Shiori Ito

Circulating microRNA-126 in patients with coronary artery disease: correlation with LDL cholesterol

BackgroundCoronary artery disease (CAD) is a major problem worldwide. Atherosclerosis and thrombosis underlying CAD involve multiple cell types. New and useful diagnostic markers are required. MicroRNAs (miRNAs) are a class of noncoding RNAs that posttranscriptionally regulate the gene expressions involved in various cellular processes. Endothelial dysfunction is implicated in early processes of athero-thrombosis. Thus, it was hypothesized that the level of vascular endothelium-enriched miRNAs would be altered in plasma samples of CAD patients.MethodsVascular endothelium-enriched miRNA (miR-126) level was analyzed in plasma from 31 patients with CAD and 36 patients without CAD (qRT-PCR analysis).ResultsMiR-126 was not significantly down-regulated or up-regulated in CAD patients. Interestingly, the level of miR-126 was significantly decreased in patients with CAD and high low-density lipoprotein (LDL) cholesterol level. In contrast, the level of miR-126 was significantly increased when LDL cholesterol was high in patients who had risk factors for CAD but did not have angiographically significant CAD.ConclusionMiR-126 was not significantly down-regulated or up-regulated in CAD patients and was not suitable for discriminating CAD patients from patients without CAD. The oppositely-directed relationship between miR-126 and LDL cholesterol in patients with or without CAD may have significant implications for identifying a potential role of miR-126 in cholesterol metabolism.

Increased plasma sphingosine-1-phosphate in obese individuals and its capacity to increase the expression of plasminogen activator inhibitor-1 in adipocytes.

ObjectivesConcentrations of plasminogen activator inhibitor-1 (PAI-1) are increased in obese individuals. One source of PAI-1 is adipocytes. Hypoxia develops within adipose tissue as it expands, presumably contributing to increased levels of sphingosine-1-phosphate (S1P). S1P is a breakdown product of sphingosine, ubiquitous in cell membranes. We have shown previously that S1P increases the expression of PAI-1 in human liver-derived cell line. In the present study, we aimed to determine whether hypoxia induces S1P in adipocytes, thereby potentially contributing to an increase in PAI-1 and hence constraints on fibrinolysis associated with obesity. Materials and methodsMouse 3T3-L1 adipocytes were exposed to CoCl2 to simulate hypoxia. Assays were performed for PAI-1 mRNA (quantitative PCR) and S1P (high-performance liquid chromatography). ResultsThe physiologic concentration of S1P increased PAI-1 mRNA expression. The S1P2 receptor antagonist attenuated the increase in PAI-1. Adipocytes expressed sphingosine kinase 1/2 (SPHK1/2) and S1P lyase, key enzymes involved in S1P production and degradation. Hypoxia increased SPHK activity and decreased S1P lyase mRNA. Hypoxia reduced cytosolic sphingosine and increased S1P release into conditioned medium. Inhibitors of ABCA1 and ABCC1 reduced the release of S1P into conditioned media. In obese patients with uncomplicated dyslipidemia and hypertension, plasma S1P was increased compared with that in nonobese and lean individuals. ConclusionHypoxia in adipose tissue of obesity can promote elaboration of S1P that binds to S1P2 receptors in an autocrine or a paracrine manner. S1P potentially contributes toward increased expression of PAI-1 and consequent constraints on fibrinolysis. S1P production and extracellular transport provide an attractive target for therapy to attenuate impaired fibrinolysis associated with obesity.

Analytical evaluation of plasma serotonin and sphingosine 1-phosphate and their clinical assessment in early atherosclerosis.

ObjectivesSerotonin stored in platelets is released into plasma on aggregation and activation in atherosclerotic diseases. Sphingosine 1-phosphate (S1P) in plasma is mainly derived from red blood cells and is responsible for the production of nitric oxide in endothelial cells and protects vasculature. The purpose of this study was to investigate the plasma levels of serotonin, S1P, and their clinical relationships with vascular endothelial function in patients with early atherosclerosis. MethodsBlood was withdrawn from patients with low-to-moderate risks of atherosclerotic diseases (n=49, 39±7 years). Platelet-poor plasma was immediately centrifuged. Serotonin levels in plasma were measured with high-performance liquid chromatography. S1P levels in plasma were measured by high-performance liquid chromatography after fluorescent derivatization with o-phthaldialdehyde. Endothelial function was assessed by endothelium-dependent flow-mediated dilation (FMD) and endothelium-independent dilation was measured by glycerol trinitrate-induced dilation using an ultrasound system. ResultsPlasma serotonin was inversely correlated with the FMD value (r=−0.287, P<0.05). Fourteen patients with dyslipidemia, who had not shown improvements after lifestyle modifications, were subsequently treated with rosuvastatin (2.5 mg/day). After 4 weeks of treatment, rosuvastatin improved lipid profiles. Rosuvastatin increased FMD, whereas glycerol trinitrate-induced dilation was unchanged. Notably, percentage decrease in plasma serotonin was inversely correlated with percentage increase in plasma S1P (r=−0.557, P<0.05). ConclusionPlasma serotonin was inversely correlated with FMD and a decrease in plasma serotonin was inversely correlated with an increase in plasma S1P after statin treatment. The results suggested that plasma levels of serotonin and S1P may be useful for the assessment of endothelial function of patients with low-to-moderate risks of atherosclerotic diseases.

