• 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.

Small extracellular vesicles are released from both healthy and disease cells to facilitate cellular communication. They have a wide variety of names including exosomes, microvesicles and microparticles. Depending on their size, very small extracellular vesicles originating from the endocytic pathway have been called exosomes and in some cases nanovesicles. Collectively, extracellular vesicles are important mediators of a wide variety of functions including immune cell development and homeostasis. Encapsulated in the extracellular vesicles are proteins and nucleic acids including mRNA and microRNA molecules. MicroRNAs are small, non-coding RNA molecules implicated in the post-transcriptional control of gene expression that have emerged as important regulatory molecules and are involved in disease pathogenesis including cancer. In some diseases, not only does the quantity and the subpopulations of extracellular vesicles change in the peripheral blood but also microRNAs. Here, we described the analysis of peripheral blood extracellular vesicles by flow cytometry and the RNA extraction from extracellular vesicles isolated from the plasma or serum to profile microRNA expression.

Extracellular vesicles (EVs) are mRNA‐containing cell fragments shed into circulation during pathophysiological events. DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) regulate gene expression by modifying DNA methylation and altering transcription. Sepsis is a systemic insult resulting in vascular dysfunction, which can lead to shock and death. We analysed plasma from ICU patients for circulating EV numbers, defined as particles isolated from 1 mL plasma at 21,000xg, and DNMTs mRNA content as prognostic markers of septic shock. Compared to plasma from critically ill patients with or without sepsis, plasma from septic shock patients contained more EVs per mL, expressed as total DNMTs mRNAs over 5 days, and more individual DNMT mRNAs at each day. A comparison of EV‐DNMT1 (maintenance methylation) with EV‐DNMT3A+DNMT3B (de novo methylation) expression correlated highly with severity, and EVs from septic shock patients carried more total DNMT mRNAs and more DNMT3A+DNMT3B mRNAs than control or sepsis EVs. Total plasma EVs also correlated with sepsis severity. EV‐DNMT mRNAs load, when coupled with total plasma EV number, may be a novel method to diagnose septic shock upon ICU admittance and offer opportunities to more precisely intervene with standard therapy or other targeted interventions to regulate EV release and/or specific DNMT activity.

No abstract