Oral Administration of Glucosamine Improves Vascular Endothelial Function by Modulating Intracellular Redox State.

Glucosamine, used to treat osteoarthritis, has been shown to have anti-inflammatory and anti-atherosclerotic effects in experimental studies. A recent cohort study has demonstrated that the use of glucosamine was significantly associated with decreased total mortality. Vascular endothelial function is a potent surrogate marker of atherosclerosis and cardiovascular mortality where oxidative stress could participate. Therefore, we investigated whether glucosamine improves vascular endothelial function and intracellular redox state. We examined the effects of oral glucosamine administration (3000 mg/day) for 4 weeks on flow-mediated vasodilation (FMD) and intraerythrocyte glutathione parameters in 20 volunteers. Nineteen age-matched volunteers served as controls. Glucosamine administration significantly increased FMD (from 7.0 ± 2.3 to 8.7 ± 2.3%, P = 0.022). In the control group, FMD did not change. Glucosamine administration significantly increased intraerythrocyte total glutathione levels (from 212.9 ± 46.2 to 240.6 ± 49.4 µmol/L, P = 0.006), intraerythrocyte reduced form of glutathione (GSH) levels (from 124.7 ± 42.6 to 155.2 ± 47.7 µmol/L; P = 0.004) and intraerythrocyte GSH/oxidized form of glutathione (GSSG) ratios (from 3.18 ± 1.64 to 3.88 ± 1.61, P = 0.04). In the control group, any glutathione parameters did not change. Moreover, a stepwise multivariate analysis revealed percent change of GSH/GSSG is the only independent predictor for those of FMD (standardized ß = 0.58, P = 0.007) in the glucosamine group. Glucosamine administration improved FMD in association with amelioration of intraerythrocyte GSH/GSSG ratios. These results suggest that oral glucosamine administration might improve vascular endothelial function by modulating intracellular redox state.

Impact of Low Diastolic Blood Pressure on Risk of Cardiovascular Death in Elderly Patients With Coronary Artery Disease After Revascularization　- The CREDO-Kyoto Registry Cohort-1.

BACKGROUND: We investigated the effects of age and low diastolic blood pressure (DBP) on cardiovascular death in patients with coronary artery disease (CAD) after coronary revascularization. METHODS AND RESULTS: Stable, chronic CAD patients after coronary revascularization in the CREDO-Kyoto registry cohort-1 were allocated to the Young (≤64 years, n=2,619), Young-Old (65-74 years, n=2,932), and Old-Old (≥75 years, n=1,629) groups. Kaplan-Meier analysis showed that the crude cumulative incidence of cardiovascular death was higher in Young-Old patients with DBP <70 mmHg (P<0.001) and in Old-Old patients with DBP <60 mmHg (P=0.017), but not <70 mmHg (P=0.629), compared with each counterpart. Low DBP did not increase cardiovascular death in young patients. After adjustments with independent predictors, DBP <60 mmHg did not increase the cardiovascular death in the Old-Old group (HR=1.579 [95% CI, 0.944-2.642], P=0.082) and DBP <70 mmHg remained a predictor in the Young-Old group (HR=1.665 [1.094-2.532], P=0.017). On multivariate stepwise Cox proportional hazard regression analysis, independent predictors for cardiovascular death in low DBP patients were creatinine clearance (CCr; inversely), prior cerebrovascular disease, and aortic disease in the Young-Old group and CCr (inversely) and malignancy in the Old-Old group. CONCLUSIONS: DBP <60 mmHg was not an independent factor for predicting cardiovascular death in Old-Old revascularized CAD patients, whereas DBP <70 mmHg remained a predictor in the Young-Old. (Circ J 2016; 80: 1232-1241).

Blood pressure variability activates cardiac mineralocorticoid receptor and induces cardiac remodeling in hypertensive rats.

