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Dextran (Dx) is a biodegradable and biocompatible polysaccharide, thus promising as a drug delivery carrier for tumor therapy. Herein, we applied mechanical energy to a high molecular weight Dx to control its molecular weight and simultaneously generate mechanoradicals. The solid-state polymerization of methacrylate- or methacrylamide derivatives initiated with Dx mechanoradicals showed polymer conversion of >95%, yielding Dx-based graft copolymers with molecular weights of approximately 30,000 g mol -1 . The Dx-based graft copolymers with hydrophobic segments formed nanoparticles with a particle size of 25-35 nm in an aqueous solution. The anti-pancreatic tumor drug 5-fluorouracil (5-FU) was covalently conjugated onto the hydrophobic segments of the amphiphilic Dx, and the nanoparticles were also prepared. The drug release profile from 5-FU-conjugated nanoparticles corresponded well to the Korsmeyer-Peppas model applied to drug release from matrix substrates, and was also immensely predicted by the Logistic and Gompertz curves. The 5-FU-conjugated nanoparticles showed cytotoxicity against the pancreatic adenocarcinoma cell lines (BxPC-3) that were not significantly inferior to the 5-FU positive group. Furthermore, the fluorescein-labeled nanoparticles internalized into BxPC-3 within 6 h and actively migrated into the cytosol. These results suggest that Dx-based graft copolymers with hydrophobic segments might be used to enhance therapeutic activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Competing Interests: Declaration of Competing Interest Dr. Kawashima receives grants from Otsuka Pharmaceutical Co., Ltd., TAIHO Phamaceutical Co., Ltd., Chugai Phamaceutical Co., Ltd., EA Pharma Co., Ltd., Eisai Co., Ltd., AbbVie Inc., and Mitsubishi Tanabe Pharma Corporation, which are unrelated to the submitted work. Dr. Ando receives grants and personal fees from Chugai Pharmaceutical Co., Ltd., Kyowa Kirin Co., Ltd., Nippon Kayaku Co., Ltd., Yakult Honsha Co., Ltd., Ono Pharmaceutical Co., Ltd., Taiho Pharmaceutical Co., Ltd., Daiichi Sankyo Company, Ltd., and Eisai Co., Ltd., grants from Mochida Pharmaceutical Co., Ltd., personal fees from Eli Lilly Japan K·K., Novartis Pharma K.K., Bayer Holding Ltd., Bristol-Myers Squibb, Sawai Pharmaceutical Co., Ltd., Roche Diagnostics K.K., MSD K.K, Astellas Pharma Inc., Otsuka Holdings Co., Ltd., Sanwa Kagaku Kenkyusho Co., Ltd., and Hisamitsu Pharmaceutical Co., Inc., which are unrelated to the submitted work. All other authors have no disclosures or conflicts of interest.

Objectives: Physical activity is associated with improvement in overall health and well-being, but robust evidence with comprehensive assessment of general health is lacking. This study aimed to clarify the effects of physical activity on intrinsic capacity among community-dwelling older adults with subjective memory concerns., Design: A single-blind randomized controlled trial compared aerobic training (AT), resistance training (RT), and combined training (AT+RT) programs for improving general health evaluated by intrinsic capacity., Setting: Toyota, Japan., Participants: Residents (65-85 years old) who screened positive for subjective memory concerns using the Kihon checklist were invited for eligibility assessment. In total, 415 community-dwelling older adults were enrolled and randomized into the AT, RT, AT+RT, and control groups., Methods: Participants in the intervention groups underwent a group training program and self-paced home training for 26 weeks. The control group received lectures about health promotion. Intrinsic capacity (IC), constructed based on locomotion, cognition, psychological function, and vitality domains, was used to assess general health at baseline, week 26, and week 52. Between-group differences were exhibited with Z-score change in individual domain and combination of all domains., Results: At baseline, mean age of all participants (47% women) was 72.3 ± 4.6 years, with a mean composited IC Z-score of -0.2 ± 0.5. Overall, AT and RT improved composite IC Z-scores by 0.17 (95% confidence interval [CI] 0.03-0.30) and 0.17 (95% CI 0.05-0.28) at week 26, respectively, but the beneficial effects waned at week 52. No significant differences in composite IC Z-scores were found in the AT+RT group at weeks 26 and 52., Conclusions and Implications: Twenty-six-week AT with self-paced home training and RT with self-paced home training improve IC among community-dwelling older adults with subjective memory concerns, but the benefits waned subsequently. It will be required to develop optimal interventions that have a continuous beneficial effect on IC among community-dwelling older adults., (Copyright © 2020 AMDA – The Society for Post-Acute and Long-Term Care Medicine. Published by Elsevier Inc. All rights reserved.)

Background & Aims: The association between dietary patterns and frailty is less investigated in Asia. We aimed to investigate the prospective associations between dietary patterns and frailty index (FI) in community-dwelling Japanese older adults aged 60 years or older., Methods: A 3-year cohort collected the data on sociodemographic information, lifestyle behaviors, comorbidities, medication history, depression status, nutrition, and physical function from 2014 to 2017. Five dietary patterns including "Mediterranean-style," "sugar and fat," "salt and pickles," "noodle and alcohol," and "protein-rich" dietary patterns were identified using principal component analysis from 20 food groups obtained using a validated food frequency questionnaire. A 54-item FI was constructed on the basis of a deficit accumulation model. This project aimed to investigate the longitudinal association between dietary pattern and frailty trajectories with annual measurements over a 3-year period using a generalized estimating equation., Result: After excluding 108 participants with incomplete data at baseline, 666 participants (56.5% female, 69.4 ± 4.4 years) were included for longitudinal analysis. Our results demonstrated that adherence score to "sugar and fat" dietary pattern" (Q4 vs. Q1: mean difference [0.017], 95% CI [0.006 to 0.029]) and "salt and pickles" dietary pattern (Q3 vs. Q1: mean difference [0.010], 95% CI [0.001 to 0.020]; Q4 vs. Q1: mean difference [0.014], 95% CI [0.003 to 0.025]) were positively associated with change in FI. Adherence score to a "protein-rich" dietary pattern was negatively associated with change in FI (Q4 vs. Q1: mean difference [-0.013], 95% CI [-0.025 to -0.002])., Conclusions: "Salt and pickles" dietary pattern and "sugar and fat" dietary pattern were positively associated with frailty. In contrast, "protein-rich" dietary pattern was negatively associated with frailty., Competing Interests: Conflict of Interest The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2020 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Clinical and research interest in sarcopenia has burgeoned internationally, Asia included. The Asian Working Group for Sarcopenia (AWGS) 2014 consensus defined sarcopenia as "age-related loss of muscle mass, plus low muscle strength, and/or low physical performance" and specified cutoffs for each diagnostic component; research in Asia consequently flourished, prompting this update. AWGS 2019 retains the previous definition of sarcopenia but revises the diagnostic algorithm, protocols, and some criteria: low muscle strength is defined as handgrip strength <28 kg for men and <18 kg for women; criteria for low physical performance are 6-m walk <1.0 m/s, Short Physical Performance Battery score ≤9, or 5-time chair stand test ≥12 seconds. AWGS 2019 retains the original cutoffs for height-adjusted muscle mass: dual-energy X-ray absorptiometry, <7.0 kg/m 2 in men and <5.4 kg/m 2 in women; and bioimpedance, <7.0 kg/m 2 in men and <5.7 kg/m 2 in women. In addition, the AWGS 2019 update proposes separate algorithms for community vs hospital settings, which both begin by screening either calf circumference (<34 cm in men, <33 cm in women), SARC-F (≥4), or SARC-CalF (≥11), to facilitate earlier identification of people at risk for sarcopenia. Although skeletal muscle strength and mass are both still considered fundamental to a definitive clinical diagnosis, AWGS 2019 also introduces "possible sarcopenia," defined by either low muscle strength or low physical performance only, specifically for use in primary health care or community-based health promotion, to enable earlier lifestyle interventions. Although defining sarcopenia by body mass index-adjusted muscle mass instead of height-adjusted muscle mass may predict adverse outcomes better, more evidence is needed before changing current recommendations. Lifestyle interventions, especially exercise and nutritional supplementation, prevail as mainstays of treatment. Further research is needed to investigate potential long-term benefits of lifestyle interventions, nutritional supplements, or pharmacotherapy for sarcopenia in Asians., (Copyright © 2019 AMDA – The Society for Post-Acute and Long-Term Care Medicine. Published by Elsevier Inc. All rights reserved.)

