Continuous inhibition of poly(ADP-ribose) polymerase does not reduce reperfusion injury in isolated rat heart.

Poly(ADP-ribose) polymerase (PARP), an enzyme that is important to the regulation of nuclear function, is activated by DNA strand breakage. In massive DNA damage, PARP is overactivated, exhausting nicotinamide adenine dinucleotide and leading to cell death. Recent studies have succeeded in reducing cellular damage in ischemia/reperfusion by inhibiting PARP. However, PARP plays an important part in the DNA repair system, and its inhibition may be hazardous in certain situations. We compared the short-time inhibition of PARP against continuous inhibition during ischemia/reperfusion using isolated rat hearts. The hearts were reperfused after 21 minutes of ischemia with a bolus injection of 3-aminobenzamide (3-AB) (10 mg/kg) followed by continuous 3-AB infusion (50 µM) for the whole reperfusion period or for the first 6 minutes or without 3-AB. At the end of reperfusion, contractile function, high-energy phosphate content, nicotinamide adenine dinucleotide content, and infarcted area were significantly preserved in the 3-AB 6-minute group. In the 3-AB continuous group, these advantages were not apparent. At the end of reperfusion, PARP cleavage had significantly proceeded in the 3-AB continuous group, indicating initiation of the apoptotic cascade. Thus, continuous PARP inhibition by 3-AB does not reduce reperfusion injury in the isolated rat heart, which may be because of acceleration of apoptosis.

Impact of low-and high-density lipoprotein cholesterol levels on carotid intima-media thickness differs by smoking status in middle-aged men.

AIM: Cigarette smoking is a strong risk factor for atherosclerotic disease; however, it remains unclear whether the impact of other risk factors differs by smoking status. The aim of this study was to investigate this issue, especially with regard to low-density and high-density lipoprotein (LDL/HDL) levels. METHODS: In total, 448 healthy, middle-aged men (aged 37 to 61) participated in this study. Smoking habits were recorded, carotid intima-media thickness (IMT) was measured by B-mode ultrasound, and serum lipids and other biochemical parameters were determined from fasted blood samples. RESULTS: Among the overall subjects, multivariate regression analyses showed that IMT was significantly associated with age (p < 0.0001 for mean IMT, p= 0.002 for max IMT), body mass index (BMI, mean IMT, p= 0.028), LDL-C levels (mean/max IMT, p= 0.001), HDL-C levels (max IMT, p= 0.022) and current smoking habit (mean IMT, p=0.012). Subgroup analyses according to smoking status revealed that LDL-C levels were significantly associated with mean/max IMT in current smokers (p=0.001) but not in ex- or nonsmokers (never smoked subjects). After adjusting for age, BMI, systolic blood pressure, hemoglobin A1c and serum lipids, mean IMT respectively increased and decreased progressively across LDL-C and HDL-C quartiles (p= 0.004 and 0.045) in the overall subjects. These associations were observed in current smokers (p= 0.01) but not in ex- or nonsmokers for LDL-C, and were observed in ex- and nonsmokers (p= 0.025, 0.017, respectively) but not in current smokers for HDL-C. CONCLUSION: The impact of LDL-C/HDL-C levels on carotid IMT differs by smoking status. These observations imply that distinct mechanisms are involved in the (anti) atherogenesis of LDL/HDL according to smoking status.

Deficiency of interleukin-1 receptor antagonist promotes spontaneous femoral artery aneurysm formation in mice.

