Yosuke Okada

Disruption of the fibroblast growth factor-2 gene results in decreased bone mass and bone formation.

Basic fibroblast growth factor (FGF-2), an important modulator of cartilage and bone growth and differentiation, is expressed and regulated in osteoblastic cells. To investigate the role of FGF-2 in bone, we examined mice with a disruption of the Fgf2 gene. Measurement of trabecular bone architecture of the femoral metaphysis of Fgf2(+/+) and Fgf2(-/-) adult mice by micro-CT revealed that the platelike trabecular structures were markedly reduced and many of the connecting rods of trabecular bone were lost in the Fgf2(-/-) mice. Dynamic histomorphometry confirmed a significant decrease in trabecular bone volume, mineral apposition, and bone formation rates. In addition, there was a profound decreased mineralization of bone marrow stromal cultures from Fgf2(-/-) mice. This study provides strong evidence that FGF-2 helps determine bone mass as well as bone formation.

Relationship between fluctuations in glucose levels measured by continuous glucose monitoring and vascular endothelial dysfunction in type 2 diabetes mellitus.

BackgroundFluctuations in blood glucose level cause endothelial dysfunction and play a critical role in onset and/or progression of atherosclerosis. We hypothesized that fluctuation in blood glucose levels correlate with vascular endothelial dysfunction and that this relationship can be assessed using common bedside medical devices.MethodsFluctuations in blood glucose levels were measured over 24 hours by continuous glucose monitoring (CGM) on admission day 2 in 57 patients with type 2 diabetes mellitus. The reactive hyperemia index (RHI), an index of vascular endothelial function, was measured using peripheral arterial tonometry (EndoPAT) on admission day 3.ResultsThe natural logarithmic-scaled RHI (L_RHI) correlated with SD (r=−0.504; P<0.001), the mean amplitude of glycemic excursions (MAGE) (r=−0.571; P<0.001), mean postprandial glucose excursion (MPPGE) (r=−0.411; P=0.001) and percentage of time ≥200 mg/dl (r=−0.292; P=0.028). In 12 patients with hypoglycemia, L_RHI also correlated with the percentage of time at hypoglycemia (r=−0.589; P= 0.044). L_RHI did not correlate with HbA1c or fasting plasma glucose levels. Furthermore, L_RHI did not correlate with LDL cholesterol, HDL cholesterol, and triglyceride levels or with systolic and diastolic blood pressures. Finally, multivariate analysis identified MAGE as the only significant determinant of L_RHI.ConclusionsFluctuations in blood glucose levels play a significant role in vascular endothelial dysfunction in type 2 diabetes.Trial registrationUMIN000007581

Advanced Glycation End Products Induce Calcification of Vascular Smooth Muscle Cells through RAGE/p38 MAPK

Background: Mönckeberg’s calcification in diabetes, known as medial artery calcification, is an independent predictor of cardiovascular mortality. However, the mechanism underlying this phenomenon remains to be elucidated. We demonstrate that advanced glycation end products (AGEs) induce calcification of vascular smooth muscle cells through the receptor for AGE (RAGE)/p38 mitogen-activated protein kinase (MAPK) signaling pathway. Methods: We detected vascular calcification by von Kossa staining. Alkaline phosphatase (ALP) activity was determined by measuring p-nitrophenol. Osteocalcin concentrations were measured using ELISA. Western blotting for protein phosphorylation and real-time RT-PCR for expression of mRNA were used. Results: AGEs induced calcification of vascular smooth muscle cells. AGEs also induced the expression of Runx2 mRNA. In addition, AGEs increased ALP activity and osteocalcin secretion. Furthermore, AGEs induced phosphorylation of p38 MAPK, and this phosphorylation was inhibited by the anti-RAGE blocking antibody. Increased ALP activity was inhibited by the p38 MAPK inhibitor or anti-RAGE blocking antibody. Furthermore, the p38 MAPK inhibitor and anti-RAGE blocking antibody both inhibited AGE-induced calcification of vascular smooth muscle cells. Diabetic serum induced calcification of smooth muscle cells and the calcification was inhibited by RAGE blocking. Conclusion: Our findings indicate that AGEs induce calcification of vascular smooth muscle cells by osteoblast-like differentiation of smooth muscle cells through RAGE/p38 MAPK.

Interleukin-1β induces differentiation of human mesenchymal stem cells into osteoblasts via the Wnt-5a/receptor tyrosine kinase-like orphan receptor 2 pathway.

