Search

Purpose: The effects of daily teriparatide (D-PTH, 20 μg/day), weekly high-dose teriparatide (W-PTH, 56.5 μg/week), or bisphosphonate (BP) on the vertebra and proximal femur were investigated using quantitative computed tomography (QCT)., Methods: A total of 131 postmenopausal women with a history of fragility fractures were randomized to receive D-PTH, W-PTH, or bisphosphonate (oral alendronate or risedronate). QCT were evaluated at baseline and after 18 months of treatment., Results: A total of 86 participants were evaluated by QCT (Spine: D-PTH: 25, W-PTH: 21, BP: 29. Hip: PTH: 22, W-PTH: 21, BP: 32. Dropout rate: 30.5 %). QCT of the vertebra showed that D-PTH, W-PTH, and BP increased total vBMD (+34.8 %, +18.2 %, +11.1 %), trabecular vBMD (+50.8 %, +20.8 %, +12.2 %), and marginal vBMD (+20.0 %, +14.0 %, +11.5 %). The increase in trabecular vBMD was greater in the D-PTH group than in the W-PTH and BP groups. QCT of the proximal femur showed that D-PTH, W-PTH, and BP increased total vBMD (+2.8 %, +3.6 %, +3.2 %) and trabecular vBMD (+7.7 %, +5.1 %, +3.4 %), while only W-PTH and BP significantly increased cortical vBMD (-0.1 %, +1.5 %, +1.6 %). Although there was no significant increase in cortical vBMD in the D-PTH group, cortical bone volume (BV) increased in all three treatment groups (+2.1 %, +3.6 %, +3.1 %)., Conclusions: D-PTH had a strong effect on trabecular bone of vertebra. Although D-PTH did not increase cortical BMD of proximal femur, it increased cortical BV. W-PTH had a moderate effect on trabecular bone of vertebra, while it increased both cortical BMD and BV of proximal femur. Although BP had a limited effect on trabecular bone of vertebra compared to teriparatide, it increased both cortical BMD and BV of proximal femur., Competing Interests: Declaration of competing interest This study was supported by Asahi Kasei Pharma Corporation and Eli Lilly Japan K.K., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: We previously reported the successful development of a computer-aided diagnosis (CAD) system for preventing retained surgical sponges with deep learning using training data, including composite and simulated radiographs. In this study, we evaluated the efficacy of the CAD system in a clinical setting., Study Design: A total of 1,053 postoperative radiographs obtained from patients 20 years of age or older who underwent surgery were evaluated. We implemented a foreign object detection application software on the portable radiographic device used in the operating room to detect retained surgical sponges. The results of the CAD system diagnosis were prospectively collected., Results: Among the 1,053 images, the CAD system detected possible retained surgical items in 150 images. Specificity was 85.8%, which is similar to the data obtained during the development of the software., Conclusions: The validation of a CAD system using deep learning in a clinical setting showed similar efficacy as during the development of the system. These results suggest that the CAD system can contribute to the establishment of a more effective protocol than the current standard practice for preventing the retention of surgical items., (Copyright © 2024 by the American College of Surgeons. Published by Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: Radioproteomics studies investigating the relationship between lesion phenotype and proteins have been progressed. The purpose of this study was to develop a radioproteomics method for discriminating between active and inactive immune checkpoint molecules based on lesion phenotype., Methods: From the public database TCGA-BRCA, mRNA and fat suppression contrast-enhanced T 1 -weighted images of 49 patients with breast cancer were selected for the experiment. Using mRNA, we defined cases with active (10 cases) and inactive (39 cases) immune checkpoint molecules. To discriminate these cases using lesion phenotype, 275 radiomics features were measured from the tumor area. After selecting 3 radiomics features by using Lasso, logistic regression was employed to discriminate between active and inactive cases of immune checkpoint molecules., Results: Evaluation of ROC analysis showed that the AUC was 0.81., Conclusion: Patients whose immune cell function is being braked by immune checkpoint molecules are likely to respond to immune checkpoint inhibitors when their activity is inhibited. Therefore, our results may be applied to predict the effects of immune checkpoint inhibitors in breast cancer treatment.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, manifesting as the progressive development of fluid-filled renal cysts. In approximately half of all patients with ADPKD, end-stage renal disease results in decreased renal function. In this study, we used CRISPR-Cas9 and somatic cell cloning to produce pigs with the unique mutation c.152_153insG (PKD1 insG/+ ). Pathological analysis of founder cloned animals and progeny revealed that PKD1 insG/+ pigs developed many pathological conditions similar to those of patients with heterozygous mutations in PKD1. Pathological similarities included the formation of macroscopic renal cysts at the neonatal stage, number and cystogenic dynamics of the renal cysts formed, interstitial fibrosis of the renal tissue, and presence of a premature asymptomatic stage. Our findings demonstrate that PKD1 insG/+ pigs recapitulate the characteristic symptoms of ADPKD., (© 2022. The Author(s).)

