MitoEVs: A new player in multiple disease pathology and treatment

Abstract

Mitochondrial damage plays vital roles in the pathology of many diseases, such as cancers, neurodegenerative diseases, aging, metabolic diseases and many types of organ injury. However, the regulatory mechanism of mitochondrial functions among different cells or organs in vivo is still unclear, and efficient therapies for attenuating mitochondrial damage are urgently needed. Extracellular vesicles (EVs) are cell‐derived nanovesicles that can deliver bioactive cargoes among cells or organs. Interestingly, recent evidence shows that diverse mitochondrial contents are enriched in certain EV subpopulations, and such mitoEVs can deliver mitochondrial components to affect the functions of recipient cells under different conditions, which has emerged as a hot topic in this field. However, the overview and many essential questions with respect to this event remain elusive. In this review, we provide a global view of mitoEVs biology and mainly focus on the detailed sorting mechanisms, functional mitochondrial contents, and diverse biological effects of mitoEVs. We also discuss the pathogenic or therapeutic roles of mitoEVs in different diseases and highlight their potential as disease biomarkers or therapies in clinical translation. This review will provide insights into the pathology and drug development for various mitochondrial injury‐related diseases.

1. INTRODUCTION

Mitochondria are central organelles of cellular bioenergetics, biosynthesis and signalling, which play critical roles in regulating substance/energy metabolism, redox status, cellular signalling and cell survival/death (Vakifahmetoglu‐Norberg & Ouchida, 2017). Their own mitochondrial DNA (mtDNA), which encodes 13 mitochondrial proteins, 2 rRNAs and 22 tRNAs, and mtDNA is susceptible to oxidative stress or other pathological factors (Wolstenholme, 1992). To date, mitochondria have been recognized as critical mediators of the immune response, inflammation and tissue regeneration since they can control cell functions through metabolic programming or the release of signalling molecules, such as mitochondrial damage‐associated molecular patterns (DAMPs) (Miliotis et al., 2019). Because of these properties, mitochondrial damage has been considered a key driving factor in various diseases, such as cancers, neurodegenerative diseases, aging, metabolic diseases and tissue injury (Supinski et al., 2020). However, the global regulatory mechanism of mitochondrial functions in physiological or diseased status among cells or organs in vivo remains elusive, which may obstruct the further development of mitochondria‐targeted therapies.

Extracellular vesicles (EVs) are cell‐derived lipid bilayer membrane small vesicles that are widely distributed in nearly all types of tissues or body fluids (Yuana et al., 2013). EVs are vital mediators of cell‐cell or organ‐organ communication since they can deliver various types of bioactive cargos (e.g., nucleic acids, proteins and lipids) to impact the signalling of the recipient cells (Anand et al., 2019; Zijlstra & Di Vizio, 2018). Due to their nanosize, intrinsic bioproperties and ability to cross biological barriers, EVs may serve as potential biomarkers or potent therapeutics for various diseases (Lou et al., 2021; Shah et al., 2018; Wiklander et al., 2019). Recently, abundant evidence indicates that diverse mitochondrial contents are enriched in certain EV subpopulations, and such mitochondrial contents‐containing EVs can carry mitochondrial components and impact the metabolic state and phenotypes of target cells (Rai et al., 2021). The production and mitochondrial contents of mitoEVs have been shown to be altered in pathological status, which makes them potential biomarkers or therapeutic targets for multiple diseases. On the other hand, mitoEVs from healthy cells can deliver functional mitochondrial fragments into target cells and restore mitochondrial biosynthesis and energy metabolism (Lin et al., 2015). The existing findings are encouraging and suggest that EV‐mediated mitochondrial component transfer may have great potential in disease diagnosis and treatment (Jang et al., 2019). However, the overview of this event and some critical questions, such as the sorting mechanism, detailed changes in mitochondrial contents, and therapeutic effects of mitoEVs in different states, remain elusive and need to be elucidated, which may provide insights into further clinical translation.

In this review, for better readership, we termed these mitochondrial contents‐containing EVs as mitoEVs, since they are highly heterogeneous and their specific sizes and markers remain elusive. We briefly introduce the biology of mitoEVs and discuss the possible sorting mechanisms of mitochondrial components into EVs and the biological effects of mitoEVs in target cells. We also discuss the different bioactivities of mitoEVs in normal or pathological states and emphasize their roles in disease diagnosis and therapy, shedding light on their clinical translational potential.

2. SUMMARY OF MITOEV BIOLOGY

2.1. General concept of EV biology

EVs are a group of highly heterogeneous vesicles and may be further divided into some subtypes based on their route of biosynthesis, size and morphology, such as exosomes (∼30–150 nm in diameter), ectosomes [including microvesicles (∼200–1000 nm) and large oncosomes (>1000 nm)] (Dixson et al., 2023) and apoptotic vesicles (∼50–2000 nm), as well as newly discovered exomeres (<50 nm) (Zhang et al., 2019) and migrasomes (Ma et al., 2015), etc. However, it is currently difficult to distinguish these EV subtypes from mitoEVs due to their overlapping sizes and/or shared biogenesis routes. For example, mitochondrial‐derived vesicles (MDVs) can fuse into multivesicular bodies (MVBs) and then be released into the extracellular space as EVs (Peng et al., 2022). Thus, it would be helpful to briefly introduce the general concept of EV biology in this review (Figure 1). Microvesicles are released into the intercellular compartment by outwards budding and fission of the membrane on the cell surface (Tricarico et al., 2017), which is initiated by alterations in plasma membrane proteins and lipid composition as well as the ATP‐dependent contraction associated with the interaction of actin and myosin and is regulated by multiple proteins, such as Ras‐related GTPase ADP‐ribosylation factor 6 (ARF6) (van Niel et al., 2018). Exosomes mainly originate from the endosomal system where the cell membrane is endocytosed to form early endosomes, and then mature into late endosomes or MVBs. During this process, the endosomal membrane invaginates to form intracellular vesicles (ILVs) in the lumen of MVBs (Mashouri et al., 2019). The components of MVBs further fuse with lysosomes or peroxisomes to degrade or fuse with the cytoplasmic membrane and release into the extracellular space. The MVB pathway is mainly mediated by the endosomal sorting complexes required for transport (ESCRT) machinery (van Niel et al., 2018). However, the detailed processes of EV biogenesis in different cell types or conditions remain controversial and need to be explored in future studies. It is generally believed that EVs can serve as regulators of intercellular communication by delivering bioactive cargos into recipient cells or the interaction between EV surface molecules and receptors on the recipient cell (Mathieu et al., 2019). The uptake mechanism of EVs by recipient cells is also complicated and mainly includes direct membrane fusion, endocytosis and ligand‐receptor interactions, which have been reviewed previously (Mathieu et al., 2019; Wang et al., 2020; van Niel et al., 2018). Although great progress has been made, more in‐depth studies related to the biogenesis routes and functions of each EV subtype are needed.

FIGURE 1.

Biogenesis and uptake of EVs. Exosomes (∼30–150 nm) are formed by endogenous budding where the cell membrane is endocytosed to form early endosomes, and then mature into late endosomes or multivesicular bodies (MVBs). MVBs further fuse with the plasma membrane and are released into the extracellular space. Microvesicles (∼200‐1000 nm) are involved in outwards budding of the plasma membrane. Mitochondrial‐derived vesicles (MDVs, ∼70–150 nm) are released into the extracellular space mainly in two ways: the MVB mediated pathway (way 1) and the microvesicle pathway (way 2). Although the details need further elucidation, MDVs can fuse into MVBs and then fuse with the plasma membrane and OPA1 and SNX9 may be involved in this process. In addition, these vesicles can also be released via the microvesicle pathway. This process can be mitophagy‐dependent or mitophagy‐independent, and LC3 and ARRDC1 are involved in. The released EVs are further taken up by recipient cells via ligand‒receptor interactions, endocytosis or direct plasma membrane fusion. Created with BioRender.com.

