Inhibition of hepatocyte growth factor/c-Met signalling abrogates joint destruction by suppressing monocyte migration in rheumatoid arthritis.

OBJECTIVES: To determine the expression of hepatocyte growth factor (HGF) in RA biological fluids, the role of HGF in monocyte migration and the therapeutic effect of the c-Met inhibitor savolitinib in an arthritis model mice. METHODS: HGF/c-Met expression in serum, SF and synovial tissues (STs) obtained from RA patients and controls, as well as RA fibroblast-like synoviocytes (FLSs), was evaluated by ELISA and immunostaining. To determine the function of HGF in RA SF, we preincubated RA SF with a neutralizing anti-HGF antibody and measured the chemotactic ability of a human acute monocytic leukaemia cell line (THP-1). Additionally, examinations were conducted of SKG mice treated with savolitinib for 4 weeks. RESULTS: HGF levels in serum from RA patients were significantly higher than those in the controls and were decreased by drug treatment for 24 weeks. Additionally, the HGF level in SF from RA patients was higher than that in SF from OA patients. HGF and c-Met expression was also noted in RA STs. Stimulation of RA FLSs with TNF-α increased HGF/c-Met expression in a concentration-dependent manner, and c-Met signal inhibition suppressed production of fractalkine/CX3CL1 and macrophage inflammatory protein-1α/CCL3. When HGF was removed by immunoprecipitation, migration of THP-1 in RA SF was suppressed. In SKG mice, savolitinib significantly suppressed ankle bone destruction on µCT, with an associated reduction in the number of tartrate-resistant acid phosphatase-positive osteoclasts. CONCLUSION: HGF produced by inflammation in synovium of RA patients activates monocyte migration to synovium and promotes bone destruction via a chemotactic effect and enhanced chemokine production.

Biological effects of anti-RANKL antibody administration in pregnant mice and their newborns.

Denosumab, a fully human monoclonal antibody that neutralizes receptor activator of nuclear factor-κB ligand (RANKL) and blocks osteoclast differentiation, has received approval in Japan for use as an anti-resorptive drug for osteoporosis and skeletal-related events (SREs) in patients with solid cancer. Denosumab is contraindicated during pregnancy, though the effects of blocking RANKL activity on pregnant mothers and their newborns are unclear. We used mice to investigate the effects of an anti-RANKL antibody on maternal and newborn health. Mothers injected with the anti-RANKL antibody had increased bone mass as compared with the controls, while osteoclast number and the level of tartrate-resistant acid phosphatase (TRAP) in serum were increased at the end of pregnancy. Newborn mice exposed to the antibody in utero were normally born, but showed increased bone mass and died within 48 h after birth. None of the newborns were found to have milk in their stomachs, suggesting that they died due to a maternal defect in lactation. Consistent with this, anti-RANKL antibody-injected mothers displayed impaired mammary gland development. However, fostering by healthy surrogate mothers rescued only 33% of the antibody-exposed newborns, suggesting that neonatal mortality was due, at least in part, to an intrinsic defect in the newborns. Our findings show that anti-RANKL antibody administration during pregnancy results in not only an undesirable increase in bone mass, but also has harmful effects on newborn survival.

Planar catechin increases bone mass by regulating differentiation of osteoclasts in mice.

OBJECTIVES: While catechins have been reported to exhibit potential to benefit osteoporosis patients, the effects of planar catechin (PCat), synthesized during the development of drugs for Alzheimer's disease, have not been clearly elucidated. Here, we examined the effects of PCat on mouse bone metabolism both in vivo and in vitro. METHODS: Six week old female mice were orally administered PCat (30 mg/kg) every other day for four weeks, and their femurs were analyzed using micro-computed tomography imaging. Osteoclasts and osteoblasts were collected from mice and cultured with PCat. Subsequently, osteoclast formation and differentiation and osteoblast differentiation were observed. RESULTS: Mice orally administered PCat displayed significantly increased femur bone mass compared to the control group. Quantitative polymerase chain reaction findings indicated that PCat addition to osteoclast progenitor cultures suppressed osteoclast formation and decreased osteoclast marker expression without affecting the proliferative potential of the osteoclast progenitor cells. Addition of PCat to osteoblast cultures increased osteoblast marker expression. CONCLUSIONS: PCat inhibits osteoclast differentiation and promotes osteoblast differentiation, resulting in increased bone mass in mice. These results suggest that PCat administration is a promising treatment option for conditions associated with bone loss, including osteoporosis.

Cholesterol and fat in diet disrupt bone and tooth homeostasis in mice.

Dyslipidemia is a condition of high levels of triglycerides and cholesterol in the blood, and high levels of cholesterol is associated with a variety of systemic diseases. The effects of a high-fat diet on bone have been reported, however, it is not clear which components of a high-fat diet affect bone. This study was conducted to examine the effects of dietary lipids and cholesterol on bone homeostasis maintenance. Eight-week-old male mice (C57BL/6 J) were fed five types of feed with different amounts of fat (14 %, 36 %) and cholesterol (0.01 %, 1.25 %, 5 %) for 12 weeks. Blood, femur, tibia, and tooth samples were examined, and serum lipid markers and bone morphology were determined using µCT and histological analysis. Additionally, bone marrow cells were obtained and cultured, and osteoclast differentiation markers analyzed using qPCR. Mice fed a diet high in both fat (36 %) and cholesterol (1.25 %) showed increased total cholesterol and low-density lipoprotein levels in blood, and decreased bone volume fraction as compared to the standard diet group. However, bone mass was unaffected in the high fat only (36 %) and high cholesterol only (1.25 %, 5 %) groups. Mice given a high fat (36%) diet also demonstrated significantly narrowed incisor pulp. In contrast, osteoclast formation was not significantly different among the groups. These results suggest that a diet with high amounts of both fat and cholesterol induces bone loss.

