A serum metabolomics-based profile in low bone mineral density postmenopausal women.

Osteoporosis is characterized as a metabolic disorder of bone tissue, and various metabolic markers are now available to support its diagnosis and evaluate treatment effects. Substances produced as end products of metabolomic activities are the correlated factors to the biological or metabolic status, and thus, metabolites are considered highly sensitive markers of particular pathological states, including osteoporosis. Here we undertook comprehensive serum metabolomics analysis in postmenopausal women with or without low bone mineral density (low BMD vs controls) for the first time using capillary electrophoresis/mass spectrometry. Among the metabolites tested, 57 were detected in sera. Levels of hydroxyproline, Gly-Gly and cystine, differed significantly between groups, with Gly-Gly and cystine significantly lower in the low BMD group and hydroxyproline, a reported marker of osteoporosis, significantly higher. Levels of TRACP5b, a bone resorption marker, were significantly higher in the low BMD group, supporting the study's validity. Taken together, our findings represent novel metabolomic profiling in low BMD in postmenopausal women.

Smad4 is required to inhibit osteoclastogenesis and maintain bone mass.

Bone homeostasis is maintained as a delicate balance between bone-resorption and bone-formation, which are coupled to maintain appropriate bone mass. A critical question is how bone-resorption is terminated to allow bone-formation to occur. Here, we show that TGFßs inhibit osteoclastogenesis and maintain bone-mass through Smad4 activity in osteoclasts. We found that latent-TGFß1 was activated by osteoclasts to inhibit osteoclastogenesis. Osteoclast-specific Smad4 conditional knockout mice (Smad4-cKO) exhibited significantly reduced bone-mass and elevated osteoclast formation relative to controls. TGFß1-activation induced expression of Irf8 and Bcl6, both of which encode factors inhibiting osteoclastogenesis, by blocking their negative regulator, Prdm1, in osteoclasts in a Smad4-dependent manner. Reduced bone-mass and accelerated osteoclastogenesis seen in Smad4-cKO were abrogated by Prdm1 deletion. Administration of latent-TGFß1-Fc to wild-type mice antagonized LPS-induced bone destruction in a model of activated osteoclast-mediated bone destruction. Thus, latent-TGFß1-Fc could serve as a promising new therapeutic agent in bone diseases marked by excessive resorption.

Vitamin D Deficiency with High Intact PTH Levels is More Common in Younger than in Older Women: A Study of Women Aged 39-64 Years.

Low serum 25-hydroxyvitamin D (25(OH)D) levels are implicated as a risk factor for hip and spine fractures. Studies of the relation between 25(OH)D levels and fractures have primarily involved elderly osteoporosis patients or patients with fractures; however, the serum 25(OH)D and parathyroid hormone (PTH) status in younger adult populations remains largely unknown. We evaluated serum 25(OH)D and intact PTH levels in 411 women aged 39-64 years who were not receiving medication for osteoporosis or other bone diseases. Serum 25(OH)D levels were positively correlated with age (P = 0.019), whereas intact PTH levels were inversely correlated with 25(OH)D levels (P < 0.001). Thus, low vitamin D levels with high intact PTH levels were more common in younger than in older women. Our data show that serum 25(OH)D insufficiency could be a more serious concern in the younger population than had been previously anticipated. Because serum 25(OH)D insufficiency is reportedly a risk factor for hip and spine fracture, the number of fracture patients could increase in the future, suggesting that we may need to correct the serum vitamin D/intact PTH status to prevent future osteoporosis.

The vitamin D analogue ED71 but Not 1,25(OH)2D3 targets HIF1α protein in osteoclasts.

