The Role of Small Extracellular Vesicles Derived from Mesenchymal Stromal Cells on Myocardial Protection: a Review of Current Advances and Future Perspectives

Abstract

Small extracellular vesicles (SEVs) secreted by mesenchymal stromal cells (MSCs) are considered one of the most promising biological therapies in recent years. The protective effect of MSCs-derived SEVs on myocardium is mainly related to their ability to deliver cargo, anti-inflammatory properties, promotion of angiogenesis, immunoregulation, and other factors. Herein, this review focuses on the biological properties, isolation methods, and functions of SEVs. Then, the roles and potential mechanisms of SEVs and engineered SEVs in myocardial protection are summarized. Finally, the current situation of clinical research on SEVs, the difficulties encountered, and the future fore-ground of SEVs are discussed. In conclusion, although there are some technical difficulties and conceptual contradictions in the research of SEVs, the unique biological functions of SEVs provide a new direction for the development of regenerative medicine. Further exploration is warranted to establish a solid experimental and theoretical basis for future clinical application of SEVs.

Introduction

Cardiomyocytes are permanent and non-renewable cells. Once irreversible myocardial damage occurs, it is difficult for the myocardium to fully recover, leading to heart disorders and end-stage heart failure, which increases the social burden and mortality rate. According to statistics, the number of patients with myocardial injury is expected to exceed 23 million worldwide by 2030 [1]. Therefore, both basic research and clinical studies on myocardial protection are urgently needed. However, current medication and surgical treatments still have limitations, such as adverse drug reactions, postoperative complications, and disease relapse [2]. Therefore, it is still imperative to find novel and effective therapeutic strategies for myocardial protection.

Mesenchymal stromal cells (MSCs) have captured significant attention in the fields of regenerative medicine and translational medicine [3, 4]. MSCs are adult stromal cells that can be cultured from virtually all tissues, including bone marrow, adipose, umbilical cord, and amniotic membrane tissues [5–7]. Due to their capacity for self-renewal and differentiation into tissue-specific cells, MSCs are crucial for tissue repair and functional recovery of organs. There is growing evidence that MSCs-mediated cardioprotection is not dependent on their ability to differentiate into functional cardiomyocytes, but rather is mainly attributed to the effect of MSCs-derived extracellular vesicles (MSCs-EVs) that promote the repair of damaged cardiomyocytes and the recovery of cardiac function. EVs were previously marked as metabolic waste and did not receive much attention. But now, as a representative of cell-free therapeutic options, EVs can exert similar therapeutic effects to MSCs and even circumvent the limitations of stromal cell transplantation, such as tumorigenicity, poor immunogenicity, and low colonization rate [8–10]. According to the International Society for Extracellular Vesicles (ISEV), EVs < 200 nm are referred to as small EVs (SEVs). EVs with a diameter of 30–150 nm, formerly called exosomes, are also included in the category of SEVs. Numerous studies have demonstrated that SEVs derived from MSCs can alleviate myocardial injury and protect cardiac function by delivering cargoes, promoting vascular regeneration, exerting anti-inflammatory effects, and reducing immune rejection [11–13]. In this review, we focused on the protective mechanisms of SEVs derived from MSCs in myocardial protection and discussed the opportunities and challenges of SEVs therapy.

SEVs Derived from MSCs

Characteristics of SEVs

As early as 1971, Aaronson et al. discovered the existence of EVs [14]. Recently, research on EVs has grown dramatically due to the increasing recognition of EVs as disease biomarkers and therapeutics. In 2014, the ISEV proposed Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines [15]. EVs can be classified into three categories: (1) microvesicles (MVs), ranging from 100 to 1000 nm in diameter [16]; (2) apoptotic bodies, which are 1 to 5 μm in diameter and secreted specifically by apoptotic cells [17]; and (3) SEVs, which have diameters ranging approximately from 30 to 150 nm. However, there are still controversies regarding the isolation, nomenclature, characterization, and function of EVs. For instance, the nomenclature for EVs such as exosome and microvesicle has contradictory definition and unclear specific biological functions. Thus, the ISEV updated the guidelines in 2018 to improve the research quality of EVs with input from over 400 international scientists. The new guidelines suggest that researchers name EVs according to their diameter, density, biochemical composition, descriptions of conditions, or cell of origin if they do not have a complete experimental and theoretical basis for granting a new name to specific EVs. Based on this, this review collectively refers to EVs with a diameter < 200 nm as SEVs [18]. Although SEVs were once regarded as waste products during cell metabolism, but now, it is well-accepted that SEVs serve as essential intercellular mediators that participate in cell repair, functional recovery, stromal cell maintenance, and other physiological processes [19].

