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 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.
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.
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.
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.
Posttranscriptional regulation of endothelial nitric oxide synthase (eNOS) expression is an important mechanism by which endothelial cells respond to various physiological and pathophysiological ...stimuli. Previously, we showed that eNOS expression was dramatically altered by the state of cell growth and that the mechanism responsible for this regulation was entirely posttranscriptional, occurring via changes in eNOS mRNA stability. The present study identifies a role for actin cytoskeleton organization in the posttranscriptional regulation of eNOS during cell growth and examines the relationship between the state of actin polymerization and eNOS expression. We identified monomeric actin (globular G-actin) as the major component of a 51-kDa ribonucleoprotein that binds to the eNOS mRNA 3′ untranslated region in UV-crosslinking analysis. Binding activity of the ribonucleoprotein complex correlated with the relative concentration of G-actin versus filamentous actin (F-actin). ENOS transcripts colocalized with cytoplasmic G-actin in cells subjected to fluorescence in situ hybridization and G-actin fluorescence staining. In subcellular fractionation studies, eNOS transcripts were enriched in the free polysomal fraction of nonproliferating cells and enriched in the cell matrix-associated polysomal fraction of proliferating cells. Furthermore, an inverse relationship between the concentration of G-actin and eNOS expression was observed in endothelial cells subjected to pharmacological alteration of their cytoskeleton; lower G/F-actin ratios correlated with increased eNOS expression. Our findings provide some insight into how endothelial cells may use the dynamic organization of the actin cytoskeleton to regulate expression of an enzyme that is crucial to vascular homeostasis.
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.
1 Division of Cardiology, Emory University School of Medicine, Atlanta, Georgia; and 2 Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia
...Submitted 8 September 2008
; accepted in final form 15 December 2008
The endothelium plays an essential role in maintaining vascular homeostasis, and it fulfills this role by modulating intracellular signaling and gene expression in response to chemical and mechanical stimuli. Assessing changes in endothelial gene expression is essential to understanding how physiological and pathophysiological processes modulate vascular homeostasis. Here we describe the use of molecular beacons to rapidly and quantitatively assess expression and 3'-polyadenylation of a gene that is important for vascular homeostasis, endothelial nitric oxide synthase (eNOS). Single- and dual-fluorescence resonance energy transfer (FRET) molecular beacon hybridization assays were developed to measure changes in mRNA levels and 3'-polyadenylation, respectively, in primary human endothelial cell cultures subjected to laminar shear stress or statin treatment. Optimized beacon hybridization assays took 15 min to perform, and eNOS mRNA levels were validated by quantitative real-time RT-PCR. Competitive inhibition assays and posttranscriptional silencing of eNOS expression were used to verify the specificity of molecular beacon fluorescence. Finally, the dual-FRET method was used to assess eNOS polyadenylation in tissues isolated from mice subjected to exercise training. These data demonstrate that molecular beacons can be used to rapidly and efficiently measure endothelial gene expression and 3'-polyadenylation. This approach could easily be adapted for studies of other endothelial genes and has promise for applications in live endothelial cells.
endothelial nitric oxide synthase; gene expression; dual-fluorescence resonance energy transfer
Address for reprint requests and other correspondence: C. Searles, Div. of Cardiology, Emory Univ. School of Medicine, Atlanta, GA 30322 (e-mail: csearle{at}emory.edu )
Abstract only Introduction: Shear-sensitive micro-RNAs play an integral role in dictating vascular wall pro-inflammatory response and development of atherosclerosis. Previously, our group and others ...have identified an inverse relationship between micro-RNA-155 (miR-155) expression and inflammation in atheroprone areas of chronic low magnitude oscillatory shear stress (OSS) in vasculature and in-vitro. Hypothesis: we hypothesized that miR-155 negatively regulates acute OSS-induced vascular inflammation and dysfunction, via modulation of the MAPK-ETS-1 pathway. Methods: 12-week old C57B/6J wild type (WT) and miR-155 knockout mice (KO) were subjected to abdominal aortic coarctation (AAC), a unique model of acute induction of OSS, for 3-7 days. Downstream acute OSS segments were compared to upstream unidirectional shear stress (USS) segments of thoracic aorta using RT-PCR, western blot and two-way ANOVA followed by Tukey’s multiple comparison analyses. Results: In WT mice, acute OSS induced vascular inflammation evidenced by upregulation of MCP-1 and VCAM-1 expression in OSS segments compared with USS. This was associated with loss of vascular barrier function as evaluated by extravasation of Evans-blue dye assay along with increased MMP-9 and MMP-3 expression. However, vascular miR-155 levels were also higher in OSS segments compared with USS (n=6-12, P<0.05). Nevertheless, miR-155 KO mice showed enhanced expression and activation of ERK and p-38 MAPKs and downstream ETS-1, VCAM-1 and MMP-9 expression in OSS segments compared with USS versus WT controls (n=3-4, P<0.05). Tail vein injections of miR-155 overexpressing lentivirus particles in WT mice after AAC resulted in further upregulation of miR-155 and abolished OSS-induced upregulation of p-38 and downstream ETS-1, VCAM-1 and MMP-9 expression in OSS segments compared with USS versus scramble controls (n=5-6, P<0.05). Conclusions: Despite the early upregulation of shear-sensitive miR-155, our data suggest that miR-155 serves as a negative feedback regulator to acute OSS-induced vascular inflammation via inhibition of p-38 and ETS-1. Further studies are in progress to evaluate the effect of exogenous miR-155 on OSS-induced oxidative stress and vascular function, which can serve as basis for developing novel miRNA-based therapeutic modalities.
