III. Embryonic development.

EVs are likely to be involved in the regulation of embryonic development, including mainte‐ nance of morphogen gradients, collective cell migration and tissue polarity. However, this still remains an emerging field with many unanswered questions, which need further investigation [15].

### IV. Tissue repair.

EVs derived from human adult mesenchymal stem cells (MSCs) have been found to prevent ischaemia-reperfusion kidney injury and improve survival in a model of lethal acute kidney injury [16]. MSC-derived EVs are reported to modify the expression of miR29c and miR150 and upregulate the expression of SDF-1, CXCR4, CXCR7, CCL2 and ANGPTL4, which are known to play essential roles in acute and chronic wounding [17].

### V. Liver homeostasis.

A comprehensive study of hepatocyte-derived EVs showed the presence of several members of cytochrome P450, uridinediphosphate-glucuronosyl-transferase (UGTs) and glutathione Stransferase (GST) protein families, supporting a role of these vesicles in the metabolism of endogenous and xenobiotic compounds [18]. Recently, it has been shown that EVs from hepatocytes were able to activate stellate cells to mediate a response to liver damage [19] and many studies support an important role of these vesicles in maintaining liver homeostasis.

### **3.2. Pathological actions of EVs**

Given their essential role in regulating biological processes, it is not surprising that EVs have a significant influence in disease pathogenesis. This has been most extensively studied in tumour biology. Several reports have indicated that EVs may be an important means of driving the formation of a pre-metastatic tumour [12, 20]. EVs can promote proliferation of their target cells, stimulate angiogenesis, induce metastasis and promote immune escape by modulating T-cell activity [21–24].

Prior to the discovery of EVs, it was known that the vesicles secreted by tumour cells retained procoagulant activity, linking cancer progression with EV-induced thrombosis [25–27]. In addition, a direct link between EVs and tumour invasion of healthy tissues was reported in 2008 [28]. It was shown that the mRNA expression of an activated mutated epidermal growth factor receptor (EGFRvII) in glioma cells can enhance vesiculation significantly and intercel‐ lular transfer of this oncoprotein to adjacent tumour cells, leading to the production of angiogenic mediators such as vascular endothelial growth factor (VEGF) [28].

Similar results were reported in another study by Skog et al. [22] showing that various miRNAs that stimulate tumour growth and angiogenesis in addition to EGFR can be transferred by human primary glioblastoma cell-derived EVs. Moreover, EVs derived from tumour cells were shown to transfer activated EGFR to endothelial cells, inducing VEGF expression and resulting in VEGF receptor activation to stimulate angiogenesis [29]. Many of the previously mentioned studies suggested that EVs can trigger tumour growth by stimulating the proliferation of cancer cells and by stimulating angiogenesis in the adjacent normal endothelial cells.

Additional data also support the association of tumour-secreted EVs in the promotion of metastasis and tumour invasion; for example, transfer of the EMMPRIN transmembrane glycoprotein, which stimulates matrix metalloproteinase (MMP) expression in fibroblasts and remodelling of the ECM [30]. Recently, it was shown that EVs derived from melanoma cells directed bone marrow cells towards a prometastatic phenotype, mediating the communication between tumour cells and normal cells [31, 32].

Furthermore, tumour-associated macrophages can secrete EVs, which contain certain miRNAs that can promote breast cancer cell invasion [33]. In addition to their role in cancer, EVs have been associated with various pathogens, including HIV-1, Epstein-Barr virus (EBV) and prions [34–36].