BACKGROUND: Hypertensive patients with large blood pressure variability (BPV) have aggravated target organ damage. Because the aldosterone/mineralocorticoid receptor (MR) system is a possible mechanism of hypertensive organ damage, we investigated in spontaneously hypertensive rats (SHRs) whether a specific MR blocker, eplerenone, would prevent BPV-induced aggravation of hypertensive cardiac remodeling. METHODS AND RESULTS: A rat model of a combination of hypertension and large BPV was created by performing bilateral sinoaortic denervation (SAD) in SHRs. SAD increased BPV without changing mean BP. SAD induced perivascular macrophage infiltration and aggravated myocardial fibrosis and cardiac hypertrophy, resulting in LV systolic dysfunction. Immunohistostaining revealed SAD-induced translocation of MRs into the nuclei (ie, MR activation) of the intramyocardial arterial medial cells and cardiac myocytes. SAD increased phosphorylation of p21-activated kinase1 (PAK1), a regulator of MR nuclear translocation. Chronic administration of a subdepressor dose of eplerenone prevented MR translocation, macrophage infiltration, myocardial fibrosis, cardiac hypertrophy, and LV dysfunction, while not affecting BPV. Circulating levels of aldosterone and cortisol were not changed by SAD. CONCLUSIONS: Eplerenone inhibited the aggravation of cardiac inflammation and hypertensive cardiac remodeling, and thereby prevented progression of LV dysfunction in SHRs with large BPV. This suggests that the PAK1-MR pathway plays a role in cardiac inflammation and remodeling induced by large BPV superimposed on hypertension, independent of circulating aldosterone.

Selective gene expression analysis of muscular and vascular components in hearts using laser microdissection method.

Background. The heart consists of various kinds of cell components. However, it has not been feasible to separately analyze the gene expression of individual components. The laser microdissection (LMD) method, a new technology to collect target cells from the microscopic regions, has been used for malignancies. We sought to establish a method to selectively collect the muscular and vascular regions from the heart sections and to compare the marker gene expressions with this method. Methods and Results. Frozen left ventricle sections were obtained from Wistar-Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHR-SP) at 24 weeks of age. Using the LMD method, the muscular and vascular regions were selectively collected under microscopic guidance. Real-time RT-PCR analysis showed that brain-type natriuretic peptide (BNP), a marker of cardiac myocytes, was expressed in the muscular samples, but not in the vascular samples, whereas α-smooth muscle actin, a marker of smooth muscle cells, was detected only in the vascular samples. Moreover, SHR-SP had significantly greater BNP upregulation than WKY (P < 0.05) in the muscular samples. Conclusions. The LMD method enabled us to separately collect the muscular and vascular samples from myocardial sections and to selectively evaluate mRNA expressions of the individual tissue component.

Low DBP may not be an independent risk for cardiovascular death in revascularized coronary artery disease patients.

OBJECTIVES: It remains controversial whether extremely low DBP is a risk for cardiovascular events in patients with coronary artery disease (CAD). Coronary revascularization therapy became prevalent in CAD patients. We sought to determine the impact of low DBP on cardiovascular events and to investigate the predicting factors in revascularized CAD patients. METHODS: We subanalyzed 7180 stable, chronic CAD patients (median follow-up period 3.6 years) of 9877 patients undergoing first coronary artery bypass graft or percutaneous coronary intervention in the registry of the Coronary REvascularization Demonstrating Outcome study in Kyoto (CREDO-Kyoto). RESULTS: Kaplan-Meier analysis revealed that unadjusted cumulative incidence of cardiovascular death was greater in patients with preprocedural DBP of less than 70 mmHg than in those with DBP of at least 70 mmHg, whereas the cumulative incidences of nonfatal myocardial infarction (MI) and of stroke were similar between the two groups. Stepwise logistic regression analysis showed that estimated glomerular filtration ratio (inversely), pulse pressure, left ventricular ejection fraction of less than 0.40, history of heart failure, prior cerebrovascular disease, and prior MI were independent risks for cardiovascular death in patients with DBP of less than 70 mmHg. After adjustments for the independent risks, the cumulative hazard ratio for cardiovascular death did not differ between patients with DBP of less than 70 mmHg and those with DBP of at least 70 mmHg. CONCLUSION: Renal insufficiency, more advanced vascular damage, and left ventricular systolic dysfunction were significant factors accounting for increased cardiovascular death in revascularized CAD patients with DBP of less than 70 mmHg. It was suggested that after adjustments for these independent risks, low DBP may not be a significant risk for cardiovascular death in revascularized CAD patients.

Enhanced cardiac inflammation and fibrosis in ovariectomized hypertensive rats: a possible mechanism of diastolic dysfunction in postmenopausal women.

