Takeshige Mori

Trans-fatty acid promotes thrombus formation in mice by aggravating antithrombogenic endothelial functions via Toll-like receptors

SCOPE Since excessive intake of trans-fatty acid (TFA) increases the risk of myocardial infarction, we investigated the effects of TFA on thrombus formation using animal and cell culture experiments. METHODS AND RESULTS C57BL/6 mice were fed a diet containing TFA or cis-fatty acid (5% each of total calories) or a chow diet for 4 weeks, and thrombus formation was induced in the carotid artery by He-Ne laser irradiation. The high-TFA diet significantly promoted thrombus formation in the carotid artery compared to the chow or cis-fatty acid diet. TFA activated the inflammatory signaling pathway in cultured endothelial cells and in mice; aortic gene expression levels of antithrombogenic molecules, including thrombomodulin and tissue factor pathway inhibitor, were decreased, and the expression levels of prothrombogenic molecules were increased in TFA-treated mice. TFA markedly upregulated the prothrombogenic molecules and downregulated the antithrombogenic molecules in endothelial cells. In addition, TFA induced phosphorylation of c-Jun N-terminal kinase, extracellular signal-regulated kinase, and nuclear factor-κB. The TFA-activated signal pathways and prothrombogenic phenotypic changes of endothelial cells were inhibited by genetic or pharmacological inactivation of Toll-like receptors 2 and 4. CONCLUSION TFA aggravates the antithrombogenic phenotypes of vascular endothelial cells via Toll-like receptors and promotes thrombus formation in mice.

β-Hydroxybutyrate elevation as a compensatory response against oxidative stress in cardiomyocytes

Recent studies have shown that the ketone body β-hydroxybutyrate (βOHB) acts not only as a carrier of energy but also as a signaling molecule that has a role in diverse cellular functions. Circulating levels of ketone bodies have been previously reported to be increased in patients with congestive heart failure (HF). In this study, we investigated regulatory mechanism and pathophysiological role of βOHB in HF. First, we revealed that βOHB level was elevated in failing hearts, but not in blood, using pressure-overloaded mice. We also measured cellular βOHB levels in both cardiomyocytes and non-cardiomyocytes stimulated with or without H2O2 and revealed that increased myocardial βOHB was derived from cardiomyocytes but not non-cardiomyocytes under pathological states. Next, we sought to elucidate the mechanisms of myocardial βOHB elevation and its implication under pathological states. The gene and protein expression levels of CoA transferase (SCOT), a key enzyme involved in ketone body oxidation, was decreased in failing hearts. In cardiomyocytes, H2O2 stimulation caused βOHB accumulation concomitantly with SCOT downregulation, implying that the accumulation of myocardial βOHB occurs because of the decline in its utilization. Finally, we checked the effects of βOHB on cardiomyocytes under oxidative stress. We found that βOHB induced FOXO3a, an oxidative stress resistance gene, and its target enzyme, SOD2 and catalase. Consequently, βOHB attenuated reactive oxygen species production and alleviated apoptosis induced by oxidative stress. It has been reported that hyperadrenergic state in HF boost lipolysis and result in elevation of circulating free fatty acids, which can lead hepatic ketogenesis for energy metabolism alteration. The present findings suggest that the accumulation of βOHB also occurs as a compensatory response against oxidative stress in failing hearts.

