Metabolomic signatures of ideal cardiovascular health in black adults.

Plasma metabolomics profiling is an emerging methodology to identify metabolic pathways underlying cardiovascular health (CVH). The objective of this study was to define metabolomic profiles underlying CVH in a cohort of Black adults, a population that is understudied but suffers from disparate levels of CVD risk factors. The Morehouse-Emory Cardiovascular (MECA) Center for Health Equity study cohort consisted of 375 Black adults (age 53 ± 10, 39% male) without known CVD. CVH was determined by the AHA Life's Simple 7 (LS7) score, calculated from measured blood pressure, body mass index (BMI), fasting blood glucose and total cholesterol, and self-reported physical activity, diet, and smoking. Plasma metabolites were assessed using untargeted high-resolution metabolomics profiling. A metabolome wide association study (MWAS) identified metabolites associated with LS7 score after adjusting for age and sex. Using Mummichog software, metabolic pathways that were significantly enriched in metabolites associated with LS7 score were identified. Metabolites representative of these pathways were compared across clinical domains of LS7 score and then developed into a metabolomics risk score for prediction of CVH. We identified novel metabolomic signatures and pathways associated with CVH in a cohort of Black adults without known CVD. Representative and highly prevalent metabolites from these pathways included glutamine, glutamate, urate, tyrosine and alanine, the concentrations of which varied with BMI, fasting glucose, and blood pressure levels. When assessed in conjunction, these metabolites were independent predictors of CVH. One SD increase in the novel metabolomics risk score was associated with a 0.88 higher LS7 score, which translates to a 10.4% lower incident CVD risk. We identified novel metabolomic signatures of ideal CVH in a cohort of Black Americans, showing that a core group of metabolites central to nitrogen balance, bioenergetics, gluconeogenesis, and nucleotide synthesis were associated with CVH in this population.

MicroRNA Detection Using a Double Molecular Beacon Approach: Distinguishing Between miRNA and Pre-miRNA.

MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate gene expression and are recognized for their roles both as modulators of disease progression and as biomarkers of disease activity, including neurological diseases, cancer, and cardiovascular disease (CVD). Commonly, miRNA abundance is assessed using quantitative real-time PCR (qRT-PCR), however, qRT-PCR for miRNA can be labor intensive, time consuming, and may lack specificity for detection of mature versus precursor forms of miRNA. Here, we describe a novel double molecular beacon approach to miRNA assessment that can distinguish and quantify mature versus precursor forms of miRNA in a single assay, an essential feature for use of miRNAs as biomarkers for disease. Using this approach, we found that molecular beacons with DNA or combined locked nucleic acid (LNA)-DNA backbones can detect mature and precursor miRNAs (pre-miRNAs) of low (< 1 nM) abundance in vitro. The double molecular beacon assay was accurate in assessing miRNA abundance in a sample containing a mixed population of mature and precursor miRNAs. In contrast, qRT-PCR and the single molecular beacon assay overestimated miRNA abundance. Additionally, the double molecular beacon assay was less labor intensive than traditional qRT-PCR and had 10-25% increased specificity. Our data suggest that the double molecular beacon-based approach is more precise and specific than previous methods, and has the promise of being the standard for assessing miRNA levels in biological samples.

PPARγ Ligands Attenuate Hypoxia-Induced Proliferation in Human Pulmonary Artery Smooth Muscle Cells through Modulation of MicroRNA-21.

Pulmonary hypertension (PH) is a progressive and often fatal disorder whose pathogenesis involves pulmonary artery smooth muscle cell (PASMC) proliferation. Although modern PH therapies have significantly improved survival, continued progress rests on the discovery of novel therapies and molecular targets. MicroRNA (miR)-21 has emerged as an important non-coding RNA that contributes to PH pathogenesis by enhancing vascular cell proliferation, however little is known about available therapies that modulate its expression. We previously demonstrated that peroxisome proliferator-activated receptor gamma (PPARγ) agonists attenuated hypoxia-induced HPASMC proliferation, vascular remodeling and PH through pleiotropic actions on multiple targets, including transforming growth factor (TGF)-ß1 and phosphatase and tensin homolog deleted on chromosome 10 (PTEN). PTEN is a validated target of miR-21. We therefore hypothesized that antiproliferative effects conferred by PPARγ activation are mediated through inhibition of hypoxia-induced miR-21 expression. Human PASMC monolayers were exposed to hypoxia then treated with the PPARγ agonist, rosiglitazone (RSG,10 µM), or in parallel, C57Bl/6J mice were exposed to hypoxia then treated with RSG. RSG attenuated hypoxic increases in miR-21 expression in vitro and in vivo and abrogated reductions in PTEN and PASMC proliferation. Antiproliferative effects of RSG were lost following siRNA-mediated PTEN depletion. Furthermore, miR-21 mimic decreased PTEN and stimulated PASMC proliferation, whereas miR-21 inhibition increased PTEN and attenuated hypoxia-induced HPASMC proliferation. Collectively, these results demonstrate that PPARγ ligands regulate proliferative responses to hypoxia by preventing hypoxic increases in miR-21 and reductions in PTEN. These findings further clarify molecular mechanisms that support targeting PPARγ to attenuate pathogenic derangements in PH.