The process of SEV formation is a complex biological process that involves several steps. It starts with endocytosis on the cell membrane, where the inward budding of early endosomes occurs. Through a series of packaging and transshipment, mature SEVs are released into the extracellular environment via outward budding. Specifically, the secretion of SEVs is regulated by two distinct molecular mechanisms: the endosomal sorting complex required for transport (ESCRT)–dependent mechanism and the ESCRT-independent mechanism. The ESCRT machinery is composed of approximately 20 proteins that assembled into 4 complexes (ESCRT-0, -I, -II, and -III) including vacuolar protein sorting–associated protein 4 (VPS4), vacuolar protein sorting–associated protein (VTA1), and programmed cell death 6–interacting protein (PDCD6IP). These complexes are critical for the recognition and transport of SEVs [20–22]. The ESCRT-0 complex precisely identifies the ubiquitinated cytoplasmic domains of transmembrane proteins and then further sorts them to the endosomal membrane [23]. The ESCRT-I and ESCRT-II complexes bind to the outside of the endosomal membrane, inducing the luminal vesicles of multivesicular bodies (MVBs). The ESCRT-III complex assembles on the outer surface of the endosomal membrane during the generation of MVBs, promoting the formation of MVBs in the nucleus [24]. On the other hand, the biogenesis of SEVs may also occur through the ESCRT-independent pathway. For instance, the classification and assembly of cargoes transported by SEVs occur in a ceramide-dependent manner, such as tetraspanin family proteins (CD81, CD82, and CD9) [25]. Recent evidence has also indicated that Ras-related protein Rab-31 (RAB31) marks an ESCRT-independent pathway in the biogenesis of SEVs [26].

Isolation of SEVs

Since the discovery of SEVs, researchers have faced challenges in stably isolating, purifying, and identifying these nanoscale biological vesicles and their subgroups. A survey published in 2016 found that the most commonly used method for isolating SEVs was ultracentrifugation, with more than 80% of EVs researchers worldwide using this method [27], though other methods such as size-exclusion chromatography are also popular. Other methods for SEV isolation include density gradient separation, precipitation, filtration, size-exclusion chromatography, and immunoaffinity interaction are still being used. Gradient centrifugation for the extraction of SEVs a is simple and low-cost method, but it has poor yield and is time-consuming [28]. SEVs obtained by precipitation and size-exclusion chromatography have low purity and high technical costs [29]. Some researchers also use commercial kits to isolate SEVs. The advantages and disadvantages of various SEV isolation methods are listed in Table 1. MISEV2018 guidelines suggest that a single or a combination of isolation methods can be selected according to the experimental design and the specificity of SEVs. Table 2 provides the details of SEV isolation methods based on recovery and specificity.

Table 1.

Major isolation methods of SEVs

Method	Principle	Time	Advantages	Disadvantages	Reference
Ultracentrifugation	Extracellular components are removed by various centrifugal forces	140–600 min	Widely used; Low cost; Suitable for large volume samples; Absence of additional chemicals; Great specificity	Equipment; Time and labor consuming; Contamination; Low recovery; Complicated; Damage of SEVs	[30–32]
Density gradient ultracentrifugation	SEVs are separated into a medium of similar density position by centrifugal force	250 min–2 days	High purity; Absence of additional chemicals; Preservation of SEVs	Equipment; Time and labor consuming; Low production; Complicated	[32–34]
Ultrafiltration	SEVs are separated by using different molecular weight cut-offs ultrafiltration membranes	130 min	Low cost; Short time; Suitable for large volume samples; Medium purity	Filter block; Loss of sample; Contamination; Low production;	[32, 35]
Size-exclusion chromatography	SEVs of different sizes exhibit various elution times passing through porous resin particles	1 mL/min	High purity; Easy operation; Preservation of SEVs; Absence of additional chemicals; Reproducibility	Relatively high cost; Time and labor consuming; Equipment	[32, 36]
Immunoaffinity capture	Binding between biomarkers such as surface antibodies of SEVs and antibody-recognized ligands	Base on antibody	Easy operation; Preservation of SEVs; High purity	High cost; Time-consuming; Contamination; Low production	[33, 35, 37]
Polymer precipitation	Reducing the solubility of SEVs	45–130 min	Low cost; Easy operation; Preservation of SEVs; High production	Contamination by non SEVs; Low quantification of SEVs	[31, 33, 35]
Microfluidic technologies	Based on different diameters of microfluidic device with microporous filtration system and immunoaffinity principle	1–14 µL/min	High production; Purity; Fast processing time; High sensitivity	High cost; Equipment; Complicated	[38–40]