OBJECTIVE Mesenchymal stem cells (MSCs) are considered to be a novel tool for the treatment of rheumatoid arthritis (RA) because of their multipotency to differentiate into osteoblasts and chondrocytes, their immunosuppressive effects, and availability. The aim of this study was to assess the mechanisms of human MSC differentiation into osteoblasts under inflammatory conditions. METHODS Human MSCs were cultured in commercialized osteogenic induction medium with inflammatory cytokines for up to 10 days. Osteoblast differentiation was detected by alkaline phosphatase staining and messenger RNA (mRNA) expression of multiple osteoblast markers. Mineralization was assessed by alizarin red S staining. RESULTS Among the various cytokines tested, interleukin-1β (IL-1β) induced differentiation of human MSCs into osteoblasts, which was confirmed by alkaline phosphatase activity, expression of RUNX2 mRNA, and strong alizarin red S staining. Among various molecules of the Wnt family, Wnt-5a and receptor tyrosine kinase-like orphan receptor 2 (Ror2), a major receptor of Wnt-5a, were significantly induced in human MSCs by IL-1β. Silencing of either WNT5A or ROR2 by small interfering RNA with 2 different sequences reduced alkaline phosphatase activity, RUNX2 expression, and alizarin red S staining of human MSCs induced by IL-1β. CONCLUSION IL-1β effectively and rapidly induced human MSC differentiation into osteoblasts and mineralization, mainly through the noncanonical Wnt-5a/Ror2 pathway. These results suggest potential benefits of IL-1β-treated human MSCs in the treatment of damaged bone as well as in the induction of self-renewal and self-repair of damaged tissue, including osseous tissue.

Advanced glycation end products increase endothelial permeability through the RAGE/Rho signaling pathway

MINT‐7301204, MINT‐7301186: RhoA (uniprotkb:P61586) physically interacts (MI:0915) with RAGE (uniprotkb:Q15109) by anti bait coimmunoprecipitation (MI:0006)

Over-expression of fibroblast growth factor-2 causes defective bone mineralization and osteopenia in transgenic mice.

Over‐expression of human FGF‐2 cDNA linked to the phosphoglycerate kinase promoter in transgenic (TgFGF2) mice resulted in a dwarf mouse with premature closure of the growth plate and shortening of bone length. This study was designed to further characterize bone structure and remodeling in these mice. Bones of 1–6 month‐old wild (NTg) and TgFGF2 mice were studied. FGF‐2 protein levels were higher in bones of TgFGF2 mice. Bone mineral density was significantly decreased as early as 1 month in femurs from TgFGF2 mice compared with NTg mice. Micro‐CT of trabecular bone of the distal femurs from 6‐month‐old TgFGF2 mice revealed significant reduction in trabecular bone volume, trabecular number (Tb.N), and increased trabecular separation (Tb.Sp). Osteoblast surface/bone surface, double‐labeled surface, mineral apposition rate, and bone formation rates were all significantly reduced in TgFGF2 mice. There were fewer TRAP positive osteoclasts in calvaria from TgFGF2 mice. Quantitative histomorphometry showed that total bone area was similar in both genotypes, however percent osteoclast surface, and osteoclast number/bone surface were significantly reduced in TgFGF2 mice. Increased replication of TgFGF2 calvarial osteoblasts was observed and primary cultures of bone marrow stromal cells from TgFGF2 expressed markers of mature osteoblasts but formed fewer mineralized nodules. The data presented indicate that non‐targeted over‐expression of FGF‐2 protein resulted in decreased endochondral and intramembranous bone formation. These results are consistent with FGF‐2 functioning as a negative regulator of postnatal bone growth and remodeling in this animal model.

Sitagliptin improves albuminuria in patients with type 2 diabetes mellitus

The aim of the present study was to determine the effect of sitagliptin on microalbuminuria in patients with type 2 diabetes mellitus.

Human Mesenchymal Stem Cells Inhibit Osteoclastogenesis Through Osteoprotegerin Production