Femoral artery aneurysms (FAAs) are very rare, and their natural history is not well understood. In this study, we sought to analyze the pathogenesis of inflammatory FAAs in interleukin-1 receptor antagonist-deficient (IL-1Ra(-/-)) B6 mice. Systolic arterial pressures and plasma lipid levels of IL-1Ra(-/-) mice and wild-type (WT) mice did not differ significantly. However, IL-1Ra(-/-) mice spontaneously developed fusiform FAAs. Real-time PCR of 9-month-old IL-1Ra(-/-) mice revealed significantly increased mRNA levels of IL-1ß (6.6-fold), tumor necrosis factor-α (TNF-α) (12.4-fold), and matrix metalloproteinase-9 (6.0-fold) compared with WT mice. Histological analysis revealed numerous inflammatory cells around the FAAs in IL-1Ra(-/-) mice, and elastin staining showed destruction of both the internal and external elastic lamina in IL-1Ra(-/-) mice. Afterward, macrophage function was studied. After lipopolysaccharide (1 µg/mL) stimulation, IL-1Ra-deficient macrophages produced much higher levels of TNF-α than those from WT mice. Finally, we performed bone marrow cell transplantation. FAAs with many inflammatory cells in the adventitia were detected in several WT mice that received bone marrow cells from IL-1Ra(-/-) mice (44%), but not from WT mice (0%). Our study is the first to demonstrate that IL-1Ra deficiency in inflammatory cells disrupts immune system homeostasis and induces inflammatory FAAs in IL-1Ra(-/-) B6 mice. We believe that these mice will provide much information about the natural history and management of FAAs.

Novel TNF-α receptor 1 antagonist treatment attenuates arterial inflammation and intimal hyperplasia in mice.

AIM: Tumor necrosis factor receptor 1 (TNFR1) participates importantly in arterial inflammation in genetically altered mice; however it remains undetermined whether a selective TNFR1 antagonist inhibits arterial inflammation and intimal hyperplasia. This study aimed to determine the effect and mechanism of a novel TNFR1 antagonist in the suppression of arterial inflammation. METHODS: We investigated intimal hyperplasia in IL-1 receptor antagonist-deficient mice two weeks after inducing femoral artery injury in an external vascular cuff model. All mice received intraperitoneal injections of TNFR1 antagonist (PEG-R1antTNF) or normal saline twice daily for 14 days. RESULTS: PEG-R1antTNF treatment yielded no adverse systemic effects, and we observed no significant differences in serum cholesterol or blood pressure in either group; however, selective PEG-R1antTNF treatment significantly reduced intimal hyperplasia (19,671±4,274 vs. 11,440±3,292 µm(2); p=0.001) and the intima/media ratio (1.86±0.43 vs. 1.34±0.36; p=0.029), compared with saline injection. Immunostaining revealed that PEG-R1antTNF inhibits Nuclear factor-κB (NF-κB), suppressing smooth muscle cell (SMC) proliferation and decreasing chemokine and adhesion molecule expression, and thus decreasing intimal hyperplasia and inflammation. CONCLUSIONS: Our data suggest that PEG-R1antTNF suppresses SMC proliferation and inflammation by inhibiting NF-κB. This study highlights the potential therapeutic benefit of selective TNFR1 antagonist therapy in preventing intimal hyperplasia and arterial inflammation.

IκBNS regulates interleukin-6 production and inhibits neointimal formation after vascular injury in mice.

AIMS: IκBNS regulates a subset of Toll-like receptor (TLR)-dependent genes including interleukin-6 (IL-6) by inhibiting nuclear factor-κB (NF-κB). IL-6 is an inflammatory biomarker for cardiovascular diseases. The aim of this study was to determine whether IκBNS changes arterial inflammation and intimal hyperplasia after vascular injury. METHODS AND RESULTS: We investigated neointimal formation in IκBNS-deficient (IκBNS(-/-); C57BL/6 background) and wild-type (IκBNS(+/+)) mice 2 weeks after cuff injury. The mean intimal area and the intima/media ratio of IκBNS(-/-) mice increased 89% (8066 ± 1141 vs. 4267 ± 1095 µm(2); P = 0.027) and 100% (0.72 ± 0.13 vs. 0.36 ± 0.09; P = 0.032) compared with IκBNS(+/+) mice. We observed significant up-regulation of TLR4 in injured arteries of IκBNS(-/-) mice. NF-κB activity in the intima of IκBNS(-/-) mice was 5.1-fold higher (P = 0.008) compared with IκBNS(+/+) mice at 7 days post-injury. IL-6 mRNA levels in injured arteries of IκBNS(-/-) mice were 1.8-fold higher (P = 0.002) compared with those of IκBNS(+/+) mice at 3 days post-injury. Vascular smooth muscle cells from IκBNS(-/-) mice showed a significant increase in cell migration compared with those from IκBNS(+/+) mice after IL-6 stimulation in the scratch-wound healing assay. Furthermore, anti-mouse IL-6 receptor antibody (MR16-1) significantly reduced intimal hyperplasia compared with control IgG injection in IκBNS(-/-) mice. These findings suggest that IL-6 participates in the development of neointimal hyperplasia after vascular injury in IκBNS(-/-) mice. CONCLUSION: IκBNS down-regulates TLR4 expression, NF-κB activity, and IL-6 production after vascular injury. IκBNS might suppress intimal hyperplasia caused by vascular inflammation such as atherosclerosis, and restenosis after angioplasty.