Table 2.

Considerations for SEVs separation/enrichment

Validity	Definition	Isolation technique
High recovery, low specificity	Recover the highest amount of EVs, whatever their nature	Precipitation kits
		Low molecular weight cutoff centrifugal filters without a further separation step
		Lengthy or very high-speed ultracentrifugation without previous lower-speed steps
Intermediate recovery, intermediate specificity	Recover mixed EVs along with some amount of free proteins, ribonucleoproteins, and lipoproteins, depending on the matrix	Size-exclusion chromatography High molecular weight centrifugal filters differential ultracentrifugation using intermediate time/speed with or without wash Tangential flow filtration membrane-affinity columns
Low recovery, high specificity	Recover a subtype (or a few subtypes) of EVs with as few non-vesicular components as possible	Subtypes of EVs can be separated by their size, their density upon either flotation or pelleting in a density gradient, their surface protein, sugar, or lipid composition, or other biophysical properties such as surface charge

Identification of SEVs

The identification of SEVs mainly focuses on their morphology, size, and surface marker proteins. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are commonly used to visualize the morphology of SEVs. Nanoparticle tracking analysis (NTA) can determine the size distribution of SEVs. Western blot and flow cytometry are applied for the identification of the surface markers of SEVs, such as CD9, CD63, and TSG101 [41].

Mechanisms of MSC-SEVs on Myocardial Protection

The transplantation of MSCs has been recognized as a promising strategy for cardiac repair after myocardial injury [42–44]. Increasing experimental studies have suggested that the therapeutic potential of MSCs may arise from their autocrine effects. Notably, recent research has also revealed that MSCs mediate cardiac repair via a paracrine manner, in which SEVs play a vital role. For example, MSCs have been demonstrated to mediate cardioprotection during myocardial ischemia–reperfusion injury (MIRI) by secreting SEVs [45]. MSCs-derived SEVs can be used alone to promote cardiac repair, independent of the biological effects of MSCs. In a rat model of myocardial infarction (MI), MSCs-derived SEVs were even significantly superior to MSCs in attenuating inflammation, inhibiting fibrosis, and improving cardiac function [46]. Here, we review the myocardial protective mechanisms of MSC-SEVs, including the effects of delivering cargo, anti-inflammatory, angiogenesis promotion, and immune regulation, as follows.

microRNAs (miRNAs) Delivered by SEVs

SEVs are known to deliver various biomolecules, including messenger RNAs (mRNAs), miRNAs, proteins, and lipids, to regulate the functions of recipient cells [47]. Among these biomolecules, miRNAs are an evolutionarily conserved class of small single-stranded noncoding RNAs (18–24 nucleotides) that bind to the 3′-untranslated region (3′-UTR) of a specific target mRNA sequence for the post-transcriptional regulation of gene expression [48]. It has been reported that more than 60% of human protein-coding genes are directly regulated by miRNAs, which can suppress gene expression by inhibiting mRNA translation and/or by promoting mRNA degradation [49, 50].