Background: Retained surgical items are a serious human error. Surgical sponges account for 70% of retained surgical items. To prevent retained surgical sponges, it is important to establish a system that can identify errors and avoid the occurrence of adverse events. To date, no computer-aided diagnosis software specialized for detecting retained surgical sponges has been reported. We developed a software program that enables easy and effective computer-aided diagnosis of retained surgical sponges with high sensitivity and specificity using the technique of deep learning, a subfield of artificial intelligence., Study Design: In this study, we developed the software by training it through deep learning using a dataset and then validating the software. The dataset consisted of a training set and validation set. We created composite x-rays consisting of normal postoperative x-rays and surgical sponge x-rays for a training set (n = 4,554) and a validation set (n = 470). Phantom x-rays (n = 12) were prepared for software validation. X-rays obtained with surgical sponges inserted into cadavers were used for validation purposes (formalin: Thiel's method = 252:117). In addition, postoperative x-rays without retained surgical sponges were used for the validation of software performance to determine false-positive rates. Sensitivity, specificity, and false positives per image were calculated., Results: In the phantom x-rays, both the sensitivity and specificity in software image interpretation were 100%. The software achieved 97.7% sensitivity and 83.8% specificity in the composite x-rays. In the normal postoperative x-rays, 86.6% specificity was achieved. In reading the cadaveric x-rays, the software attained both sensitivity and specificity of >90%., Conclusions: Software with high sensitivity for diagnosis of retained surgical sponges was developed successfully., (Copyright © 2021 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Bone metastatic lesions are classified as osteoblastic or osteolytic lesions. Prostate and breast cancer patients frequently exhibit osteoblastic-type and osteolytic-type bone metastasis, respectively. In metastatic lesions, tumor cells interact with many different cell types, including osteoblasts, osteoclasts, and mesenchymal stem cells, resulting in an osteoblastic or osteolytic phenotype. However, the mechanisms responsible for the modification of bone remodeling have not been fully elucidated. MicroRNAs (miRNAs) are transferred between cells via exosomes and serve as intercellular communication tools, and numerous studies have demonstrated that cancer-secreted miRNAs are capable of modifying the tumor microenvironment. Thus, cancer-secreted miRNAs can induce an osteoblastic or osteolytic phenotype in the bone metastatic microenvironment. In this study, we performed a comprehensive expression analysis of exosomal miRNAs secreted by several human cancer cell lines and identified eight types of human miRNAs that were highly expressed in exosomes from osteoblastic phenotype-inducing prostate cancer cell lines. One of these miRNAs, hsa-miR-940, significantly promoted the osteogenic differentiation of human mesenchymal stem cells in vitro by targeting ARHGAP1 and FAM134A Interestingly, although MDA-MB-231 breast cancer cells are commonly known as an osteolytic phenotype-inducing cancer cell line, the implantation of miR-940-overexpressing MDA-MB-231 cells induced extensive osteoblastic lesions in the resulting tumors by facilitating the osteogenic differentiation of host mesenchymal cells. Our results suggest that the phenotypes of bone metastases can be induced by miRNAs secreted by cancer cells in the bone microenvironment., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Genetically engineered pigs play an indispensable role in the study of rare monogenic diseases. Pigs harboring a gene responsible for a specific disease can be efficiently generated via somatic cell cloning. The generation of somatic cell-cloned pigs from male cells with mutation(s) in an X chromosomal gene is a reliable and straightforward method for reproducing X-linked genetic diseases (XLGDs) in pigs. However, the severe symptoms of XLGDs are often accompanied by impaired growth and reproductive disorders, which hinder the reproduction of these valuable model animals. Here, we generated unique chimeric boars composed of mutant cells harboring a lethal XLGD and normal cells. The chimeric boars exhibited the cured phenotype with fertility while carrying and transmitting the genotype of the XLGD. This unique reproduction system permits routine production of XLGD model pigs through the male-based breeding, thereby opening an avenue for translational research using disease model pigs., Competing Interests: Conflict of interest statement: H. Nagashima is a cofounder and shareholder of ChimaERA Corporation and PorMedTec Inc. H. Nakauchi is a cofounder and shareholder of iCELL Inc., ChimaERA Corporation, and ReproCELL Inc., (Copyright © 2018 the Author(s). Published by PNAS.)

Long-term antiretroviral therapy is associated with increased fracture risk, but the mechanism remains elusive. We measured serum undercarboxylated osteocalcin and pentosidine (markers of poor bone quality) in human immunodeficiency virus-infected patients treated with protease inhibitors (PIs) or an integrase strand transfer inhibitor-containing regimen. The results demonstrated significantly higher undercarboxylated osteocalcin and pentosidine in PI-treated patients. Switching to integrase strand transfer inhibitor significant decreased these markers. We also showed impaired bone mechanical properties with higher undercarboxylated osteocalcin level in PI-treated mice and inhibited osteoblast differentiation in PI-treated osteogenic cells. The results confirmed the adverse effects of PIs on bone quality and osteoblast differentiation., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The circadian clock controls many behavioral and physiological processes beyond daily rhythms. Circadian dysfunction increases the risk of cancer, obesity, and cardiovascular and metabolic diseases. Although clinical studies have shown that bone resorption is controlled by circadian rhythm, as indicated by diurnal variations in bone resorption, the molecular mechanism of circadian clock-dependent bone resorption remains unknown. To clarify the role of circadian rhythm in bone resorption, aryl hydrocarbon receptor nuclear translocator-like (Bmal1), a prototype circadian gene, was knocked out specifically in osteoclasts. Osteoclast-specific Bmal1-knockout mice showed a high bone mass phenotype due to reduced osteoclast differentiation. A cell-based assay revealed that BMAL1 upregulated nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1 (Nfatc1) transcription through its binding to an E-box element located on the Nfatc1 promoter in cooperation with circadian locomotor output cycles kaput (CLOCK), a heterodimer partner of BMAL1. Moreover, steroid receptor coactivator (SRC) family members were shown to interact with and upregulate BMAL1:CLOCK transcriptional activity. Collectively, these data suggest that bone resorption is controlled by osteoclastic BMAL1 through interactions with the SRC family and binding to the Nfatc1 promoter. © 2016 American Society for Bone and Mineral Research., (© 2016 American Society for Bone and Mineral Research.)

Osteoblastic differentiation is regulated by various factors, including hormones and transcription factors. Runt-related transcription factor 2 (Runx2) is an essential player in osteoblastogenesis and transactivates its molecular target by creating a protein complex with its hetero-dimeric partner core binding factor beta (Cbfb). However, the molecular regulation of Cbfb expression remains unknown. Here, we identified miR-145 as a crucial regulator of Cbfb expression. The expression of miR-145 increased during osteoblastogenesis, indicating that miR-145 works as an inhibitor of osteoblastogenesis. Stable expression of miR-145 decreased endogenous Cbfb expression and inhibited osteoblastogenesis, in cooperation with miR-34c. Furthermore, miR-145 decreased bone regeneration in vivo. Our results indicate that miR-145 physiologically regulates osteoblast differentiation and bone formation via Cbfb expression by forming a regulatory microRNA network., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Objective: Donepezil, an inhibitor of acetylcholinesterase (AChE) targeting the brain, is a common medication for Alzheimer's disease. Interestingly, a recent clinical study found that administration of this agent is associated with lower risk of hip fracture independently of falling, suggesting its direct effect on bone tissues as well. AChE has been reported to be involved in osteoblast function, but the role of AChE on osteoclastogenesis still remains unclear. We analyzed the effect of AChE and donepezil on osteoclastogenesis in vivo and in vitro., Methods: Cell-based assays were conducted using osteoclasts generated in cultures of murine bone marrow macrophages (BMMs) with receptor activator of nuclear factor-kappa B ligand (RANKL). The effect of donepezil was also determined in vivo using a mouse model of RANKL-induced bone loss., Results: Recombinant AChE in BMMs cultured with RANKL further promoted RANKL-induced tartrate-resistant acid phosphatase (TRAP)-positive osteoclast differentiation. RANKL also upregulated AChE expression in BMMs. RNA interference-mediated knockdown of AChE significantly inhibited RANKL-induced osteoclast differentiation and suppressed gene expression specific for osteoclasts. AChE upregulated expression of RANK, the receptor of RANKL, in BMMs. Donepezil decreased cathepsin K expression in BMMs and the resorptive function of osteoclasts on dentine slices. Donepezil decreased RANK expression in BMMs, resulting in the inhibition of osteoclast differentiation with downregulation of c-Fos and upregulation of Id2. Moreover, administration of donepezil prevented RANKL-induced bone loss in vivo, which was associated with the inhibition of bone resorption by osteoclasts., Conclusions: AChE promotes osteoclast differentiation in vitro. Donepezil inhibits osteoclast function in vitro and prevents bone loss by suppressing bone resorption in vivo, suggesting the possibility that donepezil reduces fracture risk in patients with Alzheimer's disease.