Effects of lipid metabolism on mouse incisor dentinogenesis.

Tooth formation can be affected by various factors, such as oral disease, drug administration, and systemic illness, as well as internal conditions including dentin formation. Dyslipidemia is an important lifestyle disease, though the relationship of aberrant lipid metabolism with tooth formation has not been clarified. This study was performed to examine the effects of dyslipidemia on tooth formation and tooth development. Dyslipidemia was induced in mice by giving a high-fat diet (HFD) for 12 weeks. Additionally, LDL receptor-deficient (Ldlr-/-) strain mice were used to analyze the effects of dyslipidemia and lipid metabolism in greater detail. In the HFD-fed mice, incisor elongation was decreased and pulp was significantly narrowed, while histological findings revealed disappearance of predentin. In Ldlr-/- mice fed regular chow, incisor elongation showed a decreasing trend and pulp a narrowing trend, while predentin changes were unclear. Serum lipid levels were increased in the HFD-fed wild-type (WT) mice, while Ldlr-/- mice given the HFD showed the greatest increase. These results show important effects of lipid metabolism, especially via the LDL receptor, on tooth homeostasis maintenance. In addition, they suggest a different mechanism for WT and Ldlr-/- mice, though the LDL receptor pathway may not be the only factor involved.

Seasonal trends of blood pressure during pregnancy in Japan: the babies and their parents' longitudinal observation in Suzuki Memorial Hospital in Intrauterine Period study.

OBJECTIVE: Blood pressure (BP) increases both in winter and in the last trimester of pregnancy. Some interaction seems to exist between season and gestational age. The present study observed home BP values during pregnancy with adjustment for seasonal variation and gestational age. METHODS: We observed 10353 home BP measurements from 101 normal pregnant women attending a maternity hospital in Japan. Home BP values were examined by mixed linear model adjusting for meteorological data and gestational age. RESULTS: The lowest home BP values were observed in the second trimester [mean (+/-standard deviation) systolic/diastolic BP, 101.8 +/- 7.9/59.8 +/- 5.8 mmHg at gestational week 20]. In the last trimester, home BP values gradually increased and the values after gestational week 26 were significantly higher than those at gestational week 20 (110.1 +/- 9.7/66.8 +/- 7.7 mmHg at gestational week 40). A 10 degrees C increase in daily minimum outdoor temperature was associated with a mean reduction of 2.5/2.5 mmHg (Delta systolic BP/Delta diastolic BP: 95% confidence interval, 2.3/2.4 to 2.6/2.7 mmHg) in home BP with adjustment for gestational age. The largest and smallest estimated home BP changes during pregnancy were 12.8/12.5 and 3.1/3.0 mmHg in pregnant woman who delivered in January and July, respectively. CONCLUSION: Interactions among BP, season and gestational age should be considered when evaluating BP in pregnant women. Risks associated with high BP might be underestimated in pregnant woman in summer who will deliver in winter.

Successful treatment of refractory aortitis in antineutrophil cytoplasmic antibody-associated vasculitis using tocilizumab.

A 47-year-old Japanese woman developed antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) complicated by a rare combination of aortitis and hypertrophic pachymeningitis. Despite the therapy with prednisolone and cyclophosphamide, the aortitis was not ameliorated. However, after cyclophosphamide was replaced with intravenous tocilizumab, the aortitis was improved, and the prednisolone dose was successfully tapered to 4 mg/day without elevation in C-reactive protein and myeloperoxidase ANCA (MPO-ANCA) levels. Several studies have reported that tocilizumab is effective for aortitis associated with Takayasu's arteritis and giant cell arteritis. On the other hand, we succeeded to improve the aortitis in AAV with monthly administration of tocilizumab. Moreover, we successfully controlled disease activity and enabled the tapering of prednisolone to 4 mg/day without relapses of AAV symptoms and elevated MPO-ANCA levels. It indicates that tocilizumab may be therapeutically beneficial for not only aortitis but also AAV itself. In conclusion, tocilizumab was effective in treating glucocorticoid- and cyclophosphamide-resistant AAV-associated aortitis. This is the first report demonstrating the successful treatment of AAV-associated aortitis using tocilizumab.

Phase separation of myelin sheath in Triton X-114 solution: predominant localization of the 21.5-kDa isoform of myelin basic protein in the lipid raft-associated domain.

Myelin basic protein (MBP) isoforms in the myelin sheath are known to have distinct intracellular expression patterns, which are profoundly related to functional specificity. Determining the differential localization of MBP isoforms is therefore important for understanding their pathophysiological roles. In this study, we have developed a new method for phase separation of myelin. The non-ionic detergent Triton X-114 is used to solubilize myelin sheath which then undergoes phase separation to yield four fractions. The lipid raft-associated proteins and lipids in each fraction were analysed by immunoblotting and lipid analysis, respectively. The present method gives two lipid raft-enriched fractions, one of them was found to contain only lipid raft-associated galactocerebroside and cholesterol as the major lipids. The 21.5-kDa MBP isoforms (21.5 MBP), both unphosphorylated and phosphorylated, were exclusively contained in this fraction. Phosphorylated 21.5 MBP (21.5 pMBP) has been shown to specifically disappear from demyelinated loci. The present analytical method clearly indicated that disappearance of 21.5 pMBP corresponded to demyelination and its reappearance corresponded to prevention of demyelination. Demyelination was also associated with aging and was prevented by the myelin-protecting herbal medicine, Chinpi, a type of dried citrus peel.