Diastolic dysfunction is more prevalent in individuals with hypertension, particularly postmenopausal women; however, the pathogenesis of diastolic dysfunction remains unknown. Pressure overload activates cardiac inflammation, which induces myocardial fibrosis and diastolic dysfunction in rats with a suprarenal aortic constriction (AC). Therefore, we examined the effects of bilateral ovariectomy (OVX) on left ventricle (LV) remodeling, diastolic dysfunction and cardiac inflammation in hypertensive female rats. Rats were randomized to OVX+AC, OVX and AC groups as well as a Control group receiving sham operations for both the procedures. Rats underwent OVX at 6 weeks and AC at 10 weeks (Day 0). At Day 28, OVX did not appear to affect arterial pressure, cardiac hypertrophy or LV fractional shortening in AC rats. However, OVX increased myocardial fibrosis, elevated LV end-diastolic pressure and reduced the transmitral Doppler spectra early to late filling velocity ratio in AC rats. AC-induced transient myocardial monocyte chemoattractant protein-1 expression and macrophage infiltration, both of which peaked at Day 3 and were augmented and prolonged by OVX. At Day 28, dihydroethidium staining revealed superoxide generation in the intramyocardial arterioles in the OVX+AC group but not in the AC group. NOX1, a functional subunit of nicotinamide adenine dinucleotide phosphate oxidase, was upregulated only in the OVX+AC group at Day 28. Chronic 17ß-estradiol replacement prevented the increases in macrophage infiltration, NOX1 upregulation, myocardial fibrosis and diastolic dysfunction in OVX+AC rats. In conclusion, we suggest that estrogen deficiency augments cardiac inflammation and oxidative stress and thereby aggravates myocardial fibrosis and diastolic dysfunction in hypertensive female rats. The findings provide insight into the mechanism underlying diastolic dysfunction in hypertensive postmenopausal women.

Mesenchymal stem cell-based prostacyclin synthase gene therapy for pulmonary hypertension rats.

Mesenchymal stem cells (MSCs) are the pluripotent cells, which enter the circulation and home to sites of tissue injury or inflammation. MSCs are highlighted as a potential cell vector for gene therapy. In this study, we investigated whether transplanted allogeneic MSCs preferentially accumulate in the lung in rats with pulmonary hypertension (PH) and if so to determine the efficacy of MSC-based prostacyclin synthase (PCS) gene therapy for PH. PH was induced in Lewis rats by injecting monocrotaline at 7-weeks-old (week 0). MSCs were obtained by culturing bone marrow mononuclear cells. Allogeneic MSCs were intravenously transplanted at week 2 when moderate PH had been established. PH enhanced indium-111-oxine-labeled MSC accumulation in the lungs, but not in other organs, 2.5-times and 6-times, 1 and 14 days after transplantation, respectively. Transplantation of MSCs transduced with PCS (PSC-MSCs), but not with GFP (GFP-MSCs), reduced PH, pulmonary arterial thickening, and RV hypertrophy at week 4. The lung prostacyclin production was impaired in PH rats, which was restored and maintained for long time by PCS-MSCs, but not by GFP-MSCs. The survival rate at week 7 was 100% in PCS-MSC-transplanted PH rats, whereas they were 38 and 44% in PH rats and GFP-MSC-transplanted PH rats, respectively. In conclusion, the gene-engineered MSCs would be a suitable cell vector for gene delivery specifically to the PH lung. The allogeneic PCS-MSC transplantation attenuated PH and cardiovascular remodeling, and improved the prognosis in PH rats. The MSC-based PCS gene therapy may be a promising strategy for PH treatment.

Large blood pressure variability and hypertensive cardiac remodeling--role of cardiac inflammation.

An increase in short-term blood pressure (BP) variability is a characteristic feature of hypertensive patients, especially in elderly patients. There is increasing evidence that large BP variability aggravates hypertensive target organ damage and is an independent risk factor for the cardiovascular events in elderly hypertensive patients. However, little is known about the underlying mechanism. We have created a rat model of a combination of hypertension and large BP variability by performing sinoaortic denervation (SAD) in spontaneously hypertensive rats (SHRs). SAD aggravates left ventricular (LV)/myocyte hypertrophy and myocardial fibrosis to a greater extent and impairs LV systolic function without changing mean BP in SHR. SAD upregulates cardiac monocyte chemoattractant protein-1 and transforming growth factor-beta, and induces macrophage infiltration. Cardiac angiotensinogen expression is increased and the angiotensin II type 1 receptor is activated by SAD. A subdepressor dose of angiotensin receptor blocker abolishes SAD-induced inflammatory changes and cardiac remodeling and subsequently prevents systolic dysfunction in SHR+SAD. Accordingly, it is suggested that cardiac inflammation via activation of the cardiac angiotensin II system would play a role in the aggravation of cardiac remodeling and dysfunction in hypertensives with large BP variability.

