Functional proteins of mesenchymal stem cell-derived extracellular vesicles

Extracellular vesicles (EVs) contain proteins, microRNAs, mRNAs, long non-coding RNAs, and phospholipids, and are a novel mechanism of intercellular communication. It has been proposed that the immunomodulatory and regenerative effects of mesenchymal stem/stromal cells (MSCs) are mainly mediated by soluble paracrine factors and MSC-derived EVs (MSC-EVs). Recent studies suggest that MSC-EVs may serve as a novel and cell-free alternative to whole-cell therapies. The focus of this review is to discuss the functional proteins which facilitate the effects of MSC-EVs. The first section of the review discusses the general functions of EV proteins. Next, we describe the proteomics of MSC-EVs as compared with their parental cells. Then, the review presents the current knowledge that protein contents of MSC-EVs play an essential role in immunomodulation and treatment of various diseases. In summary, functional protein components are at least partially responsible for disease-modulating capacity of MSC-EVs.


Background
Mesenchymal stem/stromal cells (MSCs) are obtainable from various human tissues and have multipotent differentiation aptitude. Due to their angiogenic, anti-apoptotic, and immunomodulatory capacities, MSCs may serve as a good candidate for regenerative medicine. Many studies including the work from our group [1] have examined the potential applications of MSCs in a variety of diseases including cardiomyopathy, kidney injuries, liver injuries, lung injuries, and cancers [2,3]. Recent studies have demonstrated that the therapeutic benefits of MSCs are dependent on their extracellular vesicles (EVs).
According to the position statement of the International Society for Extracellular Vesicles (ISEV), EVs are particles, with a lipid bilayer, that are naturally released from the cells and cannot replicate [4]. The ISEVs have also stated that two categories (categories 1 and 2) of EV markers and one category of non-EV markers (category 3) must be examined to demonstrate the presence of EVs. Category 1 includes transmembrane or glycosylphosphatidylinositolanchored proteins, indicating the lipid bilayer structure of EVs. Category 2 consists of cytosolic proteins such as enzymes, cytoskeletal proteins, and proteins actively incorporated into EVs via membrane binding. On the other hand, category 3 is composed of proteins of non-EV structures co-isolated with EVs, an indicator of impurity of the EV preparation [4]. EVs are typically classified into three subtypes according to sizes and biogenesis mechanisms: exosomes (50-150 nm), microvesicles (100-1000 nm), and apoptotic bodies (500-5000 nm). Exosomes are generated by several steps; endosomes formed from the plasma membrane, formation of intraluminal vesicles inside of multivesicular bodies (MVB) by inward budding, fusion of MVB with the plasma membrane, and secretion of internal vesicles. On the other hand, both