2-Aminobutyric acid modulates glutathione homeostasis in the myocardium

A previous report showed that the consumption of glutathione through oxidative stress activates the glutathione synthetic pathway, which is accompanied by production of ophthalmic acid from 2-aminobutyric acid (2-AB). We conducted a comprehensive quantification of serum metabolites using gas chromatography-mass spectrometry in patients with atrial septal defect to find clues for understanding myocardial metabolic regulation, and demonstrated that circulating 2-AB levels reflect hemodynamic changes. However, the metabolism and pathophysiological role of 2-AB remains unclear. We revealed that 2-AB is generated by an amino group transfer reaction to 2-oxobutyric acid, a byproduct of cysteine biosynthesis from cystathionine. Because cysteine is a rate-limiting substrate for glutathione synthesis, we hypothesized that 2-AB reflects glutathione compensation against oxidative stress. A murine cardiomyopathy model induced by doxorubicin supported our hypothesis, i.e., increased reactive oxygen species are accompanied by 2-AB accumulation and compensatory maintenance of myocardial glutathione levels. Intriguingly, we also found that 2-AB increases intracellular glutathione levels by activating AMPK and exerts protective effects against oxidative stress. Finally, we demonstrated that oral administration of 2-AB efficiently raises both circulating and myocardial glutathione levels and protects against doxorubicin-induced cardiomyopathy in mice. This is the first study to demonstrate that 2-AB modulates glutathione homeostasis in the myocardium.

Eicosapentaenoic Acid-Enriched High-Density Lipoproteins Exhibit Anti-Atherogenic Properties

BACKGROUND It has previously been reported that oral administration of purified eicosapentaenoic acid (EPA) generates EPA-rich high-density lipoprotein (HDL) particles with a variety of anti-inflammatory properties. In this study, the mechanism underlying the anti-atherogenic effects of EPA-rich HDL using reconstituted HDL (rHDL) was investigated.Methods and Results:rHDL was generated by the sodium cholate dialysis method, using apolipoprotein A-1 protein, cholesterol, and various concentrations of EPA-phosphatidylcholine (PC) or egg-PC. Increased EPA-PC contents in rHDL resulted in decreased particle size. Next, the effects of rHDL containing various amounts (0-100% of total PC) of EPA-PC on vascular cell adhesion molecule-1 (VCAM-1) expression in human umbilical vein endothelial cells (HUVECs) was examined. Cytokine-stimulated VCAM-1 expression was inhibited in a dose-dependent manner based on the amount of EPA-PC in rHDL. Surprisingly, the incubation of HUVECs with EPA-rich rHDL resulted in the production of resolvin E3 (RvE3), an anti-inflammatory metabolite derived from EPA. Incubation with EPA-PC alone did not adequately induce RvE3 production, suggesting that RvE3 production requires an endothelial cell-HDL interaction. The increased anti-inflammatory effects of EPA-rich HDL may be explained by EPA itself and RvE3 production. Furthermore, the increase in EPA-PC content enhanced cholesterol efflux. CONCLUSIONS The EPA-enriched HDL particles exhibit cardioprotective properties via the production of anti-inflammatory lipid metabolites and the increase in cholesterol efflux.

Novel mechanism of regulation of the 5-lipoxygenase/leukotriene B 4 pathway by high-density lipoprotein in macrophages

High-density lipoprotein (HDL) interacts with various cells, particularly macrophages, in functional cell-HDL interactions. Here, we found that HDL protein quality and lipid quality play critical roles in HDL functions. HDL fractions from healthy volunteers (HDLHealthy) and patients with recurrent coronary atherosclerotic disease (HDLCAD) were prepared. To analyse functional HDL-macrophage interactions, macrophages were co-incubated with each HDL, and lipid mediator production was assessed by liquid chromatography/mass spectrometry-based metabololipidomics. HDLHealthy treatment attenuated the pro-inflammatory lipid mediator production, particularly that of leukotriene (LT) B4, and this treatment enhanced lipoxin (LX) B4 and resolvin (Rv) E2 production. HDLHealthy treatment enhanced the proteasome-mediated degradation of the LTB4-producing enzyme 5-lipoxygenase (LO) in activated macrophages; however, HDLCAD did not show these anti-inflammatory effects. HDLHealthy was engulfed by macrophages via clathrin-mediated endocytosis, which was a critical step in 5-LO/LTB4 regulation. We also found that HDLCAD showed higher levels of the LTB4-producing enzymes and thus promoted LTB4 production from HDLCAD. In addition, LTB4 attenuated HDL endocytosis, HDL-mediated 5-LO degradation in macrophages, and HDL-derived augmentation of macrophage phagocytosis. These results indicated that local LTB4 produced de novo from HDLCAD regulates HDL-macrophage functional interactions and plays critical roles in dysfunctional, inflammatory HDL characteristics.