Teruyoshi Tanaka

Kudzu (Pueraria lobata) Vine Ethanol Extracts Improve Ovariectomy-Induced Bone Loss in Female Mice

Bone-loss-improving action of kudzu vine ethanol extracts (PVEE) was clarified. PVEE was composed roughly of 80% fiber, 10% puerarin, 3.6% daidzin, 2.5% 6″-O-malonyldaidzin, and the other minor isoflavones. Ten-week-old ovariectomized (OVX) mice were fed diets containing PVEE (20 mg/kg body weight/day) for 8 weeks. The bone resorption markers (urinary deoxypyridinoline and tartrate-resistant acid phosphatase activity) was elevated in OVX mice and was significantly decreased in OVX mice that consumed PVEE for 8 weeks. Consistent with the decrease in the markers, the number of matured osteoclasts in the distal femur was diminished in OVX mice fed PVEE diets. PVEE diets also suppressed the decrease in femoral bone mineral density (BMD) by OVX. PVEE showed the affinity for estrogen receptor α and β nearly 1/10000 weaker than 17β-estradiol. No hypertrophy in the uterus by the PVEE diet was observed. These results suggest that PVEE could be a promising resource for a functional food that improves osteoporosis.

Hyperglycemia Suppresses Calcium Phosphate-Induced Aneurysm Formation Through Inhibition of Macrophage Activation.

Background The aim of this study was to elucidate aspects of diabetes mellitus–induced suppression of aneurysm. We hypothesized that high glucose suppresses aneurysm by inhibiting macrophage activation via activation of Nr1h2 (also known as liver X receptor β), recently characterized as a glucose‐sensing nuclear receptor. Methods and Results Calcium phosphate (CaPO 4)–induced aneurysm formation was significantly suppressed in the arterial wall in type 1 and 2 diabetic mice. A murine macrophage cell line, RAW264.7, was treated with tumor necrosis factor α (TNF‐α) plus CaPO 4 and showed a significant increase in matrix metalloproteinase 9 (Mmp9) mRNA and secreted protein expression compared with TNF‐α alone. Elevated Mmp9 expression was significantly suppressed by hyperglycemic conditions (15.5 mmol/L glucose) compared with normoglycemic conditions (5.5 mmol/L glucose) or normoglycemic conditions with high osmotic pressure (5.5 mmol/L glucose +10.0 mmol/L mannitol). Nr1h2 mRNA and protein expression were suppressed by treatment with TNF‐α plus CaPO 4 but were restored by hyperglycemic conditions. Activation of Nr1h2 by the antagonist GW3965 during stimulation with TNF‐α plus CaPO 4 mimicked hyperglycemic conditions and inhibited Mmp9 upregulation, whereas the deactivation of Nr1h2 by small interfering RNA (siRNA) under hyperglycemic conditions canceled the suppressive effect and restored Mmp9 expression induced by TNF‐α plus CaPO 4. Moreover, Nr1h2 activation with GW3965 significantly suppressed CaPO 4‐induced aneurysm in mice compared with vehicle‐injected control mice. Conclusions Our results show that hyperglycemia suppresses macrophage activation and aneurysmal degeneration through the activation of Nr1h2. Although further validation of the underlying pathway is necessary, targeting Nr1h2 is a potential therapeutic approach to treating aneurysm.

Osteoclastogenic Differentiation of Macrophages in the Development of Abdominal Aortic Aneurysms

Objective—Arterial calcification is common and contributes to the pathogenesis of occlusive vascular disease. Similar to the dynamics of bone, it is a tightly controlled process that maintains a balance between osteogenesis and osteolysis. However, whether calcium homeostasis plays a role in the development of aneurysms has not been explored. We hypothesized that macrophages differentiate into osteoclasts in aneurysmal arteries and that protease byproducts contribute to aneurysm pathophysiology. Approach and Results—We performed histological and immunohistochemical analyses and showed that macrophages positive for several osteoclast markers, including tartrate acid phosphatase, occur in great numbers in the human aneurysmal aorta, but very few occur in the human stenotic aorta and none in the nondiseased human aorta. Moreover, in situ zymography showed elevated protease activity in these cells compared with undifferentiated macrophages. Tumor necrosis factor-&agr; and calcium phosphate stimulated this osteoclastogenic differentiation process through nuclear factor-&kgr;B, mitogen-activated protein kinases, and intracellular calcium signaling but not the receptor activator of the nuclear factor-&kgr;B ligand. Inhibition of osteoclastogenic differentiation by bisphosphonate inhibits aneurysm development in a mouse model. Conclusions—These results suggest that differentiation of macrophages into osteoclasts contributes to the pathophysiology of aneurysmal disease.

