Tmem161a regulates bone formation and bone strength through the P38 MAPK pathway.

Bone remodeling is an extraordinarily complex process involving a variety of factors, such as genetic, metabolic, and environmental components. Although genetic factors play a particularly important role, many have not been identified. In this study, we investigated the role of transmembrane 161a (Tmem161a) in bone structure and function using wild-type (WT) and Tmem161a-depleted (Tmem161aGT/GT) mice. Mice femurs were examined by histological, morphological, and bone strength analyses. Osteoblast differentiation and mineral deposition were examined in Tmem161a-overexpressed, -knockdown and -knockout MC3T3-e1 cells. In WT mice, Tmem161a was expressed in osteoblasts of femurs; however, it was depleted in Tmem161aGT/GT mice. Cortical bone mineral density, thickness, and bone strength were significantly increased in Tmem161aGT/GT mice femurs. In MC3T3-e1 cells, decreased expression of alkaline phosphatase (ALP) and Osterix were found in Tmem161a overexpression, and these findings were reversed in Tmem161a-knockdown or -knockout cells. Microarray and western blot analyses revealed upregulation of the P38 MAPK pathway in Tmem161a-knockout cells, which referred as stress-activated protein kinases. ALP and flow cytometry analyses revealed that Tmem161a-knockout cells were resistant to oxidative stress. In summary, Tmem161a is an important regulator of P38 MAPK signaling, and depletion of Tmem161a induces thicker and stronger bones in mice.

Ureter position and risk of ureteral injury during lateral lumbar interbody fusion.

BACKGROUND: Ureteral injury during lateral lumbar interbody fusion (LLIF) is uncommon. However, it is a serious complication that may require additional surgery should it occur. The objective of this study was to evaluate whether there was any change in the position of the left ureter between preoperative biphasic contrast-enhanced CT scanned in the supine position and intraoperative scanning in the right lateral decubitus position after stent placement, to assess the risk of ureteral injury in the actual surgical position. METHODS: The position of the left ureter scanned with the O-arm navigation system with the patient in the right lateral decubitus position and its position on preoperative biphasic contrast-enhanced CT images scanned with the patient in the supine position were investigated comparing their positions at the L2/3, L3/4, and L4/5 levels. RESULTS: The ureter was located along the interbody cage insertion trajectory in 25 of 44 disc levels (56.8%) in the supine position, but in only 4 (9.5%) in the lateral decubitus position. The proportion of patients in whom the left ureter was located lateral to the vertebral body (along the LLIF cage insertion trajectory) at each level was 80% in the supine position and 15.4% in the lateral decubitus position at the L2/3 level, 53.3% in the supine position and 6.7% in the lateral decubitus position at the L3/4 level, and 33.3% in the supine position and 6.7% in the lateral decubitus position at the L4/5 level. CONCLUSION: The proportion of patients in whom the left ureter was located on the lateral surface of the vertebral body when the patient was in the actual surgical position (lateral decubitus position) was 15.4% at the L2/3 level, 6.7% at the L3/4 level, and 6.7% at the L4/5 level, suggesting that caution is required during LLIF surgery.

EPLINß Is Involved in the Assembly of Cadherin-catenin Complexes in Osteoblasts and Affects Bone Formation.

Epithelial protein lost in neoplasm (EPLIN) is an actin-associated cytoskeletal protein that plays an important role in epithelial cell adhesion. EPLIN has two isoforms: EPLINα and EPLINß. In this study, we investigated the role of EPLINß in osteoblasts using EPLINß-deficient (EPLINßGT/GT ) mice. The skeletal phenotype of EPLINßGT/GT mice is indistinguishable from the wildtype (WT), but bone properties and strength were significantly decreased compared with WT littermates. Histomorphological analysis revealed altered organization of bone spicules and osteoblast cell arrangement, and decreased alkaline phosphatase activity in EPLINßGT/GT mouse bones. Transmission electron microscopy revealed wider intercellular spaces between osteoblasts in EPLINßGT/GT mice, suggesting aberrant cell adhesion. In EPLINßGT/GT osteoblasts, α- and ß-catenins and F-actin were observed at the cell membrane, but OB-cadherin was localized at the perinuclear region, indicating that cadherin-catenin complexes were not formed. EPLINß knockdown in MC3T3-e1 osteoblast cells showed similar results as in calvaria cell cultures. Bone formation markers, such as RUNX2, Osterix, ALP, and Col1a1 mRNA were reduced in EPLINß knockdown cells, suggesting an important role for EPLINß in osteoblast formation. In conclusion, we propose that EPLINß is involved in the assembly of cadherin-catenin complexes in osteoblasts and affects bone formation.