Exaggerated blood pressure variability superimposed on hypertension aggravates cardiac remodeling in rats via angiotensin II system-mediated chronic inflammation.

Hypertensive patients with large blood pressure variability (BPV) have aggravated end-organ damage. However, the pathogenesis remains unknown. We investigated whether exaggerated BPV aggravates hypertensive cardiac remodeling and function by activating inflammation and angiotensin II-mediated mechanisms. A model of exaggerated BPV superimposed on chronic hypertension was created by performing bilateral sinoaortic denervation (SAD) in spontaneously hypertensive rats (SHRs). SAD increased BPV to a similar extent in Wistar Kyoto rats and SHRs without significant changes in mean blood pressure. SAD aggravated left ventricular and myocyte hypertrophy and myocardial fibrosis to a greater extent and impaired left ventricular systolic function in SHRs. SAD induced monocyte chemoattractant protein-1, transforming growth factor-beta, and angiotensinogen mRNA upregulations and macrophage infiltration of the heart in SHRs. The effects of SAD on cardiac remodeling and inflammation were much smaller in Wistar Kyoto rats compared with SHRs. Circulating levels of norepinephrine, the active form of renin, and inflammatory cytokines were not affected by SAD in Wistar Kyoto rats and SHRs. A subdepressor dose of candesartan abolished the SAD-induced left ventricular/myocyte hypertrophy, myocardial fibrosis, macrophage infiltration, and inductions of monocyte chemoattractant protein-1, transforming growth factor-beta, and angiotensinogen and subsequently prevented systolic dysfunction in SHRs with SAD. These findings suggest that exaggerated BPV induces chronic myocardial inflammation and thereby aggravates cardiac remodeling and systolic function in hypertensive hearts. The cardiac angiotensin II system may play a role in the pathogenesis of cardiac remodeling and dysfunction induced by a combination of hypertension and exaggerated BPV.

Vitamin C reversed malfunction of peripheral blood-derived mononuclear cells in smokers through antioxidant properties.

BACKGROUND: Smoking impairs neovascularization, possibly, through the impaired function of peripheral blood-derived mononuclear cells (PB-MNCs). Thus, the mechanism of impaired function of PB-MNCs caused by chronic smoking was examined, and whether vitamin C reversed the malfunction of PB-MNCs in smokers was investigated. METHODS AND RESULTS: The cohort comprised 27 healthy male volunteers (16 smokers and 11 age-matched non-smokers). For evaluation of the colony-forming activity of PB-MNCs, the number of endothelial colony-forming units (e-CFUs) was counted in a culture assay. Migration activity of PB-MNCs was evaluated by the modified Boyden chamber method. In smokers, the number of e-CFUs was reduced to 56% and migratory activity of PB-MNCs to 40% compared with non-smokers (p<0.01). The urinary level of 8-isoprostane, an oxidative stress marker, was greater in smokers than in non-smokers (p<0.05). There was an inverse correlation between migratory activity of PB-MNCs but not between the number of e-CFUs and urinary level of 8-isoprostane. Furthermore, oral administration of vitamin C for 2 weeks ameliorated the impaired migratory activity of PB-MNCs in smokers. CONCLUSION: Chronic smoking impairs the function of PB-MNCs. Smoking-induced oxidative stress may be involved in impaired migratory activity of PB-MNCs.

Mutant MCP-1 therapy inhibits tumor angiogenesis and growth of malignant melanoma in mice.

