The emergence of publications on extracellular RNA (exRNA) and extracellular vesicles (EV) has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV) in New York City in October 2012. As part of the “ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA)”, 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments.

Vesiclepedia is a community-annotated compendium of molecular data on extracellular vesicles.

• Macrophage-derived microvesicles induced cellular differentiation in naive monocytes.• Macrophage-derived microvesicles shuttle of miRNAs to target cells.Microvesicles are small membrane-bound particles comprised of exosomes and varioussized extracellular vesicles. These are released by several cell types. Microvesicles have a variety of cellular functions from communication to mediating growth and differentiation. Microvesicles contain proteins and nucleic acids. Previously, we showed that plasma microvesicles contain microRNAs (miRNAs). Based on our previous report, the majority of peripheral blood microvesicles are derived from platelets, while mononuclear phagocytes, including macrophages, are the second most abundant population. Here, we characterized macrophage-derived microvesicles and explored their role in the differentiation of naive monocytes. We also identified the miRNA content of the macrophage-derived microvesicles. We found that RNA molecules contained in the macrophage-derived microvesicles were transported to target cells, including mono cytes, endothelial cells, epithelial cells, and fibroblasts. Furthermore, we found that miR-223 was transported to target cells and was functionally active. Based on our observations, we hypothesize that microvesicles bind to and activate target cells. Furthermore, we find that microvesicles induce the differentiation of macrophages. Thus, defining key components of this response may identify novel targets to regulate host defense and inflammation. (Blood. 2013;121(6):984-995)

MicroRNAs (miRNAs) have emerged as important regulators in the post-transcriptional control of gene expression. The discovery of their presence not only in tissues but also in extratissular fluids, including blood, urine and cerebro-spinal fluid, together with their changes in expression in various pathological conditions, has implicated these extracellular miRNAs as informative biomarkers of disease. However, exploiting miRNAs in this capacity requires methodological rigour. Here, we report several key procedural aspects of miRNA isolation from plasma and serum, as exemplified by research in cardiovascular and pulmonary diseases. We also highlight the advantages and disadvantages of various profiling methods to determine the expression levels of plasma-and serum-derived miRNAs. Attention to such methodological details is critical, as circulating miRNAs become diagnostic tools for various human diseases.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a disease of progressive lung fibrosis with a high mortality rate. In organ repair and remodeling, epigenetic events are important. MicroRNAs (miRNAs) regulate gene expression post-transcriptionally and can target epigenetic molecules important in DNA methylation. The miR-17z92 miRNA cluster is critical for lung development and lung epithelial cell homeostasis and is predicted to target fibrotic genes and DNA methyltransferase (DNMT)-1 expression. Objectives: We investigated the miR-17z92 cluster expression and its role in regulating DNA methylation events in IPF lung tissue. Methods: Expression and DNA methylation patterns of miR-17z92 were determined in human IPF lung tissue and fibroblasts and fibrotic mouse lung tissue. The relationship between the miR-17z92 cluster and DNMT-1 expression was examined in vitro. Using a murine model of pulmonary fibrosis, we examined the therapeutic potential of the demethylating agent, 59-aza-29-deoxycytidine. Measurements and Main Results: Compared with control samples, miR17z92 expression was reduced in lung biopsies and lung fibroblasts from patients with IPF, whereas DNMT-1 expression and methylation of the miR-17z92 promoter was increased. Several miRNAs from the miR-17z92 cluster targeted DNMT-1 expression resulting in a negative feedback loop. Similarly, miR-17z92 expression was reduced in the lungs of bleomycin-treated mice. Treatment with 59-aza-29-deoxycytidine in a murine bleomycin-induced pulmonary fibrosis model reduced fibrotic gene and DNMT-1 expression, enhanced miR-17z92 cluster expression, and attenuated pulmonary fibrosis. Conclusions: This study provides insight into the pathobiology of IPF and identifies a novel epigenetic feedback loop between miR-17z92 and DNMT-1 in lung fibrosis.Keywords: microRNA; miR-17z92; pulmonary fibrosis; DNA methylation; DNMT-1 Idiopathic pulmonary fibrosis (IPF) represents the most aggressive form of interstitial lung disease with a median survival of 3-5 years (1). Failure to resolve epithelial cell injury in the lung is critical to the pathogenesis of IPF (2-4). In addition, epithelialmesenchymal transition (EMT) (5), fibroblast proliferation and activation (6), and recruitment of inflammatory cells (7,8) all contribute to extracellular matrix accumulation in the lung (7). The current study focused on identifying the molecular mechanisms underlying the pathogenesis of IPF.Because changes in fibrotic gene expression (2, 9-11) and few genetic mutations have been identified in IPF (12, 13), we focused on microRNA (miRNA, miR) expression and epigenetic regulators in lung epithelial cells and fibroblasts. MiRNAs can either block translation or degrade target mRNAs (14,15). Notably, a single miRNA can regulate upward of 30 genes. MiRNAs can be encoded in intronic or exonic DNA regions and encoded in their own open reading frame and controlled by DNA promoter elements, such as DNA methylation by DNA methyltransferases (DNMTs) of CpG islands (15,16). Of the three DNMTs expressed in h...