2.2. Evidence of mitochondrial contents in EVs

To date, the presence of mitochondrial components in certain EV subpopulations seems to be a universal phenomenon in almost all cell types. Early evidence that EVs may carry mitochondrial components was first reported in 2010, and mtDNA contents were found in EVs from in vitro cultured astrocytes (U87MG), glioblastoma cells, and myoblast cells (C2C12) (Guescini, Guidolin et al., 2010; Guescini, Genedani et al., 2010). Subsequent studies from different groups also found that multiple types of mitochondrial components, such as mtDNA fragments, full‐length mtDNA, mitochondrial proteins and even intact mitochondria, were present in EVs from many types of cultured cells (Falchi et al., 2013; Rai et al., 2021; Valenti et al., 2021; Wang et al., 2020; Zhang et al., 2012). For example, EVs (∼100–800 nm in diameter) from colorectal cancer cells (SW620, LIM1863), triple‐negative breast cancer cells (MDA‐MB231), and human malignant glioma cells (U87) carry abundant mitochondrial proteins (e.g., translocase of inner mitochondrial membrane 44, aldehyde dehydrogenase 2, and pseudouridine synthase 1) (Rai et al., 2021). Likewise, mitochondria‐associated proteins (e.g., citrate synthase, malate dehydrogenase, heat shock proteins, adenylate kinase, and superoxide dismutase 2), degraded pieces of authentic mtDNA, full‐length mtDNA, and mtDNA‐transcribed RNA can be found in EVs released by neuroblastoma cells (SH‐SY5Y and NT2 cells) in vitro (Wang et al., 2020). Mitochondrial components (e.g., glutamate dehydrogenase 1, mitochondrial RNA [mtRNA] and mitochondrial genes) were detectable in EVs from breast cancer cells (Rabas et al., 2021). Furthermore, large EVs (∼1–8 µm in diameter) from primary fetal astrocytes were shown to encapsulate membrane‐potential positive mitochondria and ATP (Falchi et al., 2013), which indicates that these EVs may be functional. These findings suggest that EV‐mediated mitochondrial transfer may also be a critical messenger of cell‒cell communication.

In addition to cultured cells, mitoEVs have also been found in many types of human body fluids (e.g., plasma and sweat) under physiological or pathological conditions. For example, EVs (∼100 nm in diameter) isolated from sweat samples of rigorously trained people were enriched in mtDNA over other nucleic acid components (Bart et al., 2021). In the diseased state, plasma‐derived EVs from patients with Alzheimer's disease also contained mitochondrial proteins (e.g., transcription factor A [TFAM], voltage‐dependent anion‐selective channel 1 [VDAC1] and humanin) (Wang et al., 2020). The bronchoalveolar lavage (BAL) fluid of asthmatic patients was enriched in EVs containing mitochondria and mtDNA (Hough et al., 2018). Altogether, current studies suggest that EV‐mediated mitochondrial fractions or intact functional mitochondria transfer may occur in vitro and in vivo, and this process may be affected by the disease status. However, the underlying mechanism and biological implications of EV‐mediated mitochondrial transfer remain elusive.

Abundant evidence indicates the existence of diverse mitochondrial components in mitoEVs, but such EVs are highly heterogeneous and their specific sizes, markers and mitochondrial contents remain elusive. For example, a recent study isolated a novel subpopulation of mitoEVs (termed mitovesicles, ∼50–200 nm) from brains using a combined filtration (0.2‐µm) and density gradient centrifugation method, and these EVs have abundant mitochondrial components and ATP production ability in vitro, whereas lack of conventional EV markers (D'Acunzo et al., 2021; Kim et al., 2022). Another study found that large numbers (∼70%) of EVs isolated from MSCs contained polarized mitochondrial contents using MitoTracker and tetramethylrhodamine, methyl ester (TMRM) staining, and they showed several different clusters (∼3–400 nm, ∼600 nm and ∼1 µm), suggesting the presence of distinct subpopulations of mitoEVs (Thomas et al., 2022). Moreover, other studies have found the existence of large (>1 µm) EVs containing intact mitochondria (Islam et al., 2012; Mobarrez et al., 2019; Zhang et al., 2020). Overall, it is likely that the release of mitochondrial components via EVs is an intrinsic property of most cells, but not all EV subpopulations are enriched with mitochondrial contents. Furthermore, the secretion and properties (sizes, contents and functions) of mitoEVs may be affected by multiple factors, such as cell/tissue types of origin, states of donor cell/tissue, and isolation methods. We will briefly introduce the biology of mitoEVs in the following sections.

2.3. Summary of mitoEV biogenesis and release

Although the precise process of mitoEV biogenesis is incompletely known, current findings suggest that mitoEVs may originate from MDVs and other pathways (Figure 2). MDVs (∼70–150 nm in diameter) are generated through mitochondrial membrane budding under both steady and stress conditions, which was initially identified as a potential way for eliminating damaged mitochondrial components (Peng et al., 2022). The formation of MDVs may be mediated by the Parkinson's disease‐associated protein PINK1/Parkin‐(mitophagy)‐dependent or dynamin‐related protein 1 (DRP1)‐dependent pathway, which has been well reviewed previously (Heyn et al., 2023; Peng et al., 2022; Popov, 2022). The failed PINK1 import into mitochondria recruits Parkin to trigger mitophagy and subsequently regulate MDV formation, which is supported by translocase of outer mitochondrial membrane 20 (TOM20) and microtubule‐associated protein 1A/1B‐light chain 3 (LC3) positive EV subpopulations from platelets (De Paoli et al., 2018) and a PINK1‐dependent release of mtDNA‐containing EVs from breast cancer cells (Rabas et al., 2021). In addition, DRP1 receptors mitochondrial dynamics proteins 49 and 51 kDa and mitochondrial fission factor (MID49/MID51/MFF) can interact with DRP1 to participates in MDV biogenesis (König et al., 2021). However, some studies also suggest that DRP1 is independent of this process, which requires further classification. The fate of MDVs (lysosome‐ or peroxisome‐mediated degradation or release into extracellular space) is regulated by a complicated mechanism. During this process, some pathways are involved. For example, the transport of MDVs to lysosomes can be affected by PINK1, Parkin, Tollip or syntaxin‐17 (STX17) signalling (McLelland et al., 2016, 2014; Peng et al., 2022; Ryan et al., 2020), and transport to peroxisomes can be regulated by Vps35 and mitochondrial‐anchored protein ligase (MAPL) (Braschi et al., 2010; Mohanty et al., 2021), while the fusion of MDVs to MVBs and then release into the extracellular space as EVs may be mediated by cluster of differentiation 38 (CD38)/cyclic ADP ribose (cADPR) signalling (Suh et al., 2023), sorting nexin 9 (SNX9) signalling, optic atrophy 1 (OPA1) and inhibition by Parkin (Peng et al., 2022; Todkar et al., 2021), which have been well summarized before (Heyn et al., 2023; Peng et al., 2022; Popov, 2022; Sugiura et al., 2014). Stress conditions are likely to promote the selective incorporation of mitochondrial contents into MDVs, such as oxidative stress (McLelland et al., 2014; Todkar et al., 2021), remote ischemic preconditioning (Lv et al., 2020), hypoxia (Li et al., 2020), cannabidiol treatment (Ramirez et al., 2022), lipopolysaccharide (LPS) (Matheoud et al., 2016) and heat stress (Matheoud et al., 2016). However, whether those factors impact MDVs released into the extracellular space needs further study.

FIGURE 2.

Sorting mechanism and biological effect of mitoEVs. (a). Possible sorting mechanisms in donor cells. The formed MDVs may sort to lysosomes via PINK1/Parkin, Tollip or STX17, to peroxisomes via Vps35 and MAPL and to the extracellular space via OPA1, SNX9, DRP1 or PINK1. In certain cases, MDVs budding from mitochondria may fuse into multivesicular bodies (MVBs) and then be released into extracellular space as mitoEVs. In addition, mitoEVs also comprise new EV subtypes, mitovesicles and other pathways that may participate in mitoEV biogenesis, which need further classification. (b). The diverse roles of such EVs in target cells. Such EVs may display metabolic regulatory effects, such as bursting mitochondrial biogenesis (e.g., AMPK, PGC1), mitochondrial respiration and mtROS production, and thus mediate cell phenotypes (e.g., differentiation, angiogenesis and survival) of recipient cells. MitoEVs also have immune regulatory effects, such as the induction of proinflammatory signalling (e.g., TLR and STING), cytokine release, IFN responses and phagocytosis in immune cells. Created with BioRender.com.

