BackgroundMicroRNAs (miRNA) are small non-coding RNAs that regulate translation of mRNA and protein. Loss or enhanced expression of miRNAs is associated with several diseases, including cancer. However, the identification of circulating miRNA in healthy donors is not well characterized. Microvesicles, also known as exosomes or microparticles, circulate in the peripheral blood and can stimulate cellular signaling. In this study, we hypothesized that under normal healthy conditions, microvesicles contain miRNAs, contributing to biological homeostasis.Methodology/Principal FindingsMicrovesicles were isolated from the plasma of normal healthy individuals. RNA was isolated from both the microvesicles and matched mononuclear cells and profiled for 420 known mature miRNAs by real-time PCR. Hierarchical clustering of the data sets indicated significant differences in miRNA expression between peripheral blood mononuclear cells (PBMC) and plasma microvesicles. We observed 71 miRNAs co-expressed between microvesicles and PBMC. Notably, we found 33 and 4 significantly differentially expressed miRNAs in the plasma microvesicles and mononuclear cells, respectively. Prediction of the gene targets and associated biological pathways regulated by the detected miRNAs was performed. The majority of the miRNAs expressed in the microvesicles from the blood were predicted to regulate cellular differentiation of blood cells and metabolic pathways. Interestingly, a select few miRNAs were also predicted to be important modulators of immune function.ConclusionsThis study is the first to identify and define miRNA expression in circulating plasma microvesicles of normal subjects. The data generated from this study provides a basis for future studies to determine the predictive role of peripheral blood miRNA signatures in human disease and will enable the definition of the biological processes regulated by these miRNA.

• 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)

Background: MicroRNAs (miRNA) are small non-coding RNAs that regulate translation of mRNA and protein. Loss or enhanced expression of miRNAs is associated with several diseases, including cancer. However, the identification of circulating miRNA in healthy donors is not well characterized. Microvesicles, also known as exosomes or microparticles, circulate in the peripheral blood and can stimulate cellular signaling. In this study, we hypothesized that under normal healthy conditions, microvesicles contain miRNAs, contributing to biological homeostasis.Methodology/Principal Findings: Microvesicles were isolated from the plasma of normal healthy individuals. RNA was isolated from both the microvesicles and matched mononuclear cells and profiled for 420 known mature miRNAs by realtime PCR. Hierarchical clustering of the data sets indicated significant differences in miRNA expression between peripheral blood mononuclear cells (PBMC) and plasma microvesicles. We observed 71 miRNAs co-expressed between microvesicles and PBMC. Notably, we found 33 and 4 significantly differentially expressed miRNAs in the plasma microvesicles and mononuclear cells, respectively. Prediction of the gene targets and associated biological pathways regulated by the detected miRNAs was performed. The majority of the miRNAs expressed in the microvesicles from the blood were predicted to regulate cellular differentiation of blood cells and metabolic pathways. Interestingly, a select few miRNAs were also predicted to be important modulators of immune function. Conclusions:This study is the first to identify and define miRNA expression in circulating plasma microvesicles of normal subjects. The data generated from this study provides a basis for future studies to determine the predictive role of peripheral blood miRNA signatures in human disease and will enable the definition of the biological processes regulated by these miRNA.

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Microvesicles (MV) (also know as exosomes) are small membrane-bound vesicles released by numerous cell types that contain proteins, mRNA and microRNA. We found that MV from activated monocytes drove survival and differentiation in naïve cells. We therefore were interested in understanding the content of MV produced by activated mononuclear phagocytes. Purified peripheral blood monocytes were treated in vitro for 24 h with or without the monocyte survival factors, GM-CSF or M-CSF, respectively. Examination of monocytes and macrophages by electron microscopy or culture supernatants by flow cytometry demonstrated that monocytes produced MV, which quantitatively increased upon differentiation. Treatment with GM-CSF resulted in more MV production than M-CSF-treated monocytes. To examine whether MV from differentiated cells induced myeloid maturation, the MV were collected and added to fresh monocytes; only MV derived from GM-CSF treated cells induced differentiation of naïve monocytes into macrophages. We next hypothesized that expression of microRNA contained in the MV modulated differentiation of monocytes. Profiling of MV from GM-CSF and M-CSF derived macrophages revealed only two significantly expressed microRNAs. We found that mir-155 was significantly elevated by two-fold in MV from GM-CSF-treated cells, while mir-340 was significantly increased seven-fold in M-CSF-derived MV. Notably, mir- 223 was the highest expressed microRNA in MV from both GM-CSF and M-CSF-treated cells. Recent data suggest that expression of mir-223 regulates myeloid, granulocytic and osteoclasts differentiation, and has a role in hematopoietic stem cell proliferation. While mir-223 is present in MV from both GM-CSF and M-CSF treated cells, it is possible that the low abundance of MV produce from M-CSF-treated cells resulted in less effective concentration to induce differentiation. In this model, it is also possible that regulation of proteins targeted by the increase in mir-155 and decrease mir-340 in the GM-CSF-derived MV are responsible for myeloid differentiation. Since changes in microRNA expression including mir-223 has been reported in AML, our data suggest that myeloid-derived MV in the peripheral blood containing mir-223 may be altered contributing to leukemogenesis.