Takashi Kiyanagi

Different responses to oxidized low-density lipoproteins in human polarized macrophages

BackgroundOxidized low-density lipoprotein (oxLDL) uptake by macrophages plays an important role in foam cell formation. It has been suggested the presence of heterogeneous subsets of macrophage, such as M1 and M2, in human atherosclerotic lesions. To evaluate which types of macrophages contribute to atherogenesis, we performed cDNA microarray analysis to determine oxLDL-induced transcriptional alterations of each subset of macrophages.ResultsHuman monocyte-derived macrophages were polarized toward the M1 or M2 subset, followed by treatment with oxLDL. Then gene expression levels during oxLDL treatment in each subset of macrophages were evaluated by cDNA microarray analysis and quantitative real-time RT-PCR. In terms of high-ranking upregulated genes and functional ontologies, the alterations during oxLDL treatment in M2 macrophages were similar to those in nonpolarized macrophages (M0). Molecular network analysis showed that most of the molecules in the oxLDL-induced highest scoring molecular network of M1 macrophages were directly or indirectly related to transforming growth factor (TGF)-β1. Hierarchical cluster analysis revealed commonly upregulated genes in all subset of macrophages, some of which contained antioxidant response elements (ARE) in their promoter regions. A cluster of genes that were specifically upregulated in M1 macrophages included those encoding molecules related to nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB) signaling pathway. Quantitative real-time RT-PCR showed that the gene expression of interleukin (IL)-8 after oxLDL treatment in M2 macrophages was markedly lower than those in M0 and M1 cells. HMOX1 gene expression levels were almost the same in all 3 subsets of macrophages even after oxLDL treatment.ConclusionsThe present study demonstrated transcriptional alterations in polarized macrophages during oxLDL treatment. The data suggested that oxLDL uptake may affect TGF-β1- and NF-κB-mediated functions of M1 macrophages, but not those of M0 or M2 macrophages. It is likely that M1 macrophages characteristically respond to oxLDL.

Single Administration of α-Glucosidase Inhibitors on Endothelial Function and Incretin Secretion in Diabetic Patients With Coronary Artery Disease - Juntendo University Trial: Effects of Miglitol on Endothelial Vascular Reactivity in Type 2 Diabetic Patients With Coronary Heart Disease (J-MACH) -

Background: Post-prandial hyperglycemia, hyperlipidemia, and endothelial dysfunction play an important role in the pathogenesis of atherosclerosis. Improvement in post-prandial hyperglycemia on α-glucosidase inhibitors (α-GIs) is associated with a risk reduction of cardiovascular diseases, but the post-prandial effects of α-GIs on endothelial function and incretin secretion in type 2 diabetic patients with coronary artery disease (CAD) remain unclear. Methods and Results: The post-prandial effects of a single administration of miglitol and voglibose on endothelial function and changing levels of glucose, insulin, lipids, glucagon-like peptide (GLP)-1, and gastric inhibitory polypeptide (GIP) were compared after a standard meal loading in 11 diabetic patients with CAD, using a placebo-controlled cross-over design. The changing levels of glucose, insulin and triglycerides at 60 min were significantly lower in the miglitol group than in the voglibose and placebo groups (all P<0.01). GLP-1 levels were significantly higher at 120 min (P<0.05) and GIP levels were significantly lower at 30 min and 60 min (P<0.05) in the miglitol group compared to other treatments. The reactive hyperemia duration at 120 min was significantly maintained in the miglitol group compared to the other groups. Conclusions: A single administration of miglitol significantly improved post-prandial glucose/lipid metabolism, incretin secretion, and endothelial dysfunction in diabetic patients with CAD, suggesting that miglitol may be a useful anti-atherogenic agent (UMIN000002264).  (Circ J 2010; 74: 1471 - 1478)

Effects of calcium channel blockers on glucose tolerance, inflammatory state, and circulating progenitor cells in non-diabetic patients with essential hypertension: a comparative study between azelnidipine and amlodipine on glucose tolerance and endothelial function--a crossover trial (AGENT).