microvesicles and apoptotic bodies are larger in size than exosomes and formed by local deformation and direct outward budding from the plasma membrane [5]. In cancer research, oncosomes, which function to transfer oncogenic signals across cell boundaries, refer to microvesicles or even all EVs that are released from cancer cells [6]. Due to overlapping size, density, and membrane composition, most of the current methods of EV isolation yield a mixed EV population [7].
There is still no consensus on specific markers of EV subtypes. Flotillin, heat-shock 70 kDa proteins, and major histocompatibility complex class I and class II are common markers for all types of EVs, while CD9, CD63, and CD81 tetraspanins are generally classified as exosome markers [8].
Almost all organisms and cells are capable of producing EVs. MSC-derived EVs (MSC-EVs) bear not only the markers that are characteristic of MSCs such as CD29, CD73, CD90, CD44, and CD105, but also EV markers such as CD63, CD9, and CD81 [9]. To apply MSC-EVs in clinical studies, implementation of a good manufacturing practice-compliant protocol is necessary. To scale up the production of MSC-EVs, 3D bioreactors may be required to provide a homogeneous distribution of nutrients and oxygen while monitoring cell number, viability, and proliferation [10]. Xenogenic components such as fetal bovine serum should be substituted with human serum or human platelet lysate. For example, platelet lysate has been used to generate MSC-EVs for treating refractory graft-versus-host disease [11]. Differential ultracentrifugation has been the most widely used method for EV separation. However, ultracentrifugation has several drawbacks, including low purity and induced aggregation of EVs, and thus is not suitable for largescale purification of MSC-EVs [12]. Instead, MSC-EVs purified via precipitation with polyethylene glycol have been applied for clinical investigation [11]. It has been proposed that the ratio between particle number and protein concentration may serve as an indicator for purity in EV production [13].
MSC-EVs exert their functions via the transfer of their contents, such as proteins, mRNAs, and microRNAs (miR-NAs), to target cells. There was a horizontal transfer of insulin-like growth factor-1 receptor mRNA from EVs to cisplatin-damaged proximal tubular cells, which increased the proliferation of proximal tubular epithelial cells [14]. In mice, MSC-EVs alleviated lipopolysaccharide-induced acute lung injury via their content of angiopoietin-1 mRNA [15]. MSC-EV-mediated transfer of miRNAs is an area of active research. Transfer of miRNAs from MSC-EVs to target cells has been demonstrated as the underlying mechanisms in alleviating injury of the kidney [16], heart [17], liver [18], and brain [19]. The present review article will cover the general functions of EV proteins and proteomics of MSC-EVs and emphasize on functional proteins mediating the effects of MSC-EVs in different diseases.