Numerous experiments have shown that SEVs derived from MSCs can alleviate myocardial injury by delivering miRNAs. In a rat model of acute myocardial infarction (AMI), the left ventricular end systolic diameter (LVESD) and left ventricular end diastolic diameter (LVEDD) were significantly increased but intervention with MSC-SEVs reversed this change. While the left ventricular ejection fraction (LVEF) was reduced to 30% in AMI, after treatment with hucMSCs-derived SEVs, LVEF increased to about 60%. Furthermore, MSC-SEVs reduced infarct size from 40 to 20%, indicating a significant myocardial protective effect. Subsequent experiments revealed that miRNA-19a is poorly expressed in myocardial tissues of AMI rats. Injection of hucMSCs-derived SEVs with miRNA-19a inhibitor did not show significant myocardial protective effects, while hucMSCs-derived SEVs with miR-19a enhanced myocardial protection in AMI rats. Further research suggested that hucMSCs-derived SEVs deliver miRNA-19a to target the transcription factor SOX6, activating serine/threonine-protein kinases (AKT), inhibiting mitogen-activated protein kinase 10 (MAPK10)/caspase-3, and ultimately protecting rats from AMI [51]. Similar reports have shown that SEVs secreted by bone marrow–derived MSCs (BM-MSCs) can carry miRNA-19a/19b to injured myocardial tissues, promoting cardiac function recovery and alleviating myocardial fibrosis [52]. Other miRNAs, such as miRNA-125 [53–55], miRNA-19 [56], miRNA-21a [57], miRNA-132 [58], miRNA-182 [59], miRNA-126 [60], miRNA-25 [61], miRNA-210 [62], miRNA-671 [63], miRNA-29c [64], miRNA-486 [65], miRNA-144 [66], miRNA-149 [67], and miRNA-22 [68], have also been experimentally demonstrated to participate in myocardial protection. In these studies, miRNAs delivered into the injured myocardium by SEVs not only alleviate cell injury but also regulate apoptosis, myocardial fibrosis, and pyroptosis. A valuable study analyzed miRNA produced by three clinically grade stromal cells of EVs. While there were some miRNAs in common, each EV had a unique miRNA profiles. All EVs improved cell adhesion/migration, immune response, platelet aggregation, protein translation/stabilization, and RNA processing. This study is beneficial for further clarifying the molecular mechanisms and functions of miRNAs in SEVs [69]. Although the above studies show that SEVs can serve as cargoes for miRNA delivery, lipoproteins have also been proven to deliver miRNAs. Actually, lipoproteins are inevitably mixed with SEVs [70], making further purification of SEVs become one of the major obstacles in current SEVs research. Table 3 lists the mechanistic role of some SEVs-delivered miRNAs in myocardial protection.

Table 3.

miRNAs delivered by MSCs-derived SEVs to regulate cardiac repair

Disease model	Type of MSCs	Mechanism	Target cell	Dose	Administration	Function	Reference
SD rat AMI	HucMSCs	MiR-19a	CM	400 μg	Intravenous	Targeting SOX6 and activating AKT to protect cardiomyocytes	[51]
Mouse MI	BM-MSCs	MiR-19a/b	CM	0.5 μmol	Intravenous	Enhancing the recovery of cardiac function and reducing cardiac fibrosis	[52]
Mouse MI	BM-MSCs	MiR-125b	CM	50 μg/mL	Intramyocardial	Reducing the infarct size and promoting cardiac repair	[54]
SD rat MIRI	BM-MSCs	MiR-125b	CM	50 μg	Intramyocardial	Protecting against MIRI by targeting SIRT7	[55]
SD rat AMI	BM-MSCs	MiR-19a	CM	4 × 106 MSCs	Intramyocardial	Enhancing cardiac protection via activation of the AKT and ERK signaling pathways	[56]
Mouse MI	BM-MSCs	MiR-21a	CM	300 μg	Intramyocardial	Contributing to cardiac repair	[57]
Mouse MIRI	BM-MSCs	MiR-182	CM	50 μg	Intramyocardial	Attenuating MIRI and changing the polarization of macrophages	[59]
H/R-cell	BM-MSCs	MiR-126	HUVECs	/	Co-culture	Enhancing angiogenesis	[60]
Mouse MIRI	BM-MSCs	MiR-25-3p	CM	5 μg in 100 μL PBS	Intramyocardial	Enhancing cardiac protection by targeting pro-apoptotic proteins and EZH2	[62]
SD rat MI	BM-MSCs	MiR-210	CM	1 × 106 MSCs	Intramyocardial	Protecting cardiac injury and limiting myocyte apoptosis	[63]
Mouse MI	adMSCs	MiR-671	CM	100 μg	Intramyocardial	Alleviating myocardial infraction by inactivating TGFBR2/Smad2	[64]
Mouse MIRI	BM-MSCs	MiR-29c	CM	20 μg	Intramyocardial	Regulating autophagy under IR injury	[65]
SD rat MIRI	BM-MSCs	MiR-486-5p	CM	400 μg	Intravenous	Inhibiting apoptosis and activating the PI3K/AKT pathway	[66]
Hypoxia cell	BM-MSCs	MiR-144	H9C2	5 μg/mL	Co-culture	Promoting anti-apoptotic proteins under hypoxic conditions	[67]
H/R H9C2 model	BM-MSCs	MiR-149	H9C2	5 μg	Co-culture	Protecting myocardium against H/R injury via miR149/let-7c/Faslg	[68]
Mouse MI	BM-MSCs	MiR-22	CM	1 μg	Intramyocardial	Reducing cardiac fibrosis and inhibiting apoptosis	[69]
Mouse AMI	BM-MSCs	MiR132	CM	600 μg	Intramyocardial	Promoting angiogenesis	[70]

