Extracellular Mitochondrial Components and Effects on Cardiovascular Disease

Introduction

Besides being considered the energy plant inside cells, mitochondria are recently found to be released to extracellular space and transferred between cells. Intercellular transfer of mitochondria, including the horizontal transfer of their genome, promotes to change the bioenergy spectrum and transformation characteristics of recipient cells, thus leading to stem cell differentiation and reprogramming of stem cell activation (Torralba et al., 2016). Extracellular mitochondria can be free, encapsulated by vesicles, or as free circulating mitochondrial DNA (fc-mtDNA). Under cellular stress, free or vesicular mitochondria or mtDNA can act as a biological signal to interact with other cells, triggering inflammatory activation or cell regeneration. In some chronic diseases, the concentration of fc-mtDNA is related to the survival (Lindqvist et al., 2016; Shockett et al., 2016).

Cardiovascular disease is a serious threat to human health, especially for the middle aged and elderly people older than 50 years. It has the characteristics of high morbidity, disability rate and mortality. Even if the most advanced treatment means are applied, the number of people who die of cardiovascular disease is as high as 15 million every year in the world (López-Suárez et al., 2013; Balakumar et al., 2016). The interaction between monocytes and endothelial cells is regarded as a crucial step in vascular inflammatory diseases. Growth factors, chemokines, or cytokines affect quiescent cells by exchanging biological information in a paracrine manner (Coly and Boulanger et al., 2019). Myocardial ischemia and reperfusion (I/R) injury induces ultrastructural damage and dysfunction to mitochondria, thus leading to energy deficiency, homeostasis disruption, and oxidative stress production in cardiomyocytes.

It is still controversial whether extracellular mitochondria will be degraded or fused with endogenous mitochondria after being absorbed by recipient cells. However, in fact, they do act as signaling agents for vascular cells and are more likely to promote inflammation. The concentration of fc-mtDNA in patients with acute myocardial infarction, with poorer prognosis, is higher than that in patients with stable angina pectoris with better prognosis, and can be applied as a novel biomarker (Bliksøen et al., 2012). Since 2017, it has been reported that mitochondrial transplantation technology can be used to treat congenital heart disease and cardiac I/R injury (Emani et al., 2017). Apart from ongoing clinical trials, there is no way to use mitochondria for transplantation, so mitochondrial transplantation research is still in its infancy. In this review, we will focus on (1) the characteristics and types of extracellular mitochondria; (2) the relationship between extracellular mitochondria and molecular mechanisms of cardiovascular disease; (3) the potential of extracellular mitochondria as clinical prognostic markers; and (4) research progress and urgent problems to be solved in mitochondrial transplantation.

Characteristic of Extracellular Mitochondria

Mitochondria originate from the phagocytosis of α- proteobacterium by eukaryotic precursors and retain the double membrane structure similar to their ancestral characteristics. These semiautonomous organelles are controlled by dual genomes, including nuclear gene and mitochondrial DNA (mtDNA). Mitochondrial nuclear genes encode 22 transfer RNAs (tRNA) and 2 mitochondrial ribosomal RNAs (rRNA) necessary for the synthesis of mitochondrial proteins. The 13 essential proteins of the mitochondrial electron transport chain complex (I, III, IV, and V) are encoded by mtDNA (Kopinski et al., 2019). Mitochondria produce large amounts of ATP through oxidative phosphorylation and represent high-energy core for most eukaryotic cells. Mitochondria also possess many other functions, including calcium signaling transduction, reactive oxygen species (ROS) production, fatty acid biosynthesis, and regulation of cell apoptosis (Kastaniotis et al., 2017; Pallafacchina et al., 2018; Demine et al., 2019; Bock and Tait, 2020).

