Impact of metabolic syndrome on brachial-ankle pulse wave velocity in Japanese.

The aim of this study was to determine the effect of metabolic syndrome on brachial-ankle pulse wave velocity (baPWV) by using the new guidelines for diagnosis of this syndrome in Japan. We examined 525 men and women without a history of cardiovascular disease or cancer, and an ankle-brachial index < 0.9. The baPWV was measured using a device (Form PWV/ABI) that simultaneously monitored bilateral brachial and ankle pressure wave forms. Metabolic syndrome was defined as a waist circumference > or = 85 (90) cm in men (women) and two or more of the following risk factors: hypertension, dyslipidemia, and glucose intolerance diagnosed by a 75 g oral glucose tolerance test. The baPWV showed a significant linear relationship with waist circumference, waist-to-hip ratio, body fat, systolic and diastolic blood pressure, triglycerides, fasting glucose, 2-h-postload glucose, fasting insulin, and glycosylated hemoglobin-A1c, after adjusting for sex and age. These factors were also strongly related to fasting insulin levels. When subjects were classified into six groups based on waist circumference and the number of risk factors for metabolic syndrome (0, 1, and > or =2), we found that more risk factors clearly increased the odds ratios for an elevated baPWV in those subjects in the highest quartile of the baPWV distribution in multivariate logistic models. An increase in odds ratio was observed despite a normal waist circumference and may well have been due to increased fasting insulin and blood pressure levels. An increase in the number of risk factors for metabolic syndrome was highly correlated with an increased baPWV, probably due to insulin resistance.

Impact of diabetes on health-related quality of life in a population study in Japan.

Diabetes may impact on health-related quality of life (HRQOL). The aim of this population-based study was to confirm this influence. We examined 2135 residents aged > or =30 years in an entire community, who had no history of cancer or cardiovascular disease and did not require care for daily activity. The response rate was 87.8%. The status of diabetes, other chronic diseases and life practices were assessed by self-reported questionnaires, in which HRQOL was evaluated by the Japanese version SF-36, based on five sub-scales of the domain. Diabetes had been diagnosed in 165 (7.7%) of the study population. Comparison of data, adjusted for sex, age, living alone and employment status between non-diabetics and subjects with diabetes treated by pharmacological therapy had odds ratios (ORs) between the lowest quartiles of sub-scale scores of 1.90 for physical role, 2.51 for general health, and 1.79 for emotional role. The OR for lower general health was also increased in people using only lifestyle modification for treatment of diabetes. Although the OR for mental health was worse in the lifestyle modification group it was not increased in people with diabetes on pharmacological therapy. These associations remained almost unchanged after adjustment for the covariates. The sub-scales of physical and emotional roles and general health were decreased significantly in diabetics with duration of disease between 10 and 19 years or > or =20 years. Our study showed treatment of diabetes clearly influenced HRQOL, with this influence being dependent on the duration of diabetes and primarily affecting physical rather than mental health.

Role of metallothionein isoforms in bone formation processes in rat marrow mesenchymal stem cells in culture.

Temporal changes in mRNAs for metallothionein (MT) isoforms in subcultures of rat marrow mesenchymal stem cells (MSCs) after treatment with dexamethasone were investigated. Both MT-1 and MT-2 mRNA expression in the cultured MSCs with dexamethasone showed maximum levels at d 1, whereas ALP and osteocalcin mRNAs peaked at d 12. MT-3 mRNA was not detected in the cultured MSCs at any time. The expression level of MT-2 mRNA at d 1 was 9.4-fold higher than that of MT-1 mRNA. Finally, osteoblast differentiation and mineralization of MSCs at d 14 was inhibited by the addition of a common antisense oligonucleotide for both MT-1 and MT-2 in the culture medium during the first 4 d. The results suggest that the large amounts of MT-2 are produced in the early stage of subculture of MSCs, and this might regulate their differentiation.

Characterization of the cDNA encoding bullfrog, Rana catesbeiana, osteocalcin and two forms of the protein isolated from bone.