Deficiency of CuZn superoxide dismutase promotes inflammation and alters medial structure following vascular injury.

AIM: The anti-oxidant enzyme copper/zinc superoxide dismutase (CuZnSOD) metabolizes superoxide anion (O(2)(-)) in vascular cells. However, the role of CuZnSOD in vascular injury remains poorly understood. METHODS: Using CuZnSOD-deficient (CuZnSOD(-/-)) mice and wild-type (WT) mice, we investigated morphometric changes and the role of O(2)(-) in vascular remodeling after femoral artery injury induced by an external vascular cuff model. RESULTS: Three days post-injury, inflammatory cell infiltration increased significantly. Moreover, the percent positive area of tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in media were higher in CuZnSOD(-/-) mice than in WT mice (TNF-α: 34.8±8.4% versus 18.8±5.6%, p < 0.05, ICAM-1: 29.6±6.5% versus 11.0±2.8%, p < 0.05, VCAM-1: 23.5±7.5% versus 3.7±1.1%, p < 0.05). mRNA expression of iNOS was markedly increased in CuZnSOD(-/-) mice with cuff injury. Dihydroethidine staining revealed increased levels of vascular O(2)(-) in media from CuZnSOD(-/-) mice. Although neointimal formation remained unchanged, 14 days postinjury, we observed degeneration of the media, and the media/vessel wall ratio increased in CuZnSOD(-/-) mice (40.4±2.1% versus 26.8±1.4%, p < 0.05). Furthermore, SMemb/MHC-B-stained lesions increased markedly in CuZnSOD(-/-) mice. CONCLUSIONS: CuZnSOD-deficiency promoted inflammation, expressed adhesion molecules, and altered the structure of the media post-injury. Our results suggest that O(2)(-) participates importantly in the progression of early stage vascular inflammation, resulting in vascular remodeling in media but not neointimal formation, post-injury.

Proteasomal inhibition promotes ATP-binding cassette transporter A1 (ABCA1) and ABCG1 expression and cholesterol efflux from macrophages in vitro and in vivo.

OBJECTIVE: ATP-binding cassette transporter A1 (ABCA1) and ABCG1 are key molecules in an initial step of reverse cholesterol transport (RCT), a major antiatherogenic property of high-density lipoprotein (HDL). The ubiquitin-proteasome system (UPS) mediates nonlysosomal pathways for protein degradation and is known to be involved in atherosclerosis. However, little is known about the effects of the UPS on these molecules and overall RCT. We therefore investigated whether UPS inhibition affects ABCA1/G1 expression in macrophages and RCT in vitro and in vivo. METHODS AND RESULTS: Various proteasome inhibitors increased ABCA1/G1 expression in macrophages, translating into enhanced apolipoprotein A-I- and HDL-mediated cholesterol efflux from macrophages. ABCA1 and ABCG1 were found to undergo polyubiquitination in the macrophages and HEK293 cells overexpressing these proteins, and pulse-chase analysis revealed that proteasome inhibitors inhibited ABCA1/G1 protein degradation. In in vivo experiments, the proteasome inhibitor bortezomib increased ABCA1/G1 protein levels in mouse peritoneal macrophages, and RCT assays showed that it significantly increased the fecal (54% increase compared with saline) and plasma (23%) appearances of the tracer derived from intraperitoneally injected (3)H-cholesterol-labeled macrophages. CONCLUSIONS: The present study provided evidence that the UPS is involved in ABCA1/G1 degradation, thereby affecting RCT in vivo. Therefore, specific inhibition of the UPS pathway might lead to a novel HDL therapy that enhances RCT.

Pulmonary intravascular lymphoma diagnosed by 18-fluorodeoxyglucose positron emission tomography-guided transbronchial lung biopsy in a man with long-term survival: a case report.