TNF-α alters the release and transfer of microparticle-encapsulated miRNAs from endothelial cells.

MicroRNAs (miRNAs) encapsulated within microparticles (MPs) are likely to have a role in cell-to-cell signaling in a variety of diseases, including atherosclerosis. However, little is known about the mechanisms by which different cell types release and transfer miRNAs. Here, we examined TNF-α-induced release of MP-encapsulated miR-126, miR-21, and miR-155 from human aortic endothelial cells (ECs) and their transfer to recipient cells. ECs were treated with TNF-α (100 ng/ml) in the presence or absence of inhibitors that target different MP production pathways. MPs released in response to TNF-α were characterized by: 1) 70-80% decrease in miRNA/MP levels for miR-126 and -21 but a significant increase in pre-miR-155 and miR-155 (P < 0.05), 2) 50% reduction in uptake by recipient cells (P < 0.05), and 3) diminished ability to transfer miRNA to recipient cells. Cotreatment of donor ECs with TNF-α and caspase inhibitor (Q-VD-OPH, 10 µM) produced MPs that had: 1) 1.5- to 2-fold increase in miRNA/MP loading, 2) enhanced uptake by recipient cells (2-fold), and 3) increased ability to transfer miR-155. Cotreatment of ECs with TNF-α and Rho-associated kinase (ROCK) inhibitor (10 µM) produced MPs with features similar to those produced by TNF-α treatment alone. Our data indicate that TNF-α induced the production of distinct MP populations: ROCK-dependent, miRNA-rich MPs that effectively transferred their cargo and were antiapoptotic, and caspase-dependent, miRNA-poor MPs that were proapoptotic. These data provide insight into the relationship between MP production and extracellular release of miRNA, as well as the potential of encapsulated miRNA for cell-to-cell communication.

Angiotensin receptor type 1 single nucleotide polymorphism 1166A/C is associated with malignant arrhythmias and altered circulating miR-155 levels in patients with chronic heart failure.

BACKGROUND: Sudden cardiac death (SCD) from ventricular tachyarrhythmias accounts for approximately 450,000 annual deaths in the United States; many of these cases involve patients with chronic heart failure (HF). Prediction of which HF patients are most susceptible to SCD is difficult, and it is uncertain whether gene polymorphisms associated with HF outcomes are also linked to arrhythmic risk. METHODS: We evaluated 485 patients with chronic HF to see whether the angiotensin receptor type 1 (AT1R) 1166A/C or angiotensin-converting enzyme insertion/deletion (ACE I/D) polymorphisms were associated with a higher rate of ventricular arrhythmias requiring implantable cardioverter defibrillator (ICD) therapies over a 5-year period. We assessed the correlation between polymorphisms and antitachycardia pacing (ATP) and/or ICD shocks. RESULTS: Patients with AT1R-1166CC genotype had an increased rate of all events: ATP plus ICD shocks (P = .02). There was no association between ACE I/D genotype and ICD therapies. Furthermore, circulating levels of microRNA-155 (miR-155), a microRNA known to posttranscriptionally regulate AT1R expression, were significantly decreased in the CC compared with the AC and AA genotypes and were associated with ICD events. CONCLUSION: Our study suggests that the AT1R-1166CC genotype is associated with increased ICD therapies in patients with chronic HF, and the level of circulating miR-155 may be a potential marker for arrhythmic risk. Although these findings are novel, they will need replication and validation in larger cohorts of chronic HF patients.

MiR-21 is induced in endothelial cells by shear stress and modulates apoptosis and eNOS activity.