Although both an active form of the vitamin D metabolite, 1,25(OH)2D3, and the vitamin D analogue, ED71 have been used to treat osteoporosis, anti-bone resorbing activity is reportedly seen only in ED71- but not in 1,25(OH)2D3 -treated patients. In addition, how ED71 inhibits osteoclast activity in patients has not been fully characterized. Recently, HIF1α expression in osteoclasts was demonstrated to be required for development of post-menopausal osteoporosis. Here we show that ED71 but not 1,25(OH)2D3, suppress HIF1α protein expression in osteoclasts in vitro. We found that 1,25(OH)2D3 or ED71 function in osteoclasts requires the vitamin D receptor (VDR). ED71 was significantly less effective in inhibiting M-CSF and RANKL-stimulated osteoclastogenesis than was 1,25(OH)2D3 in vitro. Downregulation of c-Fos protein and induction of Ifnß mRNA in osteoclasts, both of which reportedly block osteoclastogenesis induced by 1,25(OH)2D3 in vitro, were both significantly higher following treatment with 1,25(OH)2D3 than with ED71. Thus, suppression of HIF1α protein activity in osteoclasts in vitro, which is more efficiently achieved by ED71 rather than by 1,25(OH)2D3, could be a reliable read-out in either developing or screening reagents targeting osteoporosis.

HIF1α is required for osteoclast activation by estrogen deficiency in postmenopausal osteoporosis.

In women, estrogen deficiency after menopause frequently accelerates osteoclastic bone resorption, leading to osteoporosis, the most common skeletal disorder. However, mechanisms underlying osteoporosis resulting from estrogen deficiency remain largely unknown. Here we show that in bone-resorbing osteoclasts, estrogen-dependent destabilization of hypoxia-inducible factor 1 alpha (HIF1α), which is unstable in the presence of oxygen, plays a pivotal role in promoting bone loss in estrogen-deficient conditions. In vitro, HIF1α was destabilized by estrogen treatment even in hypoxic conditions, and estrogen loss in ovariectomized (Ovx) mice stabilized HIF1α in osteoclasts and promoted their activation and subsequent bone loss in vivo. Osteoclast-specific HIF1α inactivation antagonized bone loss in Ovx mice and osteoclast-specific estrogen receptor alpha deficient mice, both models of estrogen-deficient osteoporosis. Oral administration of a HIF1α inhibitor protected Ovx mice from osteoclast activation and bone loss. Thus, HIF1α represents a promising therapeutic target in osteoporosis.

[Focal nodular hyperplasia that was difficult to differentiate from hepatocellular carcinoma].

A 59-year-old man had received medical treatment for alcoholic hepatopathy. He stopped drinking 3 years before visiting the hospital. On medical examination, the abdominal echo showed a hepatic mass lesion that was HBs-Ag (-) and HCV-Ab (-). Computed tomography (CT) revealed a tumor of more than 25 mm in diameter at S7 of the liver. Dynamic CT showed that it was stained in the early phase but washed out in the delay phase. Magnetic resonance imaging (MRI) showed high intensity staining of the tumor in both T1-and T2-weighted images, and it was also stained in the EOB Primovist MRI hepatobiliary phase. The findings from the images were not typical for hepatocellular carcinoma(HCC) or other benign tumors. We therefore performed an S7 partial hepatectomy. We diagnosed the tumor as focal nodular hyperplasia (FNH) by histology.

[A surgical case of solitary lymph node metastasis of hepatocellular carcinoma after nonsurgical treatment].

A 66-year-old man with multiple hepatocellular carcinomas(HCCs) underwent transcatheter arterial chemoembolization(TACE) twice and radiofrequency ablation(RFA) twice at another hospital in June 2009. In November 2010, abdominal computed tomography(CT) revealed a solitary lymph node metastasis( 23 mm in diameter) in the hepatoduodenal ligament, after which he was admitted to our hospital in December 2010. In February 2011, ethoxybenzyl diethylenetriamine pentaceric acid-enhanced magnetic resonance imaging (EOB-MRI) showed revealed a growing solitary lymph node metastasis(33 mm in diameter) and good control of the intrahepatic lesion. Positron emission tomography(PET)-CT confirmed the solitary lymph node metastasis without any other extrahepatic recurrence. We performed lymph node resection in March 2011 because of good control of the intrahepatic lesion and the lack of extrahepatic recurrence. He was discharged from our hospital 11 days after surgery with a good postoperative course. Histologically, the tumor was diagnosed as a lymph node metastasis of poorly differentiated HCC. Subsequent abdominal CT in January 2012 revealed multiple recurrent lesions, and he underwent TACE therapy in February 2012. Currently, the patient is alive 1 year 3 months after lymph node resection without any other extrahepatic recurrence.