Sarcopenia, Ectopic Fat Infiltration Into the Lumbar Paravertebral Muscles, and Lumbo-Pelvic Deformity in Older Adults Undergoing Lumbar Surgery.

STUDY DESIGN: A retrospective analysis of a prospective, non-randomized cohort dataset. OBJECTIVE: To cross-sectionally examine the prevalence of sarcopenia and the association between spine-pelvic deformity and skeletal muscle volume loss and ectopic fat infiltration into lumbar paravertebral muscles (PVMs) in patients who underwent lumbar surgery. SUMMARY OF BACKGROUND DATA: Muscle quality deterioration has been considered the main pathology of sarcopenia, reducing muscle strength directly. The qualitative deterioration as well as volume loss in PVM, which contributes significantly to core body extension, might cause aging-related spine deformity. METHODS: In total, 184 patients were included. Sarcopenia was diagnosed at baseline, and all patients underwent whole-body X-ray. The amount of fat in lumbar PVM was evaluated with the Goutallier classification in magnetic resonance imaging findings. The expression of adipogenesis- and atrophy-promoting factors in PVM was evaluated with quantitative polymerase chain reaction. RESULTS: In total, 36.1% of adults aged ≥60 years were diagnosed with sarcopenia. The values of skeletal muscle indexes of the limb and trunk were inversely correlated with the sagittal vertical axis, pelvic tilt (PT), and pelvic incidence minus lumbar lordosis (PI-LL) values. The PT and PI-LL were greater, PVM area was smaller, and Goutallier grade was greater in sarcopenic adults than in non-sarcopenic older adults. Additionally, the PVM area correlated with the LL value, and Goutallier's grade correlated with the PT and PI-LL values. Moreover, the amount of ectopic fat in PVMs inversely correlated with skeletal muscle indexes. The expression levels of atrophy gene-1 and muscle ring-finger protein-1 did not differ between the groups and did not correlate with the PVM area. In contrast, the expression of Pparg and Cebpa was upregulated in sarcopenic older adults, where it correlated with Goutallier's grade. CONCLUSION: The volume loss of skeletal muscle, including lumbar PVM, and ectopic fat infiltration into the PVM, may cause the lumbo-pelvic deformity.Level of Evidence: 3.

The impact of helicopter emergency medical services and craniocervical traction on the early reduction of cervical spine dislocation in a rural area of Japan.

BACKGROUND: Several studies have shown an association between achieving decompression of the spinal cord within a few hours and neurological recovery, even in patients with complete paralysis due to cervical spine dislocation. This study aimed to clarify the impact of helicopter emergency medical services (HEMSs) and craniocervical traction on the rapid reduction of lower cervical spine dislocation in rural Japan. METHODS: The success rate of and factors inhibiting closed reduction, the time from injury to reduction and the functional prognosis of lower cervical spine dislocations treated between July 2012 and February 2020 were retrospectively analysed. RESULTS: Fourteen patients were transported by HEMS (group H), seven by ambulance (group A) and two by themselves. Although the average traveled distance and injury severity score were significantly higher in group H (64.5 km, 28.0) than in group A (24.7 km, 18.6), there was no significant difference in the average time to admission or the time to initiation of craniocervical traction after admission between groups H (159.4 min, 52.2 min) and A (163.6 min, 53.2 min). The success rate of closed reduction was 95%, and neurological deterioration was not observed in any cases. The average traction time and weight for reduction were 30.3 min and 16.3 kg, respectively. Body size and fracture-dislocation type did not significantly affect the traction time or weight. The rate of reduction within 4 h after injury was higher in group H (79%) than in group A (33%). Inner fixations were treated an average of 5.7 days after admission. After treatment, three of nine AIS A patients recovered the ability to walk, and all three patients underwent successful closed reduction within 4 h after injury. CONCLUSION: HEMS and highly successful closed reduction contributed to the early reduction of cervical spine dislocation and can potentially improve complete paralysis.

