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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.)

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: 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.)

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.)

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.

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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.

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.

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.

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.

We evaluated the effect of a low level of hyperlipidemia and the effects of in vitro exposure to atherogenic lipoproteins (LDL, VLDL) on the vascular responsiveness of isolated porcine coronary arteries. Firstly we studied the change in vascular responsiveness induced by feeding a cholesterol-rich diet to pigs for 4 and 9 weeks (C4 and C9 pigs). The serum cholesterol level in pigs fed a cholesterol-rich diet reached 218.5 +/- 32.9 mg/dl compared with 85.5 +/- 8.4 mg/dl in the controls. Segments of the left descending coronary artery were examined. The contraction induced by KCl or prostaglandin F2 alpha was not altered significantly by hypercholesterolemia nor was the relaxation induced by the Ca2+ ionophore, A23187, or by nitroglycerin. Endothelium-dependent relaxation (EDR) evoked by high, but not low, concentrations of bradykinin was reduced in the C4 pigs as compared with those in normal animals. EDRs evoked by bradykinin, substance P, and serotonin were significantly reduced in C9 pigs. Histologically, as observed by light and electron microscopy, fatty changes or intimal thickenings were not seen in the coronary arteries of the C4 pigs. Minimal changes (intimal thickening and fragmentation of internal elastic lamina) were observed only in parts of arteries of the C9 pigs. Secondly, the direct effects of LDL and VLDL on vascular responsiveness were studied. Although preincubation with LDL inhibited the EDR caused by exposure to bradykinin and A23187 in the coronary arteries of normal and cholesterol-fed pigs, preincubation with LDL inhibited the arterial relaxation induced by exposure to substance P or serotonin in both the C4 and the C9 pigs, but not in the control animals. The degree of inhibition was especially marked in the C9 pigs. The inhibitory effect of VLDL on EDR was weaker than that of LDL. Indomethacin (5 microM) did not alter this inhibitory effect of lipoproteins. Neither LDL nor VLDL had any effect on the vascular relaxation induced by nitroglycerin. These results are consistent with the idea that endothelium-dependent arterial relaxation is attenuated even at the very early stage of cholesterol-induced atherosclerosis. Atherogenic lipoproteins may further impair the decreased EDR in the arteries of hyperlipidemic pigs by two factors: one released on stimulation with bradykinin and the calcium ionophore A23187, the other released on stimulation with substance P and serotonin.

The migration of vascular smooth muscle cells from the media into the intima and their proliferation in the intima play an important role in the pathogenesis of atherosclerosis. We examined the effects of fibrinogen and fibrin on the migration of cultured bovine aortic smooth muscle cells using a modified Boyden chamber assay. The cells migrated to a gradient of soluble fibrinogen. Checkerboard analysis indicated that the effect was largely directional in nature (chemotaxis). The cells also migrated in a dose-dependent manner to a gradient of substrate-bound fibrinogen (haptotaxis). Fibrin, converted from substrate-bound fibrinogen by thrombin, also induced haptotaxis of smooth muscle cells. These observations suggest that, by recruiting smooth muscle cells from the media into the intima, fibrinogen and fibrin may be involved in the pathogenesis of arterial intimal thickening, atherosclerosis, and the organization of a thrombus.