Makoto Ayaori

Inhibitory effect of tea flavonoids on the ability of cells to oxidize low density lipoprotein

Dietary flavonoid intake has been reported to be inversely related to mortality from coronary heart disease, and the anti-atherosclerotic effect of flavonoids is considered to be due probably to their antioxidant properties. Oxidation of low density lipoprotein (LDL) has been reported to be induced by the constituent cells of the arterial wall. Accordingly, we examined the effect of pretreatment with tea flavonoids, such as theaflavin digallate, on the ability of cells to oxidize LDL. Theaflavin digallate pretreatment of macrophages or endothelial cells reduced cell-mediated LDL oxidation in a concentration- (0-400 microM) and time- (0-4 hr) dependent manner. This inhibitory effect of flavonoids on cell-mediated LDL oxidation was in the order of theaflavin digallate > theaflavin > or = epigallocatechin gallate > epigallocatechin > gallic acid. Further, we investigated the mechanisms by which flavonoids inhibited cell-mediated LDL oxidation using macrophages and theaflavin digallate. Theaflavin digallate pretreatment decreased superoxide production of macrophages and chelated iron ions significantly. These results suggest that tea flavonoids attenuate the ability of the cell to oxidize LDL, probably by reducing superoxide production in cells and chelating iron ions.

Dipeptidyl peptidase-4 inhibitors attenuate endothelial function as evaluated by flow-mediated vasodilatation in type 2 diabetic patients.

Background Endothelial dysfunction is an independent predictor for cardiovascular events in patients with type 2 diabetes (T2DM). Glucagon like peptide‐1 (GLP‐1) reportedly exerts vasodilatory actions, and inhibitors of dipeptidyl peptidase‐4 (DPP‐4), an enzyme‐degrading GLP‐1, are widely used to treat T2DM. We therefore hypothesized that DPP‐4 inhibitors (DPP‐4Is) improve endothelial function in T2DM patients and performed 2 prospective, randomized crossover trials to compare the DPP‐4I sitagliptin and an α‐glucosidase inhibitor, voglibose (in study 1) and the DPP‐4Is sitagliptin and alogliptin (in study 2). Methods and Results In study 1, 24 men with T2DM (46±5 years) were randomized to sitagliptin or voglibose for 6 weeks without washout periods. Surprisingly, sitagliptin significantly reduced flow‐mediated vasodilatation (FMD; −51% compared with baseline, P<0.05) of the brachial artery despite improved diabetic status. In contrast, voglibose did not affect FMD. To confirm this result and determine whether it is a class effect, we conducted another trial (study 2) to compare sitagliptin and alogliptin in 42 T2DM patients (66±8 years) for 6 weeks with 4‐week washout periods. Both DPP‐4Is improved glycemic control but significantly attenuated FMD (7.2/4.3%, P<0.001, before/after sitagliptin; 7.0/4.8%, P<0.001, before/after alogliptin, respectively). Interestingly, FMD reduction was less evident in subjects who were on statins or whose LDL cholesterol levels were reduced by them, but this was not correlated with parameters including DPP‐4 activity and GLP‐1 levels or diabetic parameters. Conclusions Our 2 independent trials demonstrated that DPP‐4 inhibition attenuated endothelial function as evaluated by FMD in T2DM patients. This unexpected unfavorable effect may be a class effect of DPP‐4Is. Clinical Trial Registration URL: http://center.umin.ac.jp, Unique Identifiers: UMIN000005682 (sitagliptin versus voglibose) and UMIN000005681 (sitagliptin versus alogliptin).

