MicroRNA-20a inhibits stress-induced cardiomyocyte apoptosis involving its novel target Egln3/PHD3.

Excessive stress, e.g. due to biomechanical overload or ischemia/reperfusion is a potent inductor of cardiomyocyte apoptosis, which contributes to maladaptive remodeling. Despite substantial progress in the understanding of the molecular pathophysiology, many components of the signaling pathways underlying remodeling in general and apoptosis in particular still remain unknown. Recent evidence suggests that microRNAs (miRs) play an important role in the heart's response to increased cardiac stress. To identify novel modulators of stress-dependent remodeling, we conducted a genome-wide miR-screen of mechanically stretched neonatal rat cardiomyocytes (NRCM). Out of 351 miRs, eight were significantly regulated by biomechanical stress, including microRNA-20a, which is part of the miR17-92 cluster. Interestingly, further expression analyses also revealed upregulation of microRNA-20a in an in vitro hypoxia/"reperfusion" model. Given the potential apoptosis-modulating properties of the miR17-92 cluster, we subjected NRCM to hypoxia and subsequent reoxygenation. AdmiR-20a significantly inhibited hypoxia-mediated apoptosis in a dose-dependent fashion, while targeted knockdown of miR-20a in NRCM induced cardiomyocyte apoptosis. Mechanistically, the antiapoptotic effect of miR-20a appears to be mediated through direct targeting and subsequent downregulation of the proapoptotic factor Egln3. Thus, miR-20a is upregulated in acute biomechanical stress as well as hypoxia and inhibits apoptosis in cardiomyocytes. These properties reveal miR-20a as a cardioprotective micro-RNA and a potential target for novel therapeutic strategies to prevent cardiac remodeling.

ACBP knockdown leads to down-regulation of genes encoding rate-limiting enzymes in cholesterol and fatty acid metabolism.

The human Acyl-CoA binding protein (ACBP) is a structural and functional highly conserved protein. As an intracellular pool former and carrier of acyl-CoAs, ACBP influences overall lipid metabolism. Its nuclear abundance and physical interaction with hepatocyte nuclear factor 4alpha suggested a gene regulatory function of ACBP. To identify ACBP target genes we performed genome-wide transcript profiling under siRNA-mediated ACBP knockdown in human liver HepG2 cells. Based on a single sided permutation T-test (p<0.05) we identified 256 down-regulated and 198 up-regulated transcripts with a minimal fold change of 1.32 (log 0.5). Gene annotation enrichment analysis revealed ACBP-mediated down-regulation of 18 genes encoding key enzymes in glycerolipid (i.e. mitochondrial glycerol-3-phosphate acyltransferase), cholesterol (i.e. HMG-CoA synthase and HMG-CoA reductase) and fatty acid (i.e. fatty acid synthase) metabolism. Integration of these genes in common pathways suggested decreased lipid biosynthesis. Accordingly, saturated (16:0) and monosaturated (16:1, 18:1) fatty acids were significantly reduced to 75% in ACBP-depleted cells. Taken together, we obtained evidence that ACBP functions in lipid metabolism at the level of gene expression. This effect seems to be translated into certain metabolites. The identified 454 ACBP regulated genes present a first reference for further studies to define the ACBP regulon in mammalian cells.

Quercetin supplementation and its effect on human monocyte gene expression profiles in vivo.

Quercetin has been described as having a wide range of beneficial effects in humans, ranging from anti-carcinogenic properties to reducing the risk of CVD. Nevertheless, underlying molecular mechanisms have been mostly investigated in vitro. Here, we tested whether a daily supplementation of quercetin leads to reproducible changes in human monocyte gene expression profiles. In study I, quercetin in varying dosages was given to healthy subjects for 2 weeks. RNA from monocytes isolated at the beginning and end of the study from subjects receiving 150 mg quercetin per d was subjected to transcriptome-wide microarray analysis. In study II, a double-blind cross-over study, twenty subjects exhibiting a 'cardiovascular risk phenotype' received 150 mg quercetin or placebo daily for 6 weeks each and served as the verification group. Microarray analysis revealed a number of differentially expressed genes. The most significantly represented functional groups were those of the immune system, nucleic acid metabolism, apoptosis and O-glycan biosynthesis. Twenty-four genes were chosen for technical replication and independent verification by quantitative real-time PCR. When comparing placebo and quercetin treatment, four genes showed significantly different expression changes (C1GALT1, O-glycan biosynthesis; GM2A, glycolipid catabolism; HDGF, cell proliferation; SERPINB9, apoptosis). However, these were minimal in respect to magnitude of fold change. In conclusion, although microarray analysis revealed extensive effects of quercetin on gene expression, the employment of a placebo-controlled study design showed no comparable results for twenty-four verification targets. This emphasises the need for stringent designs in nutritional intervention studies with the aim to identify relevant changes in gene expression.

Comparative analyses of disease risk genes belonging to the acyl-CoA synthetase medium-chain (ACSM) family in human liver and cell lines.

The human ACSM1, 2A and B, 3, and 5 genes, located on chromosome 16p12-13, encode for enzymes catalyzing the activation of medium-chain length fatty acids. Association studies have linked several polymorphisms of these genes to traits of insulin resistance syndrome. In our study, ACSM transcripts showed 3 to >400-fold higher expression levels in human liver when compared to cell lines by qRT-PCR. This difference was also evident at the protein level, as shown for ACSM2. In liver, ACSM2 was the most abundant transcript, showing sixfold (vs. ACSM3) to >300-fold higher expression levels (vs. ACSM1). Mitochondrial localization of the ACSM2 protein and the presence of an N-terminal targeting sequence were shown by GFP-tagging. We have shown ACSM2B to be the predominant transcript in human liver, and genetic variations of this gene could therefore play an important role in disease susceptibility.