We investigated whether blocking of monocyte chemoattractant-1 (MCP-1) function would inhibit recruitment of tumor-associated macrophages (TAMs) and prevent tumor angiogenesis and tumor growth of human malignant melanoma. B16-F1 melanoma cells were implanted onto the back of C57BL/6 mice (Day 0). At Day 7, a dominant negative MCP-1 mutant (7ND) gene was transfected in the thigh muscle to make overexpressed 7ND protein secreted into systemic circulation. 7ND treatment inhibited TAM recruitment and partially reduced tumor angiogenesis and tumor growth. Also, 7ND treatment attenuated inductions of tumor necrosis factor-alpha (TNFalpha), interleukin-1alpha (IL-1alpha), and vascular endothelial growth factor (VEGF) in the stroma and tumor. Melanoma cells expressed not only MCP-1 but also its receptor CCR2. Accordingly, it was suggested that MCP-1 would enhance tumor angiogenesis and early tumor growth in the early stages by inducing TNFalpha, IL-1alpha, and VEGF through TAM recruitment and probably the direct autocrine/paracrine effects on melanoma cells.

Inhibition of progression and stabilization of plaques by postnatal interferon-gamma function blocking in ApoE-knockout mice.

A role of interferon-gamma is suggested in early development of atherosclerosis. However, the role of interferon-gamma in progression and destabilization of advanced atherosclerotic plaques remains unknown. Thus, the aim of this study was to determine whether postnatal inhibition of interferon-gamma signaling could inhibit progression of atherosclerotic plaques and stabilize the lipid- and macrophage-rich advanced plaques. Atherosclerotic plaques were induced in ApoE-knockout (KO) mice by feeding high-fat diet from 8 weeks old (w). Interferon-gamma function was postnatally inhibited by repeated gene transfers of a soluble mutant of interferon-gamma receptors (sIFNgammaR), an interferon-gamma inhibitory protein, into the thigh muscle every 2 weeks. When sIFNgammaR treatment was started at 12 w (atherosclerotic stage), sIFNgammaR not only prevented plaque progression but also stabilized advanced plaques at 16 w: sIFNgammaR decreased accumulations of the lipid and macrophages and increased fibrotic area with more smooth muscle cells. Moreover, sIFNgammaR downregulated expressions of proinflammatory cytokines, chemokines, adhesion molecules, and matrix metalloproteinases but upregulated procollagen type I. sIFNgammaR did not affect serum cholesterol levels. In conclusion, postnatal blocking of interferon-gamma function by sIFNgammaR treatment would be a new strategy to inhibit plaque progression and to stabilize advanced plaques through the antiinflammatory effects.

Vascular inflammation evaluated by [18F]-fluorodeoxyglucose positron emission tomography is associated with the metabolic syndrome.

OBJECTIVES: We investigated factors for carotid artery inflammation by [(18)F]-fluorodeoxyglucose (FDG) positron emission tomography (PET). BACKGROUND: Inflammation is present in some atherosclerotic plaques. The FDG-PET is capable of identifying and quantifying vascular inflammation within atherosclerotic plaques. METHODS: The FDG-PET imaging was performed in 216 consecutive patients (63 +/- 9 years, men:women 147:69) for cancer screening. Vascular inflammation in carotid atherosclerosis was quantified by measuring the standardized uptake value (SUV) of FDG into the artery. RESULTS: Multiple stepwise regression analysis revealed significant relationships between SUV and waist circumference (p < 0.001), hypertensive medication (p < 0.001), carotid intima-media thickness (p < 0.001), high-density lipoprotein cholesterol (p < 0.01, inversely), homeostasis model assessment of insulin resistance (p < 0.05), or high sensitivity C-reactive protein (p < 0.05). Age- and gender-adjusted SUV of FDG was significantly higher (p < 0.0001) in proportion to the accumulation of the number of the components of the metabolic syndrome. Thus, the metabolic syndrome was associated with increased FDG uptake in carotid atherosclerosis. CONCLUSIONS: Our present study may suggest that the metabolic syndrome is associated with inflammation in carotid atherosclerosis. (Detection of Plaque Inflammation by Positron Emission Tomography (PET); http://www.clinicaltrials.gov/ct/show/NCT00114504; NCT00114504).

Simvastatin attenuates plaque inflammation: evaluation by fluorodeoxyglucose positron emission tomography.