Bone homeostasis is maintained by bone formation and bone resorption. The traditional view of bone metabolism as a primarily endocrine regulation has been expanded in recent years following the identification of nervous system controlling bone metabolism. Especially, sympathetic and parasympathetic nervous system regulates bone formation and bone resorption. In addition, sensory nervous system also has been shown to be involved in the regulation of bone homeostasis. These studies demonstrated that nervous system is closely related to bone remodeling.

Objective: Bone mass is maintained through a balance of bone formation and resorption. This homeostatic balance is regulated by various systems involving humoral and local factors. The discovery that the anorexigenic hormone leptin regulates bone mass via neuronal pathways revealed that neurons and neuropeptides are intimately involved in bone homeostasis. Ghrelin is a stomach-derived orexigenic hormone that counteracts leptin's action. However, the physiological role of ghrelin in bone homeostasis remains unknown. In this study, through the global knockout of ghrelin receptor (Ghsr) followed by tissue-specific re-expression, we addressed the molecular basis of the action of ghrelin in bone remodeling in vivo., Methods: We performed molecular, genetic and cell biological analyses of Ghsr-null mice and Ghsr-null mice with tissue specific Ghsr restoration. Furthermore, we evaluated the molecular mechanism of ghrelin by molecular and cell-based assays., Results: Ghsr-null mice showed a low bone mass phenotype with poor bone formation. Restoring the expression of Ghsr specifically in osteoblasts, and not in osteoclasts or the central nervous system, ameliorated bone abnormalities in Ghsr-null mice. Cell-based assays revealed ghrelin induced the phosphorylation of CREB and the expression of Runx2, which in turn accelerated osteoblast differentiation., Conclusions: Our data show that ghrelin regulates bone remodeling through Ghsr in osteoblasts by modulating the CREB and Runx2 pathways.

In this paper we review the status of new applications research of the Japanese Aerospace Exploration Agency (JAXA) for global health promotion using information derived from Earth observation data by satellites in cooperation with inter-disciplinary collaborators. Current research effort at JAXA to promote global public health is focused primarily on the use of remote sensing to address two themes: (i) prediction models for malaria and cholera in Kenya, Africa; and (ii) air quality assessment of small, particulate matter (PM2.5), nitrogen dioxide (NO2) and ozone (O3). Respiratory and cardivascular diseases constitute cross-boundary public health risk issues on a global scale. The authors report here on results of current of a collaborative research to call attention to the need to take preventive measures against threats to public health using newly arising remote sensing information from space.

3-dimensional printed liver was constructed using 3D vascular imaging in a patient with intrahepatic cholangiocarcinoma who underwent major hepatectomy. The reproducibility of 3D modeling by the latest imaging has been clarified and future preoperative simulation should be adramatically changed.

Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial-to-mesenchymal transition. MiR-133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR-133-mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR-133/Snai1, is a key molecular roadblock during cardiac reprogramming., (© 2014 The Authors.)

Bone is a highly vascularized organ, thus angiogenesis is a vital process during bone remodeling. However, the role of vascular systems in bone remodeling is not well recognized. Here we show that netrin-4 inhibits osteoclast differentiation in vitro and in vivo. Co-cultures of bone marrow macrophages with vascular endothelial cells markedly inhibited osteoclast differentiation. Adding a neutralizing antibody, or RNA interference against netrin-4, restored in vitro osteoclast differentiation. Administration of netrin-4 prevented bone loss in an osteoporosis mouse model by decreasing the osteoclast number. We propose that vascular endothelial cells interact with bone in suppressing bone through netrin-4., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The ATPase 6 accessory protein 2 (ATP6AP2)/(pro)renin receptor (PRR) is essential for the biogenesis of active vacuolar H(+)-ATPase (V-ATPase). Genetic deletion of ATP6AP2/PRR causes V-ATPase dysfunction and compromises vesicular acidification. Here, we characterized the domains of ATP6AP2/PRR involved in active V-ATPase biogenesis. Three forms of ATP6AP2/PRR were found intracellularly: full-length protein and the N- and C-terminal fragments of furin cleavage products, with the N-terminal fragment secreted extracellularly. Genetic deletion of ATP6AP2/PRR did not affect the protein stability of V-ATPase subunits. The extracellular domain (ECD) and transmembrane domain (TM) of ATP6AP2/PRR were indispensable for the biogenesis of active V-ATPase. A deletion mutant of ATP6AP2/PRR, which lacks exon 4-encoded amino acids inside the ECD (Δ4M) and causes X-linked mental retardation Hedera type (MRXSH) and X-linked parkinsonism with spasticity (XPDS) in humans, was defective as a V-ATPase-associated protein. Prorenin had no effect on the biogenesis of active V-ATPase. The cleavage of ATP6AP2/PRR by furin seemed also dispensable for the biogenesis of active V-ATPase. We conclude that the N-terminal ECD of ATP6AP2/PRR, which is also involved in binding to prorenin or renin, is required for the biogenesis of active V-ATPase. The V-ATPase assembly occurs prior to its delivery to the trans-Golgi network and hence shedding of ATP6AP2/PRR would not affect the biogenesis of active V-ATPase.

The identification that nervous system controls bone metabolism through leptin knock out mice studies opened a new field in bone biology. Notably, sympathetic and parasympathetic nervous system regulate bone formation and bone resorption. In addition, sensory nervous system also has been shown to be involved in the regulation of bone homeostasis. These studies demonstrated that nervous system is closely related to bone remodeling.

