Hideki Ozasa

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.

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.

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.

Citrulline increases cholesterol efflux from macrophages in vitro and ex vivo via ATP-binding cassette transporters.

Reverse cholesterol transport (RCT) is a mechanism critical to the anti-atherogenic property of HDL. Although citrulline contributes to the amelioration of atherosclerosis via endothelial nitric oxide production, it remains unclear whether it affects RCT. This study was undertaken to clarify the effects of citrulline on expressions of specific transporters such as ATP binding cassette transporters (ABC)A1 and ABCG1, and the cholesterol efflux from macrophages to apolipoprotein (apo) A-I or HDL in vitro and ex vivo. Citrulline increased ABCA1 and ABCG1 mRNA and protein levels in THP-1 macrophages, translating into enhanced apoA-I- and HDL-mediated cholesterol efflux. In the human crossover study, 8 healthy male volunteers (age 30–49 years) consumed either 3.2 g/day citrulline or placebo for 1 week. Citrulline consumption brought about significant increases in plasma levels of citrulline and arginine. Supporting the in vitro data, monocyte-derived macrophages (MDM) differentiated under autologous post-citrulline sera demonstrated enhancement of both apoA-I- and HDL-mediated cholesterol efflux through increased ABCA1 and ABCG1 expressions, compared to MDM differentiated under pre-citrulline sera. However, the placebo did not modulate these parameters. Therefore, in addition to improving endothelium function, citrulline might have an anti-atherogenic property by increasing RCT of HDL.