General functions of EV proteins
EV proteins participate in EV biogenesis, sort EV cargo, control EV release, and facilitate interactions between EVs and recipient cells. EV biogenesis can be categorized into endosomal sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways.
ESCRT consists of 4 protein complexes, ESCRT-0, I, II,  and III, with over 20 ESCRT and accessory proteins such  as CHMP4C, VPS4B, VTA1, and ALIX. In HeLa-CIITA  cells, silencing of ESCRT-0 subunit Hrs, ESCRT-I protein  STAM1, or ESCRT-I protein TSG101 altered the size and/or protein contents of secreted EVs. The depletion of VPS4B did not affect the size and composition of EVs while elevated the secretion [20]. In head and neck squamous cell carcinoma cells, knockdown of ESCRT-0 subunit Hrs reduced the secretion of EVs per cell [21]. Biogenesis of EVs can also arise from the ESCRTindependent pathway. In human epithelial type 2 cells, depletion of key subunits of all 4 ESCRT inhibited the formation of EGF-induced EVs, but EGF-independent EVs remained intact [22]. On the other hand, tetraspanins such as CD63, CD53, CD37, CD81, and CD9, which are among the most enriched membrane proteins in EVs, play an essential role in the ESCRT-independent pathway. The release of EVs was defective from dendritic cells generated from CD9 knockout mice compared with wild type. In addition, less amount of flotillin-1 was observed in EVs from dendritic cells of the knockout mice [23]. Hurwitz et al. found that secretion of small EVs was significantly decreased in HEK293 cells with CD63 gene knockout, indicating the role of CD63 in the biogenesis of EVs [24]. The same group also reported that latent membrane protein 1, an Epstein-Barr virus-encoded oncoprotein, accumulated in CD63-positive EVs. Levels of latent membrane protein 1 were reduced after CD63 knockout in nasopharyngeal carcinoma cells [25].
Sorting of EV cargo is also regulated by both ESCRTdependent and ESCRT-independent pathways. The three early-acting ESCRT complexes (ESCRT-0, ESCRT-I, and ESCRT-II) harbored ubiquitin-binding domains and could interact with ubiquitinated cargo, sorting ubiquitinated proteins into EVs [26]. Proteins lacking ubiquitin modification could still be sorted into EVs in an ESCRTdependent pathway. ESCRT-associated protein ALIX bound with protease-activated receptor-1 and the purinergic receptor P2Y 1 via a YPX 3 L motif [27,28]. ESCRT-0 subunit Hrs interacted directly with interleukin-2 receptor β [29]. However, the downstream ESCRT-III and the Vps4 complex were required for the sorting of ubiquitin lacking proteins [28]. On the other hand, tetraspanin CD63 played a direct role in ESCRT-independent sorting of protein and EV formation in melanocytes [30]. Sorting of Argonaute 2, which is a critical RNA-induced silencing complex (RISC) component and a regulator for miRNA sorting, was controlled by KRAS-MEK signaling in colon cancer cell lines [31]. Sortilin, a sorting protein that directs target proteins to EVs, regulated its own trafficking to EVs via dimerization in HEK293 cells [32].
It is well known that EV fusion and release are facilitated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) [33]. EV release is also controlled by EV proteins such as Rab GTPases, which serve as essential regulators of membrane trafficking. The release of EVs was slightly elevated in K562 cells transfected with plasmid DNA encoding for wild-type Rab11, while overexpression of a dominant-negative Rab11 mutant blocked EV secretion [34]. Rab7 and Rab27b controlled the secretion of EVs containing miR-143 from endothelial cells induced by KLF2 [35]. Hsu et al. showed that Rab35 regulated the release of proteolipid protein in EVs of oligodendroglial cells by adjusting the docking/tethering of MVB with the plasma membrane [36]. Rab27a and Rab27b played different roles in the secretion of EVs. Rab27a silencing significantly increased the size of MVB, while the distribution of MVB was around the perinuclear region after Rab27b silencing in HeLa cells [37].
The interactions between EVs and recipient cells involve surface proteins of EVs such as integrins, tetraspanins, and ICAMs, which interact with the target cells via receptor-ligand binding. When intravenously injected, integrin α 6 β 4 -and α 6 β 1 -enriched EVs from tumors colocalized with fibroblasts and epithelial cells in lamininrich lung microenvironments. Integrin α v β 5 -expressing EVs from tumors fused with Kupffer cells in the fibronectin-rich liver. Meanwhile, integrin β 3 -abundant EVs interacted primarily with CD31+ brain endothelial cells [38]. EVs containing tetraspanin8 and its associated CD49d from an adenocarcinoma model were internalized by endothelial cells via CD106, a ligand for CD49d. Uptake of tetraspanin8-CD49d complex-containing EVs initiated angiogenesis-related gene expression in endothelial progenitors [39]. ICAM-1 is a ligand for the lymphocyte function-associated antigen 1. ICAM-1 from mature dendritic cell-derived EVs was essential for effective naive T cell priming [40]. Other EV proteins also regulate the uptake of EVs by target cells. In pancreatic ductal adenocarcinoma, the healthy pancreatic tissue surrounding the tumor released REG3β. REG3β bound to the glycoproteins on the surface of circulating EVs, resulting in reduced internalization of EVs by tumor cells [41].