Purpose: Arterial enhancement of intrahepatic cholangiocarcinoma (ICC) has been noted. To precisely identify the characteristics of tumor enhancement patterns, we examined the relationship between CT attenuation in the tumor and clinicopathological parameters or prognosis., Methods: Subjects were 42 ICC patients who had undergone hepatectomy. microvessel density (MVD) determined by CD34 staining was compared with imaging. Attenuation was calculated in images from multidetector CT of tumor and non-tumorous regions. Enhancement patterns were divided into two groups: arterial enhancement with higher attenuation (>16 HU; Hyper group, n = 12); and arterial enhancement with lower attenuation (Hypo group, n = 30)., Results: Univariate analysis identified high tumor marker level, increased size, less-differentiation, incomplete resection, increased bleeding, and lower MVD as significantly associated with poor survival (p < 0.05). Increased attenuation throughout the whole ICC correlated significantly with radiological findings and MVD. Concomitant hepatitis, well-differentiation, and smaller tumor were more significantly frequent in the Hyper group than in the Hypo group (p < 0.05). Postoperative early recurrence was significantly less frequent in the Hyper group, and overall survival was significantly better in the Hyper group (p < 0.05)., Conclusions: Increased CT attenuation correlated with ICC tumor vascularity. Increased tumor enhancement in the arterial phase was associated with chronic hepatitis, lower malignancy, and better survival.

Wnt signaling system controls diverse biological phenomena through development and adulthood of all animals. Canonical Wnt signaling is well characterized and its defect causes abnormalities in bone metabolism. Furthermore, Wnt5a, a typical non-canonical ligand, has been shown to play an important role in osteoblast differentiation. In addition, recent studies have revealed that LRP5 regulates bone mass by inhibiting duodenal synthesis of serotonin.

Semaphorin 3A (Sema3A) is a diffusible axonal chemorepellent that has an important role in axon guidance. Previous studies have demonstrated that Sema3a(-/-) mice have multiple developmental defects due to abnormal neuronal innervations. Here we show in mice that Sema3A is abundantly expressed in bone, and cell-based assays showed that Sema3A affected osteoblast differentiation in a cell-autonomous fashion. Accordingly, Sema3a(-/-) mice had a low bone mass due to decreased bone formation. However, osteoblast-specific Sema3A-deficient mice (Sema3acol1(-/-) and Sema3aosx(-/-) mice) had normal bone mass, even though the expression of Sema3A in bone was substantially decreased. In contrast, mice lacking Sema3A in neurons (Sema3asynapsin(-/-) and Sema3anestin(-/-) mice) had low bone mass, similar to Sema3a(-/-) mice, indicating that neuron-derived Sema3A is responsible for the observed bone abnormalities independent of the local effect of Sema3A in bone. Indeed, the number of sensory innervations of trabecular bone was significantly decreased in Sema3asynapsin(-/-) mice, whereas sympathetic innervations of trabecular bone were unchanged. Moreover, ablating sensory nerves decreased bone mass in wild-type mice, whereas it did not reduce the low bone mass in Sema3anestin(-/-) mice further, supporting the essential role of the sensory nervous system in normal bone homeostasis. Finally, neuronal abnormalities in Sema3a(-/-) mice, such as olfactory development, were identified in Sema3asynasin(-/-) mice, demonstrating that neuron-derived Sema3A contributes to the abnormal neural development seen in Sema3a(-/-) mice, and indicating that Sema3A produced in neurons regulates neural development in an autocrine manner. This study demonstrates that Sema3A regulates bone remodelling indirectly by modulating sensory nerve development, but not directly by acting on osteoblasts.

The physiological and beneficial actions of vitamin D in bone health have been experimentally and clinically proven in mammals. The active form of vitamin D [1α,25(OH)(2)D(3)] binds and activates its specific nuclear receptor, the vitamin D receptor (VDR). Activated VDR prevents the release of calcium from its storage in bone to serum by stimulating intestinal calcium absorption and renal reabsorption. However, the direct action of VDR in bone tissue is poorly understood because serum Ca(2+) homeostasis is maintained through tightly regulated ion transport by the kidney, intestine, and bone. In addition, conventional genetic approaches using VDR knockout (VDR-KO, VDR(-/-)) mice could not identify VDR action in bone because of the animals' systemic defects in calcium metabolism. In this study, we report that systemic VDR heterozygous KO (VDR(+/L-)) mice generated with the Cre/loxP system as well as conventional VDR heterozygotes (VDR(+/-)) showed increased bone mass in radiological assessments. Because mineral metabolism parameters were unaltered in both types of mice, these bone phenotypes imply that skeletal VDR plays a role in bone mass regulation. To confirm this assumption, osteoblast-specific VDR-KO (VDR(ΔOb/ΔOb)) mice were generated with 2.3 kb α1(I)-collagen promoter-Cre transgenic mice. They showed a bone mass increase without any dysregulation of mineral metabolism. Although bone formation parameters were not affected in bone histomorphometry, bone resorption was obviously reduced in VDR(ΔOb/ΔOb) mice because of decreased expression of receptor activator of nuclear factor kappa-B ligand (an essential molecule in osteoclastogenesis) in VDR(ΔOb/ΔOb) osteoblasts. These findings establish that VDR in osteoblasts is a negative regulator of bone mass control.

The purpose of this study was to observe variation in the local elastic distribution in aortic tissue walls under different static strain conditions, including physiological strain, by use of a scanning haptic microscope (SHM). Strain was applied by stretching aortic tissues in the circumferential direction by the simple tensile method or by the rod-insertion method to mimic in vivo internal pressure loading. SHM measurements in a saline solution at room temperature were performed on canine thoracic aorta using a glass needle probe with a diameter of ca 5 μm and a scanning area and point pitch of 160 × 80 μm and 2 μm, respectively. Under strain of 0-0.23, corresponding to internal pressure of 0-150 mmHg, wavy-shaped elastin fibers stretched until they were almost straightened, and the average elastic modulus increased almost linearly. Although there was little difference between the images obtained for the two different stretching methods, under high strain (>0.36; 250 mmHg) significant circumferential orientation of the collagen fibrils occurred with an increase in the average elastic modulus. It was concluded that the pressure resistance of the aorta under physiological strain was mainly afforded by elastin fibers; collagen fibrils contributed little except under much higher pressures.