INTRODUCTION: 18-Fluorodeoxyglucose positron emission tomography can detect the pulmonary involvement of intravascular lymphoma that presents no abnormality in a computed tomography scan. CASE PRESENTATION: We report the case of a 61-year-old Japanese man who had pulmonary intravascular lymphoma and no computed tomography abnormality. We were able to make an antemortem diagnosis of pulmonary intravascular lymphoma by transbronchial lung biopsy according to 18-fluorodeoxyglucose positron emission tomography findings. He is free of recurrent disease 24 months after chemotherapy. CONCLUSIONS: To the best of our knowledge, this is the first reported case of a long-term survivor of pulmonary intravascular lymphoma diagnosed by transbronchial lung biopsy under the guide of 18-fluorodeoxyglucose positron emission tomography.

Hemoglobin vesicle improves recovery of cardiac function after ischemia-reperfusion by attenuating oxidative stress in isolated rat hearts.

Hemoglobin vesicle (HbV) could be a useful blood substitute in emergency medicine. The aim of this study was to clarify the effects of HbV on cardiac function after ischemia-reperfusion (I/R) ex vivo. Isolated rat hearts were perfused according to the Langendorff method. An ischemia-reperfusion group (n = 6) was subjected to 25 minutes of global ischemia and 30 minutes of reperfusion. HbV (hemoglobin, 0.33 g/dL) was perfused before ischemia-reperfusion for 10 minutes (HbV group, n = 6). Hemodynamics were monitored, and tissue glutathione contents were measured. The redox state of reactive thiols in cardiac tissues was assessed by the biotinylated iodoacetamide labeling method. Left ventricular developed pressure was significantly recovered in the HbV group after 30 minutes of reperfusion (56.3 ± 2.8 mm Hg vs. ischemia-reperfusion group 27.0 ± 8.0 mm Hg, P < 0.05). Hemodynamic changes induced by HbV were similar to those observed when N-nitro-L-arginine methyl ester was perfused for 10 minutes before ischemia-reperfusion (L-NAME group). The oxidized glutathione contents of cardiac tissues significantly decreased, and biotinylated iodoacetamide labeling of thiols was maintained in both the HbV and the L-NAME groups. HbV improved the recovery of cardiac function after ischemia-reperfusion in isolated rat hearts. This mechanism is dependent on functional protection against thiol oxidation.

Serum resistin levels and cardiovascular events in patients undergoing percutaneous coronary intervention.

AIM: Resistin is an adipocytokine that may link inflammation and atherosclerosis.We studied the associations of resistin levels with cardiovascular events and restenosis. METHODS: We measured pre-procedural serum resistin levels in 140 patients with coronary artery disease undergoing elective percutaneous coronary intervention (PCI), of whom 97 had a stent. Restenosis was defined as > 50% stenosis at follow-up angiography. Patients were followed for 3 years for major adverse cardiovascular events (MACE). RESULTS: At 8±6 months after PCI, reangiography was performed in 94 (67%) patients, of whom 42 had restenosis. Between 42 patients with restenosis and 52 without restenosis, resistin (4.5±2.6 vs. 4.5±2.5 ng/mL) and Creactive protein (CRP) (median 0.70 vs. 0.70 mg/L) levels did not differ. During 3-year follow-up, MACE occurred in 24 patients (1 death, 21 unstable angina, 2 stroke). Compared with 116 patients without MACE, 24 with MACE had higher resistin (5.4±2.4 vs. 4.3±2.5 ng/mL) and CRP (1.30 vs. 0.60 mg/L) levels (p< 0.05). Patients with MACE more often had resistin >4.0 ng/mL than without MACE (75% vs. 35%, p< 0.001). Resistin correlated with CRP levels (r= 0.31). To clarify the association between MACE and resistin, patients were divided into 2 groups by resistin levels. Kaplan-Meier analysis showed a lower event-free survival rate in patients with resistin > 4.0 ng/mL than without it (p< 0.001). On multivariate analysis, resistin, but not CRP, was an independent predictor of MACE. The hazard ratio for MACE was 3.6 (95%CI=1.4-9.2) for resistin > 4.0 ng/mL. CONCLUSION: Serum resistin levels were found to be associated with further cardiovascular events in patients undergoing PCI.