An essential role for STAT6-STAT1 protein signaling in promoting macrophage cell-cell fusion.

Macrophage lineage cells such as osteoclasts and foreign body giant cells (FBGCs) form multinuclear cells by cell-cell fusion of mononuclear cells. Recently, we reported that two seven-transmembrane molecules, osteoclast stimulatory transmembrane protein (OC-STAMP) and dendritic cell-specific transmembrane protein (DC-STAMP), were essential for osteoclast and FBGC cell-cell fusion in vivo and in vitro. However, signaling required to regulate FBGC fusion remained largely unknown. Here, we show that signal transducer and activator of transcription 1 (STAT1) deficiency in macrophages enhanced cell-cell fusion and elevated DC-STAMP expression in FBGCs. By contrast, lack of STAT6 increased STAT1 activation, significantly inhibiting cell-cell fusion and decreasing OC-STAMP and DC-STAMP expression in IL-4-induced FBGCs. Furthermore, either STAT1 loss or co-expression of OC-STAMP/DC-STAMP was sufficient to induce cell-cell fusion of FBGCs without IL-4. We conclude that the STAT6-STAT1 axis regulates OC-STAMP and DC-STAMP expression and governs fusogenic mechanisms in FBGCs.

Conditional inactivation of Blimp1 in adult mice promotes increased bone mass.

Bone resorption, which is regulated by osteoclasts, is excessively activated in bone destructive diseases such as osteoporosis. Thus, controlling osteoclasts would be an effective strategy to prevent pathological bone loss. Although several transcription factors that regulate osteoclast differentiation and function could serve as molecular targets to inhibit osteoclast formation, those factors have not yet been characterized using a loss of function approach in adults. Here we report such a study showing that inactivation of B-lymphocyte induced maturation protein 1 (Blimp1) in adult mice increases bone mass by suppressing osteoclast formation. Using an ex vivo assay, we show that osteoclast differentiation is significantly inhibited by Blimp1 inactivation at an early stage of osteoclastogenesis. Conditional inactivation of Blimp1 inhibited osteoclast formation and increased bone mass in both male and female adult mice. Bone resorption parameters were significantly reduced by Blimp1 inactivation in vivo. Blimp1 reportedly regulates immune cell differentiation and function, but we detected no immune cell failure following Blimp1 inactivation. These data suggest that Blimp1 is a potential target to promote increased bone mass and prevent osteoclastogenesis.

PDGFBB promotes PDGFRα-positive cell migration into artificial bone in vivo.

Bone defects caused by traumatic bone loss or tumor dissection are now treated with auto- or allo-bone graft, and also occasionally by artificial bone transplantation, particularly in the case of large bone defects. However, artificial bones often exhibit poor affinity to host bones followed by bony union failure. Thus therapies combining artificial bones with growth factors have been sought. Here we report that platelet derived growth factor bb (PDGFBB) promotes a significant increase in migration of PDGF receptor α (PDGFRα)-positive mesenchymal stem cells/pre-osteoblastic cells into artificial bone in vivo. Growth factors such as transforming growth factor beta (TGFß) and hepatocyte growth factor (HGF) reportedly inhibit osteoblast differentiation; however, PDGFBB did not exhibit such inhibitory effects and in fact stimulated osteoblast differentiation in vitro, suggesting that combining artificial bones with PDGFBB treatment could promote host cell migration into artificial bones without inhibiting osteoblastogenesis.

Aldehyde-stress resulting from Aldh2 mutation promotes osteoporosis due to impaired osteoblastogenesis.