A full-length cDNA clone encoding osteocalcin from the bullfrog, Rana catesbeiana (bone Gla-protein, BGP) has been isolated, and the complete coding sequence for the 100-amino-acid pre-pro-osteocalcin protein was determined. The amino acid sequence of Rana catesbeiana osteocalcin, especially the mature 49-amino acid sequence, is closer to the mammalian than to the fish, Sparus osteocalcin. Rana mature osteocalcin has a similarity of 67% with human or 59% with rat osteocalcin, and only 42% with fish mature osteocalcin. The 51-amino-acid pre-pro-peptide contains the expected hydrophobic leader sequence and the dibasic Arg-Arg sequence preceding the NH2-terminal Ser of the mature 49-amino-acid Rana osteocalcin. The pro-peptide sequence also contains the expected motif of polar and hydrophobic residues, which targets vitamin K-dependent gamma-carboxylation of three specific Glu residues at positions 17, 21, and 24 in the mature protein. At the native protein expression levels, extraction from Rana cortical bone in the presence of protease inhibitor cocktail resulted in the isolation of two distinct forms of osteocalcin, P-1 and P-2, with a 3:2 distribution. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and amino acid sequence analysis of the N-terminal domain, we confirmed that P-1 is the intact 49-residue osteocalcin with N-terminal SNLRNAVFG., and that P-2 lacks four amino acids from the N-terminus, (NAVFG.). These results demonstrate the existence of a form of osteocalcin lacking four N-terminal amino acids in Rana bone, and that mature Rana osteocalcins remained highly conserved in their molecular evolution, especially with respect to the conservation of the C-terminal domain (residues 14-49).

Levels of MCP-1 and GM-CSF mRNA correlated with inflammatory cytokines mRNA levels in experimental autoimmune myocarditis in rats.

Infiltration of monocytes and T cells is known to be an essential trigger for the progression of experimental autoimmune myocarditis (EAM) in rats. Monocyte chemotactic protein-1 (MCP-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were shown to mediate the migration of monocytes and T cells into inflammatory sites and to proliferate monocytes. Thus, we evaluated levels of MCP-1 and GM-CSF mRNA in the myocardium of EAM in rats using a real time quantitative PCR method. We also examined the correlation of MCP-1 or GM-CSF mRNA levels with those of inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) in the same lesion. Levels of MCP-1, GM-CSF, TNF-alpha, IL-1beta and IL-6 mRNA increased with the progression of myocarditis which was accompanied by the accumulation of ED-1 positive cells. The MCP-1 and GM-CSF mRNA levels were positively correlated with TNF-alpha, IL-1beta and IL-6 mRNA levels in the same lesion of EAM. We also demonstrated that serum MCP-1 concentrations were increased during the active stage of EAM, and were correlated with MCP-1 mRNA levels in the myocardium of each rat. These findings suggest that elevated MCP-1 and GM-CSF may associate with the migration and proliferation of monocytes/macrophages in EAM. Thus, MCP-1 and GM-CSF may play an important role in the progression of EAM.

Osteogenic differentiation of cultured rat and human bone marrow cells on the surface of zinc-releasing calcium phosphate ceramics.

Rat and human bone marrow cells (BMCs) were cultured on a composite ceramic of zinc-containing beta-tricalcium phosphate and hydroxyapatite (ZnTCP/HAP) with a (Ca+Zn)/P molar ratio of 1.60 and varying zinc contents. After a 2-week culture of the BMCs in the presence of beta-glycerophosphate and dexamethasone, many macroscopic mineralized areas with high alkaline phosphatase (ALP) activity were seen on the ZnTCP/HAP ceramic disks. The ALP activity increased with increasing zinc content in the ceramics. The highest ALP activity was observed when the BMCs were cultured on the ceramics with 1.26 wt % zinc, and the ceramics released zinc ions at concentrations from 2.2 to 7.2 microg/mL into the culture medium. Zinc ions were incorporated into mineralized areas produced by BMCs. BMCs also were directly cultured onto the culture dish surface, and the addition of 100 microM of free ZnCl(2) (6.5 microg/mL) to the culture medium significantly increased the ALP activity of the BMCs relative to the culture medium without the ZnCl(2)addition. The maximum zinc concentration required to enhance mineralization was higher in human BMCs than in rat BMCs. The present study demonstrates the superiority of ZnTCP/HAP ceramics over TCP/HAP in supporting the osteogenic differentiation of BMCs, and thus these ceramics are safe and useful in clinical settings, such as for bone reconstructive surgery.

Recombinant growth/differentiation factor-5 (GDF-5) stimulates osteogenic differentiation of marrow mesenchymal stem cells in porous hydroxyapatite ceramic.

