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.
Background
Sudden cardiac death (SCD) due to ventricular tachyarrhythmias accounts for approximately 450,000 annual deaths in the U.S.; 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 (AT1) A1166C 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 AT1-1166 CC genotype had an increased rate of all events: ATP plus ICD shocks (p=0.02). 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 to the AC and AA genotypes and were associated with ICD events.
Conclusion
Our study suggests that the AT1R-1166 CC 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. While these findings are novel, they will need replication and validation in larger cohorts of chronic HF patients.
Angiogenesis, the process by which new blood vessels are formed, is a critical phenomenon that is activated during various stages of mammalian development. MicroRNAs (miRNAs), a class of short, single stranded, non-coding RNAs, are recognized as important regulators of angiogenesis, and the role of intracellular miRNAs in modulating angiogenesis signaling has been identified. The recent discovery of extracellular and circulating miRNAs has sparked new questions regarding their potential in modulating angiogenesis signaling not only within cells but also between cells. In this review, we discuss the characteristics of intracellular and extracellular miRNAs and decipher the potential functional roles for these molecules in regard to the angiogenic process. We summarize what is currently known about circulating miRNAs in distinct clinical populations and discuss evidence that implicates extracellular miRNAs as novel mediators of angiogenesis-associated intercellular signaling. Lastly, we offer a new perspective on the functional role of vesicle-encapsulated circulating miRNA in modulating angiogenesis signaling pathways.
Objective
Osteopontin (OPN) is a multifunctional protein found in abundance in atherosclerotic plaques. Angiotensin II (Ang II) promotes atherosclerosis by inducing adhesion and migration of vascular smooth muscle cells (VSMCs). MicroRNAs (miRNAs) are critical regulators of protein expression. However, the relationship between Ang II, miRNAs and OPN has yet to be fully explored.
Methods and results
Using cultured VSMCs, we found that Ang II increased cellular OPN protein expression 4 h after treatment by 420 ± 54% (p < 0.03) in a translation dependent manner. Sequence analysis revealed a putative binding site for mir181a and raised the possibility that miR181a is a potential regulatory mechanism for OPN expression. We demonstrated that Ang II decreased miR181a expression by 52 ± 7% (p < 0.0001) and overexpressing miR181a inhibited Ang II induced increases in OPN protein expression by 69 ± 9% (p < 0.05). Furthermore, we demonstrated that miR181a is functionally important in that overexpression of miR181a inhibited VSMCs adhesion to collagen in response to Ang II as compared to controls by 36 ± 4%. (p < 0.05)
Conclusions
These results demonstrate that miR181a regulates OPN expression and that altering miR181a expression may be a novel therapeutic approach to modulate OPN protein expression.
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, 24 hrs, 15 dynes/cm2) 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 USStreated 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.
Coronary heart disease (CHD) is characterized by abnormal intercellular communication and circulating microRNAs (miRNAs) are likely involved in this process. Here, we show that CHD was associated with changes in the transport of circulating miRNA, particularly decreased miRNA enrichment in microparticles (MPs). Additionally, MPs from CHD patients were less efficient at transferring miRNA to cultured HUVECs, which correlated with their diminished capacity to bind developmental endothelial locus-1 (Del-1). In summary, CHD was associated with distinct changes in circulating miRNA transport and these changes may contribute to the abnormal intercellular communication that underlies CHD initiation and progression.
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.
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.
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.
Coronary artery disease (CAD) is the largest killer of males and females in the United States. There is a need to develop innovative diagnostic markers for this disease. MicroRNAs (miRNAs) are a class of noncoding RNAs that posttranscriptionally regulate the expression of genes involved in important cellular processes, and we hypothesized that the miRNA expression profile would be altered in whole blood samples of patients with CAD. We performed a microarray analysis on RNA from the blood of 5 male subjects with CAD and 5 healthy subjects (mean age 53 years). Subsequently, we performed qRT-PCR analysis of miRNA expression in whole blood of another 10 patients with CAD and 15 healthy subjects. We identified 11 miRNAs that were significantly downregulated in CAD subjects (P < . 0 5). Furthermore, we found an association between ACEI/ARB use and downregulation of several miRNAs that was independent of the presence of significant CAD. In conclusion, we have identified a distinct miRNA signature in whole blood that discriminates CAD patients from healthy subjects. Importantly, medication use may significantly alter miRNA expression. These findings may have significant implications for identifying and managing individuals that either have CAD or are at risk of developing the disease.