Osteoporosis is a complex disease with various causes, such as estrogen loss, genetics, and aging. Here we show that a dominant-negative form of aldehyde dehydrogenase 2 (ALDH2) protein, ALDH2*2, which is produced by a single nucleotide polymorphism (rs671), promotes osteoporosis due to impaired osteoblastogenesis. Aldh2 plays a role in alcohol-detoxification by acetaldehyde-detoxification; however, transgenic mice expressing Aldh2*2 (Aldh2*2 Tg) exhibited severe osteoporosis with increased levels of blood acetaldehyde without alcohol consumption, indicating that Aldh2 regulates physiological bone homeostasis. Wild-type osteoblast differentiation was severely inhibited by exogenous acetaldehyde, and osteoblastic markers such as osteocalcin, runx2, and osterix expression, or phosphorylation of Smad1,5,8 induced by bone morphogenetic protein 2 (BMP2) was strongly altered by acetaldehyde. Acetaldehyde treatment also inhibits proliferation and induces apoptosis in osteoblasts. The Aldh2*2 transgene or acetaldehyde treatment induced accumulation of the lipid-oxidant 4-hydroxy-2-nonenal (4HNE) and expression of peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor that promotes adipogenesis and inhibits osteoblastogenesis. Antioxidant treatment inhibited acetaldehyde-induced proliferation-loss, apoptosis, and PPARγ expression and restored osteoblastogenesis inhibited by acetaldehyde. Treatment with a PPARγ inhibitor also restored acetaldehyde-mediated osteoblastogenesis inhibition. These results provide new insight into regulation of osteoporosis in a subset of individuals with ALDH2*2 and in alcoholic patients and suggest a novel strategy to promote bone formation in such osteopenic diseases.

Osteoclast stimulatory transmembrane protein and dendritic cell­specific transmembrane protein cooperatively modulate cell­cell fusion to form osteoclasts and foreign body giant cells.

Cell­cell fusion is a dynamic phenomenon promoting cytoskeletal reorganization and phenotypic changes. To characterize factors essential for fusion of macrophage lineage cells, we identified the multitransmembrane protein, osteoclast stimulatory transmembrane protein (OC-STAMP), and analyzed its function. OC-STAMP­deficient mice exhibited a complete lack of cell­cell fusion of osteoclasts and foreign body giant cells (FBGCs), both of which are macrophage-lineage multinuclear cells, although expression of dendritic cell specific transmembrane protein (DC-STAMP), which is also essential for osteoclast/FBGC fusion, was normal. Crossing OC-STAMP­overexpressing transgenic mice with OC-STAMP­deficient mice restored inhibited osteoclast and FBGC cell­cell fusion seen in OC-STAMP­deficient mice. Thus, fusogenic mechanisms in macrophage-lineage cells are regulated via OC-STAMP and DC-STAMP.

[A long-term survival case after two resections of the peritoneal metastasis from hepatocellular carcinoma].

A 70-year-old man with type B hepatitis had ruptured HCC in segment 5, and he underwent with TAE at other hospital in June 2007. Then, he was introduced to our hospital in July 2007. Partial hepatectomy( S5) was performed in August 2007 (pT2N0M0, Stage II). Afterward, he underwent TACE therapy twice because of multiple intrahepatic recurrences. Abdominal CT revealed a viable recurrence lesion (S5), and peritoneal dissemination (surface of S3) in June 2009. We carried out partial hepatectomy (S5), and removal of peritoneal dissemination because of good liver function and without any other extra hepatic recurrence in July 2009. Histologically, the intrahepatic lesion( S5) and the S3 surface lesion were diagnosed as moderately differentiated HCC. In July 2010, abdominal CT revealed three lesions of peritoneal dissemination (right subphrenic lesion, hepatic flexure of the colon, neighborhood of left ureter, then the second removal of peritoneal dissemination was performed. In January 2011, he had multiple lung metastatic lesions, and multiple bone metastatic lesions were occurred in March 2011, then his general condition was getting worse. In April 2011, he was dead 46 months after the first TAE therapy for ruptured HCC, or 21 months after the first resection of peritoneal dissemination. Surgical resection of peritoneal dissemination of HCC may improve a survival for patients whose intrahepatic lesion is contorollable.

Osteoclasts are dispensable for hematopoietic stem cell maintenance and mobilization.