BackgroundHypertension is associated with impaired glucose tolerance and insulin resistance. Medical treatment that interferes with various steps in the renin-angiotensin system improves glucose tolerance and insulin resistance. However, it remains unclear if long-acting calcium channel blockers (CCBs) such as azelnidipine and amlodipine affect glucose tolerance and insulin resistance in clinical practice.MethodsSeventeen non-diabetic patients with essential hypertension who had controlled blood pressure levels using amlodipine (5 mg/day) were enrolled in this study. After randomization, either azelnidipine (16 mg/day) or amlodipine (5 mg/day) was administered in a crossover design for 12-weeks. At baseline and the end of each CCB therapy, samples of blood and urine were collected and 75 g oral glucose tolerance test (OGTT) was performed. In addition, hematopoietic progenitor cells (HPCs) were measured at each point by flow cytometry and endothelial functions were measured by fingertip pulse amplitude tonometry using EndoPAT.ResultsAlthough blood pressure levels were identical after each CCB treatment, the heart rate significantly decreased after azelnidipine administration than that after amlodipine administration (P < 0.005). Compared with amlodipine administration, azelnidipine significantly decreased levels of glucose and insulin 120 min after the 75 g OGTT (both P < 0.05). Serum levels of high-sensitivity C-reactive protein (P = 0.067) and interleukin-6 (P = 0.035) were decreased. Although endothelial functions were not different between the two medication groups, the number of circulating HPCs was significantly increased after azelnidipine administration (P = 0.016).ConclusionsThese results suggest that azelnidipine treatment may have beneficial effects on glucose tolerance, insulin sensitivity, the inflammatory state, and number of circulating progenitor cells in non-diabetic patients with essential hypertension.

Serum levels of remnant lipoprotein cholesterol and oxidized low-density lipoprotein in patients with coronary artery disease

BACKGROUND Oxidized low-density lipoprotein (OxLDL) and remnant lipoprotein play a crucial role in the development of atherosclerosis. Recently, a novel method for measuring remnant cholesterol levels (remnant lipoproteins cholesterol homogenous assay: RemL-C) has been established. However, the correlation between OxLDL and remnant lipoprotein, including RemL-C, has not been fully investigated. METHODS We enrolled 25 consecutive patients with documented coronary artery disease (CAD) and 20 controls. Remnant-like particle cholesterol (RLP-C) and RemL-C were used to determine the levels of remnant lipoprotein cholesterol. Serum levels of malondialdehyde-modified LDL (MDA-LDL) and OxLDL using a monoclonal antibody DLH3 (OxPC) were used to measure the concentration of circulating OxLDL. RESULTS The CAD group had high levels of fasting glucose and glycosylated hemoglobin (HbA1c), and low levels of high-density lipoprotein cholesterol compared with the control group. Serum levels of total cholesterol or LDL cholesterol were not significantly different between the two groups. The levels of RemL-C (p = 0.035), MDA-LDL (p = 0.018), and MDA-LDL/LDL-C (p = 0.036) in the CAD group were significantly higher than those in the control group. The levels of RLP-C tended to be higher in the CAD group than those in the control group (p = 0.096). Positive correlations were demonstrated between remnant lipoprotein cholesterol and OxLDL (RLP-C and MDA-LDL/LDL-C, r = 0.45, p = 0.0024, RLP-C and OxPC, r = 0.51, p = 0.0005, RemL-C and MDA-LDL/LDL-C, r = 0.42, p = 0.0044, RemL-C and OxPC, r = 0.43, p = 0.0043). Similar trends were observed in non-diabetic subjects and in subjects without metabolic syndrome. Positive correlations were also observed between RLP-C and RemL-C (r = 0.94, p < 0.0001) and between MDA-LDL/LDL-C and OxPC (r = 0.40, p = 0.0074). CONCLUSIONS These results suggest that the association between high levels of remnant lipoprotein cholesterol and high OxLDL levels might be linked to atherogenesis in patients with CAD.