Background: The mechanism by which angiogenesis declines with aging remains largely unknown. Given that the plasma levels of adiponectin (APN) are decreased in the presence of ischemic cardiovascular disease, we explore the possible mechanisms by which APN/adiponectin receptor1 (AdipoR1) axis inactivation contributes to the decline in vascular regeneration capacity in elderly animals., Methods and Results: To study aging-related changes in the APN/AdipoR1 axis and its impact on ischemia-induced angiogenesis, a hindlimb ischemia model was applied to young and aged mice. Aging impaired ischemia-induced blood flow recovery. An ELISA showed that the aged mice had decreased plasma APN levels. Immunostaining showed lesser capillary formation in the aged mice. The aged ischemic muscles had decreased levels of AdipoR1, peroxisome proliferator activated receptor-γ (PPAR-γ), PPAR-γ co-activator 1α (PGC-1α), phospho-AMP-activated protein kinase α (p-AMPK-α), and B cell lymphoma-2 (Bcl-2) and increased levels of cleaved caspase-8 (C-caspase-8) and gp91phox/p22phox genes or/and proteins, nicotinamide adenine dinucleotide phosphate oxidase activity, superoxide production, and matrix metalloproteinase-2/-9 activity as well as increased numbers of infiltrated macrophages and leucocytes. In in vitro experiments, aged endothelial cells had negative changes in the levels of PPAR-γ, PGC-1α, p-AMPK-α, Bcl-2, and C-caspase-8 proteins in response to oxidative stress. Genetic interventions targeted toward APN and AdipoR1 negatively affected the targeted angiogenic protein levels in aged muscles and angiogenic actions and/or aged endothelial events., Conclusion: These findings indicate that aging can reduce angiogenesis in response to hypoxia via an impaired APN-AdipoR1-dependent mechanism that may be mediated by PPAR-γ/PGC-1α signaling inactivation in advanced age., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objectives: Exposure to psychosocial stress is a risk factor for cardiovascular disease. Given that dipeptidyl peptidase-4 (DPP4) regulates several intracellular signaling pathways associated with glucagon-like peptide-1 (GLP-1) metabolism, we investigated the role of DPP4 in stress-related vascular senescence and atherosclerosis in apolipoprotein E-deficient (ApoE -/- ) mice., Methods and Results: ApoE -/- mice fed a high-fat (HF) diet were randomly assigned to one of non-stress and immobilized stress groups for 12weeks. Chronic stress accelerated vascular senescence and atherosclerotic plaque growth at the aortic roots. Stressed mice had increased levels of plasma DPP4 and decreased levels of plasma GLP-1 and adiponectin (APN) and adipose APN expression. Stress increased plaque macrophage infiltration, neovessel density, and elastin fragmentation, lessened the plaque collagen content, and increased the levels of toll-like receptor-2 (TLR2), TLR4, C-X-C chemokine receptor-4, cathepsins S and K, osteopontin, peroxisome proliferator-activated receptor-α, p16 INK4A , p21, and gp91phox mRNAs and/or proteins. Stressed aortas had also increased matrix metalloproteinase-2 (MMP-2) and MMP-9 activities. DPP4 inhibition with anagliptin reversed stress-related atherosclerotic lesion formation, and this benefit was abrogated by APN blocking. In vitro, the GLP-1 receptor agonist exenatide stimulated APN expression in 3T3-L1 cells., Conclusions: These results indicate that the DPP4 inhibition-mediated benefits are likely attributable, at least in part, to attenuation of plaque inflammation, oxidative stress and proteolysis associated with GLP-1-mediated APN production in ApoE -/- mice under stress. Thus, DPP4 will be a novel therapeutic target for the treatment of stress-related cardiovascular disease., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background and Aims: Exposure to psychosocial stress is a risk factor for cardiovascular disorders. Because the glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonist prevents cardiovascular injury, we investigated the beneficial effects and mechanism of the GLP-1 analogue exenatide on stress-related vascular senescence and atherosclerosis in apolipoprotein E-deficient (ApoE -/- ) mice fed a high-fat (HF) diet., Methods: ApoE -/- mice fed the HF diet were assigned to non-stressed and immobilized-stress groups for 12 weeks. Mice fed the HF diet were divided into 2 groups and administered vehicle or exenatide for 12 weeks under stress conditions., Results: Chronic stress enhanced vascular endothelial senescence and atherosclerotic plaque growth. The stress increased the levels of plasma depeptidyl peptidase-4 activity and decreased the levels of plasma GLP-1 and both plasma and adipose adiponectin (APN). As compared with the mice subjected to stress alone, the exenatide-treated mice had decreased plaque microvessel density, macrophage accumulation, broken elastin, and enhanced plaque collagen volume, and lowered levels of peroxisome proliferator-activated receptor-α, gp91phox osteopontin, C-X-C chemokine receptor-4, toll-like receptor-2 (TLR2), TLR4, and cathepsins K, L, and S mRNAs and/or proteins. Exenatide reduced aortic matrix metalloproteinase-9 (MMP-9) and MMP-2 gene expression and activities. Exenatide also stimulated APN expression of preadipocytes and inhibited ox-low density lipoprotein-induced foam cell formation of monocytes in stressed mice., Conclusions: These results indicate that the exenatide-mediated beneficial vascular actions are likely attributable, at least in part, to the enhancement of APN production and the attenuation of plaque oxidative stress, inflammation, and proteolysis in ApoE -/- mice under chronic stress., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objectives: We investigated the differences in the physical function test results across stages from normal cognition (NC) to moderate Alzheimer disease (AD) and how risk factors of physical function decline are correlated with the physical function test results., Design: A cross-sectional study of outpatients at the Memory Disorder Outpatient Center of Japan's National Center of Geriatrics and Gerontology., Participants: We enrolled 882 individuals aged ≥65 diagnosed with NC (n = 210), amnestic mild cognitive impairment (aMCI; n = 273), mild AD (n = 181) or moderate AD (n = 197)., Measurements: We measured the participants' results for functional reach (FR), the one-leg standing (OLS) test, the Timed Up and Go (TUG) test, tandem gait (TG), and grip strength (GS). A one-way analysis of covariance (ANCOVA) was used to identify significant differences among the groups' results on the physical function tests, controlling for age, sex, educational year, Mini-Nutritional Assessment, senior activity and exercise frequency, low-density lipoprotein, body mass index, free-fat mass index, and assistance for the TUG test. Multiple regression analysis was also used to investigate the correlation between these covariates and physical function tests results., Results: The ANCOVA showed that FR, OLS, and TG were significantly worse among the individuals with aMCI, mild AD, or moderate AD compared with NC. However, TUG was significantly worse only in the moderate AD group compared with the NC, aMCI, and mild AD group. Multiple regression analysis showed that aging was correlated with poorer scores on all physical function tests, women had poorer scores on FR and GS than men, and low frequency of senior activity was significantly correlated with poorer scores on FR, OLS, and TG., Conclusion: Postural impairment and instability on TG was seen in earlier AD stages compared with instability on TUG. As were the covariates of age and sex, senior activity frequency was significantly related to 2 or more physical function tests., (Copyright © 2017 AMDA – The Society for Post-Acute and Long-Term Care Medicine. Published by Elsevier Inc. All rights reserved.)