Severe, short-term food restriction improves cardiac function following ischemia/reperfusion in perfused rat hearts.

The purpose of this study was to clarify the characteristics of improved ischemic tolerance induced by severe, short-term food restriction in isolated, perfused rat hearts. Male Wistar (8 week-old) rats were given a food intake equivalent to a 70% reduction on the food intake of ad-libitum fed rats for 11 days (FR group and AL group, respectively). After this period, hearts were isolated and perfused in the Langendorff mode, and subjected to 20 min of global ischemia followed by 30 min of reperfusion. Although the coronary flow rate in the FR group (63.0 +/- 3.1 ml/min/g dry weight) was higher than that in the AL group (47.1 +/- 1.3 ml/min/g dry weight) during preischemic perfusion, the lactate release into the coronary effluent and absolute values of +dP/dt and -dP/dt in the FR group (2422 +/- 161 and -1282 +/- 51) were inversely lower than in the AL group (2971 +/- 156 and -1538 +/- 74, respectively). An increase in ischemic contracture was suppressed in the FR group. Following reperfusion, cardiac function, high-energy phosphate content, and intracellular pH, as measured by 31P-nuclear magnetic resonance spectroscopy, had recovered to a much greater degree in the FR group than in the AL group. The serum T3 level was significantly lower in the FR group (2.7 +/- 0.1 pg/ml) than in the AL group (3.6 +/- 0.1 pg/ml), and the levels of triglycerides, free fatty acids, insulin, and glucose were also significantly lower in the FR group than in the AL group. The protein expressions of myocyte enhancer factor 2A, Na(+), K(+)-ATPase, and phospholamban in the cardiac tissue were higher in the FR group than in the AL group. These results suggested that severe, short-term food restriction improves ischemic tolerance in rat hearts via altered expression of functional proteins induced by low serum T3 levels, decreased coronary conductance, and change in metabolic flux.

Cilostazol enhances macrophage reverse cholesterol transport in vitro and in vivo.

OBJECTIVE: Recent failure of an HDL-cholesterol raising strategy using a cholesteryl ester transfer protein inhibitor highlights the importance of the anti-atherogenic function rather than plasma concentration of HDL. Cilostazol, a selective inhibitor of phosphodiesterase 3, has been widely used in patients with atherosclerotic diseases and is known to increase HDL-cholesterol. However, it remains unclear whether cilostazol enhances anti-atherogenic properties by promoting reverse cholesterol transport (RCT), a major anti-atherogenic function of HDL. METHODS AND RESULTS: We observed that treatment of THP-1 macrophages, human monocyte-derived macrophages, and RAW264.7 cells with cilostazol increased ABCA1 and ABCG1 expression in a concentration-dependent manner, translating into enhanced apoA-I- and HDL-mediated cholesterol efflux from the macrophages. However, other cyclic AMP (cAMP)-elevating agents did not increase ABCA1 gene expression in THP-1 macrophages. Cilostazol did not change intracellular cAMP levels in THP-1 macrophages and RAW264.7 cells, and a protein kinase A (PKA) inhibitor did not affect cilostazol-induced ABCA1 and ABCG1 expression. To further investigate RCT in vivo, (3)H-cholesterol-labeled and acetyl LDL-loaded RAW264.7 cells were intraperitoneally injected into mice and the appearance of the (3)H-tracer was monitored in plasma, liver, and feces. Supporting the in vitro data, cilostazol was found to significantly increase (3)H-tracer levels in both plasma and feces. CONCLUSIONS: These findings indicate that cilostazol might provide anti-atherosclerotic effects by promoting RCT through increased ABCA1/G1 expression in macrophages.

Deficiency of interleukin-1 receptor antagonist induces aortic valve disease in BALB/c mice.