To evaluate the growth/differentiation factor-5 (GDF-5) in the in vivo osteogenic potential of bone marrow mesenchymal stem cells (MSCs), we subcutaneously implanted five different kinds of hydroxyapatite (HA) ceramic implants: HA alone, GDF-5/HA composites (GDF/HA), MSCs/HA composites, the MSCs/HA composites supplemented with GDF-5 (GDF/MSCs/HA), and recombinant bone morphogenetic protein-2 (BMP/MSCs/HA). Neither the HA alone nor the GDF/HA composites exhibited any bone formation at any time after implantation. At 4 weeks, the MSCs/HA composites exhibited a certain amount of bone formation in some pore areas. In contrast, at 2 weeks, the GDF/MSCs/HA composites exhibited histologically obvious de novo bone formation together with active osteoblasts in many pore areas and additional bone formation at 4 weeks. In the de novo formed bone, neither chondrocytes nor endochondral bone was detected. The GDF/MSCs/HA composites also showed high alkaline phosphatase (ALP) and osteocalcin expression determined at both the protein and gene levels and the high level of expression was well maintained even at 4 weeks. Compared with GDF/MSCs/HA, the BMP/MSCs/HA composites exhibited excellent osteogenesis with relatively early osteoblastic phenotype expression. The results indicate that GDF-5 synergistically enhances de novo bone formation capability of MSCs/HA composite and suggest that tissue-engineered GDF/MSCs/HA composites could be used as bone graft substitutes.

[Mortality among individuals diagnosed with glucose intolerance by the 75 g oral glucose tolerance test in rural Japanese communities].

OBJECTIVE: To examine mortality among individuals confirmed to have glucose intolerance by the 75 g oral glucose tolerance test (OGTT) in rural communities. METHODS: Subjects were 1,639 residents of seven rural communities in Japan (total population size = 54,000) who underwent an OGTT as a secondary screening test for diabetes during 1987-95 and had received no medication for diabetes at baseline. OGTT results were classified as diabetes (n = 471), impaired glucose tolerance (IGT) (n = 408), or normal glucose tolerance (NGT) (n = 760), according to the recommendations of the American Diabetes Association. Indirect age-adjusted mortality rates and standardized mortality ratios (SMRs) for diabetes and IGT groups were calculated using age-stratified mortality rates for people aged 40-89 in the communities as the standard population. Relative risks of death and 95% confidence intervals (CI) for diabetes and IGT were calculated using Cox's proportional models, adjusted for age and baseline factors such as body mass index (BMI), total cholesterol, hypertension, smoking, alcohol drinking, history of diabetes, and family history of diabetes. RESULTS: During 9.4 years of follow-up, we recorded 70 deaths for the NGT, 46 for the IGT, and 71 for the diabetes group. The SMRs for IGT and diabetic men and women were not significantly increased in comparison with the standard population values. However, when analyzed in our cohort, age-adjusted relative risks of death for IGT and diabetes versus NGT men were 1.10 (9.5% CI: 0.72-1.67) and 1.54 (95% CI: 1.05-2.24), respectively, whereas, the risks among women did not significantly vary. Finally, multivariate analysis, further adjusted for the baseline factors, indicated the relative risk of mortality with diabetes in men to be significantly increased (relative risk = 1.74, 95% CI: 1.11-2.75). CONCLUSION: During follow-up, mortality rates among screened IGT and diabetes were not significantly higher than those in general. Nonetheless, we found that diabetes elevated the risk of death in our cohort, primarily in men.

Influence of the porosity of hydroxyapatite ceramics on in vitro and in vivo bone formation by cultured rat bone marrow stromal cells.

The in vitro and in vivo osteoblastic differentiation of rat bone marrow stromal cells (MSCs) was assessed on hydroxyapatite disks with 3 different porosities: 30%, 50%, and 70% (HA30, HA50, and HA70, respectively). MSCs obtained by 10-day culture of fresh bone marrow cells were subcultured for 2 weeks on 3 kinds of porous HA disks in the presence and absence of dexamethasone (Dex). After 2 weeks of subculture, alkaline phosphatase (ALP) activity and osteocalcin production of MSCs/HA composites with Dex were higher than those without, and increased with increasing porosity. The resultant bone tissue grafts "cultured-bone/HA constructs" were implanted subcutaneously into the backs of syngeneic rats, and harvested 1, 2, and 4 weeks after implantation. At 1 week, only cultured-bone/HA70 constructs exhibited expanded bone formation. At 2 and 4 weeks, active osteoblasts and progressive bone formation were observed morphologically in both cultured-bone/HA50 and HA70 constructs. At 4 weeks, bone tissue was observed even in cultured-bone/HA30 constructs. ALP activity and osteocalcin production also increased with increasing porosity and time after implantation. In this in vivo model, different scaffold porosity with similar crystal morphology of the apatite phase demonstrated marked differences in ability to support osteogenesis by implanted rat MSCs.

