MicroRNAs and Cardiovascular Disease Risk.

PURPOSE OF REVIEW: MicroRNAs (miRNAs)-short, non-coding RNAs-play important roles in almost all aspects of cardiovascular biology, and changes in intracellular miRNA expression are indicative of cardiovascular disease development and progression. Extracellular miRNAs, which are easily measured in blood and can be reflective of changes in intracellular miRNA levels, have emerged as potential non-invasive biomarkers for disease. This review summarizes current knowledge regarding miRNAs as biomarkers for assessing cardiovascular disease risk and prognosis. RECENT FINDINGS: Numerous studies over the last 10-15 years have identified associations between extracellular miRNA profiles and cardiovascular disease, supporting the potential use of extracellular miRNAs as biomarkers for risk stratification. However, clinical application of extracellular miRNA profiles has been hampered by poor reproducibility and inter-study variability that is due largely to methodological differences between studies. While recent studies indicate that circulating extracellular miRNAs are promising biomarkers for cardiovascular disease, evidence for clinical implementation is lacking. This highlights the need for larger, well-designed studies that use standardized methods for sample preparation, miRNA isolation, quantification, and normalization.

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.

The US Department of Veterans Affairs Science and Health Initiative to Combat Infectious and Emerging Life-Threatening Diseases (VA SHIELD): A Biorepository Addressing National Health Threats.

Background: The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has demonstrated the need to share data and biospecimens broadly to optimize clinical outcomes for US military Veterans. Methods: In response, the Veterans Health Administration established VA SHIELD (Science and Health Initiative to Combat Infectious and Emerging Life-threatening Diseases), a comprehensive biorepository of specimens and clinical data from affected Veterans to advance research and public health surveillance and to improve diagnostic and therapeutic capabilities. Results: VA SHIELD now comprises 12 sites collecting de-identified biospecimens from US Veterans affected by SARS-CoV-2. In addition, 2 biorepository sites, a data processing center, and a coordinating center have been established under the direction of the Veterans Affairs Office of Research and Development. Phase 1 of VA SHIELD comprises 34 157 samples. Of these, 83.8% had positive tests for SARS-CoV-2, with the remainder serving as contemporaneous controls. The samples include nasopharyngeal swabs (57.9%), plasma (27.9%), and sera (12.5%). The associated clinical and demographic information available permits the evaluation of biological data in the context of patient demographics, clinical experience and management, vaccinations, and comorbidities. Conclusions: VA SHIELD is representative of US national diversity with a significant potential to impact national healthcare. VA SHIELD will support future projects designed to better understand SARS-CoV-2 and other emergent healthcare crises. To the extent possible, VA SHIELD will facilitate the discovery of diagnostics and therapeutics intended to diminish COVID-19 morbidity and mortality and to reduce the impact of new emerging threats to the health of US Veterans and populations worldwide.

Individual Psychosocial Resilience, Neighborhood Context, and Cardiovascular Health in Black Adults: A Multilevel Investigation From the Morehouse-Emory Cardiovascular Center for Health Equity Study.

BACKGROUND: Despite well-documented cardiovascular disparities between racial groups, within-race determinants of cardiovascular health among Black adults remain understudied. Factors promoting cardiovascular resilience among Black adults in particular warrant further investigation. Our objective was to examine whether individual psychosocial resilience and neighborhood-level cardiovascular resilience were associated with better cardiovascular health in Black adults, measured utilizing Life's Simple 7 (LS7) scores. METHODS: We assessed LS7 scores in 389 Black adults (mean age, 53±10 years; 39% men) living in Atlanta, Georgia. A composite score of individual psychosocial resilience was created by assessing environmental mastery, purpose in life, optimism, resilient coping, and depressive symptoms. Neighborhood-level cardiovascular resilience was separately determined by the census tract-level rates of cardiovascular mortality/morbidity events. Generalized linear mixed regression models were used to examine the association between individual psychosocial resilience, neighborhood cardiovascular resilience, and LS7 scores. RESULTS: Higher individual psychosocial resilience was significantly associated with higher LS7 (ß=0.38 [0.16-0.59] per 1 SD) after adjustment for sociodemographic factors. Similarly, higher neighborhood-level cardiovascular resilience was significantly associated with higher LS7 (ß=0.23 [0.02-0.45] per 1 SD). When jointly examined, high individual psychosocial resilience (>median) was independently associated with higher LS7 (ß=0.73 [0.31-1.17]), whereas living in high-resilience neighborhoods (>median) was not. The largest difference in LS7 score was between those with high and low psychosocial resilience living in low-resilience neighborhoods (8.38 [7.90-8.86] versus 7.42 [7.04-7.79]). CONCLUSIONS: Individual psychosocial resilience in Black adults is associated with better cardiovascular health.