Hematopoietic stem cells (HSCs) are maintained in a specific bone marrow (BM) niche in cavities formed by osteoclasts. Osteoclast-deficient mice are osteopetrotic and exhibit closed BM cavities. Osteoclast activity is inversely correlated with hematopoietic activity; however, how osteoclasts and the BM cavity potentially regulate hematopoiesis is not well understood. To investigate this question, we evaluated hematopoietic activity in three osteopetrotic mouse models: op/op, c-Fos-deficient, and RANKL (receptor activator of nuclear factor kappa B ligand)-deficient mice. We show that, although osteoclasts and, by consequence, BM cavities are absent in these animals, hematopoietic stem and progenitor cell (HSPC) mobilization after granulocyte colony-stimulating factor injection was comparable or even higher in all three lines compared with wild-type mice. In contrast, osteoprotegerin-deficient mice, which have increased numbers of osteoclasts, showed reduced HSPC mobilization. BM-deficient patients and mice reportedly maintain hematopoiesis in extramedullary spaces, such as spleen; however, splenectomized op/op mice did not show reduced HSPC mobilization. Interestingly, we detected an HSC population in osteopetrotic bone of op/op mice, and pharmacological ablation of osteoclasts in wild-type mice did not inhibit, and even increased, HSPC mobilization. These results suggest that osteoclasts are dispensable for HSC mobilization and may function as negative regulators in the hematopoietic system.

Adaptive statistical iterative reconstruction technique for pulmonary CT: image quality of the cadaveric lung on standard- and reduced-dose CT.

RATIONALE AND OBJECTIVES: To evaluate thin-section computed tomography (CT) images of the lung reconstructed using adaptive statistical iterative reconstruction (ASIR) on standard- and reduced-dose CT. MATERIALS AND METHODS: Eleven cadaveric lungs were scanned by multidetector-row CT with two different tube currents (standard dose, 400 mA; reduced dose, 10 mA). The degree of ASIR was classified into six different levels: 0% (non-ASIR), 20%, 40%, 60%, 80%, and 100% (maximum-ASIR). The ASIR (20%, 60%, and 100%) images were compared with the ASIR (0%) images and assessed visually by three independent observers for image quality using a 7-point scale. The evaluation items included abnormal CT findings, normal lung structures, and subjective visual noise. The median scores assigned by the three observers were analyzed statistically. Quantitative noise was calculated by measuring the standard deviation in a circular region of interest on each selected image of ASIR (0%-100%). RESULTS: On standard-dose CT, the overall image quality significantly improved with increasing degree of ASIR (P ≤ .009, Wilcoxon signed-ranks test with Bonferroni correction). As ASIR increased, however, intralobular reticular opacities and peripheral vessels tended to be obscure. On reduced-dose CT, the overall image quality of ASIR (100%) was significantly better than that of ASIR (20%) (P ≤ .009). As ASIR increased, however, intralobular reticular opacities tended to be obscure. Using ASIR significantly reduced subjective and quantitative image noise on both standard- and reduced-dose CT (P < .001, Bonferroni/Dunn's method). CONCLUSION: ASIR improves the image quality by decreasing image noise. Maximum-ASIR may be needed for improving image quality on highly reduced-dose CT. However, excessive ASIR may obscure subtle shadows.

The Blimp1-Bcl6 axis is critical to regulate osteoclast differentiation and bone homeostasis.

Controlling osteoclastogenesis is critical to maintain physiological bone homeostasis and prevent skeletal disorders. Although signaling activating nuclear factor of activated T cells 1 (NFATc1), a transcription factor essential for osteoclastogenesis, has been intensively investigated, factors antagonistic to NFATc1 in osteoclasts have not been characterized. Here, we describe a novel pathway that maintains bone homeostasis via two transcriptional repressors, B cell lymphoma 6 (Bcl6) and B lymphocyte-induced maturation protein-1 (Blimp1). We show that Bcl6 directly targets 'osteoclastic' molecules such as NFATc1, cathepsin K, and dendritic cell-specific transmembrane protein (DC-STAMP), all of which are targets of NFATc1. Bcl6-overexpression inhibited osteoclastogenesis in vitro, whereas Bcl6-deficient mice showed accelerated osteoclast differentiation and severe osteoporosis. We report that Bcl6 is a direct target of Blimp1 and that mice lacking Blimp1 in osteoclasts exhibit osteopetrosis caused by impaired osteoclastogenesis resulting from Bcl6 up-regulation. Indeed, mice doubly mutant in Blimp1 and Bcl6 in osteoclasts exhibited decreased bone mass with increased osteoclastogenesis relative to osteoclast-specific Blimp1-deficient mice. These results reveal a Blimp1-Bcl6-osteoclastic molecule axis, which critically regulates bone homeostasis by controlling osteoclastogenesis and may provide a molecular basis for novel therapeutic strategies.