The authors sought to determine whether nebivolol treatment results in changes in blood pressure (BP), nitric oxide bioavailability, and vascular function in obese African Americans with recently ...diagnosed stage 1 hypertension. Forty‐three obese, hypertensive African Americans (mean BP: systolic, 148.8±14.3 mm Hg; diastolic, 90.4±8.2 mm Hg) were treated with nebivolol (5–10 mg/d) for 8 weeks. Primary outcomes were change in systolic and diastolic BP and efficacy in reaching normotensive BP. Mean systolic BP decreased by 9.2±14 mm Hg (P<.005) and diastolic BP decreased 6.8±9 mm Hg (P<.005) with 8 weeks of therapy. Significant improvements were seen in arterial compliance with nebivolol treatment as measured by aortic augmentation index (P<.005) and time to wave reflection (P=.013). Nebivolol treatment improved endothelial function as measured by flow‐mediated dilation (P<.005). Levels of erythrocyte cellular superoxide dismutase increased with nebivolol, indirectly suggesting increased bioavailability of nitric oxide (P<.005). Monotherapy with nebivolol in obese, hypertensive African Americans results in significant systolic and diastolic BP reduction by mechanisms that include improved vascular function and compliance.
Abstract only Introduction: Previously, we used plasma metabolomics profiling to identify metabolites underlying cardiovascular health (CVH) in participants of the Morehouse-Emory Cardiovascular ...(MECA) Center for Health Equity study, a cohort of Black adults living in the Atlanta area. To determine whether a health intervention could alter expression of metabolites associated with CVH, metabolomics profiles were assessed pre- and post-intervention in participants with poor CVH. Hypothesis: Metabolites previously shown to be associated with CVH in MECA participants would be altered after the health intervention. Goal: To identify metabolites that could be markers for improved CVH and therapeutic targets. Methods: Seventy-one Black adults without known cardiovascular disease and with poor CVH (AHA Life’s Simple 7 LS7 score <8), used a technology-enabled behavioral intervention platform for 6 months with or without a health coach. Metabolomics profiles were assessed pre- and post-intervention by high-resolution metabolomics profiling. A metabolome wide association study (MWAS) identified differentially expressed metabolites and enriched metabolic pathways. Metabolites were annotated by matching to an in-house library of confirmed metabolites. Changes in clinical metrics after intervention were also explored. Results: Mean age was 55 (standard deviation SD 9.0) years, 69% female. Total LS7 scores improved from 6.2 (SD 1.49) to 6.5 (SD 1.74) after the intervention. While total and subcomponent LS7 scores and clinical metrics (BMI, blood pressure, glucose and cholesterol levels) trended toward improvement, the changes were not statistically significant. MWAS identified 18 confirmed metabolites that were significantly changed after intervention, including glutamine and glutamate. Pathway analysis identified 29 enriched metabolic pathways, including those for glutamate, aspartate, asparagine, arginine and proline metabolism. Conclusion: A six-month lifestyle intervention significantly altered activity of select plasma metabolites while only modestly altering clinical metrics. These metabolites appear to be sensitive indicators of a healthier lifestyle, potentially supporting their use as markers of CVH and therapeutic targets.
The expression of the endothelial NO synthase (eNOS) is dramatically influenced by the state of cell growth. In proliferating cells, mRNA levels are increased 4-fold compared with postconfluent, ...nonproliferating cells. Nuclear run-on analysis indicated that there is no difference in the transcriptional rate of eNOS in proliferating versus postconfluent cells. The half-life of eNOS mRNA, measured after actinomycin D transcriptional arrest, was 3-fold greater in preconfluent compared with confluent endothelial cells. Using UV-cross-linking analysis, a cytoplasmic protein with an apparent molecular mass of 51 kDa was found to bind to terminal 545-nt eNOS mRNA 3-fold more in confluent cells than in proliferating cells. Further characterization of the eNOS mRNA indicated that a 43-nt sequence at the origin of the 3′-untranslated region (UTR) is critical in binding of this protein. Endothelial cells were stably transfected with a chimeric cDNA plasmid containing chloramphenicol acetyl transferase (CAT) ligated to the eNOS coding region and either the wild-type 3′-UTR (pcDNACAT/eNOS(wtUTR)) or a mutant 3′-UTR lacking the 43 nt found to bind the 51-kDa protein (pcDNACAT/eNOS(ΔUTR)). The CAT/eNOS mRNA half-life was dramatically stabilized in these latter cells as compared with cells transfected with pcDNACAT/eNOS(wtUTR)). Thus, this 43-nt region plays a critical role in destabilizing eNOS mRNA. These studies demonstrate a mechanism for modulation of eNOS expression during cell growth via interactions between the proximal 3′-UTR and a novel ≈51-kDa cytosolic protein.
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