Background/aims: Few attempts have been made to distinguish the softness of different skin layers, though specific measurement of the superficial layer would be useful for evaluating the emollient effect of cosmetics and for diagnosis of skin diseases., Materials and Methods: We developed a sensor probe consisting of a piezoelectric tactile sensor and a load cell. To evaluate it, we firstly measured silicone rubber samples with different softness. Then, it was applied to human forearm skin before and after tape-stripping. A VapoMeter and skin-surface hygrometer were used to confirm removal of the stratum corneum. A Cutometer was used to obtain conventional softness data for comparison., Results and Conclusions: Both the piezoelectric tactile sensor and the load cell could measure the softness of silicone rubber samples, but the piezoelectric tactile sensor was more sensitive than the load cell when the reaction force of the measured sample was under 100 mN in response to a 2-mm indentation. For human skin in vivo, transepidermal water loss and skin conductance were significantly changed after tape-stripping, confirming removal of the stratum corneum. The piezoelectric tactile sensor detected a significant change after tape-stripping, whereas the load cell did not. Thus, the piezoelectric tactile sensor can detect changes of mechanical properties at the skin surface. The load cell data were in agreement with Cutometer measurements, which showed no change in representative skin elasticity parameters after tape-stripping. These results indicate that our sensor can simultaneously measure the mechanical properties of the superficial skin layer and whole skin., (© 2012 John Wiley & Sons A/S.)

To expand the performance capacity of the scanning haptic microscope (SHM) beyond surface mapping microscopy of elastic modulus or topography, surface density mapping of a natural tissue was performed by applying a measurement theory of SHM, in which a frequency change occurs upon contact of the sample surface with the SHM sensor - a microtactile sensor (MTS) that vibrates at a pre-determined constant oscillation frequency. This change was mainly stiffness-dependent at a low oscillation frequency and density-dependent at a high oscillation frequency. Two paragon examples with extremely different densities but similar macroscopic elastic moduli in the range of natural soft tissues were selected: one was agar hydrogels and the other silicon organogels with extremely low (less than 25 mg/cm(3)) and high densities (ca. 1300 mg/cm(3)), respectively. Measurements were performed in saline solution near the second-order resonance frequency, which led to the elastic modulus, and near the third-order resonance frequency. There was little difference in the frequency changes between the two resonance frequencies in agar gels. In contrast, in silicone gels, a large frequency change by MTS contact was observed near the third-order resonance frequency, indicating that the frequency change near the third-order resonance frequency reflected changes in both density and elastic modulus. Therefore, a density image of the canine aortic wall was subsequently obtained by subtracting the image observed near the second-order resonance frequency from that near the third-order resonance frequency. The elastin-rich region had a higher density than the collagen-rich region.

Bone morphogenetic proteins (BMPs) inhibit myogenesis and induce osteoblastic differentiation in myoblasts. They also induce the transcription of several common genes, such as Id1, Id2 and Id3, in various cell types. We have reported that a GC-rich element in the Id1 gene functions as a BMP-responsive element (BRE) that is regulated by Smads. In this study, we analyzed and identified BREs in the 5'-flanking regions of the mouse Id2 and Id3 genes. The core GGCGCC sequence was conserved among the BREs in the Id1, Id2 and Id3 genes and was essential for the response to BMP signaling via Smads. We found a novel BRE on mouse chromosome 13 at position 47,723,740-47,723,768 by searching for conserved sequences containing the Id1 BRE. This potential BRE was found in the 5'-flanking region of a novel gene that produces a non-coding transcript, termed BMP-inducible transcript-1 (BIT-1), and this element regulated the expression of this gene in response to BMP signaling. We found that BIT-1 is expressed in BMP target tissues such as the testis, brain, kidney and cartilage. These findings suggest that the transcriptional induction of the Ids, BIT-1 and additional novel genes containing the conserved BRE sequence may play an important role in the regulation of the differentiation and/or function of target cells in response to BMPs.

Background/aims: Recent developments in radiological technology allowed acquisition of images with high spatial resolution that facilitate effective 3-dimensional (3D) reconstruction of fusion images. Present study utilized 3D cholangiography and angiography with multi-detector-row computed tomography (MDCT) to acquire information regarding operative simulations., Methodology: 3D-fusion images were evaluated in 39 patients with hepatobiliary malignancies who underwent surgical resections., Results: An aberrant branch of segment 3 over the umbilical portal vein, a large hepatoma compressed the hilar vessels, an aberrant branch of the caudate lobe vasculature in case of metastatic liver tumor with a right-sided umbilical portal vein and transected biliary leakage were clearly observed by 3D imaging system. Four patients with intrahepatic cholangiocarcinoma underwent multiple biliary stent placements and adequate placement of biliary stents was possible. In 22 patients with extrahepatic biliary carcinomas, visualization of the extent of tumor invasion by 3D-fusion images was equivalent to conventional cholangiography. In 2 patients, adequate placement of multiple stents could be visualized with this system. In 2 patients who underwent hepatectomy, more extended cancer invasion was observed than was visualized by 3D-fusion images., Conclusions: 3D fusion images were very useful for preoperative simulations in order to understand relationships between tumors and adjacent vasculatures.

Bone homeostasis is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption. Osteoclasts are multinucleated cells that are formed by mononuclear preosteoclast fusion. Fat-soluble vitamins such as vitamin D are pivotal in maintaining skeletal integrity. However, the role of vitamin E in bone remodeling is unknown. Here, we show that mice deficient in α-tocopherol transfer protein (Ttpa(-/-) mice), a mouse model of genetic vitamin E deficiency, have high bone mass as a result of a decrease in bone resorption. Cell-based assays indicated that α-tocopherol stimulated osteoclast fusion, independent of its antioxidant capacity, by inducing the expression of dendritic-cell-specific transmembrane protein, an essential molecule for osteoclast fusion, through activation of mitogen-activated protein kinase 14 (p38) and microphthalmia-associated transcription factor, as well as its direct recruitment to the Tm7sf4 (a gene encoding DC-STAMP) promoter. Indeed, the bone abnormality seen in Ttpa(-/-) mice was rescued by a Tm7sf4 transgene. Moreover, wild-type mice or rats fed an α-tocopherol-supplemented diet, which contains a comparable amount of α-tocopherol to supplements consumed by many people, lost bone mass. These results show that serum vitamin E is a determinant of bone mass through its regulation of osteoclast fusion.

Smads 1/5/8 transduce the major intracellular signaling of bone morphogenetic proteins (BMPs). In the present study, we analyzed Smad1-binding proteins in HEK293T cells using a proteomic technique and identified the protein, zinc-finger, RAN-binding domain-containing protein 2 (ZRANB2). Zranb2 interacted strongly with Smad1, Smad5, and Smad8 and weakly with Smad4. The overexpression of Zranb2 inhibited BMP activities in C2C12 myoblasts in vitro, and the injection of Zranb2 mRNA into zebrafish embryos induced weak dorsalization. Deletion analyses of Zranb2 indicated that the serine/arginine-rich (SR) domain and the glutamine-rich domain were required for the inhibition of BMP activity and the interaction with Smad1, respectively. Zranb2 was found to be localized in the nucleus; however, the SR domain-deleted mutant localized to the cytoplasm. The knockdown of endogenous Zranb2 in C2C12 cells enhanced BMP activity. Zranb2 suppressed Smad transcriptional activity without affecting Smad phosphorylation, nuclear localization, or DNA binding. Taken together, these findings suggested that Zranb2 is a novel BMP suppressor that forms a complex with Smads in the nucleus., (Copyright © 2011 Wiley Periodicals, Inc.)