OBJECTIVE: Interleukin-1 receptor antagonist (IL-1Ra), one of the most important antiinflammatory cytokines, is crucial for homeostasis of the immune system. However, the role of IL-1Ra in aortic valve stenosis (AS) remains poorly understood [corrected]. METHODS AND RESULTS: IL-1Ra-deficient (IL-1Ra(-/-)) mice on the BALB/c background showed increased aortic valve leaflet thickness compared to wild-type mice at the age of 12 weeks (P<0.001). We used peripheral T-cell transplantation to examine the role of T cells in the development of AS. T cells from IL-1Ra(-/-) but not from wild-type mice induced increased aortic valve thickness in nu/nu mice. Moreover, IL-1Ra(-/-) T cells produced much higher levels of tumor necrosis factor (TNF)-alpha in culture supernatants after anti-CD3 antibody stimulation compared to wild-type mice (P<0.001). Finally, we studied the role of TNF-alpha in the development of AS in IL-1Ra(-/-) mice by generating double-gene-deficient (TNF-alpha(-/-)/IL-1Ra(-/-)) mice. Interestingly, TNF-alpha(-/-)/IL-1Ra(-/-) mice did not have AS. CONCLUSIONS: IL-1Ra deficiency in inflammatory cells induced aortic valve inflammation and TNF-alpha participates importantly in the development of AS in IL-1Ra(-/-) mice.

Coffee consumption enhances high-density lipoprotein-mediated cholesterol efflux in macrophages.

RATIONALE: Association of habitual coffee consumption with coronary heart disease morbidity and mortality has not been established. We hypothesized that coffee may enhance reverse cholesterol transport (RCT) as the antiatherogenic properties of high-density lipoprotein (HDL). OBJECTIVE: This study was to investigate whether the phenolic acids of coffee and coffee regulates RCT from macrophages in vitro, ex vivo and in vivo. METHODS AND RESULTS: Caffeic acid and ferulic acid, the major phenolic acids of coffee, enhanced cholesterol efflux from THP-1 macrophages mediated by HDL, but not apoA-I. Furthermore, these phenolic acids increased both the mRNA and protein levels of ATP-binding cassette transporter (ABC)G1 and scavenger receptor class B type I (SR-BI), but not ABCA1. Eight healthy volunteers were recruited for the ex vivo study, and blood samples were taken before and 30 minutes after consumption of coffee or water in a crossover study. The mRNA as well as protein levels of ABCG1, SR-BI, and cholesterol efflux by HDL were increased in the macrophages differentiated under autologous sera obtained after coffee consumption compared to baseline sera. Finally, effects of coffee and phenolic acid on in vivo RCT were assessed by intraperitoneally injecting [(3)H]cholesterol-labeled acetyl low-density lipoprotein-loaded RAW264.7 cells into mice, then monitoring appearance of (3)H tracer in plasma, liver, and feces. Supporting in vitro and ex vivo data, ferulic acid was found to significantly increase the levels of (3)H tracer in feces. CONCLUSIONS: Coffee intake might have an antiatherogenic property by increasing ABCG1 and SR-BI expression and enhancing HDL-mediated cholesterol efflux from the macrophages via its plasma phenolic acids.

Late gadolinium enhanced high resolution magnetic resonance imaging reveals pathophysiological condition of cardiac sarcoidosis.

A 40-year-old man, who had been diagnosed with stage 2 pulmonary sarcoidosis, was referred to our hospital for further evaluation of dyspnea and cardiac function. The echocardiogram displayed thinning of the basal interventricular septum (IVS) and a reduced ejection fraction of 21%. Contrast-enhanced cardiac high resolution MRI (3 tesla) showed patchy subepicardial late gadolinium enhancement in the IVS, and anterior and lateral walls. There was no abnormality in the coronary angiography and the cardiac biopsy showed several small and well-defined noncaseating epithelioid granulomas. The granulomas contained multinucleated giant cells and asteroid bodies (a typical finding of sarcoidosis). Late gadolinium enhancement in high resolution MRI provided information on the pathophysiological condition of cardiac sarcoidosis very clearly, because 3 of 5 samples of endomyocardial biopsy from the septal wall of the right ventricle where late gadolinium enhancement was detected had positive findings for cardiac sarcoidosis (very high rate). These findings indicate that high resolution late gadolinium enhanced MRI might be very useful as a guide for endomyocardial biopsy in patients with cardiac sarcoidosis.

Negative correlation of anti-U1RNP antibody titers and the amount of salivary secretion with age correction.