Proteomics of MSC-EVs
An increasing number of studies have reported the proteomic signature of MSC-EVs. A summary of the data is presented in Table 1. Kim et al. identified 730 proteins in the proteomic analysis of human bone marrow (BM)-derived MSC-EVs. The MSC-EV proteins contained markers of MSCs and EVs as well as signaling molecules regulating the self-renewal and differentiation capacities of MSCs. In addition, the list included proteins participating in cell proliferation, adhesion, migration, and morphogenesis [42]. In another study of Proteins upregulated only in control MSC-EVs were associated with tissue regeneration. On the other hand, proteins upregulated only in MSC-EVs from pigs with metabolic syndrome were connected with proinflammatory pathways [51]. By analyzing datasets of published MSC-EV proteomics, an MSC-EV-specific protein signature was reported with 22 members and involved in functions such as cell adhesion and integrinreceptor interaction [52].

Functional proteins of MSC-EVs in immunomodulation
Like their parental cells, MSC-EVs have also been investigated for their potent immunomodulatory capacity. A list of functional proteins of MSC-EVs involved in immunoregulation and pathophysiology of human diseases is summarized in Table 2. Accumulating evidence has demonstrated that MSC-EVs modulate innate and adaptive immune responses through interaction with immune effector cells such as T, B, natural killer, and dendritic cells. Di Trapani et al. demonstrated that there was a direct correlation between EV uptake by immune effector cells and immunomodulation, suggesting the modulatory effects were transferable [79]. Several studies have evidenced that the immunomodulatory function of MSC-EVs could be, at least in part, facilitated by functional proteins. MSC-EVs from mouse BM were shown to produce tolerogenic molecules such as programmed death ligand-1 (PD-L1), galectin-1, and transforming growth factor β (TGF-β). MSC-EVs inhibited the proliferation of auto-reactive lymphocytes acquired from mice with experimental autoimmune encephalomyelitis. MSC-EVs also increased the secretion of anti-   inflammatory cytokines such as IL-10 and TGF-β and promoted the generation of regulatory T cells [53]. MSC-EVs derived from dog Wharton's jelly had a dosedependent suppressive effect on CD4+ T cell proliferation in vitro, which was absent after EV depletion. MSC-EVs contained TGF-β, which was likely tethered to EVs by betaglycan. EV suppression of CD4+ T cell proliferation was blocked by an antagonist for TGF-β receptor 1 and neutralizing antibodies to TGF-β [54]. In a similar study, Alvarez et al. documented that human endometrial MSC-EVs inhibited CD4+ T cell activation in stimulated lymphocytes. MSC-EVs had high active TGF-β expression compared with EV free concentrated supernatants. TGF-β neutralizing antibodies blocked the immunomodulatory activity of MSC-EVs [55]. Adamo et al. found that EVs from both resting and inflammation-primed MSCs were internalized by activated B cells. Proteins with immunomodulatory potential such as LG3BP and PTX3 were upregulated, while MOES and S10A6 were downregulated in inflammationprimed MSC-EVs compared with resting EVs. Treatment of activated B cells with inflammation-primed MSC-EVs induced a significant down-modulation of the PI3K-AKT signaling pathway and modulated actin cytoskeleton during B cell spreading [56]. Harting et al. discovered that EVs from inflammation-stimulated MSCs reduced TNF-α and IFN-γ release from activated splenocytes compared with control EVs. Both control and stimulated MSC-EVs were internalized by peripheral blood mononuclear cells. Furthermore, stimulated MSC-EVs had an elevated expression of COX2, which participated in the TNF-α inhibition [57].

Functional proteins of MSC-EVs in neurological diseases
Chen et al. reported that the treatment of MSCs with GW627368X, a prostaglandin E 2 receptor 4 (EP4) antagonist, promoted the release of MSC-EVs. The GW627368Xinduced MSC-EVs had elevated levels of anti-inflammatory cytokines and neuron-supporting proteins including IL-2, IL-10, RANTES, vascular endothelial growth factor (VEGF), and brain-derived neurotrophic factor (BDNF). In a mouse model for inducible hippocampal CA1 neuron damage, GW627368X-elicited MSC-EVs improved memory and learning deficiencies triggered by hippocampus damage. Furthermore, the induced MSC-EVs elevated the expression of genes implicated in astrocyte differentiation, bloodbrain barrier, and anti-inflammation [58]. In an in vitro model of Alzheimer's disease, MSC-EVs were transferred into neuroblastoma N2a cells and decreased both extracellular and intracellular β-amyloid peptide levels. In addition, MSC-EVs carried enzymatically active neprilysin, which is an essential molecule in degrading β-amyloid peptide in the brain [59]. In another in vitro model of Alzheimer's disease, MSC-EVs protected neurons against β-amyloid peptideinduced oxidative stress and synapse damage similar to that of parental MSCs. MSC-EVs exhibited the neuroprotective effect via the delivery of catalase [60].