It has been proposed that the release of mitochondrial contents via EVs may be a means of maintaining donor cell homeostasis. Some factors, such as oxidative stress, ionic homeostasis, mitochondrial proteases, metabolic status and mitochondrial dynamics, may affect the release and compositions of mitoEVs. For example, mitochondrial proteins are increasingly released into EVs under cold stress or oxidative stress in brown adipocytes (Rosina et al., 2022). The release of EVs carrying depolarized mitochondria from oxidatively stressed or injured donor cells is mediated by the arrestin domain‐containing protein 1 (ARRDC1) pathway (Phinney et al., 2015). The sorting of injured mitochondrial fragments into EVs was enhanced by iron chelation treatment in myotubes, while this process was unaffected by autophagy or mitophagy inhibition (Leermakers et al., 2020). Upregulation of mitochondrial Lon‐induced reactive oxygen species (ROS) can trigger mtDNA damage, as well as the secretion of EVs carrying mtDNA in cancer cells. This possible reason is that Lon‐induced mitochondrial stress drives mitoEV formation that allows such cancer cells to remove damaged or oxidized mitochondrial components into the extracellular space (Cheng et al., 2020). Mitochondrial metabolic substances, such as glutamate, can activate metabotropic glutamate receptor 3 (mGluR3) and drive Rab27‐dependent mtDNA‐containing EV release from breast cancer cells (Rabas et al., 2021). However, these reports cannot fully explain the fact that mitoEVs can also be released by cells under normal or steady state conditions. The mechanism of such EV formation may vary in different cell/tissue types and/or cell/tissue statuses.

Recently, mitovesicles, a newly discovered mitochondrial contents enriched EVs subtype from brain tissues, have been reported using an optimized isolation method (D'Acunzo et al., 2021). Interestingly, it seems that mitovesicles have completely distinct properties from canonical EVs. The mitovesicles isolated from the brain through high‐resolution density gradient were enriched with mitochondrial proteins (e.g., VDAC, COX‐IV and PDH‐E1α) while lack of common microvesicles, exosomes and endocytic markers. The distinct properties of mitovesicles suggest that they may have different biogenesis and biophysical mechanism from some conventional EV subtypes such as microvesicles and exosomes. However, this study did not reveal the exact biogenesis mechanisms of mitovesicles. Overall, current evidence indicates the unique biogenesis routes and biological properties of mitoEVs, but cannot fully rule out that the release of mitoEVs may share some common routes of conventional EVs. Thus, more in‐depth studies on the biogenesis pathways, biological features and physiological effects of mitoEVs are urgently needed. Taken together, EV‐mediated mitochondrial component delivery may exert multiple biofunctions, such as regulating cell homeostasis and the exchange of intercellular signals. Moving forwards, modulation of this process may selectively sort the desired mitochondrial components into EVs for therapeutic purposes.

2.4. Isolation and characteristic methods of mitoEVs

For different purposes, EVs can be routinely isolated from different sample types using multiple methods, such as ultracentrifugation, gradient centrifugation, poly‐ethylene glycol (PEG)‐based precipitation, and size‐exclusion chromatography, and each method has its own advantages or shortcomings, which have been well reviewed (Lou et al., 2021; Sidhom et al., 2020). However, the specific isolation methods for mitoEVs remain limited because their detailed features such as size, surface markers and density remain inconclusive. In previous studies, differential ultracentrifugation is one of the most frequently used methods for mitoEV isolation, however, this method may also collect unwanted particles and other contamination (Phinney et al., 2015; Todkar et al., 2021). Recently, several studies have reported some modified methods for isolating mitoEVs with high purity. For example, MDVs can be isolated from cell lysis using sucrose gradient centrifugation (Soubannier et al., 2012), in vitro budding from isolated mitochondria (Heyn et al., 2023) or immune‐isolation approaches based on certain proteins (e.g., TOM20 and MAPL) of MDVs (König et al., 2021). Interestingly, D'Acunzo et al. developed a new approach consisting of filtration (0.2‐µm) and iodixanol‐based high‐resolution density gradient centrifugation to isolate mitovesicles from brain tissues (D'Acunzo et al., 2021; D'Acunzo et al., 2022), and such EVs are enriched in mitochondrial proteins but lack some common protein markers of microvesicles and exosomes. This method can separate more purified mitoEVs from solid tissues and promote the further investigation of mitoEVs under physiological and pathological conditions. However, this approach is now applied only in brain tissues, and whether it is ideal for other types of biological samples (e.g., body fluid, culture medium and other tissue types) and large mitoEVs needs to be further evaluated.

Methods for general EV characterization (morphology, size and surface markers) have been proposed in the minimal information for studies of extracellular vesicles 2018 (MISEV2018) guidelines and have been well reviewed previously (Crescitelli et al., 2021; Lai et al., 2022; Théry et al., 2018). Likewise, the morphology, size and surface markers of mitoEVs can be routinely detected using nanotrack analysis (NTA), electron microscopy (EM), flow cytometry (FC) and immunoblotting. However, mitoEVs are highly heterogeneous in sizes and mitochondrial contents, and their specific size distributions and biomarkers remain elusive. Diverse mitochondrial contents (e.g., mitochondrial proteins, mtDNA, mtRNA) may exist in mitoEVs and can be detected by various methods such as immunoblotting, RT‐PCR and specific dyes (e.g., MitoTracker, TMRM). Multiple omics, such as transcriptomics, proteomics and lipidomics, have been applied to globally detect the mitochondrial contents of mitoEVs (D'Acunzo et al., 2021; Jang et al., 2019). Importantly, it is necessary to examine the impact of MitoEVs on the mitochondrial activity (e.g., mitochondrial biogenesis, redox status, dynamics and energetics) of recipient cells, since such EVs may also contain other types of cargos that can indirectly affect mitochondrial function. In the following section, we will discuss the biological effects of EV‐mediated mitochondrial delivery.

3. BIOLOGICAL EFFECTS OF EV‐MEDIATED MITOCHONDRIAL DELIVERY

Recent evidence indicates that mitochondrial components delivered by EVs from donor cells may incorporate into the mitochondrial network of recipient cells to exert biological functions. In the following section, we briefly discuss the possible roles of EV‐mediated mitochondrial transfer in recipient cells, such as metabolic regulation and immune regulation (Figure 2 and Table 1).

TABLE 1.

Biological effects of mitoEVs.

Donor cells	Bioactive contents	Recipient cells	Effect in recipient cells	Characterization	Refs
Platelets	Mitochondria	hMADS	Activated fatty acid oxidation by stimulating the TCA cycle, restored the pro‐angiogenic effects	Size: 0.2–1 µm (TEM)	(Levoux et al., 2021)
BMSCs	TFAM mRNA, mtDNA, and mitochondrial proteins (i.e., ATP5a1, COX IV)	H2O2‐treated HK‐2 cells	Rescued mitochondrial protein (TFAM/TOM20, COX IV and Sirt3) and mtDNA level, mitochondrial OXPHOS capacity and mitochondrial integrity	Size: <100 nm (TEM) Marker: HSP70, Flotillin‐1 (WB)	(Zhao et al., 2021)
BMSCs under oxidative stress	Mitochondria, mtDNA	Macrophages	Enhanced energetics	Size: 50–100 nm (TEM) Marker: MFGE8, CD9,CD63 (WB, FACS) Size: >100 nm (TEM) Marker: TSG101, ARRDC1 (WB)	(Phinney et al., 2015)
Brown adipocytes under cold stress	Damaged mitochondrial components (i.e., UPC1, mtDNA)	Brown adipocytes; bMACs	Decreased mitochondrial‐dependent thermogenesis, metabolism and adipogenesis; Increased mtROS production and antioxidant gene expression	Size: ∼50–350 nm (DLS)	(Rosina et al., 2022)
LPS‐activated THP‐1 monocytes	Mitochondria, mtRNA, mitochondrial proteins (i.e., COX IV)	Endothelial cells	Induced type I IFN and TNF responses	Size: ∼206 nm (NTA) Marker: Alix (WB)	(Puhm et al., 2019)
MEFs	Mitochondrial proteins (i.e., mtHSP70, NDUFA9, OPA1), mtDNA	RAW 264.7 cells	Enhanced IP10 secretion and IFN‐dependent genes (Rsad2 and MIfit1) expression	Size: <600 nm Marker: Alix, CD9, laminB1−, Rab11− (WB)	(Todkar et al., 2021)
Human T cells	Mitochondrial proteins (i.e., TFAM, COX I), mtDNA	Antigen‐presenting cells (i.e., dendritic cells)	Induced antiviral responses and viral infections resistance	Size: 50–200 nm (NTA) Marker: TSG101, CD81 (WB)	(Torralba et al., 2018)
BMSCs	Mitochondria	Macrophages	Enhanced phagocytosis capacity	GW4869 to block EV release, and anti‐CD44 mAb to block EV uptake	(Ko et al., 2020)

Abbreviations: bMACs: brown adipose tissue macrophages; hMADS: human multipotent adipose‐derived stem; HK‐2: human renal proximal tubular cell lines; MEFs: mouse embryonic fibroblasts.