Objectives: The aim of this study was to determine whether mild cognitive impairment (MCI) affects the development of fear of falling (FoF) in older adults., Design: Prospective cohort study., Setting: The Obu Study for Health Promotion in the Elderly, Japan., Participants: A total of 1700 community-dwelling people aged 65 years or older without FoF at baseline participated., Measurements: FoF and related variables, such as physical function, self-rated health, depression, and total number of medication doses, were investigated at baseline. Participants also underwent cognitive tests and were divided into cognitive healthy and MCI. Fifteen months after the baseline measurements, we collected information about the status of FoF and fall incidence during the 15-month follow-up., Results: At the 15-month follow-up survey, 452 participants (26.5%) reported the development of FoF. Logistic regression analysis showed that MCI (odds ratio [95% CI] = 1.41 [1.07-1.87]) and a fall incident (3.00 [2.23-4.07]) during follow-up independently predicted the development of FoF, after controlling for demographic factors, physical function, self-rated health, and depression. The odds ratio for the development of FoF in participants with both MCI and a fall incident compared with those without them was 7.34 (4.06-13.3), after controlling for confounding factors., Conclusion: MCI predicts the new onset of FoF in older adults, especially when they have experience with falling. Aside from the risk of falling, it is suggested that individuals with MCI are an at-risk population for FoF and related negative consequences in terms of postfall syndrome., (Copyright © 2015 AMDA – The Society for Post-Acute and Long-Term Care Medicine. Published by Elsevier Inc. All rights reserved.)

Background: Evidence from human and animal studies has demonstrated elevated levels of the cysteine protease cathepsin S (CatS) in hypoxic atherosclerotic lesions. We hypothesized that silencing of CatS gene would suppress ischemia-induced angiogenic action., Methods and Results: Left femoral artery ligation-induced ischemia in mice showed the increased expression and activity of CatS in the ischemic muscle. The CatS-deficiency (CatS(-/-)) mice showed impaired functional recovery following hindlimb ischemia and reduced levels of peroxisome proliferator-activated receptor-γ (PPAR-γ), phospho-Akt (p-Akt), p-endothelial nitric oxide synthase, p-extracellular signal-regulated kinase1/2 (Erk1/2), p-p38 mitogen-activated protein kinase, and vascular endothelial growth factor (VEGF) proteins, as well as reduced levels of matrix metalloproteinase-9 and macrophage infiltration in the ischemic muscles. In vitro, CatS silencing reduced the levels of these targeted essential molecules for angiogenesis and vasculogenesis. Together, the results indicated that the effects of CatS knockdown led to defective endothelial cell invasion, proliferation, and tube formation. This notion was reinforced by the finding that CatS inhibition led to a decreased PPAR-γ level and VEGF/Erk1/2 signaling activation in response to ischemia. CatS(-/-) resulted in decreased circulating EPC-like CD31(+)/c-Kit(+) cells, accompanied by the reduction of the cellular levels of PPAR-γ, p-Akt, and VEGF induced by ischemic stress. Transplantation of bone-marrow-derived mononuclear cells from CatS(+/+) mice restored neovascularization in CatS(-/-) mice., Conclusions: CatS activity controls ischemia-induced neovascularization partially via the modulation of PPAR-γ and VEGF/Akt signaling activation., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Objective: The interaction between the renin-angiotensin system and toll-like receptors (TLRs) in the pathogenesis of advanced atherosclerotic plaques is not well understood. We studied the effects of the renin inhibitor aliskiren on the progression of advanced atherosclerotic plaque in apolipoprotein E-deficient (ApoE(-/-)) mice with a special focus on plaque neovessel formation., Methods and Results: Four-wk-old ApoE(-/-) mice were fed a high-fat diet for 8 wks, and the mice were randomly assigned to one of three groups and administered a vehicle, hydralazine, or aliskiren for an additional 12 wks. Aliskiren reduced the atherosclerotic plaque area and plaque neovessel density. It increased the plaque collagen and elastin contents, and reduced plasma angiotensin II levels and plaque macrophage infiltration and cathepsin S (CatS) protein. Aliskiren also decreased the levels of AT1R, gp91phox, TLR2, monocyte chemotactic protein-1, and CatS mRNAs in the aortic roots. Hydralazine had no beneficial vascular effects, although its administration resulted in the same degree of blood pressure reduction as aliskiren. CatS deficiency mimicked the aliskiren-mediated vasculoprotective effect in the ApoE(-/-) mice, but aliskiren showed no further benefits in ApoE(-/-) CatS(-/-) mice. In vitro, TLR2 silencing reduced CatS expression induced by angiotensin II. Moreover, aliskiren or the inhibition of CatS impaired the endothelial cell angiogenic action in vitro or/and ex vivo., Conclusion: Renin inhibition appears to inhibit advanced plaque neovessel formation in ApoE(-/-) mice and to decrease the vascular inflammatory action and extracellular matrix degradation, partly by reducing AT1R/TLR2-mediated CatS activation and activity, thus regressing advanced atherosclerosis., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Background: Chronic supplementation with L-citrulline plus L-arginine has been shown to exhibit anti-atherosclerotic effects. However, the short-term action of this combination on the nitric oxide (NO)-cGMP pathway remains to be elucidated. The objective of the present study was to investigate the acute effects of a combination of oral L-citrulline and L-arginine on plasma L-arginine and NO levels, as well as on blood circulation., Methods: Rats or New Zealand white rabbits were treated orally with L-citrulline, or L-arginine, or a combination of each at half dosage. Following supplementation, plasma levels of L-arginine, NOx, cGMP and changes in blood circulation were determined sequentially., Results: L-Citrulline plus L-arginine supplementation caused a more rapid increase in plasma L-arginine levels and marked enhancement of NO bioavailability, including plasma cGMP concentrations, than with dosage with the single amino acids. Blood flow in the central ear artery in rabbits was also significantly increased by L-citrulline plus L-arginine administration as compared with the control., Conclusion: Our data show for the first time that a combination of oral L-citrulline and L-arginine effectively and rapidly augments NO-dependent responses at the acute stage. This approach may have clinical utility for the regulation of cardiovascular function in humans., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: Physicians are uncertain about what medical services should be provided to older and/or disabled patients. Better understanding of health outcome prioritization among health care providers and recipients may help the process of decision- and policy-making. For this purpose, surveys were conducted on priorities of health care outcomes for the elderly., Design: Survey research., Setting: Four groups of health care providers and four groups of health care recipients., Participants: A total of 2512 health care providers and 4277 recipients., Measurements: Questionnaires were sent to more than 8000 health care providers and more than 9000 health care recipients: geriatricians, physicians who commonly see older patients or work in long term care facilities, staff members and participants in adult day care, patients in outpatient geriatric clinics, family members of patients with dementia, and community-dwelling older adults. The questionnaire asked the subjects to rank 12 measures of health care outcomes., Results: The mean response rate was 49%. All health care provider groups considered "improvement of quality of life" the most important. In contrast, in health care recipient groups, "effective treatment of illness," "improvement of physical function," and "reduction of carer burden" were given high priority, whereas "improvement of quality of life" was perceived as less important. All the groups, including health care providers and recipients, ranked "reduction of mortality" the least important, followed by "avoiding institutional care." Stratification analysis showed that the results did not differ by sex, nursing care level, or the existence of relatives who required nursing care, whereas age slightly influenced the order of high-ranked measures., Conclusion: Priorities of health care services and their differences between providers and recipients should be taken into account in the health care of older patients and the design of health care policies and research., (Copyright © 2013 American Medical Directors Association, Inc. Published by Elsevier Inc. All rights reserved.)