OBJECTIVE: The aim of this study was to reveal whether or not the presence of anti-U1RNP antibodies is associated with a low amount of salivary secretion (ASS). SUBJECTS AND METHODS: Twenty females (mean age 49+/-12 years) who had anti-U1RNP but not ACA, anti-Ro, or anti-La antibodies (anti-U1RNP-positive group), and 65 control females (mean age 50+/-12 years) were included in this study. The saxon test was performed to measure the ASS. RESULTS: After a correction for age by ANCOVA, ASS in the anti-U1RNP-positive group was significantly lower than ASS in the control group (p <0.001). In the control group, ASS was not significantly decreased with advanced age (r=-0.140, p=0.211). In the anti-U1RNP-positive group, ASS was decreased with age, without a significant difference (r=-0.379, p=0.100). In the next analysis, we introduced 'ASS with age correction', assuming that all subjects in the anti-U1RNP-positive group were 49 years of age. A negative correlation between the titers of anti-U1RNP antibodies and the ASS with the age correction in the anti-U1RNP-positive group was noted (r=-0.520, p=0.019). The log of the antinuclear antibodies titers, or titers of rheumatoid factor was significantly correlated with the titers of anti-U1RNP antibodies, respectively (r=0.466, p=0.038 and r=0.595, p=0.006; respectively). The pathological findings of minor salivary gland biopsy in 2 subjects were compatible with Sjögren's syndrome; one subject showed moderate lymphocytic infiltration. CONCLUSION: The presence of anti-U1RNP antibodies is associated with reduced ASS.

Glycated LDL increases monocyte CC chemokine receptor 2 expression and monocyte chemoattractant protein-1-mediated chemotaxis.

BACKGROUND: Previous reports have suggested that levels of advanced glycation end product-modified LDL (AGE-LDL) increase in patients with diabetes due to elevated plasma glucose. However, understanding of the mechanisms by which AGE-LDL may accelerate atherogenesis remains incomplete. METHODS AND RESULTS: Microarray and reverse transcription real-time PCR (RT-PCR) analyses revealed that AGE-LDL significantly increased levels of CC chemokine receptor 2 (CCR2) mRNA in human macrophages compared with LDL, an effect accompanied by increased levels of CCR2 protein. Flow cytometry also showed that AGE-LDL increases CCR2 expression on the cell surface following stimulation (48h) (P<0.05). This effect appeared to depend on the receptor for AGE (RAGE), since an anti-RAGE antibody significantly blocked increased CCR2 mRNA. Functional studies demonstrated that exposure of THP-1 monocytoid cells to AGE-LDL increases chemotaxis mediated by monocyte chemoattractant protein-1 (MCP-1) up to 3-fold compared to LDL treatment (P<0.05). CONCLUSIONS: These data show that AGE-LDL can increase CCR2 expression in macrophages and stimulate the chemotactic response elicited by MCP-1. This novel mechanism may contribute to accelerated atherogenesis in diabetic patients.

Phytosterols dissolved in diacylglycerol oil reinforce the cholesterol-lowering effect of low-dose pravastatin treatment.

BACKGROUND AND AIMS: Dietary therapy using phytosterols can reinforce statin treatment; however the value of a low-dose combination of those agents remains to be investigated. Plant sterols (PS), dissolved in diacylglycerol (DAG) oil, (PS/DAG) can be effective at a relatively low dose. The objective of the present study was to examine the effect of PS/DAG oil on blood cholesterol concentrations in hypercholesterolemic outpatients on low-dose pravastatin (10 mg/day). METHODS AND RESULTS: The patients (n=61) were randomly assigned to one of three groups, who consumed TAG (control), DAG or PS/DAG oil. The average intake of PS from the PS/DAG oil during the test period was significantly higher than that for TAG and DAG oils (502 vs. 49 and 38 mg/day, P<0.05). Significant cholesterol-lowering effects from the baseline were observed in the case of the PS/DAG oil treatment alone. Changes in low-density lipoprotein (LDL) cholesterol were inversely correlated with baseline serum campesterol concentrations (r=-0.560, P<0.05), but not baseline LDL cholesterol concentrations. In addition, serum apolipoprotein B concentrations were reduced to a greater extent in subjects with high versus low levels of baseline campesterol (-13.2 mg/dL vs. -3.1 mg/dL, P<0.05). Furthermore, there was a mild, but significant reduction in serum lipoprotein (a) concentration from the baseline (-5.9 mg/dL), which was correlated with the reduction in serum apolipoprotein B concentration (r=0.596, P<0.05). CONCLUSION: A low-dose combination of PS/DAG oil and pravastatin may be a useful strategy for further ameliorating blood cholesterol and lipoprotein (a) concentrations for hypercholesterolemic patients with a low response to pravastatin.