Osteopontin Transgenic Mice Fed a High-Cholesterol Diet Develop Early Fatty-Streak Lesions

Background—Osteopontin (OPN) is a noncollagenous adhesion protein found at the site of atherosclerotic lesions. However, it has not yet been clarified whether or not OPN can promote atherosclerotic lesions. Methods and Results—We investigated the contribution of OPN to atherosclerosis by evaluating aortic sinus lesions of both OPN transgenic (Tg) and non-Tg mice fed an atherogenic diet (1.25% cholesterol) for 16 weeks. The atherosclerotic lesions were found to be significantly larger in OPN-Tg compared with those in non-Tg (17 859±2010 versus 6469±485 &mgr;m2, P <0.01). The lesions in both mice were fatty-streak lesions with an accumulation of mononuclear cells and lipids. We next investigated the production of interleukin (IL)-10 by macrophages from both mice. Compared with the non-Tg mice, a 42% (P <0.01) and 73% (P <0.001) decrease in the IL-10 production was identified in the OPN-Tg mice either without or with lipopolysaccharide. Conclusions—The expression of OPN induces fatty-streak lesion formation in mice fed an atherogenic diet and inhibits IL-10 production by macrophages, thus suggesting that OPN plays an important role in the development of fatty-streak lesions in vivo.

Endothelium-dependent flow-mediated vasodilation in the postprandial state in type 2 diabetes mellitus

This study examined the effects of fat- plus sucrose-rich meals on endothelium-dependent flow-mediated vasodilation in diabetic patients. Flow-mediated vasodilation in the postprandial state decreased significantly, and the decrease correlated inversely with the magnitude of postprandial hyperglycemia, suggesting that endothelial function in diabetic patients becomes impaired postprandially.

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 [3H]cholesterol-labeled acetyl low-density lipoprotein–loaded RAW264.7 cells into mice, then monitoring appearance of 3H tracer in plasma, liver, and feces. Supporting in vitro and ex vivo data, ferulic acid was found to significantly increase the levels of 3H 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.

Pioglitazone enhances cholesterol efflux from macrophages by increasing ABCA1/ABCG1 expressions via PPARγ/LXRα pathway: Findings from in vitro and ex vivo studies

OBJECTIVE Pioglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist, reportedly reduces cardiovascular events in diabetic patients. ATP cassette binding transporters (ABC) A1 and G1 are pivotal molecules for cholesterol efflux (ChE) from macrophages and high density-lipoprotein biogenesis, and the A1 transporter is regulated by a PPARγ-liver receptor X (LXR) pathway. Also, pioglitazone induces ABCG1 expression, though the exact mechanism remains unclear. We therefore investigated the effects of pioglitazone on ABCA1/G1 expression in vitro and ex vivo. METHODS The effects of pioglitazone on ChE and ABCA1/G1 expressions in macrophages were assessed. Then, mRNA was quantified in macrophages when PPARγ/LXR inhibition by siRNA or overexpression of oxysterol sulfotransferase was performed. ABCA1/G1 promoter activity with mutated LXR-responsive elements was also measured. As an ex vivo study, 15 type 2 diabetic patients were administered pioglitazone or placebo, and ChE assays and protein expressions were determined using macrophages cultured with the corresponding sera. RESULTS Pioglitazone increased LXRα/ABCA1/G1 expressions, which enhanced ChE from macrophages. Inhibition of PPARγ/LXR pathways revealed that LXR was primarily involved in pioglitazones transactivation of ABCA1 but only partially involved for ABCG1. Promoter assays showed that ABCG1 was regulated more by the promoter in intron 4 than that upstream of exon 1 but both promoters were responsive to LXR activation. Sera obtained after pioglitazone treatment promoted ChE and ABCA1/G1 expressions in macrophages. CONCLUSION Pioglitazone enhanced ChE from macrophages by increasing ABCA1/G1 in LXR-dependent and -independent manners. Our comparable in vitro and ex vivo results shed new light on pioglitazones novel anti-atherogenic property.