Background: Cathepsin K (CatK) is one of the most potent mammalian collagenases involved in atherosclerosis-based vascular disease. We investigated whether circulating CatK is associated with the prevalence of coronary artery disease (CAD)., Methods: Two-hundred fifty-two consecutive subjects were enrolled from among patients who underwent coronary angiography and intravascular ultrasound analyses. One-hundred thirty-two age-matched subjects served as controls. Plasma CatK, intact procollagen type I N-terminal propeptide (I-PINP), and linked carboxy-terminal telopeptide of collagen type I (ICTP) were measured., Results: Patients with CAD had higher CatK levels (44.0 ± 31.2 versus 15.5 ± 8.3 ng/mL, P < 0.001) and ICTP/I-PINP ratios (0.2 ± 0.1 versus 0.04 ± 0.03, P < 0.001) than the controls. Patients with acute coronary syndrome had higher CatK levels than those with stable angina pectoris. Overall, linear regression analysis showed that the CatK levels correlated positively with ICTP/I-PINP ratios (r = 0.41, P < 0.001). Multiple logistic regression analysis showed that CatK levels were independent predictors of CAD (odds ratio, 1.15; 95% CI, 1.07 to 1.23; P < 0.01). Furthermore, CatK levels were also correlated positively with percent plaque volumes and inversely with percent fibrous volumes by intravascular ultrasound., Conclusions: These data indicated that high levels of CatK are closely linked with the presence of CAD and that CatK serves as a novel biomarker for CAD., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Objective: Matrix metalloproteinases (MMPs) have been implicated in the process of vascular calcification. However, the exact roles of individual MMPs in vascular calcification are poorly understood. To study the putative role of MMP-2 in atherogenic calcification in vivo and in vitro, we investigate whether or not MMP-2 deficiency affects aortic atherosclerotic calcification in apolipoprotein E-deficient (Apoe(-/-)) mice and cultured smooth muscle cell (SMC) calcification., Methods and Results: The area of calcified lesions in aortic intima was significantly larger in MMP-2(+/+) Apoe(-/-) mice at 45 and 60 weeks of age than in MMP-2(-/-) Apoe(-/-) mice. In these aortic calcified atherosclerotic lesions, the expression of type II collagen, osteoprotegerin, bone morphogenetic protein (BMP)-2 and osteocalcin which are chondrocyte maker and bone-related proteins, was observed. MMP-2 deficiency reduced BMP-2 and osteocalcin expression in aortae in Apoe(-/-) mice at 30 weeks and 45-60-weeks-old, respectively. The expressions of MMP-2 and that of α-SMC actin were observed in chondrocyte-like cells of calcified lesions. Beta-glycerophosphate administration induced calcium deposition in MMP-2(+/+) aorta-derived cultured SMCs, and this calcium deposition was significantly suppressed in MMP-2(-/-) aorta-derived cultured SMCs., Conclusions: These results suggest that MMP-2 may contribute to the mechanisms of calcification associated with atherosclerosis., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Rotavirus is a common cause of severe gastroenteritis in children. It is known that rotavirus gastroenteritis may be accompanied by neurological manifestations, including encephalitis/encephalopathy and seizures. We report a case of a 4-year-old girl with clinically mild encephalopathy with a reversible splenial lesion associated with rotavirus infection. She was admitted to our hospital because of reduced level of consciousness, seizures, diarrhea, and vomiting. Fecal rotavirus antigen testing was positive. Cell counts in the cerebrospinal fluid (CSF) were normal. She had a normal serum sodium level on admission. Brain computed tomography showed no cerebral edema. However, electroencephalography showed generalized high-voltage slow waves, and diffusion-weighted magnetic resonance imaging demonstrated a transient abnormality in the splenium of the corpus callosum. We diagnosed clinically mild encephalopathy with a reversible splenial lesion associated with rotavirus infection. She recovered well and exhibited no neurological sequelae. Rotavirus RNA and antigen were not detected in the CSF, suggesting that the reversible splenial change was caused by indirect effects on the central nervous system subsequent to viral infection. Her normal serum sodium level indicates that this change can occur without hyponatremia.

Aims: Imaging mass spectrometry (IMS) enables the visualization of individual molecules present on tissue sections. We attempted to identify and visualize specific markers for aortic atherosclerotic lesions., Methods and Results: Atherosclerotic lesions were obtained from aortic roots of apolipoprotein E (ApoE)-deficient mice at 60 weeks of age and from femoral arteries of humans with peripheral artery occlusive disease. IMS was performed with a matrix-assisted laser desorption/ionization mass spectrometry time-of-flight (TOF)/TOF-type instrument. The molecular ions at m/z 671.6 and 673.6 were found to be specific molecules in the mouse and human lipid-rich regions. These molecules were assigned as cholesterol linoleate (CE 18:2) and cholesterol oleate (CE 18:1). In the case of the human samples, triacylglycerol was also localized in the lipid-rich regions. The distributions of the molecular ions at m/z 804.5 and 832.5 were the same as the distribution of both the mouse and the human SMCs. These molecules were assigned as phosphatidylcholine (PC) (diacyl 16:0/20:4) and PC (diacyl 18:0/20:4). The molecular ion at m/z 566.9 was localized in the mouse calcified regions, and the molecular ions at m/z 539.0 were localized in the human calcified regions., Conclusions: The IMS-based histopathologic examination (IbHE) revealed the characteristic peaks of lipid-rich regions, SMCs, and calcified regions in the atherosclerotic lesions. In addition, IbHE revealed the characteristic distribution of lipids in human atherosclerotic lesions. These data indicate that an IMS-based pathologic approach is of considerable value as a new histopathologic examination., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Objective: To determine whether caregiver burden is associated with subsequent all-cause mortality or hospitalization among dependent community-dwelling older care recipients., Methods: A prospective cohort study of 1,067 pairs of community-dwelling 65-year-old or older care recipients and their informal caregivers was conducted. The 1,067 pairs completed the baseline assessment including caregiver burden assessed by the Zarit Burden Interview and a 3-year follow-up for all-cause mortality and hospitalization., Results: During the 3-year follow-up, 268 recipients died and 455 were admitted to hospitals. The multivariate Cox proportional hazards model revealed that the recipients with caregivers with a baseline ZBI score in the highest quartile were 1.54 and 1.51 times more likely to show increased risks of all-cause mortality and hospitalization, respectively, in comparison with those with caregivers in the lowest quartile after adjustment for potential confounders. The highest quartile of caregiver burden was associated with all-cause mortality and hospitalization within nonusers of respite services including day-care services, home-help services, and nursing-home respite stay services. No apparent association was observed within the users of these services except for day-care services, for which users showed a statistically significant association between the highest quartile and the risk of hospitalization., Conclusions: Heavy caregiver burden is associated with mortality and hospitalization among community-dwelling dependent older adults, even after adjusting for potential confounders. The reduction of caregiver burden and improvement of caregiver well-being may not only prevent the deterioration of caregiver health but also reduce adverse health outcomes for care recipients.