Protein Cargo of MSC-SEVs

MSC-SEVs can directly affect biological processes by regulating the expression of protein levels in damaged myocardium. A study comparing proteomic analyses of myocardial infarction tissue with SEVs-treated tissue showed that MSC-SEVs significantly reduced infarct size by about 15–20% and induced significant changes in inflammatory and apoptosis-related proteins [71]. MSC-SEVs were found to modulate platelet-derived growth factor receptor-β (PDGFR-β) expression immediately and alleviate microvascular dysfunction caused by cardiac ischemia–reperfusion, as well as inhibit fibrosis development [72]. SEVs contain hundreds of proteins associated with cellular communication, inflammation, tissue repair and regeneration, and metabolism [73]. Understanding the biological role of these proteins is crucial for further elucidating the function of SEVs. Proteomic analysis of SEVs derived from bone marrow-MSC (BM-MSC) indicated that the proteins enriched in these vesicles were related to regeneration medicine that include collagen, extracellular matrix, bone regeneration, and muscle regeneration. Proteins in adipose tissue-MSC (AT-MSC) SEVs and umbilical cord-MSC (UC-MSC) SEVs were found to be related to immune response [74]. Vaka et al. reported that there were differences in the protein expression of BM-MSC-SEVs and UC-MSC-SEVs, specifically in proteins involved in actin organization, cadherin binding, and cellular adhesion, indicating the potential of MSC-SEVs proteins in repairing myocardial tissue [69]. Previous studies have also investigated the proteomics of various heart diseases [75–80], providing valuable insights into cardiac pathogenesis and identifying potential therapeutic targets. Correlating these findings with MSC-SEVs proteomics analysis could further elucidate the mechanism of action of MSC-SEVs proteins and facilitate the development of more reliable therapeutic strategies.

Pro-angiogenic

Interstitial cells in myocardial tissues, such as endothelial cells and fibroblasts, interact with myocardial cells to support normal myocardial contractility [81]. During MIRI, vascular regeneration impairment and endothelial dysfunction aggravate irreversible myocardial injury, making revascularization and angiogenesis important mechanisms for protecting the myocardium. Angiogenesis in myocardial tissues is a complex process, which requires the action of pro-angiogenic signal cascades. Some key factors, such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and angiogenin (ANG), are closely associated with vascular regeneration [82]. MSCs possess the capacity to promote angiogenesis. MSCs can directly build tubular structures and exhibit significant pro-angiogenic activity on endothelial cells in vitro. The pro-angiogenic function of MSCs is attributed to the secretion of VEGF, ANG, transforming growth factor-beta (TGF-β), nerve growth factor (NGF), and other pro-angiogenic factors by MSCs [83]. SEVs derived from MSCs have been used to improve islet transplantation outcomes due to the secretion of VEGF [84]. In human umbilical vein endothelial cells (HUVECs), the mRNA expression levels of VEGF, ANG-1, and PDGF are decreased following hypoxia injury. However, SEVs derived from BM-MSCs significantly increased the mRNA expression levels of VEGF, PDGF, and ANG-1 in hypoxic HUVECs. Other researchers have found that HUVECs-absorbed SEVs increased the tube formation of endothelial cells in vitro and enhanced the angiogenesis capacity in vivo by downregulated Ras GTPase–activating protein 1 (RASA1) expression [58]. Similarly, cardiac myocyte progenitor cells (CMPCs)–derived SEVs have powerful pro-angiogenic effects due to the presence of extracellular matrix metalloproteinases inducer (EMMPRIN) [85]. MSCs-derived SEVs can certainly have pro-angiogenic effects on repairing myocardial tissues and restoring cardiac function [86].