Hyperglycemia Suppresses RANKL-Induced Osteoclast Differentiation through LXRβ Expression in RAW264.7 Cells

There have been reports that hyperglycemia suppresses osteoclast (OCL) differentiation, although the underlying mechanism is poorly understood. Here we demonstrated that high glucose suppresses OCL differentiation through activation of liver X receptor (LXR) β, a recently reported glucose-sensing nuclear receptor. The effect of hyperglycemia on osteoclastogenesis was tested in RAW264.7 cells, a murine macrophage cell line. Cells were treated with receptor activator of NF-κB ligand (RANKL) under normoglycemic (5.5 mM glucose), normoglycemic with high osmotic pressure (5.5 mM glucose + 10.0 mM mannitol), and hyperglycemic (15.5 mM glucose) conditions. RANKL-induced osteoclastogenesis was significantly suppressed by high-glucose treatment. Mannitol treatment also significantly suppressed osteoclastogenesis, but the inhibitory effect was lower than for high-glucose treatment. The suppression of mRNA expression of Lxrβ by RANKL was significantly restored by high glucose, but not mannitol. Additionally, the deactivation of Lxrβ by siRNA attenuated high-glucose-induced suppression of osteoclastogenesis. Although further validation of the underlying pathway is necessary, targeting LXRβ is a potential therapeutic approach to treating osteoporosis.

Molecular cloning and expression profiling of procollagen α1 (I) of cultured Pacific bluefin tuna

Type I collagen is widely distributed in most organs in teleosts. It plays a role not only in intercellular adhesion, but also in molecular signaling. In this study, Pacific bluefin tuna (PBT) procollagen α1 (I) cDNA was cloned and characterized. The nine fragments of a procollagen α1 (I) chain cDNA clone were prepared and spliced together to create the complete coding region. The resulting amino acid sequence was homologous with that of other teleosts. The mRNA expression profile of PBT procollagen α1 (I) in various tissues and the phylogenetic analysis with other vertebrate procollagen α1 (I) chains suggest that PBT procollagen α1 (I) could be a precursor form of the PBT type I collagen α1 chain. In addition, its level of expression in PBT larvae and early juveniles gradually increased with somatic growth. This increase was related to the standard length, wet body weight, and protein content of each individual fish. Therefore, the expression profile of procollagen α1 (I) may be a useful indicator for somatic growth in fish larvae and juveniles.

Puerarin Suppresses Macrophage Activation via Antioxidant Mechanisms in a CaPO4-Induced Mouse Model of Aneurysm.

Aneurysm is characterized by balloon-like expansion of the arterial wall and eventual rupture of the aorta. The pathogenesis of aneurysm is associated with the degradation of matrix proteins by matrix metalloproteinases (MMPs) produced by activated macrophages. Although aneurysm is associated with significant mortality and morbidity, surgical intervention is the only proven treatment strategy. Therefore, development of therapeutic agents for aneurysm is greatly anticipated. Here, we demonstrated the protective effects of the major isoflavone puerarin, which is found in kudzu roots and vines. Aneurysms were surgically induced in ten-wk-old male mice using CaPO4. Subsequently, animals were intraperitoneally injected daily with puerarin at 2.5 mg/kg body weight or with vehicle alone for 2 wk. CaPO4-induced aneurysm was significantly suppressed by puerarin administration. In subsequent macrophage activation assays using Tumor necrosis factor (TNFα) and CaPO4 crystals in vitro, puerarin decreased Mmp9 mRNA expression and secreted protein levels. Moreover, induction of IκB, ERK, and p38 phosphorylation by TNFα and CaPO4 in macrophages was suppressed by puerarin treatments. Finally, puerarin attenuated reactive oxygen species production, following induction by TNFα and CaPO4. Taken together, the present data demonstrate that puerarin suppresses macrophage activation by inhibiting IκB, ERK, and p38 activity and reactive oxygen species production in a CaPO4-induced mouse model of aneurysm.

