Soichiro Iwaki

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.

Targeting ceramide synthase 6-dependent metastasis-prone phenotype in lung cancer cells.

Sphingolipids make up a family of molecules associated with an array of biological functions, including cell death and migration. Sphingolipids are often altered in cancer, though how these alterations lead to tumor formation and progression is largely unknown. Here, we analyzed non-small-cell lung cancer (NSCLC) specimens and cell lines and determined that ceramide synthase 6 (CERS6) is markedly overexpressed compared with controls. Elevated CERS6 expression was due in part to reduction of microRNA-101 (miR-101) and was associated with increased invasion and poor prognosis. CERS6 knockdown in NSCLC cells altered the ceramide profile, resulting in decreased cell migration and invasion in vitro, and decreased the frequency of RAC1-positive lamellipodia formation while CERS6 overexpression promoted it. In murine models, CERS6 knockdown in transplanted NSCLC cells attenuated lung metastasis. Furthermore, combined treatment with l-α-dimyristoylphosphatidylcholine liposome and the glucosylceramide synthase inhibitor D-PDMP induced cell death in association with ceramide accumulation and promoted cancer cell apoptosis and tumor regression in murine models. Together, these results indicate that CERS6-dependent ceramide synthesis and maintenance of ceramide in the cellular membrane are essential for lamellipodia formation and metastasis. Moreover, these results suggest that targeting this homeostasis has potential as a therapeutic strategy for CERS6-overexpressing NSCLC.

Modulators of induction of plasminogen activator inhibitor type-1 in HepG2 cells by transforming growth factor-β.

ObjectiveAn increased expression of plasminogen activator inhibitor type-1 (PAI-1) has been implicated in accelerating atherogenesis and coronary artery disease in patients with type 2 diabetes. Transforming growth factor (TGF)-&bgr; increases its expression. An increased PAI-1 appears to predispose also to augmented fibrosis potentially contributing to negative left ventricular remodeling and heart failure after myocardial infarction. Diabetes is well known to induce oxidative stress. To elucidate molecular mechanisms underlying an increased PAI-1 production, the effects of TGF-&bgr; and oxidative stress implicated as agonists of PAI-1 synthesis were characterized with the use of human liver-derived HepG2 cells. MethodsPAI-1 mRNA was assayed by real-time PCR, and PAI-1 protein was assayed by western blotting. PAI-1 promoter (−825 –+42 bp) activity was assessed with the luciferase assay. The role of the 3′-untranslated region was delineated with the use of luciferase constructs containing the 3′-untranslated region. Oxidative stress was measured after loading carboxy-2,7-dichlorodihydrofluorescein into cells. ResultsTGF-&bgr; increased oxidative stress, which was accompanied by increases in NADPH oxidase 3 mRNA and membrane translocation of Rac proteins. TGF-&bgr;-inducible increases in the PAI-1 promoter activity involved Smad-binding elements and a nuclear factor-&kgr;B-binding site. TGF-&bgr; did not increase the activity of the PAI-1 mRNA 3′-untranslated region. TGF-&bgr;-inducible PAI-1 expression was attenuated by simvastatin and curcumin, a natural polyphenol. ConclusionTGF-&bgr; can increase the expression of PAI-1 through multiple mechanisms involving Smad and nuclear factor-&kgr;B pathways and oxidative stress. As both oxidative stress and PAI-1 production were reduced by simvastatin and curcumin, modulation of oxidative stress and PAI-1 production are attractive targets for pharmacotherapy of cardiovascular disorders associated with an increased PAI-1 including type 2 diabetes and its associated consequences including accelerated coronary artery disease and an increased fibrosis that may exacerbate adverse left ventricular remodeling after myocardial infarction.

Increased expression of plasminogen activator inhibitor type-1 (PAI-1) in HEPG2 cells induced by insulin mediated by the 3'-untranslated region of the PAI-1 gene and its pharmacologic implications.