3.1. Metabolic regulation

As the centre of cellular energy and substance metabolism, mitochondria supply the capacity for aerobic respiration and many other metabolic reactions, while mitochondrial defects can cause serious consequences. Interestingly, EV‐mediated mitochondrial delivery has been shown to restore mitochondrial function in recipient cells. Activated platelets can transfer respiratory‐competent mitochondria to mesenchymal stem cells (MSCs) at least partly through mitoEVs (∼0.2–1 µm). Such mitochondrial transfer induces the metabolic reprogramming (enhanced tricarboxylic acid [TCA] cycle and de novo fatty acid synthesis) of MSCs, thereby increasing the proangiogenic capacity of these cells (Levoux et al., 2021). Similarly, we found the presence of mitochondrial contents (e.g., mtDNA and mitochondrial electron transport chain [ETC] proteins) in EVs from MSCs, and mitoEVs rescued mtDNA copy number and bioenergetic defects in kidney tubular cells under oxidative stress. I n vivo, such EV‐mediated mitochondrial delivery reduced mitochondrial injury and inflammation in a mouse model of ischemic kidney injury (Zhao et al., 2021). In line with our findings, a recent study found that large populations (>500 nm) of MSC‐EVs contained polarized mitochondria using TMRM staining. in vitro, such mitoEVs were taken up by stressed chondrocytes and incorporated into chondrocyte mitochondrial networks. This study again indicates that MSCs can package functional mitochondria into EVs, which can be transferred to recipient cells in the absence of direct cell‐cell interactions (Thomas et al., 2022). Additionally, EVs from human leukocyte antigen‐D positive (HLA‐DR⁺) myeloid‐derived regulatory cells can deliver polarized mitochondria to peripheral blood T cells, and the transferred mitochondria can integrate into the mitochondrial networks of the host cells, thereby maintaining the mitochondrial membrane potential and ROS generation capacity of T cells (Hough et al., 2018).

In addition, EVs carrying oxidized or damaged mitochondrial contents can also be taken up by recipient cells to impact mitochondrial function and bioenergetics in these cells. For example, mitochondria from MSCs can be phagocytosed by macrophages exposed to oxidative stress, thereby rewiring the bioenergetics and mitigating the mtROS burst in injured macrophages (Phinney et al., 2015). Adipose tissue suffering from thermogenic stress releases EVs that contain damaged mitochondria, ATP and mtDNA, and reuptake of these EVs by adipocytes leads to decreased peroxisome proliferator‐activated receptor γ and mitochondrial protein (e.g., uncoupling protein 1) expression with impaired mitochondrial function and adipogenesis of recipient cells. Meanwhile, these EVs can also be engulfed by local macrophages, thereby affecting the thermogenic function of adipose tissues (Rosina et al., 2022). Altogether, these findings suggest that delivery of mitochondrial contents via EVs can regulate metabolic states in target cells, and this effect may exert therapeutic potential in mitochondrial defect‐related diseases.

3.2. Immune regulation

The immune response plays critical roles in the infection defence and pathogenesis of many diseases, such as asthma and rheumatoid arthritis. EV‐mediated mitochondrial delivery is involved in immune modulation (e.g., pro‐ and anti‐inflammatory roles). The injured cells can release damaged mitochondrial components into the extracellular space via EVs. For example, LPS‐primed monocytes secrete EVs shedding damaged mitochondrial contents (e.g., mtRNA, ETC proteins) as DAMPs to induce interleukin (IL)‐8 secretion and the type I interferon (IFN) response in endothelial cells, and the proinflammatory capacity of EVs also depends on the mitochondrial state of donor cells (Puhm et al., 2019). Such EV release may be actively regulated in parent cells under stressed or diseased conditions. For example, mouse embryonic fibroblast cells can sort mitochondrial proteins into EVs and further drive the interferon‐γ‐inducible protein 10‐mediated immune response in macrophages (Todkar et al., 2021). EVs from Lon‐overexpressing colon cancer cells carry abundant mtDNA and immunosuppressive protein programmed death ligand 1 (PD‐L1), thereby inducing TLR9‐NF‐κB signalling in THP1 monocytes and suppressing T‐cell function (Cheng et al., 2020). Therefore, such pathogenic EV release may be a potent therapeutic target for many systemic immune disorder‐related diseases.

However, EV‐mediated mitochondrial delivery may display diverse effects under different conditions, which can also boost immune cell function and exert therapeutic effects. For example, when antigen‐presenting dendritic cells (DCs) contact T cells, T cells can deliver mitochondrial contents (e.g., mtDNA) back to DCs via EVs. Such mitochondrial transfer induced type I IFN responses and stimulator of interferon genes (STING) signalling in DCs, thereby enhancing their antiviral response via cyclic GMP‐AMP synthase/stimulator of interferon genes‐interferon regulatory factor‐3 (cGAS/STING‐IRF3) pathways (Torralba et al., 2018). MSC‐derived mitoEVs could be taken up by macrophages, which further induced an M2 phenotype and enhanced the phagocytic activity of macrophages to maintain tissue homeostasis (Ko et al., 2020). Similarly, MSC‐derived mtDNA‐containing EVs could induce nuclear translocation of NK‐κB, resulting in the inhibition of proinflammatory TLR‐associated transcripts in macrophages (Phinney et al., 2015). Taken together, EV‐mediated mitochondrial transfer can exert positive or negative effects on the immune system in different states. Therefore, a deeper understanding of this event may provide insights into disease pathology as well as disease diagnosis and treatment.

4. PATHOLOGICAL EFFECTS OF MITOEVS

Mitochondrial damage and its associated metabolic disorders are vital mediators of many diseases, and mitoEVs can participate in metabolic or immune regulation of multiple cell types under disease conditions. In this section, we briefly discuss the critical role of such EVs in the pathogenesis of various diseases, such as cancers, liver diseases, infections, cardiovascular diseases and lung diseases (Figure 3).

FIGURE 3.

Clinical translation potential of mitoEVs. MitoEVs from injured or stressed donor cells may play pathological roles in distinct diseases (inflammation, cancers, lung diseases, liver diseases, etc.) through multiple mechanisms, such as inducing an ROS burst, DAMPs release and mitochondrial defects in recipient cells. Therefore, such EVs existing in biological samples (e.g., blood and urine) may serve as potential biomarkers for multiple diseases. On the other hand, mitoEVs from healthy or stem cells also serve as promising therapeutics in various diseases (neurological diseases, lung diseases, heart diseases, sepsis, etc.) through metabolic or immune regulatory effects. BECs, brain endothelial cells; iCMs, human induced pluripotent stem cell‐derived cardiomyocytes; EPCs, endothelial progenitor cells; MSCs, mesenchymal stem cells; NSCs, neural stem cells. Created with BioRender.com.

4.1. Cancers

EV‐mediated mitochondrial transfer can occur between cancer cells or cancer cells and other cell types (Takenaga et al., 2021), and this effect has been involved in regulating the tumour microenvironment and differentiation/metastasis. For example, mtDNA‐rich EVs from breast cancer cells can increase matrix metalloproteinase (MMP) and α₅β₁ integrin expression to promote recipient breast cancer cell invasion under glutamine starvation conditions (Rabas et al., 2021). Acute myeloid leukaemia (AML) cells showed elevated mitoEVs release during cell differentiation, while inhibition of such EV formation prevented myeloid differentiation (Zhao et al., 2020). Lon‐overexpressing mouse melanoma cells released mtDNA‐rich EVs to induce cytokine production in macrophages, thereby suppressing the cytotoxic T‐cell immune response in the tumour microenvironment (Cheng et al., 2020). In addition, EV‐mediated mitochondrial delivery may also contribute to cancer drug resistance. Circulating mtDNA‐rich EVs were found in insulin therapy‐antagonized metastatic breast cancer patients, which might act as a pro‐oncogenic signal to induce endocrine therapy resistance in oxidative phosphorylation‐dependent breast cancer cells (Sansone et al., 2017). EVs released by chemoresistant triple‐negative breast cancer cells could deliver functional mitochondria to sensitive triple‐negative breast cancer cells, leading to increased chemoresistance and tumorigenesis (Abad & Lyakhovich, 2022). Likewise, EVs from tumour‐activated stromal cells delivered mitochondria to malignant glioma cells, resulting in resistance to cancer radiotherapy and chemotherapy (Salaud et al., 2020).