Mechanical forces associated with blood flow play an important role in regulating vascular signaling and gene expression in endothelial cells (ECs). MicroRNAs (miRNAs) are a class of noncoding RNAs that posttranscriptionally regulate the expression of genes involved in diverse cell functions, including differentiation, growth, proliferation, and apoptosis. miRNAs are known to have an important role in modulating EC biology, but their expression and functions in cells subjected to shear stress conditions are unknown. We sought to determine the miRNA expression profile in human ECs subjected to unidirectional shear stress and define the role of miR-21 in shear stress-induced changes in EC function. TLDA array and qRT-PCR analysis performed on HUVECs exposed to prolonged unidirectional shear stress (USS, 24h, 15 dynes/cm(2)) identified 13 miRNAs whose expression was significantly upregulated (p<0.05). The miRNA with the greatest change was miR-21; it was increased 5.2-fold (p=0.002) in USS-treated versus control cells. Western analysis demonstrated that PTEN, a known target of miR-21, was downregulated in HUVECs exposed to USS or transfected with pre-miR-21. Importantly, HUVECs overexpressing miR-21 had decreased apoptosis and increased eNOS phosphorylation and nitric oxide (NO(*)) production. These data demonstrate that shear stress forces regulate the expression of miRNAs in ECs, and that miR-21 influences endothelial biology by decreasing apoptosis and activating the NO(*) pathway. These studies advance our understanding of the mechanisms by which shear stress forces modulate vascular homeostasis.

Statin treatment and 3' polyadenylation of eNOS mRNA.

OBJECTIVE: Statins have been shown to increase endothelial nitric oxide synthase expression via enhanced mRNA stability. Because the poly(A) tail is an important determinant of transcript stability, we sought to characterize the effect of statins on eNOS mRNA 3' polyadenylation. METHODS AND RESULTS: Endothelial cells treated with statins had a time- and dose-dependent increase in eNOS transcripts with long poly(A) tails (75 to 160 adenosines). This effect was dependent on 3-hydroxy-3-methylglutaryl (HMG)-coenxyme A (CoA) reductase inhibition and was observed with both lipophilic (simvastatin) and hydrophilic (rosuvastatin) statins. In mRNA stability assays, polyadenylated eNOS transcripts from statin-treated cells were 2- to 3-fold more stable than transcripts from untreated cells. The effect of statins on eNOS polyadenylation was related to cytoskeleton organization; there was increased eNOS mRNA polyadenylation after Rho inhibition and cytochalasin D treatment. Further, we found increased phosphorylation of RNA polymerase II in statin-treated cells, suggesting that statin-induced polyadenylation involved modulation of RNA polymerase II activity. CONCLUSIONS: Our data provide insight into a mechanism by which statins enhance eNOS mRNA stability and increase eNOS protein: statins increase eNOS mRNA polyadenylation through Rho-mediated changes in the actin cytoskeleton.

Transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression.

The ability of the endothelium to produce nitric oxide is essential to maintenance of vascular homeostasis; disturbance of this ability is a major contributor to the pathogenesis of vascular disease. In vivo studies have demonstrated that expression of endothelial nitric oxide synthase (eNOS) is vital to endothelial function and have led to the understanding that eNOS expression is subject to modest but significant degrees of regulation. Subsequently, numerous physiological and pathophysiological stimuli have been identified that modulate eNOS expression via mechanisms that alter steady-state eNOS mRNA levels. These mechanisms involve changes in the rate of eNOS gene transcription (transcriptional regulation) and alteration of eNOS mRNA processing and stability (posttranscriptional regulation). In cultured endothelial cells, shear stress, transforming growth factor-beta1, lysophosphatidylcholine, cell growth, oxidized linoleic acid, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, and hydrogen peroxide have been shown to increase eNOS expression. In contrast, tumor necrosis factor-alpha, hypoxia, lipopolysaccaride, thrombin, and oxidized LDL can decrease eNOS mRNA levels. For many of these stimuli, both transcriptional and posttranscriptional mechanisms contribute to regulation of eNOS expression. Recent studies have begun to further define signaling pathways responsible for changes in eNOS expression and have characterized cis- and trans-acting regulatory elements. In addition, a role has been identified for epigenetic control of eNOS mRNA levels. This review will discuss transcriptional and posttranscriptional regulation of eNOS with emphasis on the molecular mechanisms that have been identified for these processes.