Apart from traditional functions of mitochondria, horizontal mitochondrial transfer between cells has now been discovered. This is a new mode of cell-to-cell communication: intercellular organelle transfer. Mitochondria themselves and mitochondrial genes can be released into the extracellular space, modifying functions of the recipient cells. Multiple mechanisms such as tunneling nanotubes, extracellular vesicles, gap junctions, mitochondrial ejection, or cytoplasmic fusion mediate intercellular mitochondrial transfer (Torralba et al., 2016). The horizontal transfer of mitochondria appears in different kinds of cells, including mesenchymal cells, neurons, and cardiomyocytes (Phinney et al., 2015; McCully et al., 2017; Hayakawa et al., 2018). This transfer not only occurs under pathological conditions but also plays a role in maintaining the homeostasis and development of tissues or organs under physiological conditions.

Depletion of mtDNA, inhibitors of mitochondria, cell damage, and endotoxin can trigger intercellular mitochondrial transfer. Extracellular mitochondria are capable of restoring functions of damaged cells and organs. Spees et al. (2006) found for the first time that adult stem cells and somatic cells actively transferred functional mitochondria to save cell respiration and growth. Extracellular mitochondria transported by astrocytes prevented neurons from being deprived of oxygen and glucose, thus maintaining the stability of dendritic structures (Hayakawa et al., 2016). After the liver I/R model was performed, healthy mitochondria were infused into the recipient's spleen and significantly reduced I/R injury (Lin et al., 2013). Miro1, a mitochondrial Rho-GTPase, was first put forward to enhance mitochondria transfer from mesenchymal cells to epithelial cells. Meanwhile, overexpression of Miro1 hold the anti-inflammatory effects of mesenchymal cells by decreasing the secretion of thymic stromal lymphopoietin, interleukin (IL)-25, and IL-33 (Ahmad et al., 2014). This study provides a molecular mechanism of mitochondria transfer involved in mesenchymal cell for cell repair. However, extracellular mitochondria can also play a proinflammatory role or act as “danger signals.” Otsu and colleagues found that mesenchymal cells transferred mitochondria to endothelial cells, which promoted ROS production and capillary degeneration (Otsu et al., 2009). Extracellular mitochondria released from necroptotic cells were engulfed by macrophages and dendritic cells, leading to tumor necrosis factor-alpha (TNF-α) production, promoting inflammation in macrophages (Maeda and Fadeel, 2014). Microglia released fragmented and dysfunctional extracellular mitochondria to neurons that propagated neuronal injury, while functional mitochondria in extracellular milieu were neuroprotective (Joshi et al., 2019). Extracellular mitochondria are like a double-edged sword, whether they are helpful or harmful indeed depends on context. This is closely associated with morphological integrity of functional mitochondria, especially in cell survival, oxidative stress, immune response, and organ injury.

Carriers of Mitochondria: Extracellular Vesicles

Extracellular vesicles are composed of lipid bilayers and nuclear components that wrap soluble cytoplasmic substances, and released into the extracellular space (Fu et al., 2020). Although extracellular vesicles were previously thought to be merely inert cell debris or “platelet dust,” they have recently been regarded as mediators and paracrine effectors of intercellular communication (Hargett and Bauer, 2013). Extracellular vesicles are released by almost all mammalian cells, and can carry and protect biomolecules (such as proteins, lipids, and nucleic acids) from being degraded during the transfer to recipient cells (Boulanger et al., 2017).

According to the size of the organism, the cargo, and the production mechanism, extracellular vesicles can be divided into three main populations: microvesicles, exosomes, and apoptotic bodies. Microvesicles are a major type of extracellular vesicles, which are also called microparticles in the cardiovascular field. They are phosphatidylserine-exposed membrane vesicles with a diameter of 100–1000 nm. Cytoskeleton remodeling and externalization of phosphatidylserine belong to the formation mechanisms of microvesicles, which are also one of the distinctions from other vesicles. The cytoskeleton and hydrostatic pressure maintain the balance of plasma membrane tension. The incompleteness of the cytoskeleton is critical to membrane asymmetry, mainly due to the destabilization of the actin cytoskeleton mediated by alphaIIbbeta3(αIIbβ3), promoting the coagulant microvesicles release (Cauwenberghs et al., 2006). Exosomes are the smallest extracellular vesicles (∼40–100 nm in diameter), which show a cup-shaped morphology when observed under a transmission electron microscope. They are derived from ceramide and/or endosomal sorting complexes, relying on the Endosomal Sorting Complex Responsible for Transport (ESCRT) pathway to recruit tumor susceptibility gene 101 (TSG101) and Suppressor of K+ transport growth defect 1 (SKD1) to the endosomal membrane in turn to form multivesicular body (Trajkovic et al., 2008; Abels and Breakefield, 2016). Another obvious feature of exosomes is that they are rich in lipids, especially cholesterol and tetraspanins (such as CD9, CD63, and CD81) (Ferguson and Nguyen, 2016). Apoptotic bodies are larger vesicles (50–5000 nm) containing lysed organelles and broken nuclear components, and have a high affinity for annexin V. Up to now, there are few studies on apoptotic bodies in the field of cardiovascular diseases.