TNF-α production in NKT cell hybridoma is regulated by sphingosine-1-phosphate: implications for inflammation in atherosclerosis.

ObjectivesNatural killer T (NKT) cells are unique T lymphocytes that recognize glycolipid antigen and produce various cytokines. NKT cells accelerate atherosclerosis in mice. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid and regulates T-lymphocyte trafficking. We aimed to determine the effects of S1P on the production of proinflammatory cytokine, tumor necrosis factor (TNF)-&agr;, in NKT cell hybridomas and mouse NKT cells. Materials and methodsNKT cell hybridomas and sorted mouse NKT cells were stimulated with S1P and &agr;-galactosylceramide (&agr;-GalCer), the major ligand to produce cytokines in NKT cells. TNF-&agr; mRNA expression and protein production were determined by real-time PCR and ELISA, respectively. Cell migration was assayed using chemotaxicell. Plasma S1P was measured using HPLC. ResultsHybridomas expressed S1P receptors, S1P1, S1P2, and S1P4. S1P and &agr;-GalCer increased TNF-&agr; mRNA expression and protein production. S1P enhanced TNF-&agr; induction by &agr;-GalCer. S1P receptor antagonists decreased the TNF-&agr; mRNA expression induced by S1P. FTY720, an immunosuppressive S1P receptor modulator, also decreased the TNF-&agr; mRNA expression. The migration of NKT cell hybridomas was increased by S1P. FTY720 reduced the migration induced by S1P. S1P also increased the TNF-&agr; mRNA expression in mouse NKT cells. Plasma TNF-&agr; levels in patients with high plasma S1P (≥500 nmol/l) were higher than those in patients with low S1P (<500 nmol/l). ConclusionS1P binds to S1P receptors in NKT cells and enhances TNF-&agr; production. TNF-&agr; overproduction may induce atherogenic inflammatory responses. S1P may serve as a novel therapeutic target for amelioration of vascular inflammatory diseases.

Circulating level of microRNA-126 may be a potential biomarker for recovery from smoking-related vascular damage in middle-aged habitual smokers

Background Cigarette smoking promotes vascular endothelial damage and accelerates progression of atherosclerosis. The purpose of this study was to examine whether the circulating level of vascular endothelium-enriched microRNA-126 (miR-126), which is highlighted as a regulator of gene expression, would serve as a novel biomarker for recovery from smoking-related vascular damage. Methods Middle-aged male smokers (n = 30) were enrolled and instructed to stop smoking. Their clinical profiles and laboratory findings including expression of miR-126 were investigated before and after 8 weeks of smoking cessation. Serum levels of cotinine, metabolites of nicotine, were measured to confirm smoking cessation. Endothelial function for peripheral small vessels was assessed and expressed as reactive hyperemia peripheral arterial tonometry (RH-PAT) index. The expression of miR-126 in plasma was analyzed by quantitative real-time PCR. Results At baseline, serum cotinine levels were inversely correlated with RH-PAT index (r = − 0.48, P < 0.01) and positively correlated with levels of metabolic parameters such as non-HDL cholesterol (r = 0.53, P < 0.01) and HOMA-IR (r = 0.52, P < 0.01). The RH-PAT index was not significantly changed after 8 weeks in all subjects, because only 13 subjects could attain smoking cessation. However, changes in the RH-PAT index showed a significant correlation with those in systolic blood pressure (r = − 0.54, P < 0.01). In smokers who completely attained smoking cessation (n = 13), RH-PAT index and plasma levels of miR-126 were significantly increased (P < 0.05, respectively). Conclusions Endothelial damage was improved and plasma levels of circulating miR-126 were increased after 8 weeks of smoking cessation. These findings suggested a potential use of miR-126 as a biomarker for recovery from smoking-induced vascular damage.