Extracellular vesicles in health and disease

Extracellular vesicles in physiological conditions

Extracellular vesicles are found in every body fluids, and play many important roles. In the blood, exosomes were first described as a way for the red blood cell to complete its maturation from reticulocyte to erythrocyte by excreting selectively some of its proteins like transferrin receptor [5]. Extracellular vesicles from platelets and endothelial cells are also actors of the coagulation process, representing one piece of the pro coagulant factor in plasma [18]. As an example of extracellular vesicles role in coagulation, Scott’s syndrome, a disease in which extracellular vesicles are not playing their “Phosphatidyl-Serine exposing” role due to an impaired function of scramblase, leading to a bleeding tendency [19].  In immunity, extracellular vesicles play a role by modulating antigen presenting cell (APC) responses [20], and can be secreted by APC to present antigens via MHC class II molecules [21]. Interestingly, EVs can also play a role in inhibiting immune responses, in particular in the context of pregnancy, where it induces a pro tolerant environment allowing the absence of rejection of the baby [22]. 

Extracellular vesicles in pathophysiological conditions

Extracellular vesicles very versatile properties have also a bystander effect, they can also play many roles in diseases. As an example, EVs secreted by tumors were shown to induce a pro coagulant state in the patient [4], as well as the ability to prime future metastasis sites [23]. In infections, EVs can play a role in disseminating viral agents in the body [24], and can contain viral or immune down-regulating proteins to favor leishmania infection propagation [25]. In immuno oncology, extracellular vesicles from tumour cells are transferred in a heterotypic manner from the tumour cell to its surroundings, can induce a deleterious pro tolerogenic environment [26]. Interestingly, this opens the door to the detection of these pathogenic extracellular vesicles as a diagnostic/prognostic marker. This whole “liquid biopsy” field is now flourishing [27].

Extracellular vesicles as therapeutics

Extracellular vesicles as therapeutic is mainly divided in two fields, using EV intrinsic properties to induce a phenotype of interest, or using EV as a way to transport a drug of interest. 

The interest for extracellular vesicles as a drug delivery system is mainly based on their property to be multimodal messengers, able to deliver membrane and cytosolic proteins, RNA and lipids, while not being toxic compared to synthetic vectors [28]. Their potential natural targeting properties are also discussed, especially with the “tetraspanin web” theory [29], that proposes that the complex and unique tetraspanins composition of a particular extracellular vesicles would control its fate in the body. This theory is however lacking any kind of proof at the moment.  Recently, Marie Millard et Al  (unpublished, submitted) showed that in a model tissue (cell spheroids), compared with liposomes, extracellular vesicles were more efficient at getting through the tissue, and transporting the drug deeper, a property that can be of interest for many applications, in particular in oncology for  deeply seated organs like liver, prostate, uterus, etc. Finally, the most striking finding in drug delivery was the extracellular vesicle unique ability to cross the blood brain barrier, a property that remain to be elucidated or confirmed [30]. 

Recently, the regenerative medicine / stem cell biology field encountered the extracellular vesicles field, and provided interesting data on the mechanism underlying stem cell-induced regenerative effect. In fact, the main dogma at the time was that a stem cell once in the injured organ would differentiate into the desired cell and thereby participate to tissue regeneration. However it appeared to be not the major mechanism at play.  In practice, some teams decided to look at the stem cell survival with various means like fluorescence tags and discovered that the number of cells was decreasing in a quasi-exponential way in a short period of time (1 to 3 weeks) [31]. Furthermore, experiments to find alive or even differentiated cells were mostly unsuccessful [32], but still remains the beneficial effect of stem cell injection.

The current dogma is that many stem cells, among which Mesenchymal stem cells (MSC), are acting through immune modulation and limiting differentiation of surrounding cells in ECM-secreting myofibroblasts by inhibiting TGFβ effects [33]. MSC also have a role in inhibiting oxidative stress and remodeling the extracellular matrix [34]. These four processes are mediated by different ways : direct fusion of the MSC cell with another cell, connections with the surrounding cells with Tunneling Nano Tubes (TNT) to transfer mitochondria, Ca2+, Mg2+, RNAs Proteins, secretion of soluble factors like VEGF-A HGF ANG1 SDF-1 PDGF-B IL-11 PGE2 IGF-1 TSG-6, sFRP, SDF-1, Jagged/Notch, heme-oxygenase-1, STC-1 signaling molecules or by transfer of receptors, soluble factors, RNA through exosomes and microvesicles [35].

