Microvesicles (MVs) are fragments of plasma membrane ranging from 50 nm to 1000 nm shed from almost all cell types. Besides soluble factors, MVs have been recently described as a new mechanism of cell to cell communication. In particular, MVs play a role in intercellular signals through their capacity to mediate the exchange of mRNA, microRNAs, and proteins between cells. MVs represent a heterogeneous population , differing in cellular origin, number, size and antigenic composition.
Electron microscopy of microvesicles isolated from urine of healthy donor and a prostate cancer patient.
The identification of small vesicles released by many, if not all, cell types as mechanism of cell cross-talk wasn’t a new item and it was proposed a few decades ago (Tissue factor in microvesicles shed from U87MG human glioblastoma cells induces coagulation, platelet aggregation, and thrombogenesis,1984).
Microvesicles originate directly from the plasma membrane of the cell and reflect the antigenic content of the cells from which they derive. Two vesicle-release processes were identified, each leading to the release of distinct types of signaling vesicle: the exocytosis of multivesicular bodies (MVBs), resulting in exosomes release (vesicles within the MVB lumen); and the direct budding from the plasma membrane of small vesicles (shedding vesicles) (Shedding microvesicles: artefacts no more, 2009).
Schematic representation of the two mechanisms of microvesicle release from cells.
- Exosome generation from multivesicular bodies
- Shedding vesicles bud off the plasma membrane in response to cell stimulation
The release of shedding vesicles and exosomes from the cells results to be mediate by different mechanisms.
In particular, shedding vesicle extrusion is preceded by the budding of small cytoplasmic protrusions, which then detach by fission of their stalk (Enlargeosome traffic: exocytosis triggered by various signals is followed by endocytosis, membrane shedding or both, 2007). The mechanisms of shedding vesicle sorting process remain obscure. Studies with inhibitors of cholesterol synthesis have indicated the crucial involvement of cholesterol-rich microdomains of the plasma membrane, the lipid rafts (Tissue-factor-bearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation, 2005). These microdomains, however, are also involved in the biogenesis of exosomes and cannot be considered a distinction between the two types of microvesicles.
Some release of shedding vesicles takes place from resting cells, however, the rate of the process increases dramatically upon stimulation. Ca2+ is known to induce strong shedding responses (Emission of membrane vesicles: roles in complement resistance, immunity and cancer, 2005);(Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication, 2006) in various cell types, such as dendritic cells and microglia. However, Ca2+ is not the only second messenger involved. In various cell types, in fact, phorbol ester activation of protein kinase C (PKC) is also effective. In particular, PC12 cells released shedding vesicles in response to phorbol esters and not of Ca2+ administration. Moreover, the purinergic receptors of ATP, a ligand released by many cell types, have also an important role. In dendritic cells, macrophages and microglia, activation of the purinergic receptor-channel, P2X7, was found to induce intense release (Astrocyte-derived ATP induces vesicle shedding and IL-1 b release from microglia, 2005); (Rapid secretion of interleukin-1b by microvesicle shedding, 2001); (Secretion of intracellular IL-1 receptor antagonist (type 1) is dependent on P2X7 receptor activation, 2004). In other cell types (such as PC12 and platelets), activation of the P2Y receptors coupled with the Gq protein was found to be effective.
On the other hand, the mechanism of exosome extrusion was associated to MVBs that fuse their external membrane with the plasma membrane of the cell, with the release of their segregated vesicles to the extracellular space. Moreover, the generation of exosomes, with budding, fission and segregation occurring within the MVB lumen and is governed by specific processes.
An important role for the multiprotein complex, Endosomal Sorting Complexes Required for Transport (ESCRTs), has been described in the mechanism of vesicle accumulation addressed to lysosomal degradation. Recently, the metabolism of sphingomyelin to ceramide has been shown to induce the endosome sorting into the exocytic MVBs in an oligodendrocyte cell line (Ceramide triggers budding of exosome vesicles into multivesicular endosomes, 2008) As a consequence of this sorting, specific proteins and lipids of the MVB external membrane are enriched in the exosomes, whereas others are excluded.
Also for the exosomes, the mechanism of their discharge from the cells, remain not completely understood; and it take place not only as a constitutive cell process but may be regulated by specific intracellular signals stimulating the MVB exocytosis.