Involvement of cholesterol-enriched microdomains in class A scavenger receptor-mediated responses in human macrophages

OBJECTIVE Lipid rafts are cholesterol-enriched microdomains on cell membranes. We hypothesized that these microdomains could involve modified low-density lipoprotein (LDL) uptake. METHODS AND RESULTS Co-localizations of cholesterol-enriched microdomains and CD204 during the uptake of acetyl LDL (AcLDL) and oxidized LDL were observed using Alexa488-labeled polyethylene glycol cholesteryl ester, which is a sensitive probe used to analyze the dynamics of cholesterol-rich lipid microdomains in living cells. The lipid raft disruptors, methyl-β cyclodextrin and filipin, inhibited the uptake of AcLDL. CD204 siRNA treatments significantly reduced AcLDL uptake by 80%. We also demonstrated the presence of CD204 in the detergent-resistant membrane fraction (DRM) by immunoblotting analysis. The ratio of CD204/flotillin-1 in DRM was increased 11.5-fold by modified LDL administration. The PI3 kinase inhibitor LY294002, but not the Src kinase inhibitor PP1 or the Gαi/o inhibitor pertussis toxin, inhibited modified LDL uptake. The production of interleukin (IL)-8, but not CCL2, CXCL2, CXCL3, IL-6 or tumor necrosis factor-α was increased by AcLDL administration. The AcLDL-induced IL-8 production was inhibited by LY294002 and filipin. CONCLUSIONS These data firstly demonstrated that PI3 kinase-associated cholesterol-enriched microdomains are involved in CD204-mediated modified LDL uptake in human macrophages. Cholesterol-enriched microdomains may play a critical role in inflammatory processes.

High levels of saturated very long-chain fatty acid (hexacosanoic acid; C26:0) in whole blood are associated with metabolic syndrome in Japanese men

OBJECTIVE Hexacosanoic acid (C26:0) is a saturated very long-chain fatty acid and high levels of C26:0 in red blood cells are reported to be closely related with risk factors of atherosclerosis. However, the relationship between absolute levels of C26:0 in whole blood and metabolic syndrome (MS) has not been determined. MATERIALS AND METHOD We divided 218 consecutive apparently healthy male subjects into an MS group (n=78) and a non-MS group (n=140) according to the definition of the International Diabetes Federation. The levels of C26:0 in whole blood were measured by gas liquid chromatography-mass spectrometry. RESULTS The MS group had significantly higher levels of C26:0 than the non-MS group (2.42+/-0.31mug/ml vs. 2.25+/-0.29mug/ml, P=0001). There was a significant association between the levels of C26:0 and the number of factors of MS. The levels of C26:0 positively correlated with age, blood pressure, triglyceride and fasting plasma glucose. Multivariate analysis revealed that the level of C26:0 is still an independent variable for the presence of MS after adjustment for age and each criterion of MS. CONCLUSION The absolute levels of C26:0 in whole blood appear to be associated with MS independent of its component parts.

High levels of very long-chain saturated fatty acid in erythrocytes correlates with atherogenic lipoprotein profiles in subjects with metabolic syndrome

AIM Very long chain saturated fatty acid (VLCFA) levels in erythrocytes are associated with metabolic syndrome (MS). However, the relationship between levels of the VLCFA ligonoceric acid (C24:0) in erythrocytes and the atherogenic lipoprotein profiles and inflammatory state in MS remain unclear. METHODS Based on the International Diabetes Federation (IDF) definition of MS, 195 apparently healthy males were assigned to either an MS group (n=38) or a non-MS group (n=157). Fatty acid composition of erythrocytes was determined by gas liquid chromatography. RESULTS Erythrocytes from the MS group had a significantly higher level of C24:0 than cells from the non-MS group (4.06±0.48% versus 3.88±0.34%; p=0.03). C24:0 levels were significantly correlated with several components of MS. The C24:0 levels showed a significant negative correlation with LDL and HDL particle size. Multivariate linear regression analysis showed that C24:0 levels were independently correlated with LDL particle size after adjusting for age and each MS criterion. C24:0 levels were also positively correlated with log-transformed high-sensitivity CRP levels (p=0.04). CONCLUSION C24:0 levels in erythrocytes are associated with specific atherogenic lipoprotein profiles and inflammation status in subjects with MS.