Blockade of the angiotensin II type 1 receptor increases bone mineral density and left ventricular contractility in a mouse model of juvenile Paget disease.

Juvenile Paget disease (JPD1), an autosomal-recessive disorder, is characterized by extremely rapid bone turnover due to osteoprotegerin deficiency. Its extra-skeletal manifestations, such as hypertension and heart failure, suggest a pathogenesis with shared skeletal and cardiovascular system components. In spite of this, the effects of anti-hypertensive drugs on bone morphometry remain unknown. We administered an angiotensin II type 1 receptor blocker, olmesartan (5 mg/kg/day) to 8-week-old male mice lacking the osteoprotegerin gene, with and without 1 µg/kg/min of angiotensin II infusion for 14 days. Olmesartan treatment decreased systolic blood pressure, and echocardiography showed increased left ventricular systolic contractility. Three-dimensional micro-computed tomography scans demonstrated that olmesartan treatment increased trabecular bone volume (sham, +176%; angiotensin II infusion, +335%), mineral density (sham, +150%; angiotensin II infusion, +313%), and trabecular number (sham, +407%; angiotensin II infusion, +622%) in the tibia. Olmesartan increased cortical mineral density (sham, +19%; angiotensin II infusion, +24%), decreased the cortical bone section area (sham, -16%; angiotensin II infusion, -18%), decreased thickness (sham, -18%; angiotensin II infusion, -31%), and decreased the lacunar area (sham, -41%; angiotensin II infusion, -27%) in the tibia. Similar trend was observed in the femur. Moreover, olmesartan decreased angiotensin II-induced increases in tartrate-resistant acid phosphatase concentrations in plasma, but it affected neither type I procollagen N-terminal propeptides, nor the receptor activator of nuclear factor kappa-B ligand. Our data suggest that blockade of the angiotensin II type 1 receptor improves bone vulnerability, and helps to maintain the heart's structural integrity in osteoprotegerin-deficient mice.

Hemoglobin stimulates the expression of ADAMTS-5 and ADAMTS-9 by synovial cells: a possible cause of articular cartilage damage after intra-articular hemorrhage.

BACKGROUND: ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) proteins play an important pathological role in matrix degeneration. Aggrecan degradation is a significant and critical event in early-stage osteoarthritis. To determine the effect of hemoglobin (Hb) on the ability of synovial tissues to produce ADAMTS family members, we examined the influence of Hb by synovial cells in an in vitro experimental system. METHODS: Synovial tissues were obtained from five young patients with meniscal injury under arthroscopic surgery. Primary cultures of human knee synovial cells were treated with different doses of human Hb (0, 25, 50, 100 µg/ml). The culture media were collected 24 h after Hb-treatment. In the time-course studies, cells were treated with and without 100 µg/ml Hb, and culture media were taken at 6, 12, and 24 h. To identify the proteins responsible for aggrecanase activity, Western blot analysis using antibodies against human ADAMTS-5, -8, -9, and -10; enzyme-linked immunosorbent assay (ELISA); and gene expression for ADAMTS-5 and -9 were examined. Statistical comparisons between each group were performed using paired t-tests. RESULTS: Western blot analysis revealed that Hb-treatment resulted in the expression of ADAMTS-5 and -9. Neither control group nor Hb-treated medium showed immunoreactivity against ADAMTS-8 or -10. In a dose-dependency study, the Hb-treated group showed significantly higher levels of ADAMTS-5 and -9 compared with the control (p < 0.05). There was no significant difference between 25, 50, and 100 µg/ml Hb-treated groups. In a time-course study, the ADAMTS-5 and -9 levels in the conditioned medium had significantly increased expression at 6, 12, and 24 h in the Hb-treated group (p < 0.05). Hb evoked significant expression of ADAMTS-9 mRNA at 12 and 24 h (p < 0.05). CONCLUSIONS: These findings indicate that Hb induces the expression of ADAMTS-5 and -9 by synovial cells at low doses, even at an acute phase, and suggests a possible role for Hb in cartilage damage after intra-articular hemorrhage. The results also suggest a new potential therapeutic target by inhibiting the activities of ADAMTS-5 and -9 to prevent cartilage damage after intra-articular hemorrhage.

