Kirsty Danielson

Circulating MicroRNA-30d Is Associated With Response to Cardiac Resynchronization Therapy in Heart Failure and Regulates Cardiomyocyte Apoptosis A Translational Pilot Study

Background— Biomarkers that predict response to cardiac resynchronization therapy (CRT) in heart failure patients with dyssynchrony (HFDYS) would be clinically important. Circulating extracellular microRNAs (miRNAs) have emerged as novel biomarkers that may also play important functional roles, but their relevance as markers for CRT response has not been examined. Methods and Results— Comprehensive miRNA polymerase chain reaction arrays were used to assess baseline levels of 766 plasma miRNAs in patients undergoing clinically indicated CRT in an initial discovery set (n=12) with and without subsequent echocardiographic improvement at 6 months after CRT. Validation of candidate miRNAs in 61 additional patients confirmed that baseline plasma miR-30d was associated with CRT response (defined as an increase in left ventricular ejection fraction ≥10%). MiR-30d was enriched in coronary sinus blood and increased in late-contracting myocardium in a canine model of HFDYS, indicating cardiac origin with maximal expression in areas of high mechanical stress. We examined the functional effects of miR-30d in cultured cardiomyocytes and determined that miR-30d is expressed in cardiomyocytes and released in vesicles in response to mechanical stress. Overexpression of miR-30d in cultured cardiomyocytes led to cardiomyocyte growth and protected against apoptosis by targeting the mitogen-associated kinase 4, a downstream effector of tumor necrosis factor. In HFDYS patients, miR-30d plasma levels inversely correlated with high-sensitivity troponin T, a marker of myocardial necrosis. Conclusions— Baseline plasma miR-30d level is associated with response to CRT in HFDYS in this translational pilot study. MiR-30d increase in cardiomyocytes correlates with areas of increased wall stress in HFDYS and is protective against deleterious tumor necrosis factor signaling.

Diverse human extracellular RNAs are widely detected in human plasma

There is growing appreciation for the importance of non-protein-coding genes in development and disease. Although much is known about microRNAs, limitations in bioinformatic analyses of RNA sequencing have precluded broad assessment of other forms of small-RNAs in humans. By analysing sequencing data from plasma-derived RNA from 40 individuals, here we identified over a thousand human extracellular RNAs including microRNAs, piwi-interacting RNA (piRNA), and small nucleolar RNAs. Using a targeted quantitative PCR with reverse transcription approach in an additional 2,763 individuals, we characterized almost 500 of the most abundant extracellular transcripts including microRNAs, piRNAs and small nucleolar RNAs. The presence in plasma of many non-microRNA small-RNAs was confirmed in an independent cohort. We present comprehensive data to demonstrate the broad and consistent detection of diverse classes of circulating non-cellular small-RNAs from a large population.

Meeting report: Discussions and preliminary findings on extracellular RNA measurement methods from laboratories in the NIH Extracellular RNA Communication Consortium

Extracellular RNAs (exRNAs) have been identified in all tested biofluids and have been associated with a variety of extracellular vesicles, ribonucleoprotein complexes and lipoprotein complexes. Much of the interest in exRNAs lies in the fact that they may serve as signalling molecules between cells, their potential to serve as biomarkers for prediction and diagnosis of disease and the possibility that exRNAs or the extracellular particles that carry them might be used for therapeutic purposes. Among the most significant bottlenecks to progress in this field is the lack of robust and standardized methods for collection and processing of biofluids, separation of different types of exRNA-containing particles and isolation and analysis of exRNAs. The Sample and Assay Standards Working Group of the Extracellular RNA Communication Consortium is a group of laboratories funded by the U.S. National Institutes of Health to develop such methods. In our first joint endeavour, we held a series of conference calls and in-person meetings to survey the methods used among our members, placed them in the context of the current literature and used our findings to identify areas in which the identification of robust methodologies would promote rapid advancements in the exRNA field.

Extracellular Vesicles in Heart Disease: Excitement for the Future ?

Extracellular vesicles (EV), including exosomes, microvesicles and apoptotic bodies, are released from numerous cell types and are involved in intercellular communication, physiological functions and the pathology of disease. They have been shown to carry and transfer a wide range of cargo including proteins, lipids and nucleic acids. The role of EVs in cardiac physiology and heart disease is an emerging field that has produced intriguing findings in recent years. This review will outline what is currently known about EVs in the cardiovascular system, including cellular origins, functional roles and utility as biomarkers and potential therapeutics.