Enhanced production of nitric oxide, reactive oxygen species, and pro-inflammatory cytokines in very long chain saturated fatty acid-accumulated macrophages

BackgroundDeterioration of peroxisomal β-oxidation activity causes an accumulation of very long chain saturated fatty acids (VLCSFA) in various organs. We have recently reported that the levels of VLCSFA in the plasma and/or membranes of blood cells were significantly higher in patients with metabolic syndrome and in patients with coronary artery disease than the controls. The aim of the present study is to investigate the effect of VLCSFA accumulation on inflammatory and oxidative responses in VLCSFA-accumulated macrophages derived from X-linked adrenoleukodystrophy (X-ALD) protein (ALDP)-deficient mice.ResultsElevated levels of VLCSFA were confirmed in macrophages from ALDP-deficient mice. The levels of nitric oxide (NO) production stimulated by lipopolysaccharide (LPS) and interferon-γ (IFN-γ), intracellular reactive oxygen species (ROS), and pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interluekin-6 (IL-6), and interleukin-12p70 (IL-12p70), were significantly higher in macrophages from ALDP-deficient mice than in those from wild-type mice. The inducible NO synthase (iNOS) mRNA expression also showed an increase in macrophages from ALDP-deficient mice.ConclusionThese results suggested that VLCSFA accumulation in macrophages may contribute to the pathogenesis of inflammatory diseases through the enhancement of inflammatory and oxidative responses.

Deletion of the Fc receptors γ chain preserves endothelial function affected by hypercholesterolaemia in mice fed on a high-fat diet

AIMS To clarify the role of Fc receptors (FcR) for immunoglobulin in endothelial dysfunction induced by hypercholesterolaemia, we evaluated the effect of deletion of the FcR gamma chain on endothelium-dependent relaxation and oxidative stress after 10 weeks on a high-fat diet in FcR gamma(-/-) mice compared with that in wild-type mice. METHODS AND RESULTS Plasma cholesterol levels of those on the high-fat diet were significantly increased compared with those on the normal chow diet in both groups of mice. Endothelium-dependent relaxation of the aortic ring with acetylcholine in wild-type mice was significantly reduced by the high-fat diet (ED(50): 0.22 vs. 0.43 nM, P < 0.002), whereas the relaxation in FcR gamma(-/-) mice was not inhibited (ED(50): 0.22 vs. 0.23 nM, NS). Furthermore, superoxide detection by dihydroethidium-derived fluorescence and immunohistochemical staining of p22phox expression were significantly increased in wild-type mice fed on the high-fat diet, while these oxidative stresses in FcR gamma(-/-) mice were not enhanced by the high-fat diet. Oil Red O-staining showed no significant lipid accumulation at the aortic sinus in both groups of mice. CONCLUSION This study demonstrates that the deletion of the FcR gamma chain preserves the endothelial function and attenuates oxidative stress affected by hypercholesterolaemia in FcR gamma(-/-) mice. These results indicate that FcR may play the pivotal role in endothelial dysfunction through oxidative stress induced by hypercholesterolaemia.

Disease Duration and Severity Impacts on Long-term Cardiovascular Events in Japanese Patients with Rheumatoid Arthritis

BACKGROUND Rheumatoid arthritis (RA) increases the mortality and morbidity of cardiovascular disease (CVD). However, the relationship between RA and the risk of CVD in the Japanese population remains unclear. METHODS AND RESULTS This study comprised 571 RA patients who were admitted to Juntendo University Hospital from January 1990 to December 2000. Cardiovascular events (CVEs) were defined as cardiac death, acute coronary syndrome (ACS), symptomatic stroke, and congestive heart failure. During follow-up (mean 11.7 ± 5.8 years), 7.5% of the patients died from all causes and 11.0% experienced CVEs. The morbidity of stroke and ACS was 3.6 and 2.5 per 1000 person-years, respectively. The mean RA disease duration at enrolment was significantly longer in patients who experienced CVEs than in those who did not experience CVEs (15.0 ± 12.7 years vs. 10. 8 ± 9.7 years; p = 0.01). Physical disabilities due to RA were more severe in patients who experienced CVEs than in those who did not experience CVEs. Patients with a long RA disease duration showed significantly higher event rates (p = 0.033). Cox proportional hazards analysis identified a longer RA duration as an independent risk factor for CVD (hazard ratio 1.57, 95% CI 1.09-2.30, p = 0.02). CONCLUSION Japanese RA patients showed a relatively high incidence of CVD, despite the fact that they had few coronary risk factors. The RA disease duration was an independent risk factor for CVEs.