RANKL-mediated osteoclastogenic differentiation of macrophages in the abdominal aorta of angiotensin II-infused apolipoprotein E knockout mice

Objective: Osteoclastogenic activation of macrophages (OCG) occurs in human abdominal aortic aneurysms (AAAs) and in calcium chloride‐induced degenerative AAAs in mice, which have increased matrix metalloproteinase activity. As the activity of OCG in dissecting aneurysms is not clear, we tested the hypothesis that OCG contributes to angiotensin II (Ang II)‐induced dissecting aneurysm (Ang II‐induced AAA) in apolipoprotein E knockout mice. Methods: AAAs were produced in apolipoprotein E knockout mice via the administration of Ang II. Additionally, receptor activator of nuclear factor kB ligand (RANKL)‐neutralizing antibody (5 mg/kg) was administered to one group of mice 7 days prior to Ang II infusion. Aneurysmal sections were probed for presence of RANKL and tartrate‐resistant acid phosphatase via immunohistochemistry and immunofluorescence staining. Mouse aortas were also examined for RANKL and matrix metalloproteinase 9 expression via Western blot. In vitro murine vascular smooth muscle cells (MOVAS) and murine macrophages (RAW 264.7) were analyzed for the expression of osteogenic factors via Western blot, qPCR, and flow cytometry in response to Ang II or RANKL stimulation. The signaling pathway that mediates Ang II‐induced RANKL expression in MOVAS cells was also investigated via application of TG101348, a Janus kinase 2 (JAK2) inhibitor, and Western blot analysis. Results: Immunohistochemical staining of Ang II‐induced AAA sections revealed OCG as evidenced by increased RANKL and tartrate‐resistant acid phosphatase expression compared with control mice. Immunofluorescence staining of AAA sections revealed co‐localization of vascular smooth muscle cells and RANKL, revealing vascular smooth muscle cells as one potential source of RANKL. Systemic administration of RANKL‐neutralizing antibody suppressed Ang II‐induced AAA, with significant reduction of the maximum diameter of the abdominal aorta compared with vehicle controls (1.5 ± 0.4 mm vs 2.2 ± 0.2 mm). Ang II (1 &mgr;M) treatment induced a significant increase in RANKL messenger RNA expression levels in MOVAS cells compared with the vehicle control (1.0 ± 0.2 vs 2.8 ± 0.2). The activities of JAK2 and signal transducer and activator of transcription 5 (STAT5) were also significantly increased by Ang II treatment. Inhibition of JAK2/STAT5 suppressed Ang II‐induced RANKL expression, suggesting the involvement of the JAK2/STAT5 signaling pathway. Conclusions: OCG with increased RANKL expression was present in Ang II‐induced AAA, and neutralization of RANKL suppressed AAA formation. As neutralization of RANKL has been used clinically to treat osteoporosis and other osteoclast‐related diseases, additional study of the effectiveness of RANKL neutralization in AAA is warranted. Clinical Relevance: We previously demonstrated that osteoclastogenic differentiation of macrophages (OCG) plays an important role in the development of human abdominal aortic aneurysms and murine calcium chloride‐induced degenerative abdominal aortic aneurysms. In angiotensin II‐induced dissecting aneurysm, we demonstrated the presence of OCG and its induction by receptor activator of nuclear factor &kgr;B ligand, which is a stimulator of osteoclast formation in apolipoprotein E knockout mice. Neutralization of receptor activator of nuclear factor &kgr;B ligand significantly suppressed the development of angiotensin II‐induced dissecting aneurysm, suggesting that targeting of OCG could be an effective therapeutic approach to dissecting aneurysm.