Although it has been suggested that the renin-angiotensin (RA) system and cathepsins contribute to the development and vulnerability of atherosclerotic plaque, the interaction of the RA system and cathepsins is unclear. Thus, we investigated the effects of an angiotensin II type 1 receptor (AT1) antagonist, olmesartan, on the levels of cathepsins in brachiocephalic atherosclerotic plaque and plaque stabilization in apolipoprotein E (apoE)-deficient mice receiving a high-fat diet. Under a high fat diet, treatment with olmesartan (3 mg/kg per day) maintained collagen and elastin at high levels and attenuated the plaque development and cathepsin S (Cat S) level in the atherosclerotic plaque of apoE-deficient mice. The administration of olmesartan suppressed the accumulation of macrophages in plaque. Immunoreactivities of Cat S and AT1 were observed in macrophages. The amount of Cat S mRNA and the macrophage-mediated collagenolytic and elastolytic activities in cultured macrophages were increased by exposure to angiotensin II (Ang II), and these effects were diminished by olmesartan and the NADPH-oxidase inhibitor apocynin. These results suggested that Cat S derived from macrophages is involved in the mechanisms of atherosclerotic plaque vulnerability, and AT1 blocker maintained the plaque stabilization alongside the suppression of Cat S and macrophage activities., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Although it has been demonstrated that statins stabilize atherosclerotic lesions in animal models of advanced atherosclerosis, there is little evidence to suggest that statins have a preventive effect on plaque rupture itself. In the present study, we examined the effect of fluvastatin on plaque disruption using a simple and quick method of plaque disruption in carotid artery lesions in apolipoprotein E-deficient mice. Male apolipoprotein E-deficient mice received normal chow and underwent ligation of the left common carotid artery just proximal to its bifurcation. Four weeks later, a polyethylene cuff was placed around the artery immediately proximal to the ligation site. Fluvastatin (10mg/kg per day) was given by oral gavage every day starting at 3 days before cuff placement. The administration of fluvastatin suppressed atherosclerotic plaque disruption accompanied by luminal thrombi by 31.5% compared with controls at 4 days after the cuff was placed at the ligated carotid artery. Fluvastatin administration decreased matrix metalloproteinase-9 expression, gelatinolytic activity, endothelial adhesion molecules expression and neutrophil infiltration, and increased type I collagen content in the cuffed region. In summary, fluvastatin was found to prevent plaque disruption through pleiotropic effect on acute inflammation in an animal model using apolipoprotein E-deficient mice.

Background & Aims: Recently, poor cognition and dementia have been associated with elevated homocysteine and low B vitamin concentrations. The aim of this study is to examine the association in community-dwelling older Japanese adults., Methods: Ninety-nine subjects (71 women and 28 men; mean age 75 years) were eligible for analysis after exclusion of subjects with high serum creatinine concentrations (1.3mg/dl and over) and those taking vitamin supplements. Fasting blood samples were analyzed for plasma total homocysteine, serum folate, and serum vitamin B-12. Global cognitive function was assessed using the Mini-Mental State Examination (MMSE)., Results: Multiple regression analysis revealed that homocysteine concentrations were predicted by concentrations of vitamin B-12 (p<0.001), folate (p<0.005), and creatinine (p<0.001) and age (p<0.005). Scores on the MMSE were associated with concentrations of homocysteine, vitamin B-12, and folate. The association between folate or vitamin B-12 concentrations and MMSE scores remained significant after adjusting for homocysteine concentrations. Folate concentrations, but neither homocysteine nor vitamin B-12 concentrations, were significantly associated with serum albumin concentrations., Conclusions: Reduced folate and vitamin B-12 concentrations were independently associated with cognitive decline. The correlation between folate and albumin concentrations may imply that the reduction of folate in the Japanese older population is due to nutritional deficiency.

The elastolytic activity of cathepsins in the myocardium is implicated in hypertensive heart failure (HF). Given that reactive oxygen species are also implicated in protease activation associated with cardiac remodeling, we examined the role of the reactive oxygen species-induced cathepsin activation system in cardiac remodeling during the development of hypertensive HF. Dahl salt-sensitive hypertensive rats maintained on a high-salt diet were treated with vehicle, the cathepsin inhibitor E64d, or the angiotensin receptor blocker olmesartan from 12 to 19 weeks of age. Cathepsin expression and activity were increased in the left ventricle of HF rats; olmesartan inhibited these effects, restored the balance between elastin and collagen in the left ventricle, and suppressed degradation of the elastic lamina of coronary arteries of HF rats. Furthermore, olmesartan inhibited up-regulation of NADPH oxidase subunits and activity as well as superoxide generation. These effects of olmesartan were mimicked by E64d and were accompanied by amelioration of cardiac fibrosis. Finally, olmesartan and apocynin reduced angiotensin II-induced increases in cathepsin mRNA and protein levels in cultured rat neonatal cardiac myocytes. These data suggest that cathepsins likely trigger and promote cardiac remodeling and that blocking the angiotensin II type 1 receptor attenuates cathepsin expression and activity by inhibiting the production of superoxide by NADPH oxidase, thereby attenuating cardiac remodeling and dysfunction.

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Background: There has been much interest in the role of free radicals and oxidative stress in the pathogenesis of metabolic syndrome (MetS). Cellular antioxidant enzymes such as glutathione peroxidase 1 (GPX1) play a central role in the control of reactive oxygen species., Objective: We examined whether GPX1 polymorphism (Pro198Leu) is associated with MetS as well as with each component of MetS., Design: The study was a cross-sectional analysis of randomly selected, community-dwelling Japanese persons aged 40-70 y (1128 M, 1105 F)., Results: The genotype frequencies for the GPX1 Pro198Leu polymorphism in this cohort were 0.846, 0.151, and 0.003 for CC, CT, and TT, respectively. The CT/TT genotypes had significantly higher waist-hip ratios, triacylglycerol concentrations, homeostasis model assessment for beta-cell function, and systolic and diastolic blood pressures in men (P = 0.045, 0.012, 0.011, 0.004, and 0.003, respectively) than did the CC genotype; the CC/TT genotypes also had higher insulin in both sexes (P = 0.019 for men, P = 0.010 for women) and higher body fat mass (P = 0.027) and homeostasis model assessment for insulin resistance (P = 0.008) in women. The CT/TT genotypes showed significant association with higher prevalence of MetS as defined by 2 commonly used criteria in men [odds ratio (OR): 2.02; 95% CI: 1.30, 3.15 by the International Diabetes Federation criteria; OR: 1.49; 95% CI: 1.02, 2.18 by the modified National Cholesterol Education Program criteria) but not in women. The CT/TT genotypes showed a higher prevalence of central obesity (OR: 1.93; 95% CI: 1.31, 2.85) and hypertriglyceridemia (OR: 1.52; 95% CI: 1.08, 2.15) in men but not in women; there were no differences in other components of MetS between the CC and CT/TT genotypes in either sex., Conclusion: GPX1 Pro198Leu variants are associated with the prevalence of MetS in Japanese men but not in women.

Neutrophil elastase (NE), a serine protease released from the azurophil granules of activated neutrophil, proteolytically cleaves multiple cytokines, and cell surface proteins. In the present study, we examined whether NE affects the biological abilities of angiogenic growth factors such as basic-fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). NE degraded bFGF and VEGF in a time- and concentration-dependent manner, and these degradations were suppressed by sivelestat, a synthetic inhibitor of NE. The bFGF- or VEGF-mediated proliferative activity of human umbilical vein endothelial cells was inhibited by NE, and the activity was recovered by sivelestat. Furthermore, NE reduced the bFGF- or VEGF-induced tubulogenic response of the mice aortas, ex vivo angiogenesis assay, and these effects were also recovered by sivelestat. Neutrophil-derived NE degraded potent angiogenic factors, resulting in loss of their angiogenic activity. These findings provide additional insight into the role played by neutrophils in the angiogenesis process at sites of inflammation.