Mitochondria and mtDNA have been detected in extracellular vesicles to mediate cell properties and function (Fig. 1). Mesenchymal cells suppressed cytokine production and increased phagocytic capacity of human monocyte-derived macrophages partially through CD44⁺ extracellular vesicles containing mitochondria, exhibiting an anti-inflammatory effect (Morrison et al., 2017). Extracellular vesicles also regulate signaling cascades and epigenetic changes in metastatic progression through either juxtacrine or paracrine. Hormonal therapy exerted therapeutic action by depriving estrogen receptor-independent oxidative phosphorylation of cancer cells, while extracellular vesicles harbored and transferred healthy mtDNA to these cells to rescue impaired metabolism (Sansone et al., 2017). As the second largest type of extracellular vesicles, microvesicles contain and deliver complete mitochondria to different kinds of recipient cells. Microvesicles carrying mitochondria were released by astrocytes and taken up by neurons to play a neuroprotective role (Hayakawa et al., 2016). Retinal ganglion cell axons released injured mitochondria, which were transferred by microvesicles to astrocytes for lysosomal degradation (Berridge and Neuzil, 2017).

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FIG. 1.

Schematic representation of cell-derived EVs carried mitochondria. MVs are heterogeneous vesicles with a diameter of 100 to 1000 nm. Exosomes are vesicles of ceramide and/or endosomal sorting complexes origin with 40–100 nm in diameter. Intact mitochondria or mtDNA can be contained in EVs. EV, extracellular vesicle; MV, microvesicle; mtDNA, mitochondria DNA. Color images are available online.

Exosomes carry genome and mtDNA, which act as a medium for long-distance molecular transport between cells. Mitochondria contain 100–1000 copies of maternally inherited mtDNA and induce innate immune responses through damage- related molecular patterns (DAMPs). This response can be observed in a variety of metabolic diseases characterized by chronic inflammation, including diabetes, cancer, and cardiovascular disease (Ye et al., 2017; Chen et al., 2018; Silzer et al., 2019). In the case of cell senescence, apoptosis or necrosis, mtDNA can be released into the cytoplasm or extracellular space to activate the inflammatory response and eliminate damaged cells. The accumulation of mtDNA damage promotes the release of fc-mtDNA and package it in exosomes in response to a variety of cellular pressure signals. In addition, extracellular mtDNA can be considered the result of extracellular vesicles and extracellular traps, which are also related to the increase of ROS (Cai et al., 2017; Kim et al., 2019).

Regulation of Extracellular Mitochondria Quality Control

To maintain the mitochondrial function and proper integration of intracellular signal, eukaryotic cells possess a complete quality control system (Bozi et al., 2016). Generally, mitochondrial quality control system regulates organellar homeostasis through the following mechanisms: (1) the antioxidant enzymes protect organelles from oxygen-mediated toxicity, (2) the ubiquitin proteasome system and mitochondrial unfolded protein response (UPR^mt) help the misfolded proteins to restore normal protein conformation and newly synthesized proteins to correctly fold to nuclear signal transduction, and (3) mitochondrial dynamics and mitophagy coordinate the size, shape, and clearance of mitochondria (Sugiura et al., 2014; Picca et al., 2020). In addition to supporting the migration of ions and metabolites between organelles, the mitochondrial-lysosomal axis ensures the recycling of wasted materials (Todkar et al., 2017; Wong et al., 2018). This mechanism has recently been incorporated into the mitochondrial quality control system, in that this interaction helps release extracellular vesicles containing damaged mitochondrial components (Picca et al., 2020).