Anti-inflammatory

Inflammation is a key mechanism that leads to myocardial injury. In various heart diseases, such as acute myocardial infarction, heart failure, and MIRI, the activity of pro-inflammatory factors is increased, and the activation of pathways is not conducive to myocardial repair [87–89]. TNF-α, IL-1, and IL-6 are classical pro-inflammatory factors, and reducing the activity of these factors is beneficial for myocardial repair [90–92]. In a study of AMI in vitro, the levels of pro-inflammatory cytokines including IL-1β, IL-6, and TNF-α were significantly increased in cardiomyocytes by oxygen–glucose deprivation (OGD). However, MSC-SEVs reversed this change, obviously improved cell viability, and alleviated apoptosis [61]. Inflammatory response is a critical mechanism by which the heart responds to injury for adaptive remodeling. Dysregulated inflammation leads to the excessive release of inflammatory factors and aggravates myocardial injury. Nucleotide-binding oligomerization domain–like receptor family pyrin domain–containing 3 (NLRP3) is a key factor in the inflammatory cascade, and targeting the NLRP3 inflammasome has become an emerging strategy for anti-inflammatory therapies. MIRI activates NLRP3, leading to the release of inflammatory factors such as IL-1β, IL-18, and caspase1, which eventually aggravating myocardial injury. Inhibition of NLRP3 can reduce the size of myocardial infarction and protect cardiac function [93, 94]. MSCs-derived SEVs can inhibit NLRP3 inflammasome activation and suppress inflammatory cytokine release, thereby protecting cardiomyocytes from hypoxia/reoxygenation (H/R)–induced injury [95]. MSCs-derived SEVs can also enhance the viability of cardiomyocytes in the H/R model by downregulating the levels of NLRP3 and caspase-1 [96].

Immunomodulatory Property

Recent studies have demonstrated the remarkable therapeutic efficacy of MSCs in treating a wide range of autoimmune diseases due to their immunomodulatory properties, such as inhibiting T cell proliferation, increasing the number of Treg cells, inhibiting Th cell expansion, and maintaining the balance between Th1 and Th2 cells [97]. The immunomodulatory effects of MSCs-derived SEVs have also received widespread attention [98]. The microvascular network in myocardial tissue facilitates communication between immune cells and antibodies, making the heart vulnerable to immune-mediated damage. Macrophages play a crucial role in regulating myocardial homeostasis and can be classified into two major polarization states: pro-inflammatory M1 and anti-inflammatory M2. Imbalances between these two phenotypes can lead to inflammatory conditions and cardiac injury. Disruption of macrophage metabolism can exacerbate inflammation and destroy M1/M2 homeostasis. Injections of MSCs-derived SEVs have been shown to restore cardiac function in Sprague–Dawley (SD) rats suffering from MIRI. MSCs-derived SEVs did not affect the total number of macrophages but notably decreased M1 macrophages and increased M2 macrophages. This was manifested by elevated markers of M2 macrophages, such as arginase 1 (Arg1), IL-10, CD206, and TGF-β. Furthermore, MSCs-derived SEVs inhibited the production of lipopolysaccharide (LPS)–induced IL-6 and iNOS but promoted the upregulation of IL-10 and Arg1 in vitro. This study suggested that SEVs facilitated the transformation of macrophages from M1 to M2 phenotype and reduced inflammation in MIRI [59]. MSCs-derived SEVs have inhibitory effects against immune cells, including effector T cells, macrophages, and natural killer cells [99]. Immune rejection is a serious complication of heart transplantation. How to prevent and alleviate immune rejection has always been a challenge for researchers. The biological characteristics of SEVs provide the potential to reduce immune rejection. When combined with immunosuppressive treatment, SEVs derived from donor immature dendritic cells can prolong allograft survival by inhibiting T cell activation [100]. Treatment with MSCs-derived SEVs has been shown to increase the number of Treg cells, decrease the number of CD8 + T cells, suppress the levels of pro-inflammatory factors, and elevate the levels of anti-inflammatory factors in rats receiving heart transplantation. As a result, SEVs not only alleviate immune rejection but also improve the cardiac function of heart allografts [101].