Communication between cells originally was proposed to be mediated only by molecules, such as neurotransmitters and hormones, released (or surface-exposed) by one cell and recognize by the other through receptor binding. In fact, MVs were originally considered to be inert cellular debris. In particular, vesicles observed through electron microscopy in the intercellular space were assumed to result from damage, such as necrosis, of the surrounding cells. An understanding of the nature of these structures changed when the release of small vesicles was associated to specific cell answer to different stimuli. Moreover, it is now recognized that MVs may interact with target cells through specific receptor-ligand interactions, leading to cell stimulation through the transfer of genetic information and functional proteins contained inside MVs.
In particular, upon release from their cell of origin, it seems that microvesicles do not interact with just any cell they come into contact with but, rather, only with cells that they recognize specifically. For example in the complex crosstalk between blood cells, the vesicles shed from platelets interact with macrophages and endothelial cells, but not with neutrophils; those from neutrophils interact with platelets, macrophages and dendritic cells.
Another important thing is the nature of the interaction with the target cell.
Three type of interaction have been described:
- Receptor binding and intracellular signal transduction;
- Direct fusion of the shedding vesicle with the plasma membrane of the target cells;
- Endocytic uptake of the vesicle, which can be regulated differently in various types of cell.
Microvesicles have been implicated in many cellular processes, such as angiogenesis, tumor and metastasis progression, cancer immune suppression, tumor-stroma interactions, tissue regeneration and they are potentially involved in the stem cell biology and biological actions.
The first step in MV uptake was mediated by receptor-ligand interactions. This mechanism is not completely understood and it may vary based on target cells or on MV cell source.
Recently, Bruno et al., have demonstrated as MV from bone marrow mesenchymal stem cells were incorporated by cultured tubular epithelial cells (TECs) and the MV treatment with soluble hyaluronic acid and anti-CD44 and -CD29 blocking antibodies inhibited MV incorporation in TEC, suggesting that their expression on MV surface is critical for MV internalization. Moreover, removal of surface molecules by trypsin treatment of MVs inhibited their incorporation in TECs, confirming the relevance of surface molecules in MV internalization.
The concept of MVs as signals was based on the idea that MV are released by different cell types in stress conditions, able to enhance the number of MV release and probably the MV content. Moreover, MVs contained signs (mRNA, miRNAs an proteins) that may be used only by target cells able to react though their capacity to uptake MVs.
The first MVs identified as mediators of a physiological process were those released by platelets. Over 20 years ago, these vesicles, which were then referred to as MVs were proposed to provide a membrane surface for the assembly and dissemination of pro coagulant enzyme complexes.
In particular, blood coagulation is the result of the synergism of four types of cells, that worked through the release of shedding vesicles. The first event in this cascade was the aggregation of platelets, induced by a wound of the vessel wall, that is accompanied by the shedding from the platelets of vesicles, coated by tissue factor (TF), the initiator of coagulation and thrombosis, which interact with surface ligands, such as P-selectin1, on macrophages, endothelia and platelets themselves. Shedding vesicles released by macrophages and neutrophils also have a role at this stage. In particular, the shedding vesicles released by stimulated neutrophils are highly enriched with the activated integrin Mac-1 and are, therefore, particularly effective in inducing platelet activation. Endothelial cells also release shedding vesicles addressed to macrophages and platelets.
This enables the entire coagulation system to proceed on the restricted surface of platelets, rather than in the blood fluid phase (Figure 3).
Mechanism of action of vesicle released by blood cells in the coagulation process. MV-mediated cross-talk among macrophages, neutrophils and platelets triggers coagulation (Shedding microvesicles: artefacts no more, 2009).
Microvesicles have been described to have a role not only in physiological but also in pathological conditions, such as tumor progression, inflammation and tissue damage. MVs from blood cells have a role in atherosclerosis and stroke. In particular, MVs from platelets and macrophages accumulated in the lipid core of plaques and thrombi, may alter signaling between circulating cells and cells of the vascular wall. This promotes an overall cellular dysfunction within the vascular compartment.