4.2. Liver diseases

EV‐mediated mitochondrial delivery may participate in alcoholic liver disease. In persistently overfed plus ethanol‐fed mice, the plasma levels of hepatocyte‐derived mtDNA‐rich microparticles dramatically increased, which caused liver injury by inducing endoplasmic reticulum stress and inflammation. In contrast, the release of mtDNA‐rich microparticles was further suppressed by inhibition of liver endoplasmic reticulum stress (Cai et al., 2017). Similarly, EVs from wild mouse or human hepatocytes exposed to alcohol had elevated levels of mtRNA, which further induced the release of proinflammatory factors (e.g., IL‐12A) in γδ T cells or liver Kupffer cells (Lee et al., 2020).

4.3. Infectious diseases

MitoEVs from various pathogens may affect viral load and inflammation in certain infectious diseases. For example, upon entering cells, coxsackievirus B (CVB) stimulates DRP1‐mediated mitochondrial fragmentation in host cells and then the formation and release of infectious extracellular microvesicles (EMVs). Inhibition of DRP1 blocked the release of EMVs, thereby reducing the viral load in cardiomyocytes (Sin et al., 2017). Human alveolar epithelial cells release mtDNA‐rich MVs in response to Streptococcus pneumoniae pore‐forming toxin stimulation, which in turn triggers cytokine release and proinflammatory immune activation (Nerlich et al., 2018). Therefore, targeted inhibition of EV‐mediated mitochondrial release from host cells may be a potent therapeutic strategy for infection.

4.4. Cardiovascular diseases

Obesity has been strongly associated with a high risk of cardiovascular diseases (CVDs), but the detailed mechanisms remain elusive. Adipocytes in obesity under thermal stress could release EVs carrying damaged mitochondrial components, which enter the circulation and are then taken up by cardiomyocytes, thereby inducing an ROS burst and mitochondrial dysfunction in myocardial tissues (Rosina et al., 2022). However, EVs from adipocytes preconditioned with palmitate could protect cardiac cells from acute oxidative stress in mice with heart ischemic/reperfused injury, suggesting that such EVs may have diverse bioactivities under different conditions, but the exact effects need to be further explored.

4.5. Respiratory diseases

EV‐mediated mitochondrial transfer may regulate inflammation in respiratory diseases. In airway lavage fluid, asthmatic patients had higher levels of HLA‐DR⁺ EVs than healthy controls, and such EVs play proinflammatory roles by delivering polarized mitochondria to impact the redox status of peripheral blood T cells (Hough et al., 2018). A recent study showed that cigarette extracts can stimulate mitoEV secretion in human tracheal epithelial cells in a dose‐dependent manner. Compared with healthy controls, higher levels of cell‐free mtDNA were detected in the plasma of chronic obstructive pulmonary disease (COPD) patients (Giordano et al., 2022). Thus, such EVs appear to be potential sensing molecules for cigarette exposure and the pathogenesis of COPD.

4.6. Other diseases

The radiation‐induced bystander effect (RIBE) is a phenomenon in which nonirradiated cells exhibit irradiation effects after receiving signals from nearby irradiated cells. It has been reported that EVs from human dermal fibroblast (HDF) cells and mouse serum exposed to irradiation (4 Gy) had enriched mtDNA contents and can induce DNA damage in HDFs and mouse fibroblasts (Ariyoshi et al., 2019). Thus, the release of damaged mitochondria via EVs may be a common response of cells to stress (e.g., radiation) and a possible means of exchanging danger signals between cells.

5. CLINICAL TRANSLATION OF EV‐MEDIATED MITOCHONDRIAL DELIVERY

Mitochondrial injury is a vital mediator in the pathogenesis of many diseases, and the release of damaged mitoEVs has been widely observed in many disease conditions. Such EVs may serve as possible biomarkers of diseases due to their carried information from injured parent cells or tissues. In recent years, EV‐based liquid biopsy has been increasingly studied in the diagnosis of multiple diseases. More importantly, abundant evidence indicates that mitoEVs can also impact the metabolic status of target cells and thus serve as potential therapeutics for multiple diseases.

5.1. mitoEVs as disease biomarkers

After being released into the extracellular space from donor cells, mitoEVs can enter the circulatory system. As a result, changes in the amounts or contents of circulating mitoEVs may serve as potential biomarkers to reflect different pathological states. In recent years, such EVs have shown attractive value in diagnosing or monitoring diseases. In this section, we briefly discuss the changes in mitoEVs in different diseases (Table 2).

TABLE 2.

MitoEVs as disease markers.

Diseases	Samples	Isolation methods	EVs characterization	EV contents	Changes (vs healthy control)	Detection methods	Refs
Ovarian cancer	Plasma	Commercial kit	Size: ∼150 nm (NTA, TEM)	mtDNA	Increased	qRT‒PCR	(Keseru et al., 2019)
			Marker: CD81, Flotillin1, GM130− (WB)
	Plasma	Gradient ultracentrifugation and purified by iodixanol density gradient ultracentrifugation	Size: 40–300 nm (EM) Marker:CD9, CD81, CD63, Syntenin‐1, Flotillins (Proteomic analysis)	MT‐CO2/COX6c	Increased	Western blot, ELISA	(Jang et al., 2019)
Oral squamous cell carcinoma	Plasma	Differential centrifugation	Size: ∼100 nm (TEM) Marker: Alix, CD9, TSG101, HSP70 (WB)	mtDNA	Increased	qRT‒PCR	(Cheng et al., 2020)
Melanoma	Plasma	Gradient ultracentrifugation and purified by iodixanol density gradient ultracentrifugation	Size: 40–300 nm (EM)	MT‐CO2/COX6c	Increased	Western blot, ELISA	(Jang et al., 2019)
Breast cancer	Plasma	Gradient ultracentrifugation and purified by iodixanol density gradient ultracentrifugation	Size: 40–300 nm (EM)	MT‐CO2/COX6c	Increased	Western blot, ELISA	(Jang et al., 2019)
Alzheimer's disease	Plasma	Commercial kit	Size: 70–118 nm (NTA) Marker: Alix, CD9, ApoA1, GM130− (WB)	Complex I, III, IV, V, SOD1	Decreased	ELISA	(Yao et al., 2021)
Down syndrome	Post‐mortem brain	filtration (0.2‐µm) and iodixanol‐based high‐resolution density gradient centrifugation	Size: ∼50–200 nm (NTA, cryoEM and TEM) Marker: VADC, COX‐IV, PDH‐E1α (WB)	Mitochondrial proteins (e.g., VADC, COX‐IV PDH‐E1α)	Increased	Western blot	(D'Acunzo et al., 2021)
Parkinson's disease	Plasma	Gradient ultracentrifugation	Marker: CD9, CD63, Flotillin (WB)	ATP5A, NDUFS3, NDUFB8	Decreased	Western blot	(Picca et al., 2020)
Autism spectrum disorders	Serum	Commercial kit	Size: ∼100 nm (TEM) Marker: CD9, CD81 (WB)	MtDNA‐7S	Increased	qRT‒PCR	(Tsilioni & Theoharides, 2018)
Early phase of psychosis	Blood	Commercial kit	Marker: HSP70 (IF)	COX6A2	Decreased	ELISA	(Khadimallah et al., 2021)
Aging	Plasma	Commercial kit	Size: < 6 µm (FC) Merker: CD9, CD29, CD63, CD81 (FC)	mtDNA, mitochondria	Decreased	Mito‐Tracker	(Zhang et al., 2020)
Frailty and sarcopenia	Serum	Differential ultracentrifugation	Size: <100 nm (SEM) Marker: CD9, CD63, CD81, flotillin, HNRNPA1− (WB)	ATP5A, NDUFS3 and SDHB	Decreased	Western blot	(Picca et al., 2020)
Acquired immunodeficiency syndrome	Plasma	Centrifugation	Size: 300–1000 nm (NTA, TEM) Marker: CD9 (FC)	Mitochondria	Decreased	Mito‐Tracker	(Poveda et al., 2022)
Systemic lupus erythematosus	Plasma	Differential centrifugation	Size: 0.3–3 µm (FC)	Mitochondria	Increased	Mito‐Tracker	(Mobarrez et al., 2019)
Cardiovascular disease	Plasma	Centrifugation and size‐exclusion chromatography	Size: ∼110 nm (NTA, Cryo‐EM)	MT‐COI mRNA	Decreased	qRT‒PCR	(Holvoet et al., 2016)

Abbreviations: CryoEM, cryogenic electron microscopy; ELISA: enzyme‐linked immunosorbent assay; EM, electron microscopy; FC, flow cytometry; NTA, nanoparticle tracking analysis; qRT‒PCR: quantitative reverse transcriptase polymerase chain reaction; TEM, transmission electron microscopy; WB, western blotting.

