Establishment of the MethyLight Assay for Assessing Aging, Cigarette Smoking, and Alcohol Consumption.

The environmental factors such as aging, smoking, and alcohol consumption have been reported to influence DNA methylation (DNAm). However, the versatility of DNAm measurement by DNAm array systems is low in clinical use. Thus, we developed the MethyLight assay as a simple method to measure DNAm. In the present study, we isolated peripheral blood DNA from 33 healthy volunteers and selected cg25809905, cg02228185, and cg17861230 as aging, cg23576855 as smoking, and cg02583484 as alcohol consumption biomarkers. The predicted age by methylation rates of cg25809905 and cg17861230 significantly correlated with chronological age. In immortalized B-cells, DNAm rates of two sites showed a younger status than the chronological age of donor. On the other hand, the predicted age of the patients with myocardial infarction (MI) was not accelerated. The methylation rate of cg23576855 was able to discriminate the groups based on the smoking status. The DNAm rate of cg02583484 was reduced in subjects with habitual alcohol consumption compared to that of subjects without habitual alcohol consumption. In conclusion, our MethyLight assay system reconfirms that aging, smoking, and alcohol consumption influenced DNAm in peripheral blood in the Japanese. This MethyLight system will facilitate DNAm measurement in epidemiological and clinical studies.

A novel biomarker for acute kidney injury using TaqMan-based unmethylated DNA-specific polymerase chain reaction.

There has been increasing interest in the use of circulating DNA as biomarkers for various tissue injuries, cancers, and fetal conditions. DNA methylation is a well-characterized mechanism underlying the epigenetic regulation of gene expression, and many diagnostic tests based on DNA methylation patterns have been developed. We developed a novel TaqMan-based assay for the detection of acute kidney injury using a hypomethylated promoter region of Slc22a12, a urate transporter specifically expressed in proximal tubular cells. Bisulfite sequencing analysis confirmed that the CpG islands in the promoter region of mouse Slc22a12 were preferentially hypomethylated in the kidney cortex. TaqMan minor groove binder (MGB) probes reliably discriminated the DNA fragments corresponding to the unmethylated and methylated promoter regions of Slc22a12. Plasma levels of unmethylated DNA corresponding to the Slc22a12 promoter region were undetectable at baseline and were significantly elevated after acute kidney cortex necrosis. This study showed the usefulness of the TaqMan system in discriminating methylated and unmethylated DNA fragments, and the similar strategy can be applied for establishing biomarkers for various cellular injuries or pathological conditions.

MiR-216a and miR-216b as markers for acute phased pancreatic injury.

MicroRNAs (miRNAs) are endogenous small RNAs (length, 18-ss23 nucleotides) that regulate gene expression. Recently, plasma miRNAs have been investigated as biomarkers for various diseases. In the present study, we explored cell- or tissue-specific miRNAs and assessed the applicability of miRNA profiling for identifying biomarkers of tissue injuries. miRNA analyses in various human and rat tissues identified several candidate miRNAs with possible tissue-specific expression, some of which have already been reported. In the present study, we focused on pancreas-specific miRNAs, miR-216a and miR-216b. Laser microdissection revealed that miR-216a and 216b were predominantly expressed in acinar cells of the pancreas as compared to Langerhans' islet. Plasma concentrations of miR-216a and miR-216b considerably increased in a rat model of L-arginineinduced acute pancreatitis. The current results have confirmed that miRNA expression profiling in various cells is useful for providing biomarkers for cell- or tissue-specific injuries.

MicroRNA 210 as a biomarker for congestive heart failure.

MicroRNAs (miRNAs) are endogenous small RNAs that are 18-23 nucleotides long. Recently, plasma miRNAs were reported to be sensitive and specific biomarkers of various pathological conditions. In the present study, we focused on miR-210, which is known to be induced by hypoxia and might therefore be an excellent biomarker for congestive heart failure. Plasma miR-210 levels and expression levels in mononuclear cells and skeletal muscles were elevated in Dahl salt-sensitive rats with heart failure. We also assessed miR-210 expression in patients with heart failure. The miR-210 expression levels in the mononuclear cells of patients with NYHA III and IV heart failure according to the New York Heart Association (NYHA) functional classification system were significantly higher than those with NYHA II heart failure and controls. Although no significant correlation was observed between plasma brain natriuretic peptide (BNP) and plasma miR-210 levels in patients with NYHA II heart failure, patients with an improved BNP profile at the subsequent hospital visit were classified in a subgroup of patients with low plasma miR-210 levels. Plasma miR-210 levels may reflect a mismatch between the pump function of the heart and oxygen demand in the peripheral tissues, and be a new biomarker for chronic heart failure in addition to plasma BNP concentrations.