MVs and stem cells
MVs have been studied also in stem cell biology. Recent reports have described as embryonic stem cells (ESC) and adult stem cells in different stress condition, may release MVs containing proteins and genetic information, that may promote phenotypic changes in target cells within a defined microenvironment. In fact, MVs has been described as signals involved in the interaction between the stem cells and the injured tissue. Communication among the injured cells surviving injury and stem cells resident or recruited from the bone marrow is essential to promote tissue repair (Paracrine/endocrine mechanism of stem cells on kidney repair: role of microvesicle-mediated transfer of genetic information, 2010).
Bruno et al., have recently described the potential of MVs from human bone marrow mesenchymal stem cells (MSC) to accelerate kidney repair in a mouse model of acute kidney injury (AKI), induced by intra-muscle glycerol administration (Mesenchymal stem cell-derived microvesicles protect against acute tubular injury, 2009).
The mechanism by which MVs from MSCs contribute to the repair of AKI was associated to mRNA transfer, as RNAse treatment abrogated the MV beneficial effect on AKI mice. Moreover, microarray screening on RNA from MVs, showed that they contained a specific subset of mRNA (about 239 transcripts) rather than a random sample of cellular mRNA. Several transcripts of mesenchymal cell lineages, such as neural, osteogenic, epithelial, and hematopoietic were present. Moreover, MVs contained mRNA related to several cell functions such as control of transcription, cell proliferation, and immune regulation. Complete microarray data were deposited on GEO database.
Moreover, MVs derived from human liver stem cells (HLSC) induced in vitro proliferation and apoptosis resistance of human and rat hepatocytes. In addition, when administered in vivo, MVs from HLSC, may accelerate the morphological and functional recovery of liver in a model of 70% hepatectomy in rats. This effect was associated with increase in hepatocyte proliferation and was abolished by RNase pre-treatment (Human liver stem cell-derived microvesicles accelerate hepatic regeneration in hepatectomized rats, 2009).
Furthermore Lai et al., have recently described as exosomes from human ESC-derived mesenchymal stem cells mediate cardioprotection during myocardial ischemia/reperfusion injury (MI/R). These purified exosomes reduced infarct size in a mouse model of MI/R (Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury, 2010).
All these works support the theory that MSC may mediate their protective paracrine effect not only through the release of soluble factors, as cytokines, chemokines and growth factors, but also secreting exosomes/MVs that may shuttle genetic information to target cells.
Interestingly, the hypothesis on cell cross-talk is based on the idea of a bidirectional exchange of genetic information from stem cells to injured cells or conversely from injured cells to bone marrow derived or resident stem cells.
Recent work have explored this complex hypothesis demonstrating stem cell changes mediated by signals received from injured cells and possibly involved in stem cell differentiation. Quesenberry et al. suggested that stem cell differentiation in response to specific signals, especially from injured cells may be delivered by MV-mediated transfer of genetic information (Microvesicle entry into marrow cells mediates tissue-specific changes in mRNA by direct delivery of mRNA and induction of transcription,2010). Moreover, Jang demonstrated that bone marrow cells co-cultured with injured liver cells may express liver specific genes such as albumin (Hematopoietic stem cells convert into liver cells within days without fusion, 2004). Aliotta et al. in a similar experimental setting, demonstrated that murine bone marrow cells express genes for lung-specific proteins such as Clara cell-specific protein, surfactant B, and surfactant C (Alteration of marrow cell gene expression, protein production, and engraftment into lung by lung-derived microvesicles: a novel mechanism for phenotype modulation, 2007). Moreover, they found that the injured lung conditioned medium also induced lung-specific gene expression in MSC and that this activity resided in MVs released in the cell supernatant. These MVs were shown to contain high levels of lung-specific mRNA and to deliver this mRNA to MSC, suggesting that MV derived from injured tissue might mediate MSC phenotype change during physiologic tissue repair.
All these new studies have tried to understand the complex machinery of cell communication taking place during the different stages of tissue damage and regeneration. Moreover they suggest an important role of MVs in the transfer of RNA-based genetic information from stem cells/precursors to injured cells mediating microenvironment modification and tissue regeneration (Paracrine/endocrine mechanism of stem cells on kidney repair: role of microvesicle-mediated transfer of genetic information, 2009).