E XTRACELLULAR VESICLES AS REGENERATIVE CELL - FREE APPROACH

In 1946, Cargaff and West observed pro-coagulated particles in platelet isolations, which Wolf referred to as “platelet dust” according to their appearance in electron microscopic images [81,82]. Today, these vesicular structures with a lipid bilayer are called EVs.

In total, three types of EVs can be distinguished according to their biogenesis and size: apoptotic bodies, microvesicles, and exosomes (Figure 3) [83–89]. The largest EVs are apoptotic bodies (diameter (d) > 1000 nm), which occur as consequence of the programmed cell death in which the plasma membrane dissembles. Microvesicles range in their size between 100 to 1000 nm and therefore occupy the middle position in terms of diameter. This subset is budded directly from the plasma membrane into the extracellular space. The smallest EVs are the exosomes

8 with diameters less than 100 nm. They are generated intracellularly in the endosomal compartment, where exosomes are called intraluminal bodies contained in multivesicular bodies (MVBs). When MVBs eventually fuse with the plasma membrane, exosomes are released into the extracellular space. Although major progress was achieved in the research field of EVs, distinct proteins are still missing to distinguish between exosomes and microvesicles.

Nevertheless, a number of EV-associated proteins were identified [85]. It includes next to transmembrane or plasma membrane anchored proteins, such as integrins or representatives from the tetraspanin family (e.g. CD9), also cytosolic proteins, like heat shock proteins (HSPs), syntenin, or annexins to name just a few. Apoptotic bodies additionally contain typical cell organelle proteins, such as calnexin, cytochrome C, or cytokeratin 18. Moreover, recent studies showed that the isolation by differential centrifugation purifies EVs together with a non-EV compartment, which includes next to apo-lipoproteins A1/2, also fibronectin or the RNA binding protein Argonaute-2 [90–92].

Figure 3: Schematic illustration of the biogenesis of the three EV types.

EVs can be differentiated by their biogenesis and size into apoptotic bodies, microvesicles and exosomes. During apoptosis, the plasma membrane dissembles, which leads to the formation of apoptotic bodies (> 1,000 nm). A viable cell buddes microvesicles (100-1,000 nm) directly from the plasma membrane, whereas exosomes (<100 nm) are formed in endosomes as intraluminal bodies (ILB) contained in multivesicular bodies (MVBs). Fusion of MVBs finally release exosomes into the extracellular space.

Interestingly, not only the most investigated mammalian cells but also parasites, gram-negative bacteria and fungi were observed to release these vesicular structures into the extracellular space [93,94]. The reason for such an omnipresent behaviour lies in the capability of EVs to function as intercellular communicator. Multiple processes, such as differentiation, angiogenesis, or migration, were shown to be influenced in recipient cells upon interaction with EVs and their transported molecules [95–97]. It has to be highlighted that EVs do not only carry lipids and proteins but also RNA molecules, such as messenger (mRNA) or micro RNA (miRNA), as well as small signalling molecules [88,89,98–102].

At present, EVs are evaluated for their potential as medical tool, including as biomarkers for diseases or as therapeutic option. In context of CVDs, several studies could already prove that EVs from MSCs, CPCs, and cardiac fibroblasts are capable to improve cardiac function in rodent MI disease models [37,40,100–103]. In these studies, mechanistic studies were included that demonstrated that the cardio protection could be attributed to EV-mediated reduction of fibrosis, inhibition of apoptosis, support of angiogenesis, and modulation of immune responses.

As such, it was discovered that MSC-EVs diminished T cell proliferation[104–107], the release of pro-inflammatory cytokines, such as IFNy or IL-1ß [104–107], or enhanced the frequency of regulatory T cells [107,108] in induced immune reactions in vitro. Also APCs gained a tolerogenic

9 or anti-inflammatory phenotype when treated with EVs derived from MSCs as observed for dendritic cells, monocytes as well as macrophages [109–111]. Herein, this phenotype included an amplified expression of programmed cell death 1 ligand 1 (PD-L1), the macrophage mannose receptor (CD206) on their cell surface, while, in contrast, other surface proteins, such as the co-stimulatory molecule CD86, HLA-DR or activation markers, like CD83, were significantly reduced.

Yet, no satisfying answer can be given on how EVs from regenerative cells facilitate their beneficial effects. Some studies indicate that their transported RNA molecules have a superior role. The anti-apoptotic or proliferative inducing effect of MSC-EVs could be shown to be abolished when EVs were treated with RNase [112]. Likewise, Zou and colleagues could provide evidence that the RNase treatment of MSC-EVs also eliminated the pro-angiogenic effect [113].

In the last years, several miRNAs transported by EVs were identified as potential candidates to trigger beneficial effects, such as miRNA 126, miRNA 146 or miRNA 149 to name just a few [16]. But also mRNAs can be transferred from an EV to a recipient cell. Herein, it was demonstrated that murine cells expressed human proteins, like the DNA-directed RNA polymerase II 23 kDA polypeptide (POLR2E) or the small ubiquitin-related modifier 1 (SUMO-1), when treated with human MSC-EVs [112]. Additionally, EVs were shown to transport proteins with crucial enzymatic function for immunomodulation. Clayton et al. showed that EVs from different human cancer cells (colon, breast and prostate) own ATP hydrolytic activity via their transported ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and CD73 . Moreover, it was revealed that this enzymatic activity of EVs contribute to the modulation of induced immune responses, like the decrease of T cell proliferation or IL-2 production, in vitro [114]. Also the pro-angiogenic effect of EVs was shown to be influenced by their transported proteins, such as VEGF or other molecules [95,113]. Up to now, the field of EV transported lipids is scarely studied, but future investigations will elucidate whether lipids have an impact on beneficial therapeutic effects or on the delivery to certain target cells. Additionally, mechanistic analysis becomes increasingly complex when considering that the cell source and especially the milieu during the biogenesis of EVs determines which molecules will be transported. For example, EVs from MSCs cultivated under normoxic versus hypoxic conditions revealed that hypoxia enhances the pro-angiogenic feature of these generated EVs significantly in comparison to their normoxic counterpart [115]. Likewise, a pro-inflammatory stimulation with IL-3 favoured the angiogenesis promoting features of EVs from endothelial cells [116].

Investigations will also elucidate key molecules of these EVs and hopefully provide insight into the underlying mechanism for the various beneficial effects needed for treating CVDs. Most importantly, EVs derived from regenerative cells provide an attractive alternative for cell therapy. One advantage is that EVs do not, in contrast to their originating regenerative cell, bear the risk of teratoma formation. Additionally, it is proposed that EVs could be used as an

“emergency” therapeutic tool, since they are storable and thus immediately available for administration to the patient without any time-consuming preparation. Nevertheless, before CVD patients can be treated with EVs from regenerative cells, certain challenges have to be explored. This includes question about dosing, delivery routes, and identity markers of EVs, but also innovations for isolation procedures that allow obtaining large amounts of EVs as well as evaluation of potential off-target effects of administrated EVs.

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