Variations in microscopic elastic structures along the entire length of canine aorta were evaluated by use of a scanning haptic microscope (SHM). The total aorta from the aortic arch to the abdominal aorta was divided into 6 approximately equal segments. After embedding the aorta in agar, it was cut into horizontal circumferential segments to obtain disk-like agar portions containing ring-like samples of aorta with flat surfaces (thickness, approximately 1 mm). The elastic modulus and topography of the samples under no-load conditions were simultaneously measured along the entire thickness of the wall by SHM by using a probe with a diameter of 5 μm and a spatial resolution of 2 μm at a rate of 0.3 s/point. The elastic modulus of the wall was the highest on the side of the luminal surface and decreased gradually toward the adventitial side. This tendency was similar to that of the change in the elastin fiber content. During the evaluation of the mid-portion of each tunica media segment, the highest elastic modulus (40.8 ± 3.5 kPa) was identified at the thoracic section of the aorta that had the highest density of elastic fibers. Under no-load conditions, portions of the aorta with high elastin density have a high elastic modulus.

The most recommendable MRI sequence for the morphologic assessment of the cartilage damage due to osteoarthritis (OA) is gradient echo fat suppression T1 weighted image (SPGR: Spoiled gradient recalled acquisition in the steady state, FLASH: Fast low-angle shot). For the detection of the cartilage degeneration before the morphological change, T2 mapping, dGEMRIC (delayed Gd-DTPA2-Enhanced MRI of Cartilage), and T1ρ mapping are suggested. Subchondral bone structural changes such as bone sclerosis and cyst are deeply related to the etiology of OA, and quantitative evaluation for them using MDCT (Multi-Detector row CT) and 3TMRI are suggested.

Prostate cancer development is associated with hyperactive androgen signaling. However, the molecular link between androgen receptor (AR) function and humoral factors remains elusive. A prostate cancer mouse model was generated by selectively mutating the AR threonine 877 into alanine in prostatic epithelial cells through Cre-ERT2-mediated targeted somatic mutagenesis. Such AR point mutant mice (ARpe-T877A/Y) developed hypertrophic prostates with responses to both an androgen antagonist and estrogen, although no prostatic tumor was seen. In prostate cancer model transgenic mice, the onset of prostatic tumorigenesis as well as tumor growth was significantly potentiated by introduction of the AR T877A mutation into the prostate. Genetic screening of mice identified Wnt-5a as an activator. Enhanced Wnt-5a expression was detected in the malignant prostate tumors of patients, whereas in benign prostatic hyperplasia such aberrant up-regulation was not obvious. These findings suggest that a noncanonical Wnt signal stimulates development of prostatic tumors with AR hyperfunction.

Bone morphogenetic proteins (BMPs) induce osteoblastic differentiation in myogenic cells via the phosphorylation of Smads. Two types of Smad phosphatases--small C-terminal domain phosphatase 1 (SCP1) and protein phosphatase magnesium-dependent 1A--have been shown to inhibit BMP activity. Here, we report that SCP1 inhibits the osteoblastic differentiation induced by BMP-4, a constitutively active BMP receptor, and a constitutively active form of Smad1. The phosphatase activity of SCP1 was required for this suppression, and the knockdown of SCP1 in myoblasts stimulated the osteoblastic differentiation induced by BMP signaling. In contrast to protein phosphatase magnesium-dependent 1A, SCP1 did not reduce the protein levels of Smad1 and failed to suppress expression of the Id1, Id2, and Id3 genes. Runx2-induced osteoblastic differentiation was suppressed by SCP1 without affecting the transcriptional activity or phosphorylation levels of Runx2. Taken together, these findings suggest that SCP1 may inhibit the osteoblastic differentiation induced by the BMP-Smad axis via Runx2 by suppressing downstream effector(s).

The traditional view of bone metabolism as a primarily endocrine activity has been expanded in recent years following the identification of nervous system controlling bone metabolism by leptin studies. Especially, hypothalamic appetite regulating-peptides, such as NPY, CART and NMU have been demonstrated to be bone-regulating neuropeptides. Recently, other neuropeptides, such as serotonin and oxytocin, are reported to be associated with bone metabolism.

Excessive accumulation of bone marrow adipocytes observed in senile osteoporosis or age-related osteopenia is caused by the unbalanced differentiation of MSCs into bone marrow adipocytes or osteoblasts. Several transcription factors are known to regulate the balance between adipocyte and osteoblast differentiation. However, the molecular mechanisms that regulate the balance between adipocyte and osteoblast differentiation in the bone marrow have yet to be elucidated. To identify candidate genes associated with senile osteoporosis, we performed genome-wide expression analyses of differentiating osteoblasts and adipocytes. Among transcription factors that were enriched in the early phase of differentiation, Id4 was identified as a key molecule affecting the differentiation of both cell types. Experiments using bone marrow-derived stromal cell line ST2 and Id4-deficient mice showed that lack of Id4 drastically reduces osteoblast differentiation and drives differentiation toward adipocytes. On the other hand knockdown of Id4 in adipogenic-induced ST2 cells increased the expression of Ppargamma2, a master regulator of adipocyte differentiation. Similar results were observed in bone marrow cells of femur and tibia of Id4-deficient mice. However the effect of Id4 on Ppargamma2 and adipocyte differentiation is unlikely to be of direct nature. The mechanism of Id4 promoting osteoblast differentiation is associated with the Id4-mediated release of Hes1 from Hes1-Hey2 complexes. Hes1 increases the stability and transcriptional activity of Runx2, a key molecule of osteoblast differentiation, which results in an enhanced osteoblast-specific gene expression. The new role of Id4 in promoting osteoblast differentiation renders it a target for preventing the onset of senile osteoporosis., Competing Interests: The authors have declared that no competing interests exist.