Functional proteins of MSC-EVs in dermatological diseases
Results from clinical trials have shown that MSCs were effective in the treatment of epidermolysis bullosa, a genetic skin condition resulting from a lack of type VII collagen production, by improving the reconstruction of the basement membrane and healing of cutaneous wound [80]. In vitro, MSC-EVs transferred both type VII collagen protein and mRNA to fibroblasts from a patient with epidermolysis bullosa [61]. In a rat model of burn injury, Zhang et al. found that MSC-EVs delivered Wnt4 to skin cells, promoted wound healing via activation of Wnt/β-catenin signaling, and inhibited skin cell apoptosis via activation of the AKT pathway. The effects of MSC-EVs on skin cells were abolished by knockdown of Wnt4 expression [62]. The same group also demonstrated that MSC-EVs restricted excessive cell proliferation and expansion in the tissue remodeling phase via delivering of 14-3-3ζ to skin cells. Under high cell density conditions, 14-3-3ζ induced YAP phosphorylation, which inhibited Wnt/β-catenin signaling [63]. In an in vitro model of wound healing, Shabbir et al. demonstrated that MSC-EVs were taken up by fibroblasts and promoted the growth and migration of fibroblasts from normal and diabetic chronic wounds in a dose-dependent manner. MSC-EVs also enhanced the angiogenesis of human umbilical vein endothelial cells. Additionally, MSC-EVs were enriched with transcriptionally active STAT3, an inducer for growth factors [64].

Functional proteins of MSC-EVs in pulmonary diseases
Ahn et al. demonstrated that MSC-EVs were as effective as MSCs in alleviating neonatal hyperoxic lung injury in a rat model. The protective effects were lost in MSC-EVs with VEGF-knockdown, indicating the protection was mediated by the transfer of VEGF [65]. In an in vitro model of LPS-induced endothelial cell injury, Wang et al. found that treatment with MSC-EVs reduced endothelial paracellular and transcellular permeabilities, and the effect was abolished after knockdown of hepatocyte growth factor (HGF) in MSCs. MSC-EVs also diminished endothelial apoptosis and elevated the expression of junction proteins VE-cadherin and occludin. In addition, treatment with MSC-EVs decreased IL-6 level and elevated IL-10 level in the media of endothelial cells via an HGF-dependent manner [66]. Using an ex vivo lung perfusion model, Gennai et al. discovered that MSC-EVs promoted alveolar fluid clearance, reduced lung weight gain, and improved lung compliance. Anti-CD44 antibody blocked the effects of MSC-EVs, indicating the role of CD44 in mediating internalization of the MSC-EVs into the injured cells in the lung [67]. Hu et al. revealed that the administration of MSC-MVs restored the permeability of injured human lung microvascular endothelial cells. MSC-EVs prevented F-actin reorganization and restored the function of junction proteins ZO-1 and VE-cadherin in injured endothelial cells. Pretreatment of MSC-EVs with an anti-CD44 antibody reduced the beneficial effect, indicating that the internalization of MSC-EVs into the endothelial cells is an essential process [68].

Functional proteins of MSC-EVs in renal diseases
In a pig model of renal artery stenosis, treatment with MSC-EVs restored renal blood flow and alleviated renal inflammation and fibrosis. Fragments of EVs colocalized with kidney tubular cells and macrophages, suggesting internalization of EVs. The protective effects were abrogated in pigs treated with IL-10-depleted MSC-EVs [69]. In a mouse model of ischemia/reperfusion-induced renal injury, MSC-EVs suppressed macrophage function and attenuated renal injury. MSC-EVs were enriched with C-C motif chemokine receptor-2 (CCR2). Knockdown of CCR2 abolished the protective effect of MSC-EVs [70]. In a rat model of type I diabetes, Jiang et al. documented that human urine MSC-EVs alleviated diabetes-induced kidney injury by inhibiting the apoptosis of podocytes and tubular epithelial cells and increasing the proliferation of glomerular endothelial cells. MSC-EVs contained angiogenesis factors such as VEGF, TGF-β, and angiogenin [71].