Interestingly, stem cell derived extracellular vesicles in the cell culture supernatant, once concentrated by ultracentrifugation had a similar effect compared to parental cells in various organs [35], and were therefore considered to be major players of this paracrine effect. Nowadays, the stem cell derived extracellular vesicles effect has been investigated in most organs [36] with interesting results. 

The current paradigm is that stem cells releasing EVs elicit a paracrine effect for a small period of time before dying, that is beneficial to the injured tissue, and that it allows the surrounding cells to resolve inflammation, and to repair the injured tissue in a more efficient way. 

Knowing that MSC-EVs are able to recapitulate their parental cell effect, for instance, it is reported in the literature that MSC EVs induce a proangiogenic effect by transferring miR-125a [37], reduce fibrosis via miR-22 [38], enhances cell survival via miR-221 [39], modulate immune response by expressing PD-L1, galectin-1 and membrane bound TGF-β [40], just to name a few.

The comparison between the injection of MSC and the injection of MSC-derived EVs give a real advantage to EVs and unique opportunities like off the shelf availability, low immunogenicity [41] and no risks of anarchic differentiation or pulmonary embolism [42] (due to the large size of MSCs). MSC EVs were for example demonstrated to induce a downregulation of H₂O₂ production by neutrophils, limiting differentiation of monocytes in Dendritic Cells (DCs), limiting the cytotoxic effect of Natural Killer (NK) and T cells, or limiting the cell activation/polarization and proliferation. 

An interesting way to use extracellular vesicles is their use in enzyme replacement therapies (ERT). The goal of these therapies is to deliver in the body an enzyme that is lacking, usually due to a genetic mutation. Interestingly, some enzymes can probably be delivered or protected by EVs, especially in storage disorders touching lysosomes or endosomes, where extracellular vesicles can easily be targeted. 

However, major obstacles are still to be faced to get in the clinic with an interesting product and we will now describe major hurdles the field has to face in our opinion to achieve EV use in a therapeutic way. 

[5] C. Harding, J. Heuser, P. Stahl, Endocytosis and intracellular processing of transferrin and colloidal gold-transferrin in rat reticulocytes: demonstration of a pathway for receptor shedding, European journal of cell biology, 35 (1984) 256-263.

[18] A.P. Owens, N. Mackman, Microparticles in Hemostasis and Thrombosis, Circulation research, 108 (2011) 1284-1297.

[19] H.J. Weiss, W.J. Vicic, B.A. Lages, J. Rogers, Isolated deficiency of platelet procoagulant activity, The American Journal of Medicine, 67 (1979) 206-213.

[20] F.-M. Lola, G.-V. Cristina, R.-G. Eva, S.-M. Francisco, F. Hortensia, Immunomodulatory role of microRNAs transferred by extracellular vesicles, Biology of the Cell, 107 (2015) 61-77.

[21] B.S. I, B.B.W. M, A. Marian, H.A.J. R, W. Marca, S. Willem, MHC class II-associated proteins in B-cell exosomes and potential functional implications for exosome biogenesis, Immunology and Cell Biology, 88 (2010) 851-856.

[22] M. Tong, L.W. Chamley, Placental Extracellular Vesicles and Feto-Maternal Communication, Cold Spring Harbor Perspectives in Medicine, 5 (2015) a023028.

[23] d.A.P. Brunna, A.C.d.L. Felipe, S.F. Sara, P.F.d.S. Leandro, C.B.T. Paula, A.G. Vivian, F. Wagner, R.G. Luiz, J.B.S. Marcelo, The multifaceted role of extracellular vesicles in metastasis: Priming the soil for seeding, International Journal of Cancer, 140 (2017) 2397-2407.

[24] D.G. Meckes, Exosomal Communication Goes Viral, Journal of Virology, 89 (2015) 5200-5203.

[25] K. Hassani, M. Olivier, Immunomodulatory Impact of Leishmania-Induced Macrophage Exosomes: A Comparative Proteomic and Functional Analysis, PLoS Neglected Tropical Diseases, 7 (2013) e2185.

[26] T.L. Whiteside, Exosomes and tumor-mediated immune suppression, The Journal of Clinical Investigation, 126 (2016) 1216-1223.

[27] S. Halvaei, S. Daryani, Z. Eslami-S, T. Samadi, N. Jafarbeik-Iravani, T.O. Bakhshayesh, K. Majidzadeh-A, R. Esmaeili, Exosomes in Cancer Liquid Biopsy: A Focus on Breast Cancer, Molecular Therapy. Nucleic Acids, 10 (2018) 131-141.