Effect of quercetin on paraoxonase 2 levels in RAW264.7 macrophages and in human monocytes--role of quercetin metabolism.

There is increasing evidence that the intracellular antioxidant enzyme paraoxonase 2 (PON2) may have a protective function in the prevention of atherogenesis. An enhancement of PON2 activity by dietary factors including flavonoids is therefore of interest. In the present study we determined the effect of quercetin on paraoxonase 2 levels in cultured murine macrophages in vitro and in overweight subjects with a high cardiovascular risk phenotype supplemented with 150 mg quercetin/day for 42 days in vivo. Supplementation of murine RAW264.7 macrophages in culture with increasing concentrations of quercetin (1, 10, 20 micromol/L) resulted in a significant increase in PON2 mRNA and protein levels, as compared to untreated controls. Unlike quercetin, its glucuronidated metabolite quercetin-3-glucuronide did not affect PON2 gene expression in cultured macrophages. However the methylated quercetin derivative isorhamnetin enhanced PON2 gene expression in RAW264.7 cells to similar extent like quercetin. Although supplementing human volunteers with quercetin was accompanied by a significant increase in plasma quercetin concentration, dietary quercetin supplementation did not change PON2 mRNA levels in human monocytes in vivo. Current data indicate that quercetin supplementation increases PON2 levels in cultured monocytes in vitro but not in human volunteers in vivo.

Dysbindin is a potent inducer of RhoA-SRF-mediated cardiomyocyte hypertrophy.

Dysbindin is an established schizophrenia susceptibility gene thoroughly studied in the context of the brain. We have previously shown through a yeast two-hybrid screen that it is also a cardiac binding partner of the intercalated disc protein Myozap. Because Dysbindin is highly expressed in the heart, we aimed here at deciphering its cardiac function. Using a serum response factor (SRF) response element reporter-driven luciferase assay, we identified a robust activation of SRF signaling by Dysbindin overexpression that was associated with significant up-regulation of SRF gene targets, such as Acta1 and Actc1. Concurrently, we identified RhoA as a novel binding partner of Dysbindin. Further phenotypic and mechanistic characterization revealed that Dysbindin induced cardiac hypertrophy via RhoA-SRF and MEK1-ERK1 signaling pathways. In conclusion, we show a novel cardiac role of Dysbindin in the activation of RhoA-SRF and MEK1-ERK1 signaling pathways and in the induction of cardiac hypertrophy. Future in vivo studies should examine the significance of Dysbindin in cardiomyopathy.

Supplementation with the reduced form of Coenzyme Q10 decelerates phenotypic characteristics of senescence and induces a peroxisome proliferator-activated receptor-alpha gene expression signature in SAMP1 mice.

Our present study reveals significant decelerating effects on senescence processes in middle-aged SAMP1 mice supplemented for 6 or 14 months with the reduced form (Q(10)H(2), 500 mg/kg BW/day) of coenzyme Q(10) (CoQ(10)). To unravel molecular mechanisms of these CoQ(10) effects, a genome-wide transcript profiling in liver, heart, brain and kidney of SAMP1 mice supplemented with the reduced (Q(10)H(2)) or oxidized form of CoQ(10) (Q(10)) was performed. Liver seems to be the main target tissue of CoQ(10) intervention, followed by kidney, heart and brain. Stringent evaluation of the resulting data revealed that Q(10)H(2) has a stronger impact on gene expression than Q(10), primarily due to differences in the bioavailability. Indeed, Q(10)H(2) supplementation was more effective than Q(10) to increase levels of CoQ(10) in the liver of SAMP1 mice. To identify functional and regulatory connections of the "top 50" (p<0.05) Q(10)H(2)-sensitive transcripts in liver, text mining analysis was used. Hereby, we identified Q(10)H(2)-sensitive genes which are regulated by peroxisome proliferator-activated receptor-alpha and are primarily involved in cholesterol synthesis (e.g. HMGCS1, HMGCL and HMGCR), fat assimilation (FABP5), lipoprotein metabolism (PLTP) and inflammation (STAT-1). These data may explain, at least in part, the decelerating effects on degenerative processes observed in Q(10)H(2)-supplemented SAMP1 mice.

Association analysis between the prostaglandin E synthase 2 R298H polymorphism and body mass index in 8079 participants of the KORA study cohort.

CONTEXT: The H-allele of the R298H polymorphism in the prostaglandin E synthase 2 (PTGES2) gene was associated with lower risk of diabetes type 2. AIM: To explore the association between the PTGES2 R298H SNP and body mass index (BMI). METHODS: We analyzed the R298H SNP (rs13283456) and three haplotype single-nucleotide polymorphisms (rs884115, rs10987883, and rs4837240) covering a 20 kb gene region in population-based surveys of the Kooperative Gesundheitsforschung in der Region Augsburg study cohort with 8079 participants. RESULTS: A statistically significant difference in BMI between the heterozygous PTGES2 R298H genotype and the homozygous R/R genotype was found in males but not in females. Males with the R/H genotype showed a decrease in BMI of -0.30 BMI units (95% CI: -0.55, -0.04, p = 0.02) in comparison to R/R males. A haplotype comprising the minor allele of PTGES2 R298H showed a significant decrease of -0.23 BMI units in males (-0.45, -0.02; p = 0.04) but not in females. Other haplotypes and haplotype single-nucleotide polymorphisms were not significantly associated with BMI. CONCLUSION: We found a marginal but significant influence of the PTGES2 298H SNP on BMI in a large population-based study.