Development of an efficient screening system to identify novel bone metabolism-related genes using the exchangeable gene trap mutagenesis mouse models.

Despite numerous genetic studies on bone metabolism, understanding of the specific mechanisms is lacking. We developed an efficient screening system to identify novel genes involved in bone metabolism using mutant mouse strains registered with the Exchangeable Gene Trap Clones (EGTC) database. From 1278 trap clones in the EGTC database, 52 candidate lines were selected in the first screening, determined based on "EST profile", "X-gal", "Related article", and "Novel gene". For the second screening, bone morphometric analysis, biomechanical strength analysis, bone X-gal staining, etc. were performed on candidate lines. Forty-two male trap lines (80.8%) showed abnormalities with either bone morphometric analysis or biomechanical strength analysis. In the screening process, X-gal staining was significantly efficient (P = 0.0057). As examples, Lbr and Nedd4 trap lines selected using the screening system showed significant bone decrease and fragility, suggesting a relationship with osteoblast differentiation. This screening system using EGTC mouse lines is extremely efficient for identifying novel genes involved in bone metabolism. The gene trap lines identified as abnormal using this screening approach are highly likely to trap important genes for bone metabolism. These selected trap mice will be valuable for use as novel bio-resources in bone research.

Angiotensin II Stimulation of Cardiac Hypertrophy and Functional Decompensation in Osteoprotegerin-Deficient Mice.

Circulating and myocardial expressions of receptor activator of nuclear factor-κb ligand and osteoprotegerin are activated in heart failure; however, it remains to be determined their pathophysiological roles on left ventricular structure and function in interaction with renin-angiotensin system. We conducted experiments using 8-week-old osteoprotegerin(-/-) mice and receptor activator of nuclear factor-κb ligand-transgenic mice to assess whether they affect the angiotensin II-induced left ventricular remodeling. Subcutaneous infusion of angiotensin II to osteoprotegerin(-/-) mice progressed the eccentric hypertrophy, resulting in left ventricular systolic dysfunction for 28 days, and this was comparable with wild-type mice, showing concentric hypertrophy, irrespective of equivalent elevation of systolic blood pressure. The structural alteration was associated with reduced interstitial fibrosis, decreased procollagen α1 and syndecan-1 expressions, and the increased number of apoptotic cells in the left ventricle, compared with wild-type mice. In contrast, angiotensin II infusion to the receptor activator of nuclear factor-κb ligand-transgenic mice revealed the concentric hypertrophy with preserved systolic contractile function. Intraperitoneal administration of human recombinant osteoprotegerin, but not subcutaneous injection of anti-receptor activator of nuclear factor-κb ligand antibody, to the angiotensin II-infused osteoprotegerin(-/-) mice for 28 days ameliorated the progression of heart failure without affecting systolic blood pressure. These results underscore the biological activity of osteoprotegerin in preserving myocardial structure and function during the angiotensin II-induced cardiac hypertrophy, independent of receptor activator of nuclear factor-κb ligand activity. In addition, the antiapoptotic and profibrotic actions of osteoprotegerin that emerged from our data might be involved in the mechanisms.