Cardiovascular Risk and Resilience Among Black Adults: Rationale and Design of the MECA Study.

Background Cardiovascular disease incidence, prevalence, morbidity, and mortality have declined in the past several decades; however, disparities persist among subsets of the population. Notably, blacks have not experienced the same improvements on the whole as whites. Furthermore, frequent reports of relatively poorer health statistics among the black population have led to a broad assumption that black race reliably predicts relatively poorer health outcomes. However, substantial intraethnic and intraracial heterogeneity exists; moreover, individuals with similar risk factors and environmental exposures are often known to experience vastly different cardiovascular health outcomes. Thus, some individuals have good outcomes even in the presence of cardiovascular risk factors, a concept known as resilience. Methods and Results The MECA (Morehouse-Emory Center for Health Equity) Study was designed to investigate the multilevel exposures that contribute to "resilience" in the face of risk for poor cardiovascular health among blacks in the greater Atlanta, GA, metropolitan area. We used census tract data to determine "at-risk" and "resilient" neighborhoods with high or low prevalence of cardiovascular morbidity and mortality, based on cardiovascular death, hospitalization, and emergency department visits for blacks. More than 1400 individuals from these census tracts assented to demographic, health, and psychosocial questionnaires administered through telephone surveys. Afterwards, ≈500 individuals were recruited to enroll in a clinical study, where risk biomarkers, such as oxidative stress, and inflammatory markers, endothelial progenitor cells, metabolomic and microRNA profiles, and subclinical vascular dysfunction were measured. In addition, comprehensive behavioral questionnaires were collected and ideal cardiovascular health metrics were assessed using the American Heart Association's Life Simple 7 measure. Last, 150 individuals with low Life Simple 7 were recruited and randomized to a behavioral mobile health (eHealth) plus health coach or eHealth only intervention and followed up for improvement. Conclusions The MECA Study is investigating socioenvironmental and individual behavioral measures that promote resilience to cardiovascular disease in blacks by assessing biological, functional, and molecular mechanisms. REGISTRATION URL: https://www.clini​caltr​ials.gov. Unique identifier: NCT03308812.

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.

Pleomorphic Structures in Human Blood Are Red Blood Cell-Derived Microparticles, Not Bacteria.

BACKGROUND: Red blood cell (RBC) transfusions are a common, life-saving therapy for many patients, but they have also been associated with poor clinical outcomes. We identified unusual, pleomorphic structures in human RBC transfusion units by negative-stain electron microscopy that appeared identical to those previously reported to be bacteria in healthy human blood samples. The presence of viable, replicating bacteria in stored blood could explain poor outcomes in transfusion recipients and have major implications for transfusion medicine. Here, we investigated the possibility that these structures were bacteria. RESULTS: Flow cytometry, miRNA analysis, protein analysis, and additional electron microscopy studies strongly indicated that the pleomorphic structures in the supernatant of stored RBCs were RBC-derived microparticles (RMPs). Bacterial 16S rDNA PCR amplified from these samples were sequenced and was found to be highly similar to species that are known to commonly contaminate laboratory reagents. CONCLUSIONS: These studies suggest that pleomorphic structures identified in human blood are RMPs and not bacteria, and they provide an example in which laboratory contaminants may can mislead investigators.

Platelets confound the measurement of extracellular miRNA in archived plasma.

