The prevailing view on EVs’ classification depends on the diameter and origin. Several subtypes of EVs have been identified, such as exosomes, membrane vesicles, apoptotic bodies, and MVs (O'Brien et al., 2020). Exosomes are released from cells via the endolysosomal pathway. Exosomes are formed by inward budding of the limiting membrane of multivesicular endosomes (MVEs). The diameter of exosomes is 30–50 nm and MVs (also referred to as ectosomes, 50–1000 nm diameter), budded directly from the plasma membrane. The apoptotic bodies (1–5 μm diameter) are derived from the apoptotic cell membrane (Han et al., 2021) (Figure 1).

EVs size is crucial for the composition, tissue biodistribution efficiency, and intracellular trafficking in the application of CVDs therapy (Théry et al., 2006; Théry et al., 2018; Cabeza et al., 2020). For example, larger aggregates are more likely to be associated with membrane recycling or lysosomal degradation (Lim and Gleeson 2011). However, smaller vesicles (diameter <100 nm), were taken up via clathrin- or caveolae-mediated endocytosis (Costa Verdera et al., 2017). Therefore, smaller EVs may be more efficiently delivered into the cell. In the cardiac environment, especially for systemically administered EVs, the volume of EVs is critical relative to the successfully penetration into the heart tissue. So that EVs can be effectively absorbed by the relevant cell types (Liu et al., 2020).

The surface potential of EVs is another important property. The EVs potential depends on the sugar composition of the plasma membrane (Akagi et al., 2014), which is rich in phosphate groups. In other words, the global negative charge is the norm for EVs. The changes in surface charge can be used to infer the stability of EVs in suspension. With the reduction of repulsive force, EVs accumulate mostly. The surface potential of EVs is the key to the interaction between EVs and many potential ligands, as well as their uptake by target cells (Ayala et al., 2013; de Abreu et al., 2020). In addition, more and more studies have shown that EVs play important roles in hemostasis and thrombosis due to the exposure to negatively charged procoagulant phospholipids (PPL) (Francula-Zaninovic and Nola 2018).

For the structure of EVs, there is some structural similarity between cells and vesicles, both of which are lipid bilayer structure and negative potential. EVs are less susceptible to the penetration of small solutes due to their high cholesterol content (de Abreu et al., 2020). Benefiting from the external structure, EVs ensure the safe and efficient transmission from internal content to target cells. More interestingly, membrane composition differs from different types of EVs.

The biological contents of EVs consist of various bioactive substances, including nucleic acids (DNA and RNA), proteins (biogenesis factors, enzymes), lipids, and metabolites (Jeppesen et al., 2019). The microRNA (miRNA), transfer RNAs (tRNAs), messenger RNA (mRNA) and fragmented mRNAs, long-stranded non-coding RNAs (lncRNAs), and circRNA are all found in EVs though the concentrations of RNA are relatively low (Li J et al., 2021). Proteins, such as Membrane surface markers (annexins and GTPases), lysosomal-associated membrane proteins 1 (LAMP1 and LAMP2), heat shock proteins (HSP 70 and HSP 90), tetraspanins proteins (CD9, CD63, CD37, CD53, CD81, and CD82), phospholipases, and other lipid-related proteins, are used to identify and isolate cell-type-specific EVs (Loyer et al., 2018). As the major part of EVs, RNAs and proteins don’t exist in the cytoplasm randomly (Valadi et al., 2007). Compared with lncRNAs and miRNA, circRNA is rarely studied, which is likely to become the next hot molecule for exosomes detection due to its unique stability, tissue specificity, timing, and disease specificity (Shi et al., 2020) (Figure 2). In addition to directly cell-cell contact or the transport of secreted molecules, EVs also participate in intercellular communication. By containing and transporting various bioactive molecules to target cells, EVs could affect biological behaviors and gene phenotypes through several molecular pathway’s regulation.