Diurnal Variations of Circulating Extracellular Vesicles Measured by Nano Flow Cytometry

The identification of extracellular vesicles (EVs) as intercellular conveyors of biological information has recently emerged as a novel paradigm in signaling, leading to the exploitation of EVs and their contents as biomarkers of various diseases. However, whether there are diurnal variations in the size, number, and tissue of origin of blood EVs is currently not known, and could have significant implications when using EVs as biomarkers for disease progression. Currently available technologies for the measurement of EV size and number are either time consuming, require specialized equipment, or lack sufficient accuracy across a range of EV sizes. Flow cytometry represents an attractive alternative to these methods; however, traditional flow cytometers are only capable of measuring particles down to 500 nm, which is significantly larger than the average and median sizes of plasma EVs. Utilizing a Beckman Coulter MoFlo XDP flow cytometer with NanoView module, we employed nanoscale flow cytometry (termed nanoFCM) to examine the relative number and scatter distribution of plasma EVs at three different time points during the day in 6 healthy adults. Analysis of liposomes and plasma EVs proved that nanoFCM is capable of detecting biologically-relevant vesicles down to 100 nm in size. With this high resolution configuration, we observed variations in the relative size (FSC/SSC distributions) and concentration (proportions) of EVs in healthy adult plasma across the course of a day, suggesting that there are diurnal variations in the number and size distribution of circulating EV populations. The use of nanoFCM provides a valuable tool for the study of EVs in both health and disease; however, additional refinement of nanoscale flow cytometric methods is needed for use of these instruments for quantitative particle counting and sizing. Furthermore, larger scale studies are necessary to more clearly define the diurnal variations in circulating EVs, and thus further inform their use as biomarkers for disease.

Diet and adipose tissue distributions: The Multi-Ethnic Study of Atherosclerosis

BACKGROUND AND AIMS Dietary quality affects cardiometabolic risk, yet its pathways of influence on regional adipose tissue depots involved in metabolic and diabetes risk are not well established. We aimed to investigate the relationship between dietary quality and regional adiposity. METHODS AND RESULTS We investigated 5079 individuals in the Multi-Ethnic Study of Atherosclerosis (MESA) who had food-frequency questionnaires and measurement of pericardial fat and hepatic attenuation at the baseline study visit in MESA, as well as a subgroup with imaging for visceral and subcutaneous fat (N = 1390). A dietary quality score (DietQuality) was constructed to include established food group constituents of a Mediterranean-type diet. Linear models estimated associations of dietary score as well as its constituents with regional adiposity. Baseline mean age was 61 (± 10) years, and approximately half of the participants (47%) were male. Those with a higher DietQuality score were generally older, female, with a lower body mass index, C-reactive protein, and markers of insulin resistance. After adjustment, a higher DietQuality score was associated with lower visceral fat (lowest vs. highest dietary score quartile: 523.6 vs. 460.5 cm(2)/m; P < 0.01 for trend), pericardial fat (47.5 vs. 41.3 cm(3)/m; P < 0.01 for trend), lesser hepatic steatosis (by hepatic attenuation; 58.6 vs. 60.7 Hounsfield units; P < 0.01 for trend), but not subcutaneous fat (P = 0.39). Greater fruits, vegetables, whole grains, seeds/nuts and yogurt intake were associated with decreased adiposity, while red/processed meats were associated with greater regional adiposity. CONCLUSION A higher quality diet pattern is associated with less regional adiposity, suggesting a potential mechanism of beneficial dietary effects on diabetes, metabolic, and cardiovascular risk.

High Throughput Sequencing of Extracellular RNA from Human Plasma.