Pex11α deficiency impairs peroxisome elongation and division and contributes to nonalcoholic fatty liver in mice.

Hepatic triglyceride (TG) accumulation is considered to be a prerequisite for developing nonalcoholic fatty liver (NAFL). Peroxisomes have many important functions in lipid metabolism, including fatty acid ß-oxidization. However, the pathogenic link between NAFL and peroxisome biogenesis remains unclear. To examine the molecular and physiological functions of the Pex11α gene, we disrupted this gene in mice. Body weights and hepatic TG concentrations in Pex11α(-/-) mice were significantly higher than those in wild-type (WT) mice fed a normal or a high-fat diet. Hepatic TG concentrations in fasted Pex11α(-/-) mice were significantly higher than those in fasted WT mice. Plasma TG levels increased at lower rates in Pex11α(-/-) mice than in WT mice after treatment with the lipoprotein lipase inhibitor tyloxapol. The number of peroxisomes was lower in the livers of Pex11α(-/-) mice than in those of WT mice. Ultrastructural analysis showed that small and regular spherically shaped peroxisomes were more prevalent in Pex11α(-/-) mice fed normal chow supplemented without or with fenofibrate. We observed a significantly higher ratio of empty peroxisomes containing only PMP70, a peroxisome membrane protein, but not catalase, a peroxisome matrix protein, in Pex11α(-/-) mice. The mRNA expression levels of peroxisomal fatty acid oxidation-related genes (ATP-binding cassette, subfamily D, member 2, and acyl-CoA thioesterase 3) were significantly higher in WT mice than those in Pex11α(-/-) mice under fed conditions. Our results demonstrate that Pex11α deficiency impairs peroxisome elongation and abundance and peroxisomal fatty acid oxidation, which contributes to increased lipid accumulation in the liver.

Classification of various muscular tissues using miRNA profiling.

MicroRNAs (miRNAs) are endogenous small RNAs of 18-23 nucleotides that regulate gene expression. Recently, plasma miRNAs have been investigated as biomarkers for various diseases. In the present study, we explored whether miRNA expression profiling of various muscle cells may be useful for the diagnosis of various diseases involving muscle necrosis. miRNA expression profiling was assessed by miRNA array and real-time reverse-transcriptase polymerase chain reaction by using a reverse primer of a stem loop structure. Profiling of various muscle cells of mouse, including cardiac muscles, skeletal muscles, and vascular and visceral smooth muscles, indicated that profiling of miR-1, miR-133a, miR-133b, miR-145, miR-206, miR-208a, miR-208b, and miR499 were adequate to discriminate muscle cells. miR-145 was remarkably highly expressed in smooth muscles. miR-208a and miR-499 were highly expressed in cardiomyocytes. miR-133a was highly expressed in fast-twitch skeletal muscles. miR-206 and miR-208b were expressed in the slow-twitch skeletal muscles, and they can likely discriminate fast- and slow-twitch types of skeletal muscle cells. We observed that brown fat adipose cells had an miRNA expression profile very similar to those of skeletal muscle cells in the mouse. Plasma concentrations of miR-133a and miR-145 were extremely useful in diagnosing skeletal muscle necrosis in a mouse model of Duchenne muscular dystrophy and colon smooth muscle necrosis in a rat ischemic colitis model, respectively. In the present study, we investigated the miRNA expression profiles of various muscular tissues. Our results suggest that expression profiling would be useful for the diagnosis of various diseases such as muscular necrosis.

Assessment of plasma miRNAs in congestive heart failure.

BACKGROUND: MicroRNAs (miRNAs) are endogenous small RNAs that are 21-25 nucleotides in length. Recently, plasma miRNAs have been reported to be sensitive and specific biomarkers of various tissue injuries and pathological conditions. The goal of this study was to assess plasma miRNA profiles and to identify plasma miRNAs that are differentially expressed in patients with heart failure. METHODS AND RESULTS: A total of 33 patients with ischemic heart diseases and 17 asymptomatic controls were recruited. In 10 patients with heart failure, miRNAs were assessed at both NYHA IV and III. miRNA array analyses were found to be not appropriate for plasma miRNA profiling. The plasma concentrations of well-characterized miRNAs (miR-126, 122 and 499) were assessed by a real-time reverse transcription-polymerase chain reaction using an artificial small RNA as an internal standard. Plasma concentrations of miR-126 were negatively correlated with age and logBNP. In 10 patients with heart failure, plasma concentrations of miR-126 were up-regulated with improvement of the NYHA class from IV to III. CONCLUSIONS: The plasma concentration of miR-126 was negatively correlated with age and NYHA class, and could be a useful biomarker for heart failure.