OBJECTIVE Mesenchymal stem cells (MSCs) have been proposed to be a useful tool for treatment of rheumatoid arthritis (RA), not only because of their multipotency but also because of their immunosuppressive effect on lymphocytes, dendritic cells, and other proinflammatory cells. Since bone destruction caused by activated osteoclasts occurs in RA, we undertook the present study to investigate the effect of MSCs on osteoclast function and differentiation in order to evaluate their potential use in RA therapy. METHODS Human MSCs and peripheral blood mononuclear cells were cultured under cell-cell contact-free conditions with osteoclast induction medium. Differentiation into osteoclast-like cells was determined by tartrate-resistant acid phosphatase staining and expression of osteoclast differentiation markers. RESULTS The number of osteoclast-like cells was decreased and expression of cathepsin K and nuclear factor of activated T cells c1 (NF-ATc1) was down-regulated by the addition of either MSCs or a conditioned medium obtained from MSCs. Osteoprotegerin (OPG) was constitutively produced by MSCs and inhibited osteoclastogenesis. However, osteoclast differentiation was not fully recovered upon treatment with either anti-OPG antibody or OPG small interfering RNA, suggesting that OPG had only a partial role in the inhibitory effect of MSCs. Moreover, bone-resorbing activity of osteoclast-like cells was partially recovered by addition of anti-OPG antibody into the conditioned medium. CONCLUSION The present results indicate that human MSCs constitutively produce OPG, resulting in inhibition of osteoclastogenesis and expression of NF-ATc1 and cathepsin K in the absence of cell-cell contact. Therefore, we conclude that human MSCs exert a suppressive effect on osteoclastogenesis, which may be beneficial in inhibition of joint damage in RA.

Alendronate Protects Premenopausal Women from Bone Loss and Fracture Associated with High-dose Glucocorticoid Therapy

Objective We assessed the efficacy of bisphosphonate in premenopausal women (n = 47) commencing high-dose glucocorticoid (GC) therapy in protection against induced bone loss and bone fracture. Methods Subjects had just developed systemic autoimmune diseases and were randomized to be treated with 1 mg/kg/day prednisolone and alfacalcidol 1 μg/day alone (alfacalcidol group; n = 22), or prednisolone and alfacalcidol 1 μg/day with alendronate 5 mg/day (alendronate group; n = 25), each for 18 months. Results The percentage changes in lumbar spine bone mineral density (BMD) after 6 months of the therapy were −10.5% ± 0.8% in the alfacalcidol group, but only −2.1% ± 1.2% in the combined group. The rate of bone loss in the lumbar spine was significantly lower in the combined group than in the alfacalcidol group at 6 months. At 12 months of treatment, the percentage change in lumbar spine BMD was increased by 1.7% ± 1.4% in the combined group, but decreased by 9.9% ± 1.9% in the alfacalcidol group; the difference was significant. Bone fracture occurred at 12 months or later in 4 patients of the alfacalcidol groups, but not in the combined group, even at up to 18 months. Conclusion Our results indicate that alendronate with alfacalcidol can maintain BMD and protects against high-dose GC-induced bone loss and bone fracture.

Short-Term Treatment of Recombinant Murine Interleukin-4 Rapidly Inhibits Bone Formation in Normal and Ovariectomized Mice

Estrogen deficiency contributes to an increase in bone resorption and bone formation characterized by a high rate of bone turnover. Interleukin-4 (IL-4) is a rapid and potent inhibitor of bone resorption. We examined the short term in vivo effects of recombinant murine IL-4 (rmIL-4) on bone remodeling in normal and ovariectomized mice. Eight-week-old mice were randomized into the following five groups: (1) sham-operated mice (sham); (2) sham-operated mice infused with rmIL-4; (3) ovariectomized mice (ovx); (4) ovx infused with rmIL-4; and (5) ovx replaced by 10 or 20 microg of 17beta-estradiol (E2) for 14 or 28 days after ovariectomy, respectively. rmIL-4 at a dose of 5 microg/day was infused into ovx and sham for 3 days prior to sacrifice. Analyses were performed 14 and 28 days after operation. An increase in serum alkaline phosphatase and urinary deoxypyridinoline levels induced by ovariectomy was inhibited by the 3-day infusion of rmIL-4. In ovx, serum and urinary IL-6 levels were also increased significantly 14 days after ovariectomy, which were restored by E2 but not by rmIL-4. Histomorphometrical analysis of trabecular bone revealed that the 3-day infusion of rmIL-4 inhibited the high rate of bone turnover induced by ovariectomy, such as an increase in the osteoclastic surface (Oc.S/BS), number of osteoclasts per mm bone surface (N.Oc/BS), mineralized surface per mm bone surface (MS/BS), and bone mineral apposition rate (MAR). A significant decrease in the bone volume (BV/TV) observed in ovx was not modulated by a 3-day infusion of rmIL-4 prior to sacrifice. In sham, rmIL-4 also caused a significant decrease in the Oc.S/BS, N.Oc/BS, MS/BS, and MAR, but the BV/TV was not modulated by rmIL-4. We conclude that short term infusion of rmIL-4 in vivo rapidly inhibits not only bone resorption but also its formation in both sham-operated and ovariectomized growing mice, resulting in a low rate of bone turnover without modulating bone volume.