The presence and relative stability of extracellular RNAs (exRNAs) in biofluids has led to an emerging recognition of their promise as ‘liquid biopsies’ for diseases. Most prior studies on discovery of exRNAs as disease-specific biomarkers have focused on microRNAs (miRNAs) using technologies such as qRT-PCR and microarrays. The recent application of next-generation sequencing to discovery of exRNA biomarkers has revealed the presence of potential novel miRNAs as well as other RNA species such as tRNAs, snoRNAs, piRNAs and lncRNAs in biofluids. At the same time, the use of RNA sequencing for biofluids poses unique challenges, including low amounts of input RNAs, the presence of exRNAs in different compartments with varying degrees of vulnerability to isolation techniques, and the high abundance of specific RNA species (thereby limiting the sensitivity of detection of less abundant species). Moreover, discovery in human diseases often relies on archival biospecimens of varying age and limiting amounts of samples. In this study, we have tested RNA isolation methods to optimize profiling exRNAs by RNA sequencing in individuals without any known diseases. Our findings are consistent with other recent studies that detect microRNAs and ribosomal RNAs as the major exRNA species in plasma. Similar to other recent studies, we found that the landscape of biofluid microRNA transcriptome is dominated by several abundant microRNAs that appear to comprise conserved extracellular miRNAs. There is reasonable correlation of sets of conserved miRNAs across biological replicates, and even across other data sets obtained at different investigative sites. Conversely, the detection of less abundant miRNAs is far more dependent on the exact methodology of RNA isolation and profiling. This study highlights the challenges in detecting and quantifying less abundant plasma miRNAs in health and disease using RNA sequencing platforms.

Extracellular RNAs Are Associated With Insulin Resistance and Metabolic Phenotypes

OBJECTIVE Insulin resistance (IR) is a hallmark of obesity and metabolic disease. Circulating extracellular RNAs (ex-RNAs), stable RNA molecules in plasma, may play a role in IR, though most studies on ex-RNAs in IR are small. We sought to characterize the relationship between ex-RNAs and metabolic phenotypes in a large community-based human cohort. RESEARCH DESIGN AND METHODS We measured circulating plasma ex-RNAs in 2,317 participants without diabetes in the Framingham Heart Study (FHS) Offspring Cohort at cycle 8 and defined associations between ex-RNAs and IR (measured by circulating insulin level). We measured association between candidate ex-RNAs and markers of adiposity. Sensitivity analyses included individuals with diabetes. In a separate cohort of 90 overweight/obese youth, we measured selected ex-RNAs and metabolites. Biology of candidate microRNAs was investigated in silico. RESULTS The mean age of FHS participants was 65.8 years (56% female), with average BMI 27.7 kg/m2; participants in the youth cohort had a mean age of 15.5 years (60% female), with mean BMI 33.8 kg/m2. In age-, sex-, and BMI-adjusted models across 391 ex-RNAs in FHS, 18 ex-RNAs were associated with IR (of which 16 were microRNAs). miR-122 was associated with IR and regional adiposity in adults and IR in children (independent of metabolites). Pathway analysis revealed metabolic regulatory roles for miR-122, including regulation of IR pathways (AMPK, target of rapamycin signaling, and mitogen-activated protein kinase). CONCLUSIONS These results provide translational evidence in support of an important role of ex-RNAs as novel circulating factors implicated in IR.

Potential functional applications of extracellular vesicles: a report by the NIH Common Fund Extracellular RNA Communication Consortium

The NIH Extracellular RNA Communication Programs initiative on clinical utility of extracellular RNAs and therapeutic agents and developing scalable technologies is reviewed here. Background information and details of the projects are presented. The work has focused on modulation of target cell fate by extracellular vesicles (EVs) and RNA. Work on plant-derived vesicles is of intense interest, and non-mammalian sources of vesicles may represent a very promising source for different therapeutic approaches. Retro-viral-like particles are intriguing. Clearly, EVs share pathways with the assembly machinery of several other viruses, including human endogenous retrovirals (HERVs), and this convergence may explain the observation of viral-like particles containing viral proteins and nucleic acid in EVs. Dramatic effect on regeneration of damaged bone marrow, renal, pulmonary and cardiovascular tissue is demonstrated and discussed. These studies show restoration of injured cell function and the importance of heterogeneity of different vesicle populations. The potential for neural regeneration is explored, and the capacity to promote and reverse neoplasia by EV exposure is described. The tremendous clinical potential of EVs underlies many of these projects, and the importance of regulatory issues and the necessity of general manufacturing production (GMP) studies for eventual clinical trials are emphasized. Clinical trials are already being pursued and should expand dramatically in the near future.

miRNA Signatures of Insulin Resistance in Obesity

Extracellular microRNAs (miRNAs) represent functional biomarkers for obesity and related disorders; this study investigated plasma miRNAs in insulin resistance phenotypes in obesity.