ObjectiveInsulin increases, through several molecular mechanisms, expression of plasminogen activator inhibitor-1 (PAI-1), the major physiologic inhibitor of fibrinolysis. This phenomenon has been implicated as a cause of accelerated coronary artery disease and the increased incidence of acute coronary syndromes associated with type 2 diabetes. We have previously reported that physiologic and pharmacologic concentrations of insulin induce PAI-1 synthesis in human HepG2 cells and that simvastatin can attenuate its effects. This study was performed to further elucidate mechanisms responsible for the insulin-induced PAI-1 production. MethodsConcentrations of PAI-1 mRNA were determined by real-time PCR, and PAI-1 protein was assayed by western blotting. PAI-1 promoter (−829 to +36 bp) activity was assayed with the use of luciferase reporter assays. The potential role of the 3′-untranslated region (UTR) in the PAI-1 gene was assayed with the use of luciferase constructs containing the 3′-UTR. Oxidative stress was measured by loading cells with carboxy-2,7 dichlorodihydrofluorescein. ResultsInsulin increased PAI-1 promoter activity, PAI-1 mRNA, and accumulation of PAI-1 protein in the conditioned media. Insulin-inducible PAI-1 promoter activity was attenuated by simvastatin. Experiments performed with luciferase reporters containing the 3′-UTR showed that insulin increased luciferase activity through this region. Insulin also increased oxidative stress. Both insulin-inducible luciferase activity through the 3′-UTR and oxidative stress were attenuated by simvastatin. ConclusionInsulin can increase PAI-1 expression through multiple mechanisms including induction mediated by the 3′-UTR of the PAI-1 gene. Accordingly, beneficial pleiotropic effects of statins on coronary artery disease may be attributable, in part, to attenuation of overexpression of PAI-1 mediated by the 3′-UTR in syndromes of insulin resistance (such as the metabolic syndrome) and type 2 diabetes.

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.

Sphingosine kinase 1 expression is downregulated during differentiation of Friend cells due to decreased c-MYB

Sphingosine kinase 1 (SPHK1) overexpression in malignant cells has been reported. Mouse Friend cells showed higher SPHK1 but not SPHK2 expression compared with other mouse cell lines. A Sphk1 promoter analysis demonstrated the region between -53bp and the first exon as the minimal promoter. Further promoter truncation revealed the importance of a MYB-binding site. EMSA using this region as the probe demonstrated one band containing c-MYB protein, and its intensity decreased during erythroid differentiation with hexamethylane bisacetamide (HMBA), a potent inducer of erythroid differentiation of Friend cells. ChIP assay also revealed in vivo binding of c-MYB. c-MYB overexpression and siRNA for c-Myb affected SPHK1 expression, confirming the important regulatory role of c-MYB in SPHK1 expression. HMBA reduced c-MYB expression rapidly. Induced differentiation by HMBA caused a marked and rapid reduction of SPHK1 mRNA, protein and enzyme activity leading to the rapid decrease of cellular sphingosine 1-phosphate level. Moreover, terminally differentiated cells did not resume SPHK1 expression. Compared with original Friend cells, stable overexpression of wild-type SPHK1 showed higher cell proliferation, resistance to cell death by serum depletion. Interestingly, HMBA-induced differentiation of these cells was delayed but not completely suppressed. In contrast, SPHK inhibitor and its siRNA inhibited cell growth and enhanced HMBA-induced differentiation significantly, suggesting that SPHK1 delayed HMBA-induced differentiation by its cell proliferation-promoting activity. Effects of pertussis toxin, a G-protein-coupled receptor inhibitor, and S1P receptor antagonist on Friend cell growth and differentiation were negligible, suggesting the importance of the intracellular SPHK1/S1P signaling in Friend cells.

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.

Necrostatin-7 suppresses RANK-NFATc1 signaling and attenuates macrophage to osteoclast differentiation

Osteoclasts play a crucial role in osteolytic bone diseases, such as osteoporosis, rheumatoid arthritis, periodontitis, Pagets disease of bone and bone metastatic tumors. Therefore, controlling osteoclast differentiation and function has been considered a promising therapeutic strategy. Here, we show that necrostatin (Nec)-7, an inhibitor of programmed necrosis, strongly suppressed receptor activator of nuclear factor (NF)-κB ligand (RANKL)-induced osteoclastogenesis and bone resorption, without compromising macrophage colony-stimulating factor (M-CSF)-supported survival and growth of osteoclast precursor cells. Accordingly, Nec-7 significantly decreased the levels of RANKL-induced osteoclastogenic marker genes, such as cathepsin K. Mechanistically, Nec-7 neither affected MAPK nor NF-κB activation; however, it strongly inhibited the RANKL receptor (RANK) to nuclear factor of activated T cells c1 (NFATc1) signaling. Lentiviral expression of RANK in bone marrow-derived macrophages significantly restored osteoclastogenesis and NFATc1 amplification in Nec-7-treated cells. In this study, we revealed that Nec-7-sensitive pathways are crucially involved in osteoclast formation and function. Investigation of the molecular mechanism(s) through which Nec-7 inhibits RANK-NFATc1 signaling axis may lead to the development of new therapeutic strategies for bone disease.