Mitochondria possess the ability to produce their own vesicles to transfer mitochondrial proteins and lipids to other intracellular organelles. Mitochondrial-derived vesicles are generated by small vesicles (70–150 nm of diameter) rich in mitochondria cargo possibly undergo degradation in lysosomes. These vesicles can be identified by mitochondria marker staining of both matrix and outer membrane. Aggregation of phosphatase and tensin homolog-induced kinase 1 (PINK1) with ubiquitination of Parkin is required for mitochondrial-derived vesicle biogenesis and transition to lysosome (Picca et al., 2018). Under oxidative stress, hyperfused mitochondria and excessive accumulation of protein were observed along with mitochondrial-derived vesicle increase (Youle and Narendra, 2011).

Mitophagy triggered by mitochondrial depolarization ensure functional organelle network within cells by autophagosome engulfing severe damaged mitochondria. However, mitochondrial-derived vesicle places emphasis on disposing mildly damaged mitochondria independent of autophagy or mitochondrial fission. Mitochondrial-derived vesicle containing outer membrane mitochondrial-anchored protein ligase shuttles to the peroxisome and prevents clearance of entire organelles. Endophilin B1, Rab GTPases, and PIP-related microdomains may be involved in mitochondrial-derived vesicle transport and then contribute to biosynthesis and endocytosis (Sugiura et al., 2014). Microvesicles containing mitochondria were generated by alveolus-attached bone marrow-derived stromal cells and transferred to the alveolar epithelium for protective effects (Islam et al., 2012).

Clearance of depolarized mitochondria under mitochondrial stress has emerged as an alternative mitochondrial quality control pathway. Mitochondria released into extracellular space are predominantly in free form (500 nm–1.5 μm) rather than in membrane-surrounded vesicles (Choong et al., 2020). This finding is consistent with the previous research that free mitochondria and mitoparticles derived from astrocyte spanned a range of sizes from 300 to 1100 nm (Hayakawa et al., 2016). Extracellular mitochondria as well as mitochondria proteins were also detected in cerebrospinal fluid after subarachnoid hemorrhage (Chou et al., 2017). Besides mitochondrial-derived vesicle, extracellular mitochondria released in free form is probably an appreciable mechanism to dispose unwanted mitochondria when the scavenging ability of mitophagy reaches its limit.

Extracellular mtDNA Transfer: Cell fc-mtDNA and Mitochondrial DAMPs

mtDNA is organized in the structure of discrete condensed spots called nucleoids, and mitochondrial transcription factor A is the only recognized protein that packages mtDNA in mammalian nucleoid strictly, which is essential for mtDNA transcription. It has been generally considered that mtDNA molecules are identical at birth (homoplasmy); however, variant mtDNA (heteroplasmy) involving noncoding mtDNA D-loop has been found to be maternally inherited. Abnormal electronic transport chains, defective mtDNA protection, and repair mechanisms together lead to highly variable mtDNA (Gustafsson et al., 2016). Once the harmful mutation exceeds the threshold level, it will cause increased production of oxidative free radicals, defects in ATP synthesis, and cell death. Mutant mtDNA and progressive energy decline of mitochondria result in infection, diabetes, and cardiovascular diseases (Fang et al., 2018; Moriyama et al., 2019; Castellani et al., 2020).