EVs have been proposed to transfer membrane encapsulated cargoes from donor to acceptor cells. However, the mechanism of EV-content release within acceptor cells remains debated. There is no consensus on the uptake mode of EVs, whether is receptor-dependent or not. High-resolution microscopy or new living cell reporter genes are needed for the research on EVs-content delivery within target cells (Sung et al., 2020), and reporter gene assay could also be used to measure the EVs membrane fusion efficiency during cargo delivery to receptors (Somiya and Kuroda 2021). Currently, studies have shown the way that EVs enter cells including receptor-mediated endocytosis, clathrin interaction, lipid raft interaction, phagocytosis, micropinocytosis, and possible directing fusion (Kalluri and LeBleu 2020). In addition, several pieces of research have shown that most EVs may not be absorbed by uninjured or chronically damaged heart tissues, but by non-cardiac cells (Yi et al., 2020; Kang et al., 2021).

EVs are a group of heterogeneous natural particles that can be used for CVDs therapy. More and more evidence highlights that EVs exhibit potential therapeutic function in CVDs (Sánchez-Alonso et al., 2018). Certain properties in these endogenous vesicles enable them to survive in the extracellular space, bypass biological barriers, and transport their biologically active molecular cargo to recipient cells (Nawaz and Fatima 2017; Kalluri and LeBleu 2020). The biological function of EVs depends on the state of donor cells and can vary during the different microenvironments (Genschmer et al., 2019). EVs containing miRNAs and proteins regulate multiple functions in target cells, including maintaining cardiovascular balance and health, inducing pathological changes in CVDs. Therefore, the fascinatingly complex features of EVs should also be taken into consideration in clinical applications (Han et al., 2021). EVs carried with miRNA-21 can effectively inhibit apoptosis and restore cardiac function in vivo and in vitro (Song et al., 2019). The therapeutic benefits of EVs in CVDs have also been confirmed by large animal models such as pigs and nonhuman primates (Li Q et al., 2021; Yao et al., 2021). The therapeutic effect of EVs has also been evaluated in several kinds of diseases through small animal models, including MI (Couto et al., 2017), hindlimb ischemia (Prabhu et al., 2017), and stroke (Tian et al., 2018). Studies have shown that EVs from different sources can trigger a variety of cardioprotective effects (Figure 3) (Benjamin et al., 2017; Jie et al., 2017; Fu et al., 2020). EVs isolated from the plasma of healthy volunteers can protect myocardium from ischemic reperfusion (I/R) injury or promote angiogenesis in the ischemic limb injury in animals (Vicencio et al., 2015; Aday et al., 2021). Increasing evidence suggests that the effects of EVs on target cells are mainly dependent on miRNAs and proteins transferred by EVs (Benjamin et al., 2017). Cardiomyocytes release EVs with high expression of miR-217, which act on fibroblasts and promote the proliferation of fibroblasts. These results indicate that miR-217 plays an important role in cardiac hypertrophy and dysfunction (Nie et al., 2018). Cardiomyocytes can promote cardiac fibroblast proliferation and myofibroblast differentiation by releasing EVs containing a high level of miR-208a (Yang et al., 2018). Related studies have also shown that EVs derived from platelets containing polyubiquitin, which can reduce platelet aggregation and inhibit the expression of CD36 through ubiquitination, thereby inhibiting the formation of atherosclerotic thrombosis (Srikanthan et al., 2014). EVs can act as a drug and ideal drug carrier in therapy for their benefit on circulation, immune rejection and cellular toxicity. As drug carrier, EVs exhibit potentials on protecting bioactive cargoes from degradation and higher transmission efficiency, compare with common liposomes (Barile and Vassalli 2017).

