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.

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.

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.

miR-23a is decreased during muscle atrophy by a mechanism that includes calcineurin signaling and exosome-mediated export.

Skeletal muscle atrophy is prevalent in chronic diseases, and microRNAs (miRs) may play a key role in the wasting process. miR-23a was previously shown to inhibit the expression of atrogin-1 and muscle RING-finger protein-1 (MuRF1) in muscle. It also was reported to be regulated by cytoplasmic nuclear factor of activated T cells 3 (NFATc3) in cardiomyocytes. The objective of this study was to determine if miR-23a is regulated during muscle atrophy and to evaluate the relationship between calcineurin (Cn)/NFAT signaling and miR-23a expression in skeletal muscle cells during atrophy. miR-23a was decreased in the gastrocnemius of rats with acute streptozotocin-induced diabetes, a condition known to increase atrogin-1 and MuRF1 expression and cause atrophy. Treatment of C2C12 myotubes with dexamethasone (Dex) for 48 h also reduced miR-23a as well as RCAN1.4 mRNA, which is transcriptionally regulated by NFAT. NFATc3 nuclear localization and the amount of miR-23a decreased rapidly within 1 h of Dex administration, suggesting a link between Cn signaling and miR-23a. The level of miR-23a was lower in primary myotubes from mice lacking the α- or ß-isoform of the CnA catalytic subunit than wild-type mice. Dex did not further suppress miR-23a in myotubes from Cn-deficient mice. Overexpression of CnAß in C2C12 myotubes prevented Dex-induced suppression of miR-23a. Finally, miR-23a was present in exosomes isolated from the media of C2C12 myotubes, and Dex increased its exosomal abundance. Dex did not alter the number of exosomes released into the media. We conclude that atrophy-inducing conditions downregulate miR-23a in muscle by mechanisms involving attenuated Cn/NFAT signaling and selective packaging into exosomes.

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.

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.

In vitro quantification of specific microRNA using molecular beacons.

MicroRNAs (miRNAs), a class of non-coding RNAs, have become a major focus of molecular biology research because of their diverse genomic origin and ability to regulate an array of cellular processes. Although the biological functions of miRNA are yet to be fully understood, tissue levels of specific miRNAs have been shown to correlate with pathological development of disease. Here, we demonstrate that molecular beacons can readily distinguish mature- and pre-miRNAs, and reliably quantify miRNA expression. We found that molecular beacons with DNA, RNA and combined locked nucleic acid (LNA)-DNA backbones can all detect miRNAs of low (<1 nM) concentrations in vitro, with RNA beacons having the highest detection sensitivity. Furthermore, we found that molecular beacons have the potential to distinguish miRNAs that have slight variations in their nucleotide sequence. These results suggest that the molecular beacon-based approach to assess miRNA expression and distinguish mature and precursor miRNA species is quite robust, and has the promise for assessing miRNA levels in biological samples.

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.

Using information theory to assess the communicative capacity of circulating microRNA.

The discovery of extracellular microRNAs (miRNAs) and their transport modalities (i.e., microparticles, exosomes, proteins and lipoproteins) has sparked theories regarding their role in intercellular communication. Here, we assessed the information transfer capacity of different miRNA transport modalities in human serum by utilizing basic principles of information theory. Zipf Statistics were calculated for each of the miRNA transport modalities identified in human serum. Our analyses revealed that miRNA-mediated information transfer is redundant, as evidenced by negative Zipf's Statistics with magnitudes greater than one. In healthy subjects, the potential communicative capacity of miRNA in complex with circulating proteins was significantly lower than that of miRNA encapsulated in circulating microparticles and exosomes. Moreover, the presence of coronary heart disease significantly lowered the communicative capacity of all circulating miRNA transport modalities. To assess the internal organization of circulating miRNA signals, Shannon's zero- and first-order entropies were calculated. Microparticles (MPs) exhibited the lowest Shannon entropic slope, indicating a relatively high capacity for information transfer. Furthermore, compared to the other miRNA transport modalities, MPs appeared to be the most efficient at transferring miRNA to cultured endothelial cells. Taken together, these findings suggest that although all transport modalities have the capacity for miRNA-based information transfer, MPs may be the simplest and most robust way to achieve miRNA-based signal transduction in sera. This study presents a novel method for analyzing the quantitative capacity of miRNA-mediated information transfer while providing insight into the communicative characteristics of distinct circulating miRNA transport modalities.

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.

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.

Laminar shear stress modulates phosphorylation and localization of RNA polymerase II on the endothelial nitric oxide synthase gene.

OBJECTIVE: In endothelial cells exposed to unidirectional laminar shear stress, endothelial nitric oxide synthase transcription (eNOS), mRNA stability, and protein levels are enhanced. We have previously demonstrated that these changes are associated with increased 3' polyadenylation of eNOS mRNA. Here, we investigated the effect of laminar shear stress on the phosphorylation and localization of RNA polymerase (Pol) II, the enzyme primarily responsible for coordinating transcription and posttranscriptional processing. METHODS AND RESULTS: Using Western and chromatin immunoprecipitation analyses, Pol II phosphorylation and localization on the eNOS gene were assessed in bovine aortic endothelial cells exposed to laminar shear stress. Total Pol II (phosphorylated and unphosphorylated) levels were increased 65% in response to laminar shear stress. This was associated with an increase in Pol II phosphoserine 2, but no change in levels of the unphosphorylated or phosphoserine 5 isoforms. Quantitative chromatin immunoprecipitation analysis showed that laminar shear stress enhanced binding of Pol II phosphoserine 2 to the 3' end of the eNOS gene, particularly exon 26, which encodes the 3'UTR. Treatment of cells with DRB attenuated laminar shear stress-induced Pol II phosphorylation, eNOS 3' polyadenylation, and eNOS expression. CONCLUSIONS: These data suggest that laminar shear stress enhances eNOS mRNA 3' polyadenylation by modulating phosphorylation and localization of Pol II.

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.

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.

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.

Laminar shear stress and 3' polyadenylation of eNOS mRNA.

The 3' poly(A) tail is important in messenger RNA stability and translational efficiency. In somatic tissues, 3' polyadenylation of mRNAs has been thought to largely be a constitutively active process. We have reported that laminar shear stress causes a brief increase in endothelial nitric oxide synthase (eNOS) transcription, followed by a prolonged increase in eNOS mRNA stability. We sought to determine whether shear stress and other stimuli affected eNOS 3' polyadenylation in endothelial cells. Under basal (static) conditions, eNOS mRNA possessed short 3' poly(A) tails of <25 nt. In contrast, laminar shear stress increased expression of eNOS transcripts with long poly(A) tails. ENOS transcripts with longer poly(A) tails had prolonged half-lives (6 hours in static cells versus 18 hours in sheared cells). Polysome analysis revealed that eNOS mRNA from sheared cells was shifted into more translationally active polysome fractions compared with eNOS mRNA from static cells. Shear-induced lengthening of the eNOS 3' poly(A) tail was the result of increased nuclear polyadenylation. Furthermore, hydrogen peroxide and HMG Co-A reductase inhibitors, other stimuli known to modulate eNOS expression posttranscriptionally, also induced eNOS 3' poly(A) tail lengthening. These results support the concept that shear stress modulates eNOS mRNA stability and translation via increased 3' polyadenylation. We suggest that mRNA 3' polyadenylation is a posttranscriptional mechanism used by endothelial cells to regulate gene expression.