[28] X. Zhu, M. Badawi, S. Pomeroy, D.S. Sutaria, Z. Xie, A. Baek, J. Jiang, O.A. Elgamal, X. Mo, K.L. Perle, J. Chalmers, T.D. Schmittgen, M.A. Phelps, Comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from HEK293T cells, Journal of Extracellular Vesicles, 6 (2017) 1324730.

[29] S. Rana, S. Yue, D. Stadel, M. Zöller, Toward tailored exosomes: The exosomal tetraspanin web contributes to target cell selection, The International Journal of Biochemistry & Cell Biology, 44 (2012) 1574-1584.

[30] L. Alvarez-Erviti, Y. Seow, H. Yin, C. Betts, S. Lakhal, M.J.A. Wood, Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes, Nature Biotechnology, 29 (2011) 341.

[31] S. Liu, J. Zhou, X. Zhang, Y. Liu, J. Chen, B. Hu, J. Song, Y. Zhang, Strategies to Optimize Adult Stem Cell Therapy for Tissue Regeneration, International Journal of Molecular Sciences, 17 (2016) 982.

[32] G. YONG-JIAN, Molecular Mechanisms for Cardiovascular Stem Cell Apoptosis and Growth in the Hearts with Atherosclerotic Coronary Disease and Ischemic Heart Failure, Annals of the New York Academy of Sciences, 1010 (2003) 687-697.

[33] M. Di Nicola, C. Carlo-Stella, M. Magni, M. Milanesi, P.D. Longoni, P. Matteucci, S. Grisanti, A.M. Gianni, Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli, Blood, 99 (2002) 3838-3843.

[34] F. Arslan, R.C. Lai, M.B. Smeets, L. Akeroyd, A. Choo, E.N.E. Aguor, L. Timmers, H.V. van Rijen, P.A. Doevendans, G. Pasterkamp, S.K. Lim, D.P. de Kleijn, Mesenchymal stem cell-derived exosomes increase ATP levels, decrease oxidative stress and activate PI3K/Akt pathway to enhance myocardial viability and prevent adverse remodeling after myocardial ischemia/reperfusion injury, Stem Cell Research, 10 (2013) 301-312.

[35] J.L. Spees, R.H. Lee, C.A. Gregory, Mechanisms of mesenchymal stem/stromal cell function, Stem Cell Research & Therapy, 7 (2016) 125.

[36] I.M. Bjørge, S.Y. Kim, J.F. Mano, B. Kalionis, W. Chrzanowski, Extracellular vesicles, exosomes and shedding vesicles in regenerative medicine – a new paradigm for tissue repair, Biomaterials Science, 6 (2018) 60-78.

[37] X. Liang, L. Zhang, S. Wang, Q. Han, R.C. Zhao, Exosomes secreted by mesenchymal stem cells promote endothelial cell angiogenesis by transferring miR-125a, Journal of Cell Science, 129 (2016) 2182-2189.

[38] S. Keshtkar, N. Azarpira, M.H. Ghahremani, Mesenchymal stem cell-derived extracellular vesicles: novel frontiers in regenerative medicine, Stem Cell Research & Therapy, 9 (2018) 63.

[39] B. Yu, M. Gong, Y. Wang, R.W. Millard, Z. Pasha, Y. Yang, M. Ashraf, M. Xu, Cardiomyocyte protection by GATA-4 gene engineered mesenchymal stem cells is partially mediated by translocation of miR-221 in microvesicles, PloS one, 8 (2013) e73304.

[40] A. Mokarizadeh, N. Delirezh, A. Morshedi, G. Mosayebi, A.-A. Farshid, K. Mardani, Microvesicles derived from mesenchymal stem cells: Potent organelles for induction of tolerogenic signaling, Immunology Letters, 147 (2012) 47-54.

[41] G. Zheng, R. Huang, G. Qiu, M. Ge, J. Wang, Q. Shu, J. Xu, Mesenchymal stromal cell-derived extracellular vesicles: regenerative and immunomodulatory effects and potential applications in sepsis, Cell and Tissue Research, (2018).

[42] S. Rani, A.E. Ryan, M.D. Griffin, T. Ritter, Mesenchymal Stem Cell-derived Extracellular Vesicles: Toward Cell-free Therapeutic Applications, Molecular Therapy, 23 (2015) 812-823.