT-PCR. Tumor necrosis factor (TNF)-alpha mRNA expression was detected by RT-PCR in otosclerotic bone and was correlated with preoperative audiologic findings and measles virus positivity. RESULTS: Among 154 clinically otosclerotic patients, 99 stapes footplate specimens contained measles virus RNA. TNF-alpha mRNA was detectable in 88 virus-positive and in 6 virus-negative stapes footplates. There was no detectable TNF-alpha mRNA expression in virus negative cases. CONCLUSION: The etiologic role of measles virus in the pathogenesis of otosclerosis should be considered. Detection of TNF-alpha mRNA demonstrates activated osteoclast functions and inflammatory pathways in otosclerotic stapes footplates associated with measles virus presence. Virus-associated and virus-negative pathomechanisms of otosclerosis should be distinguished.

Increased activity of the diastrophic dysplasia sulfate transporter in otosclerosis and its inhibition by sodium fluoride.

HYPOTHESIS: This study investigates the function of the diastrophic dysplasia sulfate transporter (DTDST) in otosclerotic bone and the effect on it of sodium fluoride (NaF). BACKGROUND: Otosclerosis is a localized bone dystrophy with increased bone turnover. DTDST is implicated in the regulation of the bone turnover. MATERIALS AND METHODS: Primary cultures of cells were obtained from the stapes and external auditory canal (EAC) of 26 patients with otosclerosis and from nine control patients. Sulfate uptake was quantified under basal conditions and with NaF. The NaF signaling pathways were investigated using forskolin and verapamil. RESULTS: The relative initial rates of sulfate uptake and the apparent Vmax values were: otosclerotic stapes > EAC > control stapes = control EAC. The sulfate uptake by the otosclerotic stapes was correlated with the loss of sensorineural hearing. The amounts of DTDST mRNA (RNase protection assay) in the four subgroups did not differ. NaF (10(-6)M, 1 hr) inhibited sulfate uptake by the otosclerotic stapes and EAC cells but not by control samples. CONCLUSION: The authors believe that whether the increased DTDST activity is a cause or an effect of otosclerosis, it appears to be a specific target for NaF treatment.

Otosclerotic stapes: morphological and microchemical correlates. An electron microscopic and x-ray analytical investigation.

A total of 32 otosclerotic stapes is thin-sectioned without decalcification and examined using transmission and scanning electron microscopes, with a nondispersive x-ray analyzer attached to the latter. These otosclerotic stapes are classified as spongiotic, sclerotic, or preotosclerotic, accoring to their pathologic characteristics and state of mineralization. Either diffuse or patchy demineralization in the ground substance appears to be the initial stage of otosclerosis, and this area coincides with preotosclerotic lesions (also known as blue mantle) in light microscopy. Therefore, it is interpreted that demineralization precedes the destruction of ground substance in the preotosclerotic lesion. Bone mineral deposits in new otosclerotic bone appear to be related to the collagen fibrils that are embedded in the ground substance. No mineral deposit could be seen without the ground substance deposition; therefore, it is suggested that this ground substance is the single most important factor in the poor mineralization of the otosclerosis. The sclerotic lesions are well mineralized and show a typical pattern of hydroxyapatite by x-ray diffraction study. We could not confirm the notion that the sclerotic lesion is hypermineralized as compared to the normal stapes. The spongiotic lesions are poorly mineralized, with low calcium salt. Using the Ca/P ratio and x-ray diffraction pattern as criteria, it was determined that spongiotic lesions belong to unstable, immature bone.

Non-collagen proteins of stapedial bone matrix in perilymph of otosclerotic patients.

Non-collagen proteins extracted from the otosclerotic stapes footplate, superstructure and temporal cortical bone were compared with the protein patterns of normal and otosclerotic perilymph by analytical isotachophoresis (ITP). Normal and otosclerotic perilymph yielded basically identical isotachophoretic subfractions. Thirteen subfractions were detected moving as anions at pH 9.6 in the normal perilymph, versus 16 subfractions of similar character in the otosclerotic perilymph. Of these 16 protein subfractions, one (which was not detected in the normal perilymph) could be found in the ITP-gram of the stapes footplate non-collagen proteins, but not of the stapes superstructure or temporal cortical bone. Our results support the concept that protein can enter the inner ear fluid spaces from the otosclerotic bone. The biological significance of these proteins has not yet been clarified.

Active otosclerotic foci in the stapes. An electron microscopic study.

A total of 12 undecalcified and decalcified otosclerotic stapes containing active spongiotic lesions were examined using an electron microscope. Evidence of osteolytic osteolysis was seen in all specimens, but osteoclasts were observed in only four. The advancing front in the spongiotic lesion was 'moth-eaten' due to the demineralization of canaliculi and lacunae. The demineralizing process appeared to be initiated by degranulation of lysosomes by the degenerating osteocytes. As bone resorption continued, poorly mineralized new bone was laid down by osteoblasts which showed mitochondrial swelling. The possible implication of this mitochondrial swelling is discussed in relation to the pathology of the otosclerosis.

Effect of 17 beta-estradiol on diastrophic dysplasia sulfate transporter activity in otosclerotic bone cell cultures and SaOS-2 cells.