The objective of this study was to make an elasticity distribution image of natural arteries in a liquid environment at high resolution at the micrometer level and at a wide area at the sub-square millimeter level by improving the scanning haptic microscope (SHM), developed previously for characterization of the stiffness of natural tissues. The circumferential sections (thickness, 1.0 mm) of small-caliber porcine arteries (approximately 3-mm diameter) were used as a sample. Measurement was performed by soaking a probe (diameter, 5 microm; spatial resolution, less than 2 microm) in saline solution at an appropriate depth. The vascular tissues were segregated by multi-layering a high elasticity region with mainly elastin (50.8 +/- 13.8 kPa) and a low one with mainly collagen and smooth muscle cells (17.0 +/- 9.0 kPa), as observed previously in high humidity conditions. The elasticity was measured repeatedly with little change for over 4 h in a liquid environment, which enabled observation with maintenance of high precision of a large area of at least 1,200 x 100 microm, whereas the elasticity was increased with time by the dehydration of samples with shrinkage in the air, in which an averaged elasticity in the overall area was approximately doubled within 2 h. This simple, inexpensive system allows observation of the distribution of the surface elasticity at the extracellular matrix level of vascular tissues in a liquid environment close to the natural one.

Both BMPs and Wnts play important roles in the regulation of bone formation. We examined the molecular mechanism regulating cross-talk between BMPs and Wnts in the osteoblastic differentiation of C2C12 cells. Canonical Wnts (Wnt1 and Wnt3a) but not non-canonical Wnts (Wnt5a and Wnt11) synergistically stimulated ALP activity in the presence of BMP-4. Wnt3a and BMP-4 synergistically stimulated the expression of type I collagen and osteonectin. However, Wnt3a did not stimulate ALP activity that was induced by a constitutively active BMP receptor or Smad1. Noggin and Dkk-1 suppressed the synergistic effect of BMP-4 and Wnt3a, but Smad7 did not. Overexpression of beta-catenin did not affect BMP-4-induced ALP activity. By contrast, inhibition or stimulation of GSK3beta activity resulted in either stimulation or suppression of ALP activity, respectively, in the presence of BMP-4. Taken together, these findings suggest that BMPs and canonical Wnts may regulate osteoblastic differentiation, especially at the early stages, through a GSK3beta-dependent but beta-catenin-independent mechanism., (Copyright 2010 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Bone morphogenetic proteins (BMPs) induce ectopic bone formation in muscle tissue in vivo and convert myoblasts such that they differentiate into osteoblastic cells in vitro. We report here that constitutively active Smad1 induced osteoblastic differentiation of C2C12 myoblasts in cooperation with Smad4 or Runx2. In floxed Smad4 mice-derived cells, Smad4 ablation partially suppressed BMP-4-induced osteoblast differentiation. In contrast, the BMP-4-induced inhibition of myogenesis was lost by Smad4 ablation and restored by Smad4 overexpression. A nuclear zinc finger protein, E4F1, was identified as a possible component of the Smad4 complex that suppresses myogenic differentiation in response to BMP signaling. In the presence of Smad4, E4F1 stimulated the expression of Ids. Taken together, these findings suggest that the Smad signaling pathway may play a dual role in the BMP-induced conversion of myoblasts to osteoblastic cells.

Phosphorylation of Smad1/5/8 at carboxyl-terminal serine residues by type I receptors activates downstream bone morphogenetic protein (BMP) signaling. Protein phosphatase magnesium-dependent 1A (PPM1A) has been shown to suppress BMP activity by dephosphorylating phospho-Smads. We report here that PPM1A suppresses BMP signaling via a novel mechanism. PPM1A inhibited a constitutively activated Smad1 mutant lacking BMP receptor phosphorylation sites. PPM1A reduced the protein levels not only of Smad1 but also of Smad5 and Smad8. A proteasome inhibitor blocked the inhibitory effects of PPM1A on Smad1, but the Smurf-binding motif in the Smad1 linker region was not involved in this inhibition. The phosphatase activity of PPM1A is essential for inhibition. Taken together, these findings suggest that through the dephosphorylation of unidentified substrate(s), PPM1A inhibits BMP signaling by decreasing Smad protein levels via the proteasome pathway. Moreover, knockdown of endogenous PPM1A stimulated osteoblastic differentiation, suggesting that PPM1A may physiologically suppress BMP signaling via Smads., (Copyright 2010 American Society for Bone and Mineral Research.)

Objective: : We demonstrated that the tactile mapping system (TMS) has a high degree of spatial precision in the distribution mapping of surface elasticity of tissues or organs., Methods: : Samples used were a circumferential section of a small-caliber porcine artery (diameter: ∼3 mm) and an elasticity test pattern with a line and space configuration for the distribution mapping of elasticity, prepared by regional micropatterning of a 14-μm thick gelatin hydrogel coating on a polyurethane sheet. Surface topography and elasticity in normal saline were simultaneously investigated by TMS using a probe with a diameter of 5 or 12 μm, a spatial interval of 1 to 5 μm, and an indentation depth of 4 μm., Results: : In the test pattern, a spatial resolution in TMS of <5 μm was acquired under water with a minimal probe diameter and spatial interval of the probe movement. TMS was used for the distribution mapping of surface elasticity in a flat, circumferential section (thickness: ∼0.5 mm) of a porcine artery, and the concentric layers of the vascular wall, including the collagen-rich and elastin-rich layers, could be clearly differentiated in terms of surface elasticity at the spatial resolution of <2 μm., Conclusions: : TMS is a simple and inexpensive technique for the distribution mapping of the surface elasticity in vascular tissues at the spatial resolution <2 μm. TMS has the ability to analyze a complex structure of the tissue samples under normal saline.

Although microRNAs (miRNAs) are involved in many biological processes, the mechanisms whereby miRNAs regulate osteoblastic differentiation are poorly understood. Here, we found that BMP-4-induced osteoblastic differentiation of bone marrow-derived ST2 stromal cells was promoted and repressed after transfection of sense and antisense miR-210, respectively. A reporter assay demonstrated that the activin A receptor type 1B (AcvR1b) gene was a target for miR-210. Furthermore, inhibition of transforming growth factor-beta (TGF-beta)/activin signaling in ST2 cells with SB431542 promoted osteoblastic differentiation. We conclude that miR-210 acts as a positive regulator of osteoblastic differentiation by inhibiting the TGF-beta/activin signaling pathway through inhibition of AcvR1b.