Modified SEVs Derived from Engineered MSCs

MSCs can be selectively modified on demand, and SEVs derived from modified MSCs can precisely target key regulatory pathways and cytokines with biological effects than ordinary SEVs. SEVs produced by MSCs under different culture conditions also have distinct biological functions. SEVs produced by MSCs under hypoxic conditions, for example, can promote the functional recovery of ischemic tissues [102]. In a mouse model of MI induced by ligation of the left anterior descending artery, the effects of MSCs-derived SEVs cultured under normal and hypoxic conditions were compared. MSCs-derived SEVs cultured under hypoxic conditions have a stronger effect on cardiac function recovery, as evidenced by significantly increased cardiac ejection fraction, decreased cardiac left ventricular internal diameter (LVID), and effectively reduced MI size compared with the MSCs-derived SEVs cultured under normal conditions. By comparing the miRNAs transported by normal SEVs and hypo-SEVs, miRNA-125b was found to be significantly enriched in hypo-SEVs. Down-regulation of miRNA-125b weakened the myocardial protection of hypo-SEVs, while overexpression of miRNA-125b-5p inhibited the expression of p53 in ischemic myocardium of MI mice. Blocking the apoptosis of cardiomyocytes is conducive to myocardial protection [54]. Heme oxygenase-1 (HO-1) plays a critical role in myocardial protection by promoting cell survival, repressing cardiomyocyte apoptosis, and enhancing angiogenesis [103]. Hemin is an HO-1 inducer. Researchers had discovered that both SEVs derived from MSCs and SEVs derived from MSCs pre-treated with Hemin could significantly improve cardiac function and reduce fibrosis in MI, but the latter exhibited better myocardial protection. Further miRNA sequencing showed a higher expression pattern of miRNA-183-5p in Hemin-MSCs-SEVs than MSCs-SEVs. miR-183-5p could target high-mobility group box 1 (HMGB1), and miRNA-183-5p knockdown partially reversed the effects of Hemin-MSCs-SEVs in attenuating mitochondrial fission and repressing cell senescence of cardiomyocytes in MI [104].

Angiogenesis is essential for myocardial tissue repair following MI. Hypoxia inducible factor (HIF)–1α, a key transcriptional regulator under hypoxia conditions, regulates the expression of numerous genes related to angiogenesis. HIF-1α promoted the secretion of SEVs by MSCs. SEVs released by HIF-1α-overexpressing MSCs (HIF-MSCs) induced angiogenesis in human endothelial cells. Further study have found that the expression of HES family transcription factor 1(HES1) and helix-loop-helix transcription factor (Hey2) were higher in HUVECs treated with HIF-MSCs-derived SEVs than those treated with MSCs-derived SEVs [105]. Similarly, HIF-1α-overexpressing SEVs were superior to control SEVs in preserving cardiac function and promoting angiogenesis in the SD rat model of AMI [106].

Prospect and Limitation of MSCs-Derived SEVs

The paracrine function of MSCs has made them a promising option for tissue repair and regenerative medicine (Fig. 1). As one of the key paracrine effectors, SEVs are essential for cell-to-cell communication by carrying bioactive substances [107]. MSCs-derived SEVs have unique advantages. Firstly, MSCs may contain mutated DNA that contributes to tumorigenesis, but the existing studies do not find evidence to support the idea that MSCs-derived SEVs lead to the development of tumors. Secondly, SEVs are smaller than MSCs, making them able to easily pass through capillaries, while MSCs are unable to cross the capillary bed and can cause blockage. Thirdly, the infusion dose of MSCs is rapidly reduced after transplantation, whereas MSCs-derived SEVs can attain a larger circulating dose.

Fig. 1.

Biological characteristics of SEVs. In MSCs, the invaginated cell membrane forms vesicles, and then a variety of proteins, nucleic acids, lipids, and other substances are absorbed by the vesicles to form early endosomes and then MVBs have two pathways: one is to secrete to the extracellular space and release SEVs; another pathway is intracellular lysosomal binding degradation. There are three main ways for cardiomyocytes to absorb SEVs: membrane fusion directly, membrane endocytosis, and receptor binding on the cardiomyocyte membrane. SEVs have a double-membrane structure and are rich in DNA, miRNA, mRNA, polypeptides, enzymes, and other biological active substances. The membrane of SEVs contains tetraspanins, fusion proteins, integrins, etc.