Glucocorticoid Receptor Regulates ATP-Binding Cassette Transporter-A1 Expression and Apolipoprotein-Mediated Cholesterol Efflux from Macrophages

Objective—The ATP-binding cassette transporter-A1 (ABCA1) regulates cholesterol efflux from cells and is involved in high-density lipoprotein metabolism and atherogenesis. The objective of this study was to investigate the effect of dexamethasone (Dex) and other glucocorticoid receptor (GR) ligands on apolipoprotein AI–mediated cholesterol efflux from macrophages and ABCA1 expression in them. Methods and Results—Dex, a GR agonist, decreased ABCA1 mRNA levels in a dose- and time-dependent fashion, and RU486, a GR antagonist, reversed the inhibitory effect of Dex. The effects of Dex and RU486 on ABCA1 protein levels and apolipoprotein AI–mediated cholesterol efflux from the macrophages were consistent with these changes in mRNA levels. Transfected RAW264.7, together with a human ABCA1 promoter–luciferase construct, inhibited transcriptional activity by Dex and overexpression of human GR. Transrepression by GR was not mediated by liver X receptor (LXR), because there were no differences in the effects of the GR ligands on promoter activity between a reporter construct with mutations at the LXR binding site and one without the mutations, and no changes were brought about in ABCG1 and ABCG4 expression by GR ligands. Conclusions—Our results showed that GR ligands affected ABCA1 expression and cholesterol efflux from macrophages, which are regulated by GR through a LXR-independent mechanism.

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 3H-cholesterol-labeled macrophages. Conclusion— 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.

ATP-Binding Cassette Transporter A1 Gene Transcription Is Downregulated by Activator Protein 2α Doxazosin Inhibits Activator Protein 2α and Increases High-Density Lipoprotein Biogenesis Independent of α1-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 &agr;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 &agr;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 AP2&agr; inhibited ABCA1 transcription in a dose-dependent fashion. Mutation in the AP2-binding site caused increase of the basal promoter activity and canceling both the transactivation by doxazosin and the trans-repression by AP2&agr;. Doxazosin had no effect on ABCA1 mRNA level in HepG2 cells, which lack endogenous AP2&agr;, and it reversed the inhibitory effect of AP2&agr; expression in this type of cells. Chromatin immunoprecipitation and gel shift assays revealed that doxazosin reduced specific binding of AP2&agr; to the ABCA1 promoter, as it suppressed phosphorylation of AP2&agr;. Finally, doxazosin increased ABCA1 expression and plasma HDL in mice. We thus concluded that AP2&agr; negatively regulates the ABCA1 gene transcription. Doxazosin inhibits AP2&agr; activity independent of &agr;1-adrenoceptor blockade and increases the ABCA1 expression and HDL biogenesis. AP2&agr; is a potent pharmacological target for the increase of HDL.

Beneficial effect of gemfibrozil on the chemical composition and oxidative susceptibility of low density lipoprotein: a randomized, double-blind, placebo-controlled study

Previous reports have shown that administration of fibrates can reduce coronary events and also improve plasma lipid levels. Oxidative modification of low density lipoprotein has been implicated in the pathogenesis of atherosclerosis, and the resistance of low density lipoprotein (LDL) to in vitro oxidation has been found to be correlated with the extent of atherosclerosis. We performed a double-blind, placebo-controlled intervention trial to establish whether gemfibrozil could improve resistance of LDL to oxidation in patients with hyperlipidemia. Patients were randomly assigned to treatment with gemfibrozil (450 mg, twice a day, n = 10) or placebo (n = 9) for 8 weeks. Blood samples were obtained after an overnight (12 h) fast. Gemfibrozil administration significantly reduced total plasma cholesterol and triglyceride levels and changed the LDL from small, dense particles (pattern B, < or = 25.5 nm) to larger, more buoyant particles (pattern A, > 25.5 nm). Gemfibrozil significantly increased the lag time of LDL oxidation in vitro by 18.2% from 45.5 +/- 8.0 min at week 0 to 53.4 +/- 11.4 min at week 8, but did not change LDL vitamin E and beta-carotene concentrations. Surprisingly, gemfibrozil significantly decreased LDL lipid peroxides by -33.1% and increased the LDL vitamin E/lipid peroxide ratio by 67.6% from 1.3 +/- 0.5 at week 0 to 2.1 +/- 0.9 at week 8. These results demonstrate that gemfibrozil treatment can render LDL less susceptible to oxidative modification while reducing plasma cholesterol and triglyceride and improving LDL subclass pattern. This antioxidative effect of gemfibrozil on LDL may be one of the factors which could delay the progression of atherosclerosis.