Extracellular miRNAs are detectable in biofluids and represent a novel class of disease biomarker. Although many studies have utilized archived plasma for miRNA biomarker discovery, the effects of processing and storage have not been rigorously studied. Previous reports have suggested plasma samples are commonly contaminated by platelets, significantly confounding the measurement of extracellular miRNA, which was thought to be easily addressed by additional post-thaw plasma processing. In a case-control study of archived plasma, we noted a significant correlation between miRNA levels and platelet counts despite post-thaw processing. We thus examined the effects of a single freeze/thaw cycle on microparticles (MPs) and miRNA levels, and show that a single freeze/thaw cycle of plasma dramatically increases the number of platelet-derived MPs, contaminates the extracellular miRNA pool, and profoundly affects the levels of miRNAs detected. The measurement of extracellular miRNAs in archived samples is critically dependent on the removal of residual platelets prior to freezing plasma samples. Many previous clinical studies of extracellular miRNA in archived plasma should be interpreted with caution and future studies should avoid the effects of platelet contamination.

Peroxisome Proliferator-Activated Receptor γ and microRNA 98 in Hypoxia-Induced Endothelin-1 Signaling.

Endothelin-1 (ET-1) plays a critical role in endothelial dysfunction and contributes to the pathogenesis of pulmonary hypertension (PH). We hypothesized that peroxisome proliferator-activated receptor γ (PPARγ) stimulates microRNAs that inhibit ET-1 and pulmonary artery endothelial cell (PAEC) proliferation. The objective of this study was to clarify molecular mechanisms by which PPARγ regulates ET-1 expression in vitro and in vivo. In PAECs isolated from patients with pulmonary arterial hypertension, microRNA (miR)-98 expression was reduced, and ET-1 protein levels and proliferation were increased. Similarly, hypoxia reduced miR-98 and increased ET-1 levels and PAEC proliferation in vitro. In vivo, hypoxia reduced miR-98 expression and increased ET-1 and proliferating cell nuclear antigen (PCNA) levels in mouse lung, derangements that were aggravated by treatment with the vascular endothelial growth factor receptor antagonist Sugen5416. Reporter assays confirmed that miR-98 binds directly to the ET-1 3'-untranslated region. Compared with littermate control mice, miR-98 levels were reduced and ET-1 and PCNA expression were increased in lungs from endothelial-targeted PPARγ knockout mice, whereas miR-98 levels were increased and ET-1 and PCNA expression was reduced in lungs from endothelial-targeted PPARγ-overexpression mice. Gain or loss of PPARγ function in PAECs in vitro confirmed that alterations in PPARγ were sufficient to regulate miR-98, ET-1, and PCNA expression. Finally, PPARγ activation with rosiglitazone regimens that attenuated hypoxia-induced PH in vivo and human PAEC proliferation in vitro restored miR-98 levels. The results of this study show that PPARγ regulates miR-98 to modulate ET-1 expression and PAEC proliferation. These results further clarify molecular mechanisms by which PPARγ participates in PH pathogenesis and therapy.

An accurate, precise method for general labeling of extracellular vesicles.

Extracellular, membrane vesicles (microvesicles, exosomes) are secreted by cells and may serve as mediators of intercellular communication. Methods for detecting them by flow cytometry have included the use of agents that fluorescently stain vesicle membrane, or fluorescent antibodies that target specific cell-of-origin antigens. However, these methods may falsely detect cell debris or require prior cell-of-origin knowledge. Here, we demonstrate the suitability of calcein AM for detection of intact extracellular vesicles (EVs) by flow cytometry.•Calcein AM is non-fluorescent until it passively enters EVs, after which it is activated and becomes fluorescent and EV-impermeant.•Permeabilized/lysed EVs label positive with antibodies and lipophilic membrane stain, whereas no labeling was observed with calcein. In contrast to methods that use antibodies or membrane stains, calcein AM allows for the differentiation between intact EVs and debris.•Calcein AM can be used for detection of intact EVs from numerous cell types.

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.

Identification of therapeutic covariant microRNA clusters in hypoxia-treated cardiac progenitor cell exosomes using systems biology.