Background & Aims: Although it is not uncommon for there to be frail older people living in the community, who do not know their weight and/or height, the health-related outcomes of those older remains unknown. We examined whether missing these anthropometries are a predictor of mortality or hospitalization during a 2-year follow-up period in community-dwelling older people using various community-based services., Methods: This study was a prospective cohort analysis of 952 community-dwelling elderly. Data included the clients' demographic characteristics, basic activities of daily living (ADL), comorbidity, and anthropometric measurements at baseline. Analysis of mortality and hospitalization over the 2-year period was conducted using multivariate Cox proportional hazards models., Results: Among the 952 participants, 342 and 292 had missing data for height and weight at baseline, respectively. Multivariate Cox proportional hazards models adjusting for potential confounders showed that the lack of data on weight was associated with 2-year mortality (hazard ratio, HR:1.54, 96% CI:1.09-1.79) as well as hospitalization (HR:1.34, 95% CI:1.01-1.79) during the 2-year follow-up, although the lack of height measurement was not associated with these adverse outcomes., Conclusions: Older people living in the community with unavailable weight data appear to be more likely to have a high risk of mortality and hospitalization.

Background & Aims: It remains controversial whether mid-arm anthropometric measurements (MAAMs) are reflected with physical impairment or useful predictors of mortality in the frail elderly. We examined the following hypotheses: (1) MAAMs in frail community-dwelling elderly are lower than those of independent elderly, (2) the lower MAAMs are associated with physical function impairment, and (3) are independent predictors of 2-year mortality., Methods: This study was composed of cross-sectional and prospective cohort analyses of 957 community-dwelling elderly. Data included the clients' demographic characteristics, comorbidity, activities of daily living (ADL), and MAAMs at baseline. The mean scores of MAAMs of participants were compared with Japanese Anthropometric Reference Data. Survival analysis of 2-year mortality was conducted using multivariate Cox proportional hazards models., Results: Significantly lower arm muscle area (AMA) and higher triceps skinfold (TSF) levels were observed in most of the age groups of the study participants than those of the standard Japanese population. ADL function was correlated with AMA but not with TSF, both of which were independent risk factors for 2-year mortality in the participants (highest tertile versus lowest, AMA, HR:2.03, 95%CI:1.36-3.02; TSF, HR:1.89, 95%CI:1.30-2.75)., Conclusions: AMA and TSF were independent risk factors for 2-year mortality in the community-dwelling frail elderly.

To examine real age-related changes in the prevalence of metabolic syndrome, we studied longitudinal changes in the prevalence of metabolic syndrome in a single cohort of individuals. The participants included 112,960 Japanese (70,996 men, 14-94 years and 41,946 women, 17-85 years), who had received annual examinations between 1989 and 2004. Metabolic syndrome was defined according to the Japan Metabolic Syndrome Criteria Study Group and the US National Cholesterol Education Program (NCEP) guidelines. Overweight was defined as BMI>or=25 kg/m(2). Longitudinal changes indicated a birth cohort effect in the prevalence rate of metabolic syndrome with a lower or higher prevalence in the younger birth cohort than in the older for females or males, respectively. The estimation of the age-specific prevalence of metabolic syndrome demonstrated that in males, the prevalence of metabolic syndrome increased up to 50 decades of life for the Japanese and 60 decades of life for the NCEP criteria. In females, the prevalence increased with age up to 80 years old for both criteria. The estimated secular trends suggested that the prevalence rate of metabolic syndrome decreased in females and increased in males during study periods.

Background and Aims: Although serum albumin is well known as a marker of nutritional status, it has remained unclear whether impaired physical function affects serum albumin concentrations in older people. We examined whether hypoalbuminemia can be used as a marker of malnutrition in elderly subjects with various levels of physical impairment., Methods: A total of 262 elderly subjects without acute illness were enrolled from various geriatric settings. For the nutritional assessment, serum albumin, total cholesterol, anthropometric measurements, and subjective global assessment (SGA) were determined. Physical function was evaluated by rating score of activity of daily living (ADL)., Results: As a whole, participants' serum albumin levels correlated with various nutritional parameters including anthropometric measurements and levels of serum total cholesterol as well as the SGA evaluation. However, after adjusting for age and gender, serum albumin levels in participants with a low ADL function did not correlate with nutritional parameters. Approximately 80% participants with low ADL function who were evaluated as being well nourished according to SGA evaluation had serum albumin levels lower than 35 g/l., Conclusions: The utility of serum albumin and the traditional cutoff (35 g/l) in older people with low ADL function is questionable even among those without inflammation.

Background and Aims: Although malnutrition is common in the geriatric population, the relationship between frail elderly with various care needs and nutritional status remains unknown. The purpose of this study was to analyze the association between subjects with higher care needs and poorer nutritional status in the Japanese community-dwelling frail elderly., Methods: A total of 281 community-dwelling elderly subjects from day-care centers (81.9+/-7.2yr of age mean+/-SD; 72 men and 209 women) who were eligible for Long-Term Care Insurance were enrolled in this study to evaluate their nutritional status using the mini-nutritional assessment. The levels of care needs of participants were classified into six levels according to the Long-Term Care Insurance program., Results: According to the mini-nutritional assessment classification, 39.9%, 51.2%, and 8.9% of the participants were assessed as well-nourished, at-risk of malnutrition, and malnourished, respectively. There were significant differences among the six groups with regard to the nutritional status; subjects with higher care needs were associated with poorer nutritional status. In the higher care needs group, more than half of the subjects did not know their weight change during 3-month intervals., Conclusions: The population of elderly with higher care needs in the community is associated with a higher prevalence of malnutrition.

To observe the effect of smoking habit on age-related serum lipid levels, we examined a large cohort of Japanese cross-sectionally and longitudinally. The participants included 103,648 Japanese men and women 17-94 years of age, who had received annual health examinations from 1989 to 2003. In cross-sectional analysis, total and LDL cholesterol levels of smokers were lower than those of nonsmokers up to an elderly age in men and up to middle age in women. Smoking was associated with decreased HDL cholesterol levels up to the 65-74 years age group in men and 55-64 years in women. The triglyceride levels were higher in smokers in both genders than those of nonsmokers below 55-64 years. In the longitudinal analysis, although smoking was associated with lower total and LDL cholesterol up to 60 years of age in women, beyond the sixties an inverted association was observed. The associations of smoking with lower LDL cholesterol levels in men and lower HDL cholesterol in both genders were fairly consistent at any given age. The increase of triglyceride levels in female smokers remained rather constant between 25 and 75 years, whereas the increase in triglyceride levels in male smokers was greater with older ages up to middle age. These results suggest that the effect of smoking on the serum lipid levels is dependent on age and gender.

Smooth muscle cell (SMC) migration from the tunica media to the intima, a key event in neointimal formation, requires proteolytic degradation of elastin-rich extracellular matrix barriers. Although cathepsin S (Cat S) is overexpressed in atherosclerotic and neointimal lesions, its exact role in SMC behavior remains primarily unresolved. We examined the involvement of Cat S on SMC migration through an extracellular matrix barrier and its localization in SMCs. A selective Cat S inhibitor and the endogenous inhibitor cystatin C significantly attenuated SMC invasion across elastin gel. Western blotting and cell surface biotinylation analysis demonstrated localization of the 28-kd active form of Cat S on the SMC surface, consistent with its role in the proteolysis of subcellular matrices. Treatment with interferon-gamma or interleukin-beta1 significantly augmented the ability of SMC membranes to degrade elastin along with a significant increase in the level of active Cat S compared with controls. Immunofluorescence and confocal microscopy showed a punctuated pattern of Cat S clusters at the periphery of SMCs; further studies demonstrated partial co-localization of Cat S and integrin alphanubeta3 at the cell surfaces. These findings demonstrate that active Cat S co-localizes with integrin alphanubeta3 as a receptor on the SMC surface, playing an important role in the invasive behavior of SMCs.