A number of nuclear complexes modify chromatin structure and operate as functional units. However, the in vivo role of each component within the complexes is not known. ATP-dependent chromatin remodeling complexes form several types of protein complexes, which reorganize chromatin structure cooperatively with histone modifiers. Williams syndrome transcription factor (WSTF) was biochemically identified as a major subunit, along with 2 distinct complexes: WINAC, a SWI/SNF-type complex, and WICH, an ISWI-type complex. Here, WSTF(-/-) mice were generated to investigate its function in chromatin remodeling in vivo. Loss of WSTF expression resulted in neonatal lethality, and all WSTF(-/-) neonates and approximately 10% of WSTF(+/-) neonates suffered cardiovascular abnormalities resembling those found in autosomal-dominant Williams syndrome patients. Developmental analysis of WSTF(-/-) embryos revealed that Gja5 gene regulation is aberrant from E9.5, conceivably because of inappropriate chromatin reorganization around the promoter regions where essential cardiac transcription factors are recruited. In vitro analysis in WSTF(-/-) mouse embryonic fibroblast (MEF) cells also showed impaired transactivation functions of cardiac transcription activators on the Gja5 promoter, but the effects were reversed by overexpression of WINAC components. Likewise in WSTF(-/-) MEF cells, recruitment of Snf2h, an ISWI ATPase, to PCNA and cell survival after DNA damage were both defective, but were ameliorated by overexpression of WICH components. Thus, the present study provides evidence that WSTF is shared and is a functionally indispensable subunit of the WICH complex for DNA repair and the WINAC complex for transcriptional control.

Bone morphogenetic proteins (BMPs) induce osteoblastic differentiation of myoblasts via binding to cell surface receptors. Repulsive guidance molecules (RGMs) have been identified as BMP co-receptors. We report here that DRAGON/RGMb, a member of the RGM family, suppressed BMP signaling in C2C12 myoblasts via a novel mechanism. All RGMs were expressed in C2C12 cells that were differentiated into myocytes and osteoblastic cells, but RGMc was not detected in immature cells. In C2C12 cells, only DRAGON suppressed ALP and Id1 promoter activities induced by BMP-4 or by constitutively activated BMP type I receptors. This inhibition by DRAGON was dependent on the secretory form of the von Willbrand factor type D domain. DRAGON even suppressed BMP signaling induced by constitutively activated Smad1. Over-expression of neogenin did not alter the inhibitory capacity of DRAGON. Taken together, these findings indicate that DRAGON may be an inhibitor of BMP signaling in C2C12 myoblasts. We also suggest that a novel molecule(s) expressed on the cell membrane may mediate the signal transduction of DRAGON in order to suppress BMP signaling in C2C12 myoblasts.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by congenital malformation of the great toes and by progressive heterotopic bone formation in muscle tissue. Recently, a mutation involving a single amino acid substitution in a bone morphogenetic protein (BMP) type I receptor, ALK2, was identified in patients with FOP. We report here that the identical mutation, R206H, was observed in 19 Japanese patients with sporadic FOP. This mutant receptor, ALK2(R206H), activates BMP signaling without ligand binding. Moreover, expression of Smad1 and Smad5 was up-regulated in response to muscular injury. ALK2(R206H) with Smad1 or Smad5 induced osteoblastic differentiation that could be inhibited by Smad7 or dorsomorphin. Taken together, these findings suggest that the heterotopic bone formation in FOP may be induced by a constitutively activated BMP receptor signaling through Smad1 or Smad5. Gene transfer of Smad7 or inhibition of type I receptors with dorsomorphin may represent strategies for blocking the activity induced by ALK2(R206H) in FOP.

This study aimed to map the elasticity of a natural artery at the micron level by using a tactile mapping system (TMS) that was recently developed for characterization of the stiffness of tissue slices. The sample used was a circumferential section (thickness, approximately 1 mm) of a small-caliber porcine artery (diameter, approximately 3 mm). Elasticity was measured with a probe of diameter 1 microm and a spatial resolution of 2 microm at a rate of 0.3 s per point, without significant sample invasion. Topographical measurements were also performed simultaneously. Wavy regions of high elasticity, layered in the circumferential direction, were measured at the tunica media, which was identified as an elastin-rich region. The Young's modulus of the elastin-rich region in the media was 50.8 +/- 13.8 kPa, and that of the elastin-rich region of the lamina elastica interna was 69.0 +/- 12.8 kPa. Both these values were higher than the Young's modulus of the other regions in the media, including smooth muscle cells and collagen fibrils (17.0 +/- 9.0 kPa). TMS is simple and inexpensive to perform and allows observation of the distribution of the surface elastic modulus at the extracellular matrix level in vascular tissue. TMS is expected to be a powerful tool in evaluation of the maturation and degree of reconstruction in the development of tissue-engineered or artificial tissues and organs.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant congenital disorder characterized by progressive heterotopic bone formation in muscle tissues. A common mutation among FOP patients has been identified in ALK2, ALK2(R206H), which encodes a constitutively active bone morphogenetic protein (BMP) receptor. Recently, a unique mutation of ALK2, ALK2(G356D), was identified to be a novel mutation in a Japanese FOP patient who had unique clinical features. Over-expression of ALK2(G356D) induced phosphorylation of Smad1/5/8 and activated Id1-luc and alkaline phosphatase activity in myoblasts. However, the over-expression failed to activate phosphorylation of p38, ERK1/2, and CAGA-luc activity. These ALK2(G356D) activities were weaker than those of ALK2(R206H), and they were suppressed by a specific inhibitor of the BMP-regulated Smad pathway. These findings suggest that ALK2(G356D) induces heterotopic bone formation via activation of a BMP-regulated Smad pathway. The quantitative difference between ALK2(G356D) and ALK2(R206H) activities may have caused the phenotypic differences in these patients.

Although various microRNAs regulate cell differentiation and proliferation, no miRNA has been reported so far to play an important role in the regulation of osteoblast differentiation. Here we describe the role of miR-125b in osteoblastic differentiation in mouse mesenchymal stem cells, ST2, by regulating cell proliferation. The expression of miR-125b was time-dependently increased in ST2 cells, and the increase in miR-125b expression was attenuated in osteoblastic-differentiated ST2 cells induced by BMP-4. The transfection of exogenous miR-125b inhibited proliferation of ST2 cells and caused inhibition of osteoblastic differentiation. In contrast, when the endogenous miR-125b was blocked by transfection of its antisense RNA molecule, alkaline phosphatase activity after BMP-4 treatment was elevated. These results strongly suggest that miR-125b is involved in osteoblastic differentiation through the regulation of cell proliferation.

BMPs bound to specific receptors expressed on cell membrane of target cells, then activate Smad-dependent intracellular signaling. Wnt signaling through LRP5 receptor has been genetically shown to be involved in bone formation in humans. Recently, it was shown that Smad1 degradation requires phosphorylation by GSK3beta, which is suppressed by Wnt signaling. These findings suggest that a cross-talk between BMP and Wnt through Smad1 plays an important role on bone formation.