In this study, we hypothesized that miRNAs circulate in plasma microvesicles, platelets, and PBMC of normal human volunteers in the peripheral blood. We have characterized peripheral blood miRNA patterns in healthy humans. We found significant differences in miRNA expression between plasma microvesicles, platelets, and PBMC. To date, numerous studies have established the ability for miRNAs to regulate many cellular functions. However, these studies largely imply that the miRNA resides within its host cell to elicit an effect 
. Our data indicate that the miRNAs are also contained in plasma microvesicles that may influence homeostasis.
The defined mechanism or cellular signals that regulate microvesicle production is unknown. Some suggest that the vesicle “hijacks” the cytoplasm prior to release from the cell through membrane blebbing 
. This paradigm suggests that packaging of the miRNAs in the microvesicle may be random. Alternatively, endosomal trafficking may modulate the formation of microvesicles 
. Since P-bodies in the cytoplasm co-localized with miRNAs 
, we are exploring whether P-bodies may regulate the transfer and sorting of miRNAs into vesicles. There are other reports of tumor cell fragments containing miRNAs in the plasma and/or serum 
. Recently Taylor and colleagues found circulating tumor microvesicles in the peripheral blood of patients with ovarian cancer but not healthy individuals 
. These microvesicles express miRNAs found in the primary tumor.
It is conceivable that the miRNAs in the plasma microvesicles circulate to tissue targets. Further examination of the highest expressed miRNAs in plasma microvesicles (), demonstrate that many of these miRNA are predicted to regulate hematopoiesis and cellular differentiation. For instance, expression of miR-223 is predicted to influence myeloid, granulocyte and osteoclast differentiation 
. miR-223 also appears to have a role in hematopoietic stem cell proliferation 
. Interestingly, miR-223 expression is lost in acute myelogenous leukemia (AML) 
. miR-24 expression is regulated by TGF-β, a potent positive and negative regulator of hematopoiesis 
. Both miR-24 and miR-16 regulate red cell production 
, while miR-16 also modulates lymphoid development 
. Loss of miR-16 expression has been extensively examined in chronic lymphocytic leukemia (CLL) 
. In contrast, downregulation of miR-126 expression occurs during megakarocyte differentiation 
Many miRNAs expressed in the plasma microvesicles also regulate the progression of the cell cycle proteins 
. miR-222 targets p27Kip1 
while miR-24 suppresses p16 (INK4a) 
. Increased expression of miR-16 results in the accumulation of cells in G0/G1 phase of the cell cycle 
To date, there are no known functions for miR-484 which is the second highest expressed miRNA in the plasma microvesicles fraction based on normalized expression (). Using IPA analysis, miR-484 appears to regulate hematopoiesis, as well as cellular differentiation, proliferation and growth similar to many of the highly expressed miRNAs in the plasma microvesicles. Another of the highly expressed miRNAs, miR-146a is predicted to modulate NK cell signaling and IL-4 signaling pathways. Similarly, miR-146a is also predicted to regulate immune functions 
. Based on IPA analysis examining gene ontology of targets, the top associated networks predicted to be influenced by miR-146a expression are cellular proliferation, immune and lymphatic system development and function. In addition, this miRNA is predicted to regulate innate immune responses. From the analysis, we found that LPS/IL-1 and toll-like receptor signaling are among the top five canonical pathways predicted to be regulated by miR-146a.
Comparison of differentially expressed miRNAs between the plasma microvesicles and the PBMC indicated that miR-486 was the highest differentially expressed in the plasma microvesicles (). Little is known about this miRNA, except that it is expressed in glioblastoma cells and may function in brain cell differentiation 
. Analyzing the predicted targets from Sanger miRBase with the IPA software, the top five canonical pathways predicted to be regulated by miR-486 are those in phenylalanine and cyanoamino acid metabolism, insulin receptor signaling, antigen presentation, and pentose phosphate pathways. Another miRNA differentially expressed in the plasma microvesicles is miR-328. Recently, Wang and colleagues reported that miR-328 is important in regulating tumor development and invasion by targeting CD44 expression in epithelial carcinoma cell lines 
. Overexpression of miR-328 inhibits cell migration and adhesion and preventing them to expand into the tumor.
The majority of the plasma microvesicles from normal individuals are derived from blood cells. We did detect a small percentage of microvesicles derived from endothelial cells. While human endothelial progenitor cells can produce microvesicles in vitro 
, detection of the endothelial derived vesicles in circulation is of interest. We would predict that the endothelial-derived microvesicles may increase upon endothelial cell damage. Recently, elevation of endothelial-derived microvesicles has been reported in patients with pulmonary arterial hypertension 
, however their content is currently unknown. Likewise, the detection of tissue specific miRNAs and microvesicles in the peripheral blood may be a frequent event upon tissue damage. The detection of tumors exosomes (microvesicles) have been found in the peripheral blood to contain miRNAs 
. Interestingly, comparing this paper's findings with our data, 18 elevated tumor-derived miRNAs were not detected in the plasma from our normal donors (Table S1
While it has been reported that miRNAs are detected in the plasma 
, this is the first study to profile miRNAs from the plasma microvesicles in a normal healthy population. Since our study was limited to Caucasians, we do not know the influence of race or ethnicity. In this study, we also examined whether miRNA expression in the plasma microvesicles was influenced by age. Surprisingly, we observed no difference between gender and age in our study. These are important factors as we and others examine miRNA expression from the plasma and/or serum in various diseases to serve as diagnostic biomarkers.
Our findings indicate that miRNAs circulate in the plasma and PBMC of normal donors. These miRNAs are predicted to regulate homeostasis of hematopoietic cells and of metabolic function. Investigations are currently underway to understand if specific cells are targeted by the plasma microvesicles and determine the influence of these microvesicle-containing miRNA on target cell mRNA expression. We predict that the plasma microvesicles may be selective in their target cells. For instances, platelet-derived microparticles transfer tissue factor to monocytes but not neutrophils 
. Thus, we predict that the defined function of each subpopulation of the plasma microvesicles and their miRNAs will be important factors in the regulation of immune responses and hematopoiesis.