5.1.1. Cancers

To date, the early diagnosis of cancers is a huge challenge in the clinic. The approaches for early diagnosis of cancers include physical examination, imaging examination, tissue biopsy and liquid biopsies (De Rubis et al., 2019). EVs can carry information that reflects the state of the parent cells, which makes them a potential biomarker for cancers. In plasma EVs, the levels of mtDNA copy number were higher in ovarian cancer patients than in healthy subjects and were further increased in patients with advanced stages (e.g., FIGO III and FIGO IV) (Keseru et al., 2019). Oral squamous cell carcinoma patients showed higher mtDNA levels in plasma EVs than healthy subjects, and the mtDNA levels in plasma EVs were positively correlated with circulating IFN‐γ and PD‐L1 levels (Cheng et al., 2020). Thus, such mitoEVs may serve as a tool for assessing the efficiency of anti‐PD‐L1 therapy in the clinic. Likewise, EVs from melanoma patient biopsies had abundant mitochondrial proteins (COX6c, SLC25A22 and MT‐CO2), and the levels of MT‐CO2/COX6c in plasma mitoEVs were higher in malignant patients (melanoma, ovarian and breast cancer) than healthy controls (Jang et al., 2019). Although the sensitivity/specificity of mitoEVs for cancer diagnosis remains elusive, such EVs may provide a means to monitor the progression and therapeutic response of multiple cancers.

5.1.2. Aging‐related diseases

Aging‐related diseases are strongly associated with oxidative stress and mitochondrial injury. However, due to clinical heterogeneity, the efficacy of current diagnostic approaches (e.g., brain imaging and biochemical assays) for these diseases is not ideal (Kalaria & Hase, 2019), and novel noninvasive biomarkers are needed. Interestingly, the levels of mtDNA in circulating EVs were shown to decline with age (Lazo et al., 2021). Additionally, the decline in functional mitochondria of EVs from immune cells in plasma was strongly associated with aging (Zhang et al., 2020). In elderly adults with frailty and sarcopenia (PF&S), mitochondrial markers in EVs such as ATP5A, NDUFS3 and SDHB were decreased, and the levels of mitochondrial protein in serum EVs may serve as a predictor to distinguish them with or without PF&S (Picca et al., 2020). The decline of mitochondrial contents in circulating EVs is also closely associated with neurological disorders. For example, plasma EVs from patients with Alzheimer's disease had lower levels of mitochondrial ETC complexes I/III/IV and ATP synthase than healthy controls (Yao et al., 2021). In Down syndrome mouse model and human, a higher number of mitovesicles and altered EV compositions (lower UQCRC2, VDAC and SDHB proteins while higher mtDNA) were observed in brain tissues compared to controls (D'Acunzo et al., 2021). In plasma EVs from Parkinson's syndrome (PD) patients, the levels of mitochondrial proteins (ATP5A, NDUFS3 and NDUFB8) were lower than those in EVs from healthy controls. NDUFS3 in plasma EVs may be a potential predictor of PD, since it had a high sensitivity (93.8%) and a high specificity (91.7%) to distinguish PD when combined with a panel of other molecules (Picca et al., 2020). The changes in the mitochondrial contents of EVs may reflect the degree of mitochondrial defects and provide a means to predict the progression of aging‐related diseases.

5.1.3. Psychiatric disorders

Psychiatric disorders, such as autism spectrum disorder (ASD) and depressive disorder, are a type of mind abnormality that makes it difficult to distinguish between the real and the unreal world. Current clinical diagnosis for these diseases mainly depends on physical examination and neuroimaging methods, but the timely diagnosis for certain psychiatric disorders is not ideal. Recent studies indicate that mitoEVs may be possible biomarkers for these diseases. For example, plasma EVs from major depressive disorder patients had abundant mitochondrial proteins (e.g., NRF2, CYPD, MFN2, LETM1), whose levels were lowered after antidepressant (selective serotonin reuptake inhibitor) treatment (Goetzl et al., 2021). Children with ASD showed higher levels of mtDNA7S‐enriched EVs in their serum than normotypic controls (Tsilioni & Theoharides, 2018). In blood exosomes, early phase psychosis (EPP) patients showed increased miR‐137 and reduced mitochondrial COX6A2 compared to healthy controls. Combining high levels of exosomal miR‐137 and low levels of COX6A2 could predict mitochondrial injury, poor neurocognitive performance and social functioning in psychosis patients (Khadimallah et al., 2021). Such mitoEVs may serve as potential biomarkers of psychiatric diseases, but their efficacy needs to be validated in large cohorts.

5.1.4. Autoimmune diseases

Systemic lupus erythematosus (SLE) is an autoimmune disease frequently leading to multiorgan complications, especially in the kidney. Current clinical diagnosis mainly depends on antibody testing, tissue biopsy and genetic analysis. Recently, the potential diagnostic role of circulating EVs in immune diseases has increasingly attracted scientific attention. The levels of mitochondria‐containing microparticles (mitoMPs) increased in the plasma of SLE patients and were positively correlated with high disease activity, as well as the levels of anti‐dsDNA antibodies and inflammatory cytokines (e.g., IL‐8 and IP‐10) (Mobarrez et al., 2019). Patients with activated SLE showed higher levels of mitoMPs and IgG‐positive mitoMPs than healthy controls (Mobarrez et al., 2019). These results suggest that mitoEVs may have pathological roles in SLE and reflect the degree of disease activation to some extent.

5.1.5. Cardiovascular diseases

Cardiovascular diseases, such as coronary artery disease (CAD), are mainly caused by major vascular injury and insufficient blood supply to the heart. CAD is a leading cause of patient mortality worldwide, but early diagnosis of CAD is still difficult. Although the specific reasons remain elusive, the potential role of EVs as biomarkers for CAD has been investigated. In plasma EVs of CAD patients, the levels of mtRNA (MT‐COI) were lower in patients with new cardiovascular events than in those without new cardiovascular events. According to the Kaplan‒Meier survival analysis, CAD patients in the lowest tertile of MT‐COI levels in plasma EVs developed cardiovascular malignant events earlier (Holvoet et al., 2016). These findings suggest that circulating mitoEVs may be a possible predictor for the outcome of cardiovascular diseases.

5.1.6. Infectious diseases

Infectious diseases caused by various pathogens (e.g., viruses and bacteria) are huge public problems worldwide. Changes in the mitochondrial contents of circulating EVs have been found in infectious diseases. For example, acquired immune deficiency syndrome (AIDS), caused by human immunodeficiency virus (HIV) infection, is a chronic and life‐threatening disease affecting millions of people. In plasma platelet‐derived microvesicles, AIDS patients had lower levels of mitochondrial content and mitochondrial density than uninfected controls. Moreover, AIDS patients receiving antiretroviral therapy showed lower levels of mitoEVs in plasma than elite controllers and viremic controllers, however, these should be further examined as biomarkers in treated and untreated HIV infection (Poveda et al., 2022). Altogether, these reports suggest that such EVs are possible biomarkers of multiple diseases, but their clinical translational potential indeed requires more trials.

5.2. Therapeutic effects of mitoEVs

On the other hand, mitoEVs produced by multiple types of healthy cells can also restore mitochondrial function, energy metabolism and immune homeostasis in recipient cells under disease conditions, which indicates the favourable clinical translational potential of these EVs. In this section, we will discuss the therapeutic role of mitoEVs in various preclinical disease models (Table 3).

TABLE 3.

Therapeutic potential of mitoEVs.