Background & Aims: Malnutrition is a widespread but largely unrecognized problem in aged people. Although absolute total lymphocyte count (TLC) has been proposed as a useful indicator of nutritional status, there is little evidence that low TLC levels reflect malnutrition in the elderly. To examine whether TLC is a suitable marker of malnutrition in the elderly., Methods: A total of 161 elderly subjects (44 males and 117 females, mean age+/-SD: 77.9+/-7.4; range: 65-95 years) were enrolled from geriatric clinical settings. The participants were categorized according to severely low, low, or normal TLC. Anthropometry measurements, serum albumin, total cholesterol levels, and total score on the mini-nutritional assessment (MNA) were determined., Results: There were no significant differences among the three TLC groups with regard to anthropometry measurements, serum albumin, total cholesterol levels, or MNA score. There was a significant negative correlation of TLC with age, but not with other nutritional markers. The clinical nutritional screening tool, MNA score, was well correlated with all of the nutritional parameters used in the present study except for TLC., Conclusion: TLC is not a suitable marker of malnutrition in the elderly.

The matrix-degrading activity of several proteases are involved in the accelerated breakdown of extracellular matrix associated with vascular remodeling during the development of atherosclerosis and vascular injury-induced neointimal formation. Previous studies have shown that the potent elastolytic cysteine proteases, cathepsins S and K, are overexpressed in atherosclerotic lesions in human and animal models. However, the role of these cathepsins in vascular remodeling remains unclear. In the present study, the expressions of cathepsin S and K and their inhibitor cystatin C were examined during arterial remodeling using a rat carotid artery balloon-injury model. The increase in both cathepsin S and K mRNA levels was observed from day 1 and day 3 through day 14 following the induction of balloon injury, respectively. Western blotting analysis revealed that both cathepsin S and K protein levels also increased in the carotid arteries during neointima formation, coinciding with an increase elastolytic activity assayed using Elastin-Congo red, whereas, no significant change in the expressions of cystatin C mRNA and protein was observed during follow-up periods after injury. Immunohistochemistry, Western blot, and in situ hybridization showed that the increase of cathepins S and K and the decrease of cystatin C occurred preferentially in the developing neointima. These findings suggest that cathepsin S and K may participate in the pathological arterial remodeling associated with restenosis.

Although epigallocatechin-3-gallate (EGCg), a dominant component of green tea catechins, has been demonstrated to have anti-gelatinase properties, the molecular mechanisms by which EGCg blocks gelatinolytic activities remain unknown. We investigated whether EGCg may affect matrix metalloproteinase-2 (MMP-2) binding to native and denatured-type I collagen, and binding to the tissue inhibitor of metalloproteinase-2 (TIMP-2). Here, we report that EGCg forms a reversible complex with MMP-2, resulting in the inhibition of gelatinolytic activity of MMP-2. EGCg had no effect on the MMP-2 binding to immobilized native and denatured-type I collagen, but significantly enhanced pro- and activated MMP-2 binding to TIMP-2, as assessed by immunoprecipitation. These findings provide a new understanding of the molecular mechanisms underlying the inhibitory effect of EGCg on the gelatinolytic activity of MMP-2.

Epidemiological studies suggest that green tea consumption is associated with a reduced risk of cardiovascular disease. Antioxidative properties of green tea flavonoids, catechins, have been believed to be involved in the antiatherogenic effect of green tea, since catechins inhibit low density lipoprotein oxidation. The migration of vascular smooth muscle cells (SMCs) from the tunica media to the subendothelial region is a key event in the development and progression of atherosclerosis and post-angioplasty vascular remodeling. Matrix metalloproteinases (MMPs) play a key role in these processes of SMC migration. In the present study, we investigated the effect of catechins on the gelatinolytic activity of MMP-2 that was derived from cultured bovine aortic SMCs. We also investigated the effect of catechins on the SMC invasion through the reconstituted basement membrane barrier. A major constituent of green tea catechins, (-)-epigallocatechin gallate (EGCG), inhibited the gelatinolytic activity of MMP-2 and concanavalin A (ConA)-induced pro-MMP-2 activation without the influence of membrane-type MMP expression in SMCs. EGCG also inhibited the SMC invasion through the basement membrane barrier in a concentration-dependent manner without any influence of SMC migration across the basement membrane protein thin-coated filter. The antagonistic effects of other catechins, namely (-)-epigallocatechin (EGC) and (-)-epicatechin gallate (ECG), on gelatinolytic activity of MMP-2, ConA-induced pro-MMP-2 activation, or PDGF-BB-directed SMC invasion were much less pronounced than those of EGCG. Also, (+)-catechin and (-)-epicatechin failed to show any effect. These findings may suggest that the anti-invasive and anti-metalloproteinase activities involve at least part of the anti-atherogenic action of catechin in accordance with the antioxidant properties of catechin.

To determine the recent secular trends in serum lipid levels and characterize their influence on the aging process, we examined a large cohort of Japanese cross-sectionally and longitudinally. The participants included 80331 Japanese men and women 20-79 years of age, who had received annual health examinations from 1989 to 1998. In cross-sectional analysis, an increase in total and LDL cholesterol as well as triglyceride levels was observed in the population during the period of 1989-1998. The longitudinal changes showed that total and LDL cholesterol increased with age in men between the birth cohorts of the 1920s and 1960s. In women, these cholesterol levels increased in the 1930s and younger cohorts. HDL cholesterol decreased in men of all birth cohorts. However, HDL cholesterol increased in women of the 1940s and younger cohorts. Triglyceride levels increased in men of the 1940s and younger cohorts but decreased in the 1930s and older. Triglyceride levels increased in women of the 1930s and younger. Longitudinal analysis also suggested a birth cohort effect except for the triglyceride level for women. These results suggest that Japanese serum lipid levels continue to increase and that there exist birth cohort effects regarding serum lipid levels in the Japanese population.

Although Rho, a small GTPase, has been demonstrated to play an important role in the smooth muscle contraction and relaxation, little is known about the involvement of Rho protein in smooth muscle cell (SMC) migration. In this study the role of Rho-Rho kinase pathway was examined in SMC migration induced by platelet-derived growth factor (PDGF) and lysophosphatidic acid (LPA). C3 transferase, a specific inhibitor of Rho, blocked SMC migration induced by PDGF and LPA. Y-27632, a specific inhibitor of Rho kinase, a direct target molecule of Rho, inhibited PDGF and LPA-induced SMC migration in a concentration dependent manner. Although rapid increase in myosin light chain (MLC) phosphorylation in SMC treated with LPA was observed, no enhanced MLC phosphorylation was detected in response to PDGF. Y-27632 suppressed LPA-induced as well as basal level of MLC phosphorylation. ML-9, a specific inhibitor of myosin light chain kinase (MLCK), inhibited PDGF and LPA-induced SMC migration without the suppression of MLC phosphorylation at 5 min incubation, suggesting that MLCK may contribute to SMC migration via mechanism other than MLC phosphorylation. These results suggest that Rho-Rho kinase pathway is implicated in SMC migration and that different signaling pathways downstream of Rho-Rho kinase may be involved in LPA and PDGF-induced SMC migration. MLC phosphorylation via Rho-Rho kinase pathway appears to be implicated in LPA-dependent SMC migration. Whereas PDGF-mediated SMC migration is independent of increased MLC phosphorylation and other target molecules downstream of Rho-Rho kinase seem to be involved.