Functional proteins of MSC-EVs in cardiovascular diseases
In a rat model of acute myocardial infarction, EVs derived from MSCs with AKT overexpression improved cardiac function through the promotion of angiogenesis. The MSC-EVs with AKT overexpression displayed a higher level of platelet-derived growth factor D (PDGF-D) compared with control MSC-EVs. Transfection of PDGF-D-siRNA into MSCs abolished the angiogenesis effect of MSC-EV in endothelial cells [72]. Gonzalez-King et al. demonstrated that Jagged1 was more abundant in hypoxia-inducible factor 1-alpha (HIF1-α) overexpressing MSC-EVs than in EV control. HIF1-α overexpressing MSC-EVs enhanced angiogenesis as revealed in endothelial tube formation assay in vitro and Matrigel plug assay in vivo. In addition, HIF1-α-induced angiogenesis effects were abolished by prior incubation of MSC-EVs with an anti-Jagged 1 antibody [73]. Vrijsen et al. reported that MSC-EVs were efficiently taken up by endothelial cells, enhanced endothelial vessel formation in vitro, and induced angiogenesis in Matrigel plug assay in vivo. Elevated expression of extracellular matrix metalloproteinase inducer (EMMPRIN) was revealed in MSC-EVs. Pro-angiogenic effect was abolished by knockdown of EMMPRIN [74]. In addition, Lopatina et al. discovered that PDGF stimulated the release of EVs from adipose-derived MSCs. EVs derived from PDGF-treated MSCs had elevated expression of proangiogenic c-kit and stem cell factor (SCF) and enhanced angiogenesis in vitro and in vivo. Blockade of SCF or c-kit with specific antibodies abrogated the proangiogenic activity of the MSC-EVs [75]. Furthermore, Wysoczynski et al. found that human cardiac MSC-EVs promoted cell migration, tube formation, and survival of human umbilical vein endothelial cells in vitro. There was an enrichment of angiopoietins 1 and 2 in MSC-EVs as revealed by cytokine array analysis. Inhibition of Tie2, a receptor for angiopoietins 1 and 2, abolished cell migration induced by MSC-EVs [76].

Functional proteins of MSC-EVs in other diseases
Cystinosis is a rare disease caused by homozygous mutations of the cystinosin gene, resulting in the accumulation of intralysosomal cystine in the body. MSC-EVs alleviated the accumulation of cystine in skin fibroblasts from cystinosis patients in vitro. MSC-EVs also transferred wild-type cystinosin to the skin fibroblasts [77]. Mao et al. reported that ubiquitin protein ligase E3 component n-recognin 2 (UBR2) was enriched in MSC-EVs with p53 deficiency. MSC-EVs with p53 deficiency could be internalized into murine foregastric carcinoma cells, resulting in the overexpression of UBR2. Additionally, UBR2 activated the Wnt/ β-catenin signaling pathway to promote the growth and metastasis of gastric cancer [78].

Conclusions
It has been well established that MSC-EVs have the same immunomodulatory and anti-inflammatory effects as their parental cells. Compared with MSCs, MSC-EVs offer several advantages including lower immunogenicity and an improved safety profile. Therefore, MSC-EVs may serve as an alternative to whole-cell therapy for the treatment of a myriad of diseases. The therapeutic effects of MSC-EVs are at least partially mediated by delivering functional proteins to the recipient cells. MSC-EVs obtained from different origins and preparation methodologies have similar beneficial effects, indicating similar protein compositions of MSC-EVs. Proteomic analysis in MSC-EVs has identified hundreds of proteins in key biological processes such as EV biogenesis, cellular structure, tissue repair and regeneration, and inflammatory response. Approximately 40 of the functional proteins responsible for the protective effects of MSC-EVs are listed in the present review. However, research in functional proteins of MSC-EVs faces several challenges. Foremost, the sorting mechanisms of proteins into MSC-EVs are not clear. The existing sorting pathways apply mainly to proteins with post-translational modifications. Secondly, there is a lack of methodology to efficiently target MSC-EVs to specific tissue types, although targeting attempts have been made by altering EVs via chemical modification and manipulating parental cells via genetic modification. Lastly, it is unknown whether functional miRNAs and proteins work independently or synergistically, giving that miRNAs are also transferred from MSC-EVs to target cells. Further studies are warranted to evaluate MSC-EV therapeutics based on the delivery of functional proteins.