RATIONALE: Myocardial infarction is a leading cause of death in developed nations, and there remains a need for cardiac therapeutic systems that mitigate tissue damage. Cardiac progenitor cells (CPCs) and other stem cell types are attractive candidates for treatment of myocardial infarction; however, the benefit of these cells may be as a result of paracrine effects. OBJECTIVE: We tested the hypothesis that CPCs secrete proregenerative exosomes in response to hypoxic conditions. METHODS AND RESULTS: The angiogenic and antifibrotic potential of secreted exosomes on cardiac endothelial cells and cardiac fibroblasts were assessed. We found that CPC exosomes secreted in response to hypoxia enhanced tube formation of endothelial cells and decreased profibrotic gene expression in TGF-ß-stimulated fibroblasts, indicating that these exosomes possess therapeutic potential. Microarray analysis of exosomes secreted by hypoxic CPCs identified 11 miRNAs that were upregulated compared with exosomes secreted by CPCs grown under normoxic conditions. Principle component analysis was performed to identify miRNAs that were coregulated in response to distinct exosome-generating conditions. To investigate the cue-signal-response relationships of these miRNA clusters with a physiological outcome of tube formation or fibrotic gene expression, partial least squares regression analysis was applied. The importance of each up- or downregulated miRNA on physiological outcomes was determined. Finally, to validate the model, we delivered exosomes after ischemia-reperfusion injury. Exosomes from hypoxic CPCs improved cardiac function and reduced fibrosis. CONCLUSIONS: These data provide a foundation for subsequent research of the use of exosomal miRNA and systems biology as therapeutic strategies for the damaged heart.

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.

Shear sensitive microRNAs and atherosclerosis.

Atherosclerosis, the leading cause of morbidity and mortality in developed nations, is a chronic inflammatory disease of arteries. In large and medium-sized vessels, the atherosclerotic burden is focal and non-random, despite the systemic nature of risk factors. This observation has prompted numerous studies over the past two decades that have evaluated the relationship between blood flow, endothelial function and plaque localization. The recent discovery of microRNAs (miRNAs) that are sensitive to distinct flow conditions has added a new layer of complexity to the pathophysiology of atherosclerosis, but may ultimately help us better understand the disease process. In this manuscript we will briefly review the most commonly used in vitro and in vivo model systems developed to study the relationship between flow, endothelial function and plaque development. We will also provide a brief summary of shear sensitive miRNAs that have been shown to modulate inflammatory signaling pathways and atherosclerotic burden through changes in the endothelial gene expression.

MiRNA-155 targets myosin light chain kinase and modulates actin cytoskeleton organization in endothelial cells.

Previously, we identified a microRNA (miRNA) signature for endothelial cells (ECs) subjected to unidirectional shear stress (USS). MiR-155, a multifunctional miRNA that has been implicated in atherosclerosis, was among the shear stress-responsive miRNAs. Here, we examined the role of miR-155 in modulating EC phenotype and function. In vitro, increased miR-155 levels in human ECs induced changes in morphology and filamentous (F)-actin organization. In addition, ECs transfected with miR-155 mimic were less migratory and less proliferative and had less apoptosis compared with control ECs. In mouse aorta, miR-155 expression was increased in the intima of thoracic aorta, where blood flow produces steady and unidirectional shear stress, compared with the intima of the lower curvature of the aortic arch, which is associated with oscillatory and low shear stress. These differences in miR-155 expression were associated with distinct changes in EC morphology and F-actin. The effects of miR-155 in vitro were mediated through suppression of two key regulators of the EC cytoskeleton organization: RhoA and myosin light chain kinase (MYLK). A novel direct interaction between miR-155 and the MYLK 3'UTR was verified by luciferase-MYLK 3'UTR reporter assays. Furthermore, the intensity of immunofluorescence staining for RhoA and MYLK in mouse aorta correlated inversely with miR-155 expression. In conclusion, a prominent effect of the multifunctional miR-155 in ECs is modulation of phenotype through alterations in RhoA, MYLK expression, and actin cytoskeleton organization.