OBJECTIVES: We investigated whether simvastatin attenuates plaque inflammation by using 18F-fluorodeoxyglucose positron emission tomography (18FDG-PET) co-registered with computerized tomography. BACKGROUND: Inflammation plays a key role in progression and destabilization of atherosclerotic plaque. 18F-fluorodeoxyglucose PET is a promising tool for visualizing inflammation of atherosclerotic plaque. Antiinflammatory action is one of the pleiotropic effects of statins. METHODS: Forty-three consecutive subjects, who underwent 18FDG-PET for cancer screening and had 18FDG uptakes in the thoracic aorta and/or the carotid arteries, were randomized to either statin group receiving simvastatin (n = 21) or diet group receiving dietary management only (n = 22). The maximum standardized uptake values (SUVs) were measured in individual plaques, and were averaged for analysis of the subjectwise results. The responses were assessed after 3-month treatments. RESULTS: Positron emission tomography revealed 117 and 123 18FDG-positive plaques in the statin and diet groups, respectively. Simvastatin, but not diet alone, attenuated plaque (18)FDG uptakes and decreased the SUVs (p < 0.01). Simvastatin reduced low-density lipoprotein cholesterol (LDL-C) by 30% (p < 0.01) and increased high-density lipoprotein cholesterol (HDL-C) by 15% (p < 0.01), whereas LDL-C and HDL-C levels were not changed in the diet group. In the statin group, the decrease in the SUV was well correlated with the HDL-C elevation (p < 0.01) but not with the LDL-C reduction. CONCLUSIONS: 18F-fluorodeoxyglucose PET visualized plaque inflammation and simvastatin attenuated it. The LDL-C-independent effects of simvastatin may participate in the beneficial effect. 18F-fluorodeoxyglucose PET has a potential for visually monitoring plaque inflammation and the therapeutic effectiveness of statins.

Roles of endogenous monocyte chemoattractant protein-1 in ischemia-induced neovascularization.

OBJECTIVES: We sought to investigate the role of endogenous monocyte chemoattractant protein (MCP)-1 in ischemia-induced neovascularization. BACKGROUND: Roles of inflammatory changes including macrophage infiltration are suggested in ischemic neovascularization. METHODS: Unilateral hindlimb ischemia was induced by excising surgically the entire femoral artery and vein in mice. Immediately after operation, plasmid deoxyribonucleic acid encoding a dominant negative mutant of MCP-1 (7ND) or the empty plasmid (mock) was injected into the ipsilateral thigh adductor muscle. RESULTS: In mock-treated mice, MCP-1 was upregulated transiently in ischemic hindlimb peaking at day 3. Serial laser Doppler blood flow (LDBF) analysis showed an abrupt decrease in blood flow, followed by a recovery to the near-normal levels in mock-treated mice; 7ND treatment had no effects on the initial decrease in LDBF but deteriorated the recovery. At day 3, macrophage infiltration and inductions of tumor necrosis factor (TNF)-alpha and vascular endothelial growth factor (VEGF) were prominent in the ischemic adductor muscle in mock-treated mice; 7ND treatment significantly reduced macrophage infiltration and suppressed TNF-alpha and VEGF inductions in response to ischemia. At day 21, postmortem angiography and anti-CD31 immunohistostaining revealed well-developed collateral vessels and capillary formation, respectively, in the ischemic muscle of mock-treated mice; 7ND overexpression remarkably suppressed the collateral vessel formation and capillary formation. CONCLUSIONS: Endogenous MCP-1 may play a role in ischemia-induced neovascularization by recruiting macrophages that activate TNF-alpha and VEGF inductions.

Soybean isoflavones eliminate nifedipine-induced flushing of tail skin in ovariectomized mice.

Hot flushes are one of the most frequent symptoms in menopausal women. We investigated effect of soybean isoflavones (Soyaflavone HG) on nifedipine-induced flushing in ovariectomized mice. Ovariectomy markedly aggravated nifedipine-induced increase in tail skin temperature. Soyaflavone HG (10 mg/kg, p.o., once a day for 5 days) inhibited nifedipine-induced flushing in ovariectomized mice. The inhibitory effect of Soyaflavone HG was significantly reversed by an estrogen-receptor antagonist, ICI 182,780, suggesting that Soyaflavone HG prevents nifedipine-induced flushing partially through estrogen receptors. We presented the experimental evidence suggesting that soybean isoflavones including Soyaflavone HG have the benefits for menopausal hot flushes.