Telmisartan enhances cholesterol efflux from THP-1 macrophages by activating PPARgamma.

AIM: The ATP binding cassette transporters A1 and G1 (ABCA1/G1) and scavenger receptor class B type I (SR-BI) are key molecules in cholesterol efflux and atherogenesis. These genes are regulated by peroxisome proliferator-activated receptor gamma (PPARgamma) and liver X receptor (LXR). Telmisartan is an angiotensin type 1 receptor blocker which has been reported to act as a ligand for PPARgamma. We investigated whether PPARgamma-activating ARBs affect the expression of these genes and cholesterol efflux from macrophages. METHODS AND RESULTS: Telmisartan increased ABCA1, ABCG1 and SR-BI mRNA levels in THP-1 macrophages in a dose- and time-dependent fashion. It also increased their protein levels and enhanced apoA-I- and HDL-mediated cholesterol efflux from macrophages. The knockdown of PPARgamma by siRNA abolished the telmisartan-induced expression of these genes. The knockdown of LXRalpha resulted in the complete and partial abolishment of telmisartan-induced ABCA1 and ABCG1 expression, respectively. We also demonstrated that telmisartan-induced SR-BI expression was dependent on the PPARgamma pathway but not on the LXRalpha pathway. A luciferase assay using an ABCA1 promoter construct showed that telmisartan activated ABCA1 transcription, which was abolished if the LXR binding element was mutated, indicating that increased ABCA1 transcription by telmisartan is LXR-dependent. CONCLUSION: Our results showed that telmisartan enhanced both apoA-I- and HDL-mediated cholesterol efflux from macrophages by increasing ABCA1, ABCG1 and SR-BI expression via PPARgamma-dependent and LXR-dependent/independent pathways.

ATP-binding cassette transporter A1 gene transcription is downregulated by activator protein 2alpha. Doxazosin inhibits activator protein 2alpha and increases high-density lipoprotein biogenesis independent of alpha1-adrenoceptor blockade.

ATP-binding cassette transporter A1 (ABCA1) is a rate-limiting factor for high-density lipoprotein (HDL) biogenesis. The ABCA1 gene expression is known to be upregulated by various transcriptional factors. However, negative regulation factors would be better targets for pharmacological modulation of HDL biogenesis. Doxazosin, an alpha(1)-adrenoceptor blocker, increased ABCA1 mRNA, its protein, and apolipoprotein A-I-mediated HDL biogenesis in THP-1 macrophages and CHO-K1 cells, independent of alpha(1)-adrenoceptor blockade. Analysis of the human ABCA1 promoter indicated that the region between the positions -368 and -147 that contains an activator protein (AP)2-binding site responsible for the effects of doxazosin. Overexpression of AP2alpha inhibited ABCA1 transcription in a dose-dependent fashion. Mutation in the AP2-binding site caused increase of the basal promoter activity and cancelling both the transactivation by doxazosin and the trans-repression by AP2alpha. Doxazosin had no effect on ABCA1 mRNA level in HepG2 cells, which lack endogenous AP2alpha, and it reversed the inhibitory effect of AP2alpha expression in this type of cells. Chromatin immunoprecipitation and gel shift assays revealed that doxazosin reduced specific binding of AP2alpha to the ABCA1 promoter, as it suppressed phosphorylation of AP2alpha. Finally, doxazosin increased ABCA1 expression and plasma HDL in mice. We thus concluded that AP2alpha negatively regulates the ABCA1 gene transcription. Doxazosin inhibits AP2alpha activity independent of alpha(1)-adrenoceptor blockade and increases the ABCA1 expression and HDL biogenesis. AP2alpha is a potent pharmacological target for the increase of HDL.