Diseases	EV origins	Isolation methods	Characterization	Contents	Models	Effects	Refs
Severe emphysema	BMSCs	Differential ultracentrifugation	Marker: CD63, CD81(FC) Size: ∼150–250 nm (NTA)	Mitochondria	Elastase instilled mice	Reduced TGF‐β and IL‐1β levels, linear intercept between alveolar walls and the neutrophil cell count in lung tissue; Increased PAT/PET ratio and reduce the right ventricular area, which reduces cardiorespiratory dysfunction.	(Antunes et al., 2021)
Acute lung injury	BMSCs	Optical imaging	Size: 1–2 µm (IF)	Mitochondria	LPS‐induced ALI mice	Decreased leukocytosis, albumin leakage in the bronchoalveolar lavage; Induced lamellar body exocytosis and alveolar ATP production.	(Islam et al., 2012)
	Adipose‐derived MSCs	Differential ultracentrifugation	Size: 50–150 nm (NTA, TEM) Marker: CD9, CD63, TSG101 (WB)	Mitochondria, mtDNA	LPS‐induced MH‐S macrophage; LPS‐induced ALI mice	Restored the macrophage mitochondrial function, inflammatory response (decreased IL‐6, TNF‐α, IL‐1β); Alleviated lung inflammation and lung damage.	(Xia et al., 2022)
Acute respiratory distress syndrome	BMSCs	Differential ultracentrifugation	Size: 70–100 nm (NTA, EM) Marker: CD44, CD63 (FC)	Mitochondria	LPS‐induced HSAECs, HPMECs and PCLSs; LPS‐induced lung injury mice	Decreased IL‐8 level, alleviated mitochondrial function, barriers integrity and inflammatory response in vitro; Attenuated lung injury and lung mitochondrial function in vivo	(Su et al., 2021)
Anthracycline‐induced cardiomyopathy	BMSCs	Size‐based filtration	Size: 30–800 nm (NTA, TEM) Marker: CD9 (FC)	Mitochondria	DOX‐induced injury iCM; breast cancer survivors received DOX treated	Recovered cardiomyocyte viability, physiology and mitochondrial biogenesis and function in iCM; Improved myocardial function and remodelling in patients.	(O'Brien et al., 2021)
Myocardial infarction (MI)	Human iCM	Differential ultracentrifugation	Size: 100–600 nm (NTA, TEM) Marker: β1‐integrin, CD63 (FC)	Mitochondria; PGC‐1a and ERRγ mRNA	Hypoxia‐induced iCM; Artery ligated mice	Improved mitochondrial bioenergetics and biogenesis in iCM; Prevented cardiac remodelling of MI mice model	(Ikeda et al., 2021)
Sepsis	BMSCs	Differential centrifugation	Size: 100–1000 nm (TEM, NTA) Marker: Annexin V, CD29, CD73, CD90, CD105, CD45− (FC)	Mfn2, PGC‐1α and mitochondria	Cecal ligated and  punctured rats; LPS‐induced IEC‐6 cells	Improved intestinal barrier dysfunction in sepsis rats; Restored mitochondrial dynamic balance in IEC‐6 cells	(Zheng et al., 2021)
Autoimmune encephalomyelitis	Neural stem cells	Differential ultracentrifugation, ultrafiltration, sucrose gradient ultracentrifugation and commercial kit	Size: 80–1000 nm (TEM, NTA)Marker: TSG101, PDC6IP, CD9, Golga2− (WB)	Mitochondrial proteins (ND2, ND5, etc.), mtDNA and mitochondria	MOG‐induced mice	Ameliorated neuroinflammation of autoimmune encephalomyelitis mice	(Peruzzotti‐Jametti et al., 2021)
Brain ischemic stroke	hCMEC/D3 cells	Differential ultracentrifugation	Size: 100–250 nm (DLS)	Mitochondria, ATP5A	OGD‐treated BECs	Increased ATP level and maximal OCR and ECAR of BECs	(D'Souza et al., 2021)
Acute kidney injury	BMSCs, hUC‐MSCs	Differential ultracentrifugation	Size: ∼50–500 nm (NTA, TEM)	TFAM mRNA, mtDNA, ATP5a1, COX IV, TOM20	LPS‐induced HK‐2 cells; Bilateralrenal pedicle clamped and reperfusion mice	Attenuated mitochondrial dysfunction of HK‐2 cells; Attenuated renal dysfunction, inflammatory response and mitochondrial damage of AKI mice	(Zhao et al., 2021)
			Marker: Alix, TSG101, HSP70 (WB)
Ischemic renal injury	STC‐like cells	Differential ultracentrifugation	Size: 100–300 nm (NTA) Marker: CD24, CD133, CD29, CD9, CD81 (WB)	Mitochondria	AMA‐induced injured TECs; Unilateral renal artery stenosis mice	Restored oxidative stress, and mtDNA level and DRP1 expression in injured TECs; Restored murine renal haemodynamics and function	(Zou et al., 2018)
Liver ischemia/reperfusion injury	hUC‐MSCs	Differential ultracentrifugation	Size: 100–700 nm (NTA, TEM)	Mitochondria	Liver IRI mice	Reduced ALT, AST and LDH level, attenuated liver injury and NETs formation	(Lu et al., 2022)
			Marker: Alix, CD63, CD9, HSP70, GRP95− (WB)

Abbreviations: ALI, acute lung injury; ALT, alanine transaminase; AMA, antimycin‐A; AST, aspartate transaminase; BECs, brain endothelial cells; BMSC, bone marrow‐derived stromal cells; DOX, doxorubicin; hCMEC/D3, human cerebral microvascular endothelial cell line; HSAECs, human small airway epithelial cells; HPMECs, human pulmonary microvascular endothelial cells; hUC‐MSCs, human umbilical cord mesenchymal stem cells; iCM, induced pluripotent stem cell‐derived cardiomyocytes; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; MOG, myelin oligodendrocyte glycoprotein; MSCs, mesenchymal stem cells; OGD, oxygen‐glucose deprivation; PAT/PET ratio, pulmonary artery acceleration time/pulmonary artery ejection time ratio; PCLSs, precision cut lung slices; STC‐like cells, intrinsic renal scattered tubular cells; TECs, tubular epithelial cells.

5.2.1. Lung diseases

Lung diseases, such as acute lung injury (ALI), acute respiratory distress syndrome (ARDS) and silicosis, are serious disorders with high lethality worldwide, and mitochondrial injury has been recognized as a vital pathogenic factor of lung injury. Thus, efficient therapies for lung injury therapy are urgently required in the clinic. To date, mitoEVs from healthy cells (mainly stem cells) have been shown to play therapeutic roles in lung injury models by regulating the metabolic state and phenotypes of immune cells. For example, MSC‐derived EVs (MSC‐EVs) delivered mitochondria to improve the oxygen consumption rate (OCR) and mtDNA and ATP levels in alveolar macrophages (AMs) in vitro (Antunes et al., 2021; Xia et al., 2022). In LPS‐induced ALI mice, MSC‐EVs can deliver mitochondria to the alveolar epithelium, thereby restoring ATP production and reducing bronchoalveolar lavage leukocytosis and lung inflammation (Islam et al., 2012; Xia et al., 2022). Additionally, mtDNA‐containing MSC exosome treatments limited Ly6C^hi monocyte infiltration and cytokine production in lung tissues of silica‐exposed mice and thus prevented lung fibrosis (Phinney et al., 2015).

In addition to immune cells, MSC‐EVs can also impact the metabolic state of other lung tissue cells by shedding functional mitochondria (Sinclair et al., 2016; Su et al., 2021). For example, MSC‐EV treatments could restore mitochondrial function and barrier integrity in pulmonary microvascular endothelial cells, airway epithelial cells and human lung slices exposed to endotoxin or ARDS patient plasma through mitochondrial transfer (Sinclair et al., 2016). Although the detailed mechanism is still unclear, mitochondrial shedding by MSC‐EVs can be integrated into the mitochondrial networks of recipient cells and thus induce mitophagy and mitochondrial biosynthesis (Su et al., 2021). in vivo, such MSC‐EVs could restore mitochondrial respiration and alleviate lung injury in a mouse model of LPS‐induced ALI (Su et al., 2021). Collectively, mitoEVs from stem cells may serve as potential therapies for lung diseases, and clinical trials are needed to assess their safety and efficacy.

5.2.2. Cardiovascular diseases

Anthracycline‐induced cardiomyopathy (AIC) is one of the leading causes of lethality in cancer patients with chemotherapy, but efficient therapeutics for AIC are still lacking in the clinic. A recent study showed that treatment with larger MSC‐derived mitoEVs (diameter > 200 nm) rescued the mitochondrial function and cell viability of iPSC‐derived cardiomyocytes (iCMs) under doxorubicin stimulation. This effect was associated with EV‐mediated peroxisome proliferator‐activated receptor‐gamma coactivator‐1 alpha (PGC‐1α) expression in injured iCMs. Conversely, mitochondrial disruption (MPP⁺ treatment) impaired the therapeutic potency of such EVs (O'Brien et al., 2021). Another study also found that mitoEVs from mature iCMs could reduce hypoxia‐induced cardiomyocyte injury by enhancing mitochondrial biosynthesis and bioenergetics in damaged cardiomyocytes, and PGC‐1α might be a key mediator of this rescue effect. I n vivo, iCM‐EV treatments improved cardiac function post myocardial infarction by restoring mitochondrial biogenetics and biogenesis (Ikeda et al., 2021). These findings suggest that mitoEVs may represent an innovative cell‐free therapeutic strategy for the treatment of cardiovascular diseases.