Computed tomography values calculation and volume histogram analysis for various computed tomographic patterns of diffuse lung diseases.

OBJECTIVE: The aim of this study was to determine the computed tomography (CT) values of various pulmonary abnormalities in cubic region of interest (ROI) and square ROI and evaluate the CT findings by histogram analysis in the ROI. METHODS: The study included 89 patients with the following 8 pulmonary CT patterns: normal lung, ground-glass attenuation, fine reticular opacity, coarse reticular opacity, honeycombing, airspace consolidation, nodular opacity, and emphysema. Cubic and square ROIs were selected in each CT pattern, and 5 values (contrast, variance, entropy, skewness, and kurtosis) were calculated. RESULTS: In the histogram of ground-glass attenuation, fine reticular opacity, and coarse reticular opacity, peaks had moved to the right compared with the normal lung. Only emphysema had higher contrast and lower entropy than the normal lung (P < 0.001). The other abnormalities had lower contrast and higher entropy than the normal lung. CONCLUSIONS: In conclusion, the shapes of histograms were characteristic of various abnormalities of the lung, and the values reflected the histogram quantitatively.

MCP-1 expressed by osteoclasts stimulates osteoclastogenesis in an autocrine/paracrine manner.

Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that plays a critical role in the recruitment and activation of leukocytes. Here, we describe that multinuclear osteoclast formation was significantly inhibited in cells derived from MCP-1-deficient mice. MCP-1 has been implicated in the regulation of osteoclast cell-cell fusion; however defects of multinuclear osteoclast formation in the cells from mice deficient in DC-STAMP, a seven transmembrane receptor essential for osteoclast cell-cell fusion, was not rescued by recombinant MCP-1. The lack of MCP-1 in osteoclasts resulted in a down-regulation of DC-STAMP, NFATc1, and cathepsin K, all of which were highly expressed in normal osteoclasts, suggesting that osteoclast differentiation was inhibited in MCP-1-deficient cells. MCP-1 alone did not induce osteoclastogenesis, however, the inhibition of osteoclastogenesis in MCP-1-deficient cells was restored by addition of recombinant MCP-1, indicating that osteoclastogenesis was regulated in an autocrine/paracrine manner by MCP-1 under the stimulation of RANKL in osteoclasts.

Commercially available computer-aided detection system for pulmonary nodules on thin-section images using 64 detectors-row CT: preliminary study of 48 cases.

RATIONALE AND OBJECTIVES: Most studies of computer-aided detection (CAD) for pulmonary nodules have focused on solid nodule detection. The aim of this study was to evaluate the performance of a commercially available CAD system in the detection of pulmonary nodules with or without ground-glass opacity (GGO) using 64-detector-row computed tomography compared to visual interpretation. MATERIALS AND METHODS: Computed tomographic examinations were performed on 48 patients with existing or suspicious pulmonary nodules on chest radiography. Three radiologists independently reported the location and pattern (GGO, solid, or part solid) of each nodule candidate on computed tomographic scans, assigned each a confidence score, and then analyzed all scans using the CAD system. A reference standard was established by a consensus panel of different radiologists, who found 229 noncalcified nodules with diameters > or = 4 mm. True-positive and false-positive results and confidence levels were used to generate jackknife alternative free-response receiver-operating characteristic plots. RESULTS: The sensitivity of GGO for 3 radiologists (60%-80%) was significantly higher than that for the CAD system (21%) (McNemar's test, P < .0001). For overall and solid nodules, the figure-of-merit values without and with the CAD system were significantly different (P = .005-.04) on jackknife alternative free-response receiver-operating characteristic analysis. For GGO and part-solid nodules, the figure-of-merit values with the CAD system were greater than those without the CAD system, indicating no significant differences. CONCLUSION: Radiologists are significantly superior to this CAD system in the detection of GGO, but the CAD system can still play a complementary role in detecting nodules with or without GGO.