OBJECTIVE: Diastrophic dysplasia sulfate transporter (DTDST) is involved in the regulation of bone turnover, and its activity in otosclerosis has been shown to be abnormally high. Taking into account the role of estrogens in the progression of otosclerosis, the possible effect of estrogens on DTDST was investigated in otosclerotic bone cell cultures and in SaOS-2, a human osteoblastic cell line. MATERIAL AND METHODS: Primary bone cell cultures of stapes and external auditory canal (EAC) bone were obtained from 33 patients with otosclerosis and 18 control patients undergoing cerebellopontine angle tumor surgery. These cultures were assessed in parallel with SaOS-2 cells. Estrogen receptors (ERs) were detected using reverse transcriptase polymerase chain reaction. DTDST activity was assessed by sulfate uptake at baseline and after 24 h of incubation with 17 beta-estradiol at concentrations ranging from 10(-12) to 10(-6) M. RESULTS: Stapes and EAC cultures predominantly expressed mRNA of ER alpha, while ER beta expression was predominant in SaOS-2 cells. In stapes and EAC cultures no modification of DTDST activity was observed with 10(-8) M 17 beta-estradiol. In SaOS-2 cells, DTDST activity was inhibited by 17 beta-estradiol (93.5+/-9.21 vs 83.6+/-8.83 pmol/mg protein/5 min, n=29; mean of differences=10.0+/-3.22, paired t-test, p<0.01). CONCLUSION: DTDST activity is regulated by estrogens in SaOS-2 cells, but not in primary cell cultures from stapes and EAC. This difference in the regulation mechanisms may be related to the type of estrogen receptor expressed.

Differences in otosclerotic and normal human stapedial osteoblast properties are normalized by alendronate in vitro.

OBJECTIVE: Identify and compare phenotypic properties of osteoblasts from patients with otosclerosis (OSO), normal bones (HOB), and normal stapes (NSO) to determine a possible cause for OSO hypermineralization and assess any effects of the bisphosphonate, alendronate. STUDY DESIGN: OSO (n = 11), NSO (n = 4), and HOB (n = 13) cultures were assayed for proliferation, adhesion, mineralization, and gene expression with and without 10(-10)M-10(-8)M alendronate. SETTING: Academic hospital. METHODS: Cultures were matched for age, sex, and passage number. Cell attachment and proliferation + alendronate were determined by Coulter counting cells and assaying tritiated thymidine uptake, respectively. At 7, 14, and 21 days of culture + alendronate, calcium content and gene expression by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were determined. RESULTS: OSO had significantly more cells adhere but less proliferation than NSO or HOB. Calcification was significantly increased in OSO compared to HOB and NSO. NSO and HOB had similar cell adhesion and proliferation rates. A dose-dependent effect of alendronate on OSO adhesion, proliferation, and mineralization was found, resulting in levels equal to NSO and HOB. All cultures expressed osteoblast-specific genes such as RUNX2, alkaline phosphatase, type I collagen, and osteocalcin. However, osteopontin was dramatically reduced, 9.4-fold at 14 days, in OSO compared to NSO. Receptor activator of nuclear factor κB ligand/osteoprotegerin (RANKL/OPG), important in bone resorption, was elevated in OSO with decreased levels of OPG levels. Alendronate had little effect on gene expression in HOB but in OSO increased osteopontin levels and decreased RANKL/OPG. CONCLUSIONS: OSO cultures displayed properties of hypermineralization due to decreased osteopontin (OPN) and also had increased RANKL/OPG, which were normalized by alendronate.

No evidence for the expression of renin-angiotensin-aldosterone system in otosclerotic stapes footplates.

INTRODUCTION: Recent studies have reported genetic associations between with single nucleotide polymorphism (SNP) of the several genes of the renin-angiotensin-aldosterone (RAA) system in otosclerosis without the confirmation of RAA system expression in human stapes footplates. There are conflicting results. These results are conflicting because RAA system expression has been attributed exclusively to neural, vascular, and renal tissues, exclusively. MATERIALS AND METHODS: Ankylotic stapes footplates (n = 20), cortical bone fragments (n = 10), and human kidney tissue specimens (n = 10) were processed to hematoxylin-eosin (HE) staining and RAA system-specific immunofluorescent assay (IFA), respectively. RESULTS: Histologic diagnosis of otosclerosis was established in all ankylotic stapes footplates. Histologically active- (n = 13) and inactive (n = 7) foci of otosclerosis were consequently characterized by negative immunoreactions for renin, angiotensin converting enzyme (ACE), angiotensin-II (AT-II), and angiotensin-II receptor (AT-IIR), consequently. In cortical bones, a considerable RAA system expression was observed confirmed in the perivascular bone marrow progenitor cells. Kidney specimens, applied as positive controls, showed intense RAA system-specific immunoreactions. CONCLUSION: Concerning current observations, the 4 studied members of RAA system that did not display active expression were not expressed at protein level in otosclerotic stapes footplates. This phenomenon was independent from the histologic activity of otosclerosis. Between these conditions, the etiologic role of RAA system is questionable in the pathogenesis of otosclerosis.