In consideration of that cardiac is lack of regenerative capacity following MI. Stem cell therapy has recently been applied to improve cardiac repairs in research. Early studies have found that stem cells, especially modified stem cells, show significant therapeutic potential in CVDs. During therapy, the differentiation degree can determine the efficacy of stem cells (Mangi et al., 2003; Kawamoto et al., 2006). Gnecchi’s group also found that the higher expression of Akt in mesenchymal stem cells (MSCs) the shorter recovery time cardiac function has, which means that modified stem cells are optimized for CVDs therapy (Gnecchi et al., 2006). Stem cells have a great potential for tissue regeneration and repair. However, stem cells have the ability of self-renew and proliferate indefinitely. The clinic application of stem cells is limited due to the teratoma risk (Nawaz et al., 2016). Recent findings elucidate exchange of genetic information utilizing persistent bidirectional communication mediated by EVs could regulate stemness, self-renewal, and differentiation of stem cells (Nawaz et al., 2016). Studies found that MSCs transplantation accelerated angiogenesis and improved cardiac repair after MI (Müller et al., 2018; Liang et al., 2021). Subsequently, the mechanism of MSCs mediated paracrine has been accepted and validated in exploring the principal mechanism of stem cells for CVDs therapy (Fan et al., 2020). It has emerged that the paracrine functions of MSCs could, at least in part, be mediated by EVs. EVs have significant potential as a novel alternative to CVDs. Studies have also found that native EVs used for CVDs could be derived from MSCs (Gollmann-Tepekylü et al., 2019), cardiac progenitor cells (CPCs), cardiosphere-derived cells (CDCs) (Couto et al., 2017), embryonic (ESCs), induced pluripotent stem cells (iPSCs) (Adamiak et al., 2018), dendritic (DCs) (Liu et al., 2016), and endothelial progenitor cells (EPCs). Compared to cell-based therapies, EVs may exhibit a superior safety profile such as a lower propensity to trigger innate and adaptive immune responses and the inability to form tumors directly (Rani et al., 2015). Moreover, the isolation of EVs from stem cells is potentially sustainable and reproducible. Compared with cells, EVs can be stored with high efficiency safely and easily (Ran et al., 2015).

MSC-derived EVs (MSC-EVs), derived from different origins such as bone marrow, adipose tissues, umbilical cord and heart, have exhibited comprehensive protection and reparation effects on cardiovascular (Racchetti and Meldolesi 2021). MSC-EVs can reduce cardiomyocyte apoptosis and cardiac fibrosis, but promote angiogenesis via the transfering bioactive miRNA, lncRNA, and protein cargos into targeted cells (Peng et al., 2020; Gca et al., 2021). CDC and CPC-derived EVs (CDC-EVs and CPC-EVs) have also been extensively used in MI or I/R injury (Barile et al., 2016; Romain et al., 2016). Studies had proved that CDC-EVs was safe and effective during repairing heart tissue damaged in HF (Raj and Mohsin 2017; Ibrahim et al., 2019). Other studies have shown that CDC-EVs can also reduce infiltration and inhibit cardiomyocyte apoptosis via transferring Y RNA fragment (EV-YF1) and miRNA-181 to the macrophages (Couto et al., 2017). Importantly, miR-147, miR-18, miR-133, miR-206, miR-10, miR-142, miR-146a were enriched in CDC-EVs and performed protective effects (Ibrahim et al., 2014). In addition, CPC-derived exosomes have also exhibited cardiac protection by reducing cell apoptosis and poor remodeling (Xiao et al., 2016). Another study found that human CPC-EVs reduced myocardial infarction by reducing cardiomyocyte death and promoting angiogenesis (Wu et al., 2020).

The iPSCs-derived EVs (iPSCs-EVs) also provide a cell-free system to avoid the risks associated with direct cell transplantation (Chandy et al., 2020). Regarding iPSC-EVs, miRNA is also an important functional component. As reported, miR-19, miR-20, miR-126, miR-130, and miR-17 derived from iPSCs exert a powerful effect on promoting angiogenesis, adjusting hypoxia, and oxidative stress. In addition, bioinformatics analyses showed that miRNA in iPSC-EVs can prove the cellular functional state to inhibit apoptosis through regulating Wnt, phosphatidylinositol-3 kinase/protein kinase B (PI3K-Akt), and mitogen-activated protein kinase (MAPK) pathways (Adamiak et al., 2017).