Plasma miR-208 as a biomarker of myocardial injury.

BACKGROUND: MicroRNAs (miRNAs) are endogenous small RNAs of 21-25 nucleotides that can pair with sites in 3' untranslated regions in mRNAs of protein-coding genes to downregulate their expression. Recently, circulating miRNAs have been reported as promising biomarkers for various pathologic conditions. We assessed the hypothesis that miRNAs may leak into the circulating blood from injured cells and thereby serve as biomarkers for identifying the injured cell type. METHODS: We used isoproterenol-induced myocardial injury in rats as a model and miRNA array analyses to identify candidate miRNAs specifically produced in the ventricles of the heart. Individual miRNA concentrations were measured by real-time reverse-transcription PCR. Plasma cardiac troponin I (cTnI) concentrations were measured with an ELISA. RESULTS: Array analyses revealed miR-208 to be produced exclusively in the heart, and we selected this miRNA as a possible biomarker of myocardial injury. Plasma concentrations of miR-208 increased significantly (P < 0.0001) after isoproterenol-induced myocardial injury and showed a similar time course to the concentration of cTnI, a classic biomarker of myocardial injury. CONCLUSIONS: The plasma concentration of miR-208 may be a useful indicator of myocardial injury. Our results suggest that profiling of circulating miRNAs may help identify promising biomarkers of various pathologic conditions.

Effects of the Y chromosome on cardiovascular risk factors in Japanese men.

Excess cardiovascular risk in men compared with women has been suggested to be partly explained by effects of the Y chromosome. However, inconsistent results have been reported on the Y chromosome's genetic influence on blood pressure and lipid levels. The purpose of the present study was to settle the question whether genetic variants of the Y chromosome influence cardiovascular risk factors using a large epidemiological cohort, the Suita study. Possible influences of the Y chromosome polymorphisms (Y chromosome Alu insertion polymorphism [YAP], M175 and SRY+465) on cardiovascular risk factors were assessed in 974 Japanese men. The frequency of the YAP(+) allele in our study sample was 0.31. The prevalence of hypertension tended to be higher in YAP(+) than in YAP(-) men, and this tendency was found to be stronger among men aged 65 years or older. Men with the YAP(+) genotype had higher levels of high density lipoprotein (HDL) cholesterol compared with those with the YAP(-) genotype, even after adjustment for age, body mass index, and daily ethanol and cigarette consumption (57.0+/-14.6 mg/dL vs. 54.2+/-14.2 mg/dL, nominal p=0.011, adjusted p=0.0062). However, these observed nominal associations disappeared after adjusting for multiple testing (Bonferroni). No association was detected between the YAP genotype and myocardial infarction. Similarly, none of the associations with M175 and SRY+465 attained significance when multiple testing was taken into account. In conclusion, Y chromosome polymorphisms (YAP, M175 and SRY+465) do not appear to be associated with cardiovascular risk factors in Japanese men. Studies using much larger sample sizes and/or additional independent samples will be required for definitive conclusions.

Validation of the association of genetic variants on chromosome 9p21 and 1q41 with myocardial infarction in a Japanese population.

BACKGROUND: Recent large-scale genome-wide association studies have identified several loci associated with the risk of coronary artery disease (CAD). The aim of the present study was to examine whether the previously reported CAD-associated single-nucleotide polymorphisms (SNPs) confer susceptibility to myocardial infarction (MI) in a study population of 2,475 controls and 589 cases of MI. The effect of the CAD-associated SNPs on cardiovascular risk factors in the control group was also investigated. METHODS AND RESULTS: Significant associations were observed between 2 SNPs, rs1333049 on chromosome 9p21 and rs17465637 on chromosome 1q41, and MI, with odds ratios adjusted for age, sex, diabetes, hypertension and smoking habit of 1.47 (95% confidence interval (CI), 1.15-1.89; corrected p=0.006) and 1.45 (95%CI, 1.15-1.83; corrected p=0.006) for rs1333049 and rs17465637, respectively. None of the genotypes was associated with body mass index, plasma lipid profile, blood pressure, glucose, or hemoglobin A1c. The genotypes also had no effect on the marker of inflammation (C-reactive protein) or atherosclerosis (mean and maximum carotid intima-media thickness). CONCLUSIONS: Although the underlying mechanisms are not clearly understood, the previously reported association between the 2 SNPs (rs1333049 and rs17465637) and MI was reproduced in this Japanese sample.