5.2.3. Neurological diseases

Neurological diseases are central and peripheral nervous system disorders that affect the brain and neurons, and mitochondrial injury plays a critical role in the development of these diseases. Currently, efficient treatment of neurological disorders remains challenging in the clinic. It has been found that neuronal cells can release mitoEVs, which may be promising therapeutics for neurological disease (Falchi et al., 2013; Guescini, Genedani et al., 2010). For example, neural stem cell (NSC)‐derived EVs carry abundant mitochondrial proteins and functional mitochondria. in vitro, such EVs can be taken up by mononuclear phagocytes via endocytosis and then fuse with host mitochondrial networks, thereby suppressing the proinflammatory state of these cells. in vivo, delivery of functional mitochondria by NSC‐EVs reduced neuroinflammation and disability in mice with autoimmune encephalomyelitis (Peruzzotti‐Jametti et al., 2021). In addition to NSCs, EVs from other cell types (brain microvascular endothelial cells and macrophages) also carry polarized mitochondria. Such EV treatments can restore mitochondrial function and increase the survival of brain endothelial cells under ischemic injury (D'Souza et al., 2021), suggesting that they might be an efficient strategy to cure central nervous system disorders.

5.2.4. Kidney diseases

Acute kidney injury (AKI), characterized by a rapid decline in kidney function, is a common form of organ failure that occurs in the intensive care unit (ICU), and mitochondrial injury is a hallmark of AKI. Current clinical therapies, such as renal replacement therapy, cannot markedly improve the long‐term outcomes of AKI patients. Recently, stem cell (mainly MSC)‐derived EVs have shown therapeutic effects to attenuate AKI, and mitochondrial transfer may be a novel mechanism. For example, we have found that MSC‐EVs carry functional mitochondrial components, such as mtDNA and ETC proteins, which can decrease mitochondrial dysfunction and inflammation in AKI mice by restoring TFAM signalling and mtDNA stability in injured renal tubular epithelial cells (TECs) (Zhao et al., 2021). Similarly, in the context of kidney ischemic injury, renal resident scattered tubular cell (STC‐like cell)‐derived EVs can deliver mitochondrial components to damaged TECs, thereby restoring their redox balance, mitochondrial membrane potential and mitochondrial protein expression. in vivo, such EV treatments restored oxygenation and mitochondrial pathways (e.g., COXI, DRP1) and alleviated renal fibrosis in stenotic kidney (STK) mice (Zou et al., 2018). These studies suggest that mitoEVs may be potent therapeutics for other kidney diseases.

5.2.5. Liver diseases

Ischemic liver injury is a major cause of early organ dysfunction after liver surgery or liver transplantation. Inflammatory responses, such as neutrophil extracellular trap (NET) formation, can promote liver damage during the liver ischemic stage (Huang et al., 2015). However, the efficacy of clinical therapies for ischemic liver injury is not ideal. Stem cell‐derived EVs have emerged as novel potent therapies for multiple liver diseases. For example, human umbilical cord‐derived MSC‐derived EVs (hUC‐MSC‐EVs) can prevent NET formation by delivering functional mitochondria, which preserve membrane potential and inhibit mitochondrial permeability transition pore initiation and mtROS and cytoplasmic mtDNA release in neutrophils. in vivo, such EV treatments protected liver function (reduced alanine transaminase, aspartate transaminase and lactate dehydrogenase levels) from ischemia‒reperfusion injury (IRI) (Lu et al., 2022). These studies provide insights into the links between mitochondrial damage and liver injury and suggest the potential of mitoEVs in the treatment of liver diseases.

5.2.6. Sepsis

Sepsis is the primary cause of death in the ICU. However, efficient sepsis therapy is currently limited, and novel therapeutic approaches are needed. In an LPS‐induced rat sepsis model, MSC‐derived microvesicles (MMVs) have been shown to improve mitochondrial functions (oxidative phosphorylation and mitochondrial dynamic balance) and intestinal barrier function. These beneficial effects may be due to the functional mitochondrial proteins (e.g., MFN2 and PGC‐1α) carried by MMVs, which promoted mitochondrial fusion and biogenesis in injured intestinal epithelial cells (Zheng et al., 2021). Taken together, mitoEV treatment can preserve mitochondrial function and thus serve as a potent strategy for multiple diseases.

6. CONCLUSION AND FUTURE PERSPECTIVES

Notably, abundant evidence suggests that EV‐mediated mitochondrial delivery can occur in both physiological and pathological states, and this process appears to be a novel means of mitochondrial quality control and intercellular communication. Moreover, it may have promising diagnostic and therapeutic potential for various diseases. However, some important questions need to be answered in future studies.

To date, mitoEVs have been widely observed in many types of biological samples, but whether the changes in amounts or contents of such EVs can reflect the signals (metabolic or energy status, etc.) or merely as byproducts of mitophagy of donor cells remain elusive. Some studies suggest that mitochondrial quality control pathways may selectively sort mitochondrial fractions into EVs (Cheng et al., 2020; Phinney et al., 2015; Rabas et al., 2021), while Parkin may direct these fractions into lysosomes (Todkar et al., 2021). In addition to these effects, whether other mechanisms are involved in determining the fate of mitochondrial contents in EVs needs to be further explored. Furthermore, it has been found that mitochondrial components delivered by EVs can be integrated into the mitochondrial network of the recipient cell (Crewe et al., 2021; Ikeda et al., 2021), but the detailed mechanism of the mitochondrial fusion process in target cells remains unclear.

Although stress or disease conditions may promote the release of mitochondrial fractions via EVs (Leermakers et al., 2020; Rosina et al., 2022), the mitochondrial components in EVs may vary under different conditions. In fact, mitoEVs may have diverse roles (e.g., pro‐ or anti‐inflammatory, pathogenic or curative) due to their different origins. Thus, the differences in the mitochondrial contents of EVs between the normal state and pathological state need to be explored. Preliminary studies have shown that such EVs may be biomarkers of multiple diseases, but whether their changes indeed correlate with disease course, treatment outcome or prognosis requires more clinical evidence. Additionally, blocking pathological EV transfer may be a possible strategy for the treatment of multiple diseases, but there is currently no specific EV inhibitor for in vivo use. Moreover, mitoEVs from healthy cells (e.g., stem cells) have shown potential in attenuating mitochondrial damage and organ injury in multiple disease models. However, the specific role of mitochondrial contents carried by EVs still needs to be deeply evaluated, since such EVs also contain other cargos (proteins, nucleic acids, etc.). In addition, whether EVs from different cell types have different mitochondrial contents and which EV subtypes are enriched with functional mitochondria remain elusive. In fact, mitochondrial contents, especially mtDNA, are susceptible to oxidative stress damage. For therapeutic purposes, it is important to establish highly efficient EV isolation and preservation methods that can maintain the integrity and biofunction of mitoEVs. To evaluate the safety and efficacy of such EVs, more preclinical studies using different disease models are required before clinical trials.

In conclusion, EV‐mediated mitochondrial delivery widely exists in vivo and may exert different roles in normal or pathological conditions. Therefore, it is imperative to explore the sorting mechanisms of such EVs and their detailed mode of action in different disease states, as well as to develop highly efficient EV production methods. More studies are required to assess the exact value of mitoEVs in disease diagnosis and treatment before future clinical applications.

AUTHOR CONTRIBUTIONS

Xiyue Zhou: Conceptualization; Writing – original draft; Writing – review & editing. Shuyun Liu: Writing – review & editing. Yanrong Lu: Writing – review & editing. Meihua Wan: Writing – review & editing. Jingqiu Cheng: Writing – review & editing. Jingping Liu: Conceptualization; Funding acquisition; Supervision; Writing – review & editing.

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no conflicts of interest.

Zhou, X. , Liu, S. , Lu, Y. , Wan, M. , Cheng, J. , & Liu, J. (2023). MitoEVs: a new player in multiple disease pathology and treatment. Journal of Extracellular Vesicles, 12, e12320. 10.1002/jev2.12320