By searching www.ClinnicalTrial.gov, it has been found that there are several clinical studies reporting on the role of MSCs-derived SEVs in other diseases, such as dry eye, T1DM, and periodontitis [99]. However, relatively little is known about the effect of SEVs on cardiovascular diseases. There are two ongoing clinical trials (NCT04356300, NCT05669144) evaluating the effect of MSCs-derived SEVs on cardiovascular diseases, specifically related to aortic dissection and patients undergoing coronary artery bypass grafting (CABG) surgery. The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has become an unprecedented global health crisis [108–110]. Due to the unique biological activity of SEVs, several clinical trials are been conducted to explore the therapeutic effect of SEVs on SARS-CoV-2-infected patients [111, 112].

Currently, there is still controversy regarding the basic research of MSCs-derived SEVs. Unlike MSCs, which have the ability to proliferate, MSCs-derived SEVs cannot be replicated or reproduced. The primary issues that need to be addressed are the potency and therapeutic dose of SEVs. As shown in Table 3, each study used different doses of MSCs-derived SEVs, which may be related to differences in the sources of SEVs, extraction methods, and usage routes. Therefore, it is necessary to establish a unified standard for extracting SEVs. Furthermore, although MSCs-derived SEVs have shown great myocardial protection effects, the specific biological mechanisms still need to be explored. As mentioned earlier, multiple studies have shown that SEVs can alter biological processes in the heart muscle by delivering miRNAs, but some scholars have found that the content of miRNA in SEVs is very low, as little as 0.9% of the total RNA. This means that 100 µg of MSC exosomes only contains 60 ng of miRNA. Therefore, if miRNA from SEVs is a key factor in the biological mechanism, it depends on the high concentration and abundance of SEVs. Additionally, there are many treatments that target miRNA, such as siRNA. Moreover, the cost of extracting SEVs is high. If SEVs are only used as a tool for miRNA delivery, it may result in economic losses and waste. Furthermore, it is speculated that the main biological role of SEVs may be due to the protein cargo they carry [113–115].

Despite the potential of MSCs-derived SEVs in regenerative medicine, several key issues remain to be addressed before extensive clinical application. Firstly, SEVs of different sizes may have different therapeutic effects on cardiac regeneration and repair. And thus, accurately isolating SEVs of the required size is one of the current technical challenges. Secondly, there may be a possibility of mutual invasion and contamination between SEVs from different sources, which could affect their therapeutic efficacy. Thirdly, while studying a single SEV can provide a method to deeply understand the biological characteristics and mechanisms of SEVs, currently, there is a lack of experimental techniques to study a single SEV. Finally, SEVs produced under different conditions can be used to improve cardiac injury under different conditions, And obtaining targeted SEVs accurately is also a challenge for current research.

Conclusion

This review discusses the therapeutic potential of MSCs-derived SEVs for myocardial protection. The publication of numerous studies on the cardioprotective effect of MSCs-derived SEVs has contributed to the understanding of the composition and function of SEVs. At present, new techniques and evaluation criteria for SEVs are urgently needed. Preclinical research on SEVs is still in its preliminary stage, and stronger evidence is required to support the application of SEVs as a novel biological agent in clinical practice.

Author Contribution

Hongkun Wu generated the concept for this review; Xingkai Qian and Guiyou Liang performed the literature search; Hongkun Wu drafted the manuscript; Xingkai Qian and Guiyou Liang critically edited the manuscript. All authors reviewed and approved the final version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (No. 82170286, No. 81960051), Basic Research Program of Guizhou Province (QKHJC-ZK[2022] YB376), The Science and Technology Fund Project of Guizhou Provincial Health Commission (gzwkj2022-088), Project of Guizhou Provincial education Commission, YJSKYJJ[2021]145. YJSKYJJ[2021]146. GSZYQN[2021]NO.13.

Data Availability

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Code Availability

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Declarations

Human and animal rights

This article does not contain any studies with animals performed by any of the authors. This article does not contain any studies with human participants or animals performed by any of the authors.

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Conflict of Interest

The authors declare no competing interests.