Matrix metalloproteinase-2 (MMP-2) plays critical roles in cell migration through the breakdown of the extracellular matrix. Cell movements require dynamic actin reorganization, which is controlled by Rho family GTPases. In order to examine the relation between MMP-2 regulation and actin reorganization, we used several inhibitors of Rho family GTPases. Treatment of smooth muscle cells with Clostridium difficile toxin B known to inactivate Rho family GTPases activated MMP-2. However, neither C3 transferase, a Rho inhibitor, nor Y-27632, a specific inhibitor of Rho-kinase, induced MMP-2 activation. Treatment with C3 transferase and Y-27632 caused morphological changes into the round and stellate shape, respectively, by inhibition of actin stress fiber formation. In addition, toxin B treatment induced expression and processing of MT1-MMP, a major activator of MMP-2. Taken together, we suggest the involvement of Rho family GTPases, although inhibition of neither Rho nor Rho-kinase is sufficient, in the activation of MMP-2 through expression and activation of MT1-MMP., (Copyright 2000 Academic Press.)

Cultured vascular endothelial cells derived from bovine aorta (BAECs) can survive and proliferate in the condition of two-dimensional monolayer culture in the presence of serum without any specific growth factors. When BAECs were embedded in collagen lattice, they underwent apoptotic death within 2 days unless the cultures were repeatedly supplied with angiogenic growth factor such as fibroblast growth factor-2 (FGF-2). Supplementation with FGF-2 induced endothelial cell differentiation, resulting in capillary-like tube formation inside collagen lattice. Following tube formation, withdrawal of FGF-2 induced disruption of the tube structures associated with the characteristic apoptotic cell death. These effects of FGF-2 were regulated by tyrosine phosphorylation, but not mediated through protein kinase C pathway. This model of endothelial cell apoptosis inside collagen lattice may represent in vivo endothelial cell-matrix interaction during angiogenesis process, indicating that apoptotic death of endothelial cells may regulate angiogenesis and the regression of vessels.

While endothelial nitric oxide synthase has been reported to be expressed in the endothelial cells of normal and atherosclerotic vessels, there are few reports about inducible nitric oxide synthase (iNOS). We investigated the expression of iNOS and its relation to inflammatory cells in atheroma. New Zealand White rabbits were fed 1 of 4 diets: (i) normal diet for 9 weeks; (ii) normal plus 1% cholesterol diet (atherogenic diet) for 9 weeks; (iii) atherogenic diet for 9 weeks, then normal diet for 9 weeks; (iv) atherogenic diet for 9 weeks, then the normal diet for 36 weeks. The aortas were examined by immunohistochemical staining for anti-iNOS antibody, as well as antibodies for macrophages, T lymphocytes, and muscle actin. No iNOS was detected in normal aortas, intimal thickenings, or fatty streaks. Although iNOS was detected in necrotic cores of advanced plaque, it was not seen in smooth muscle-derived cells or endothelial cells but was found in some macrophage-derived cells and in T lymphocytes. In regressive atherosclerotic aortas, iNOS was detected only in necrotic cores, not in macrophage-derived cells but in T lymphocytes. These findings suggest that T lymphocytes and some macrophages induce iNOS through cytokine production in atheroma. This is the first report of iNOS expression in atheromatous plaque.

To determine the mechanism of the antiatherosclerotic effect of estrogen, we investigated the effect of estrogen on endothelial nitric oxide synthase (NOS-3). Preincubation with a physiologic concentration of 17 beta-estradiol (10(-12)-10(-8) M) over 8 hours significantly enhanced the activity of NOS-3 in endothelial cells of cultured human umblical vein (HUVEC) and of bovine aortas (BAEC). 17 beta-estradiol also enhanced the release of nitric oxide (NO) as measured by an NO selective meter and NO2-/NO3-, metabolites of NO, from endothelial cells. Western blot showed a similar effect of 17 beta-estradiol on NOS-3. The estrogen receptor antagonists, tamoxifen and ICI182780, each inhibited the effect of 17 beta-estradiol by 80%. The effect of 17 beta-estradiol gradually decreased in cells beyond the 10th passage and was not significant in cells beyond the 16th passage. Immunocytochemistry showed the existence of estrogen receptor in HUVEC and BAEC (less than 5 passages) and the sparseness of the existence in BAEC beyond the 16th passage. Estrogen increases NOS-3 via a receptor-mediated system, and estrogen receptor, which appeared to be altered by cell senescence, could be important in the release of NO from endothelium.

Estrogen exhibits a variety of actions, including immuno-modulatory effects, in vivo and in vitro. The mechanism by which estrogen exerts its anti-inflammatory effect is not yet understood. We investigated the possible mechanisms of estradiol acting via the polymorphonuclear leukocytes (PMNs), which are important in the immune response. The agent, 17 beta-estradiol, but not 17 alpha-estradiol, significantly reduced PMNs chemotaxis to FMLP in a dose-dependent manner (control vs estrogen 10(-10)-(-6) M, P < 0.05). Physiological concentrations of estradiol significantly reduced the chemotaxis of PMNs (10(-10) mol). Pre-incubation with clomiphene or tamoxifen which are estrogen receptor antagonists, eliminated the inhibitory effect of 17 beta-estradiol on the chemotaxis of PMNs, restoring it to the control level. These observations suggest that 17 beta-estradiol suppressed the chemotaxis of PMNs by a receptor-dependent mechanism. In addition, the level of estradiol in human plasma, which PMNs were drawn, showed a close, inverse correlation with the PMNs chemotaxis to FMLP (r = -0.821 p < 0.001). Estrogen may modify the activity of neutrophils during the normal menstrual cycle, not only during pregnancy, and influence inflammation.

Estrogen is known to retard the development of atherosclerosis and to work in the brain, but the mechanism of hormonal action is completely unknown. We investigated the effect of estrogen on the activity of neuronal constitutive nitric oxide synthase (NNOS). A low concentration of estrogen (10(-10)(-7) M) enhanced the activity of homogenates of the cytosol fraction of rabbit cerebellums and also that of partially purified NNOS, and high dose (10(-6)(-5) M) attenuated them. The study using estrogen receptor antagonists, tamoxifen, clomiphene, and ICI182780 suggested that estrogen receptor did not relate significantly to those effects of 17 beta-estradiol. 17 alpha-estradiol or progesterone did not change significantly it in low doses, although moderately inhibited it in high doses. Estrogen enhanced the fluorescence of dansyl-calmodulin in low doses and attenuated it in high doses, suggesting that estrogen affects Ca(2+)-calmodulin directly. This study demonstrated that estrogen has a biphasic effect on the activity of NNOS through a Ca(2+)-calmodulin.

For anthraquinone and its mono- and dihydroxyl derivatives, and for their semiquinone anion radicals, semiempirical MNDO (moderately neglected differential overlap)/H calculations were performed and an attempt was made to correlate the calculated quantum quantities with the genotoxic and the antitumor activities. From these calculations, it was found that the electrophilic frontier electron densities (fE beta) on the beta position in anthraquinones were distinctly correlated with the genotoxic activity. On the other hand, calculations for the semiquinone anion radicals revealed the great stabilizations by the intramolecular hydrogen bond of the carbonyl group with alpha-hydroxyl groups. It has been suggested that this type of stabilization may be associated with the antitumor activity of several hydroxylanthraquinone derivatives.