Cardiac hypertrophy is exacerbated in aged mice lacking the osteoprotegerin gene.

AIMS: Osteoprotegerin (OPG) may play a role in the progression of cardiac hypertrophy and heart failure. However, its pathophysiological role in changes in cardiac structure and function with ageing remains to be elucidated. METHODS AND RESULTS: We conducted experiments using 2.5- and 12-month-old OPG(-/-) mice and age-matched wild-type (WT) mice and compared the morphology and function of the left ventricle (LV). Both 2.5- and 12-month-old OPG(-/-) mice showed a higher systolic blood pressure and a greater heart weight/body weight ratio than age-matched WT mice. Twelve-month-old OPG(-/-) mice had a significantly larger LV chamber and reduced wall thickness compared with age-matched WT mice, and contractile function was decreased. The morphological differences were accompanied by an increase in the number of apoptotic cells and activation of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) in the LV of 12-month-old OPG(-/-) mice. Correspondingly, OPG small interfering RNA induced the expressions of TRAIL and cleaved caspase-3 in cultured cardiac myocytes. In addition, these mice revealed a decrease in interstitial fibrosis, activation of matrix metalloproteinase (MMP)-2 and tissue inhibitors of MMP-1 and -2, and inactivation of procollagen α1 synthesis. Moreover, intraperitoneal administration of recombinant OPG to either 2.5- or 12-month-old OPG(-/-) mice for 28 days led to partial improvement of LV structure and function without affecting systolic blood pressure. CONCLUSION: These results suggest that OPG plays a role in preserving myocardial structure and function with ageing through a reduction in apoptosis and preservation of the matrix structure. In addition, this appears to be independent of effects on the vasculature.

Segmental copy number loss in the region of Semaphorin 4D gene in patients with acetabular dysplasia.

Acetabular dysplasia (AD) appears to be a multi-factorial disease, which may involve both genetic and environmental factors and whose pathogenesis remains obscure. The present study aims to identify a genetic variation that might confer risk of AD. We performed whole-genome screening of a copy number variation (CNV) using a deCODE-Illumina CNV beadchip with 20 female AD patients and 131 control subjects. Subsequently, Agilent's region-targeted high-density oligonucleotide tiling microarray was used to analyze 64 female AD patients and 32 female control subjects. By sequential analyses, we found a copy number loss in 18 of 64 AD patients, but none in the 32 controls. The loss occurred within a 472 kb region on 9q22.2, which harbors the gene for Semaphorin 4D (Sema4D; 18/64 vs. 0/32, p = 4.81 × 10(-4) , OR = 25.86). We suggest that a copy number loss of the Sema4D gene region may play a role in the etiology of AD.

Roles of the endoplasmic reticulum stress transducer OASIS in fracture healing.

A new type of endoplasmic reticulum (ER) stress transducer, Old Astrocyte Specifically Induced Substance (OASIS), which is induced by bone morphogenetic protein-2 (BMP2), has been reported to activate the transcription of type I collagen and contribute to the secretion of bone matrix proteins in osteoblasts. Here, we examined the role of OASIS in fracture healing using the fracture models in wild-type (WT) and OASIS(-/-) mice. We found that the expression of OASIS mRNA was induced after fracture. Micro-computed tomography indicated that the callus density of OASIS(-/-) mice was less than that of WT mice, and the newly formed bone in OASIS(-/-) mice exhibited a decrease of the bone volume by bone morphometric analysis. Biomechanically, the callus bone strength of OASIS(-/-) mice was inferior to that of WT mice. Based on RT-PCR, in situ hybridization, immunohistochemical, and electrophoretic analyses, it was clarified that the synthesis of type I collagen was impaired in OASIS(-/-) mice. Electron microscopic analysis revealed that OASIS(-/-) osteoblasts in the fracture callus contained the abnormal expansion of the ERs, similar to OASIS(-/-) osteoblasts in the normal skeletal development. Thus, OASIS may play a role in bone formation through the expression of type I collagen and the secretion of bone matrix proteins in fracture healing.