ESC-derived EVs can also augment cardiac function effectively in infarcted hearts through enhancing neovascularization, cardiomyocyte survival and proliferation, but inhibiting fibrosis in cardiac. This beneficial effect of ESC-derived EVs was linked to miR-294 was delivery from ESC to CPCs specifically, then increased survival, cell cycle progression, and proliferation (Adamiak and Sahoo 2018). The study showed that human CD34⁺-positive EPCs exhibited the potential on CVDs therapy (Sahoo et al., 2011; Sahoo and Losordo 2014) and promote proangiogenic paracrine activity in ischemic limb tissues (Prabhu et al., 2017). Further studies shown that EPCs-derived EVs (EPCs-EVs) could increase the formation of new blood vessels and improved left ventricular function in patients with MI (Yue et al., 2020). In addition, EPCs-EVs could also enhance blood vessel formation by promoting the transformation of fibroblasts into endothelial cells (Huang, et al., 2021; Ke, et al., 2021). DC-derived exosomes were involved in activation ECs by TNF-α and NF-kB signaling pathways in human umbilical vein endothelial cells (Jadli et al., 2021).

In conclusion, EVs were identified as the major component of stem cell secretome responsible for the observed increase in cardiac function. The contents of EVs play key roles in CVDs therapy, and their effects can be summarized as follows: 1) Inhibit apoptotic; 2) Reduction of oxidative stress; 3) Reduction of fibrosis; 4) Regulation of autophagy; 5) Reduction of inflammatory response; 6) promotion of angiogenesis; 7) Stabilization of mitochondrial membrane potential. As reported, the stem cell-derived EVs in CVDs therapy included MSC-EVs, CDC-EVs, iPSC-EVs and DC-EVs. They can help to carry different microRNAs to cardiac develop their therapy function (Table 1).

The potential of EVs is limited in multiple factors, including bioactivity, biodistribution, targeting, intracellular trafficking, and internalization. Variations and limitations in EVs isolation techniques, basic characterization, and precise dosing regimens can affect study results. The expected biological effects of EVs are mostly produced from internalization of recipient cells through endocytosis pathways (Mulcahy et al., 2014). Numerous studies have found that intravenously administered EVs are rapidly cleared by macrophages and accumulated in mononuclear phagocyte system (MPS) organs such as the liver, spleen, and lung (Chen, Wang et al., 2021). Compared with intracoronary or intravenous administration, intramyocardial administration of EVs can increase the lifetime of EVs in heart. Results showed that intramyocardial delivery of EVs can improve left ventricular ejection fraction and reduce the infarct size, regardless of its source (de Abreu, Fernandes et al., 2020). However, intramyocardial delivery of EVs is complex in a clinical catheterization (Gallet et al., 2017). Targeted technology can increase the accumulation and decrease the application dose of EVs in the cardiovascular system. The strategy of using specific biomolecules to increase the content of EVs may be the key to its successful clinical application. Currently, three strategies for targeted delivery of therapeutic EVs to the heart have been reported: 1) encapsulation of EVs in hydrogels, 2) genetic engineering of EVs, and 3) two-step EV delivery. In summary, three strategies can shorten the time that EVs take to reach their therapeutic targets and significantly reduce off-target effects, thereby improve therapeutic efficacy (Chen et al., 2021). To improve the efficacy of native EVs in CVDs, researchers have also developed technologies to improve the biological activity and stability of EVs in the cardiovascular system. The bioengineered EVs can be obtained by modulating the source of cells, genetics, metabolic engineering, and chemical or physiological methods (Huang et al., 2019; Hao et al., 2020). Cardiac homing peptide (CHP) was used to conjugate with EVs with a special linker. Modified EVs exhibited a longer lifetime in myocardial tissue as well as better functional status in the heart after injecting intravenously (Wen et al., 2019). The protein or peptide modified lipid is physically incorporated into the EVs membrane, or the linker is chemically coupled to the functional groups on the surface of the EVs. Compared with traditional bio-combination technology, the modified lipid is fast, more selective, and efficient. Chemical structure modification can change Evs’ surface and targeted epitopes’ density effectively, regardless of the source of the cell. In addition, the chemical method can be carried out during the purification process of EVs. Therefore, it is more suitable for clinical application (de Abreu et al., 2020). In conclusion, the modified EVs were enriched in therapeutically relevant compounds, and decorated with surface epitopes that improved their cardiac targeting and pharmacokinetics. Therapies based in modulated EVs exhibits improvement on cardiac function through decreasing in inflammation, cardiomyocyte death, fibrosis and infarct size, as well as increasing angiogenesis.