Association of body mass index, body fat, and weight gain with inflammation markers among rural residents in Japan.

Because obesity is thought to play a key role in atherosclerosis through the low-grade chronic inflammation, the present study was designed to investigate associations of body mass index (BMI), body fat, and weight gain with optimized inflammation markers in 1,053 residents who were 40 years of age and older from a rural community (total population = 3,940 in 2000) in Japan. People reporting having a cold and those who did not undergo blood examinations were excluded. C-reactive protein (CRP), fibrinogen, serum albumin, and white blood cell (WBC) count were used as the markers for inflammation, body fat was calculated by a conventional method, and weight change since the age of 20 was assessed. The BMI and body fat significantly increased with CRP quartile, and its correlation coefficients to BMI or body fat were relatively high. Similar associations were found for fibrinogen, serum albumin and WBC. Multivariate-adjusted analysis found a high concentration of CRP was significantly associated with obesity, but attenuated the association in other markers. In an analysis restricted to people aged 40-69 years, body fat levels were more strongly associated with CRP and fibrinogen than with BMI only. Furthermore, only CRP concentrations were significantly elevated according to weight gain. Strong associations of CRP concentration with BMI, body fat, and weight gain were found among elderly Japanese, but not with fibrinogen, serum albumin or WBC.

Enhancement of cell viability in cryopreserved rat vascular grafts by administration of regenerating gene (REG) inducers.

The regenerative capacity of viable cells remaining in cryopreserved vascular allografts is still unclear. Recently, the regenerating gene (REG) has been documented to play an important role in various regenerating tissues. Here we show the possibility of REG induction for the enhancement of cryopreserved vascular allograft viability. Cryopreserved rat aortae were isografted or allografted heterotopically. Fresh isografts were also tested. The transplants were retrieved 3, 6, 9, and 12 days after implantation and the intragraft REG mRNA was measured by a real-time quantitative reverse transcriptional polymerase chain reaction method. REG expression was not detected before implantation. REG expression in cryopreserved isografts gradually increased after transplantation, whereas in fresh isografts or cryopreserved allografts it decreased over time after initial expression. Daily administration of 0.5 g/kg nicotinamide (an agent known to be a potent inducer of REG) induced intragraft REG mRNA in cryopreserved allografts (p < 0.05) accompanied by augmentation of the intragraft cell population. Daily administration of 0.5 mg/kg FK506 (an immunosuppressant) induced intragraft REG mRNA both in cryopreserved isografts and allografts (p < 0.01). We conclude that REG-inductive therapy shows promise as a novel strategy for enhancing the viability of vascular allografts. Moreover, FK506 may be involved in tissue regeneration as well as immunosuppression.

In vitro effects of Habu snake venom on cultured mesangial cells.

BACKGROUND: Habu snake venom (HSV)-induced glomerulonephritis is a unique model showing a progressive course of mesangial proliferation. To elucidate the in vitro effects of HSV, we examined whether HSV itself could have direct effects on the cultured mesangial cells, such as cell proliferation and activation of chemokine gene expression. METHODS: The incorporation of 5-[(125)I]iodo-2'-deoxyuridine was measured with a gamma-counter, and gene expressions of growth factors, chemokines and cytokines were evaluated by a real time quantitative PCR. RESULTS: We demonstrated that excessive or continuous HSV stimulation decreased a mesangial cell viability. However, adequate and temporary HSV stimulation induced proliferation of mesangial cells in vitro along with a significant elevation of monocyte chemoattractant protein-1 (MCP-1) mRNA levels. In addition to these in vitro results, we showed that MCP-1 mRNA levels increased in renal cortices of glomerulonephritis induced by HSV. Immunohistochemistry also showed a positive staining for MCP-1 in the marginal area of glomerulus with mesangiolysis. CONCLUSIONS: These data suggest that HSV itself may elicit direct biological effects on mesangial cells which may participate in pathophysiology of glomerulonephritis induced by HSV.