Cumulative mtDNA mutations with age can induce cell senescence, apoptosis, or even necrosis and ultimately proinflammatory responses. To initiate immune responses, mtDNA needs to be liberated from mitochondrial matrix and transferred to nearby organelles, the cytosol, and extracellular space. Released mtDNA can be seen in the form of nucleoids, double- or single-stranded molecules, and small fragments. Inner mitochondrial membrane is the place where ROS are mostly generated by respiratory complexes, and mtDNA is also located there. Due to this structural feature, mtDNA is highly susceptible to ROS, which suggests oxidative stress can be one of the main mtDNA leakage causes (Tian et al., 2016). Under oxidative stress, the opening of mitochondrial permeability transition pore promotes Ca2⁺ release, and subsequently mtDNA liberation can be observed. Interestingly, only small mtDNA fragments shorter than 700 bp in length were released and they are responsible for mitochondrial gene (MTCO1, MTND3, and MTCYB) codification (García and Chávez, 2007). Widely accepted as an intracellular danger sensor, Toll-like receptor 9 (TLR9) can be activated by DNA containing unmethylated cytosine-phosphodiester-guanine (CpG) motifs and the interaction of itself from cell endogenous source with mtDNA. This interaction may be attributed to damaged mtDNA accumulation resulting from the impairment of mitophagy under deoxyribonuclease II (DNase II) deletion condition (Pohar et al., 2017). Besides, during intrinsic mitochondrial apoptosis, mtDNA release induced IFN response and TBK1/interferon regulatory factor pathway dependent on Bax/Bak pore (Kawasaki and Kawai, 2014).

Fragments of mitochondrial mtDNA released to extracellular space usually become cell-free fc-mtDNA in plasma. Since mtDNA contains abundant hypomethylated CpG motifs similar to its bacterial ancestor, great attention has been attached that fc-mtDNA triggers inflammation. mtDNA activates neutrophils and mammalian immunes through TLR9, Nod-like receptor family pyrin domain-containing 3 (NLRP3), and STING signaling (Ding et al., 2014). Neutrophils function in innate immune responses by killing bacteria, but recently, they have also embraced this role in the form of neutrophil extracellular traps (NETs). NETs are netlike extracellular structures composed of DNA and proteins, whose formation requires fully mature neutrophils to guarantee the ability to kill both bacteria and fungi. Ribonucleoprotein-containing immune complexes from systemic lupus erythematosus and surgical trauma-induced proinflammatory NETs production bound to mtDNA in respective ways (McIlroy et al., 2014; Wang et al., 2015). Plasma DNA was also isolated for quantitative real-time PCR (qPCR) to measure mtDNA damage in Intensive Care Unit (ICU) patients. Elevated mtDNA level more than or equal to 3200 copies had increased odds of dying within 28 days of ICU admission, indicating that fc-mtDNA is associated with ICU mortality and predict an increased risk of 3.5-year death (Nakahira et al., 2013). The systematic theory of fc-mtDNA origin and release has not been fully elucidated, but there is growing evidence support that it is positively correlated with postinjury inflammation initiation. The patterns of fc-mtDNA release without cell necrosis marker variation showed two peaks: early and 5-day postinjury (Simmons et al., 2013). Whether inflammatory process can be prevented when fc-mtDNA is below the minimum concentration necessary for catalyzing inflammation needs to be further investigated, as cell-free DNA from mitochondria seems to be a promising therapeutic intervention.

In 1994, Polly Matzinger proposed that, besides distinguishing self from nonself components, immune system was primarily driven by dangerous signals (Matzinger, 1994). These endogenous molecules originate from extracellular matrix, plasma membrane, nucleus, and mitochondria after apoptotic or necrotic cells occur. This category of molecules is referred as DAMPs, in that they are no longer shielded by integrated cell structure and released to circulation or nearby tissues for immune system activation. DAMPs are recognized by specific pattern recognition receptors of the innate immune system, including TLRs, NOD-like receptors, RIG-I-like receptors, and purinergic receptors (De Lorenzo et al., 2018). DAMPs belong to noninfectious inflammatory responses and focus on potentially damaging or danger to the host, and they are recognized by the same cellular receptors for pathogen-associated molecular patterns. According to the endosymbiont hypothesis, mitochondria like their bacterial ancestor contain unmethylated CpG DNA and N-Formyl peptides for translation (Korimová et al., 2018). N-Formyl peptides and mtDNA released by injury mitochondria within cells to the extracellular space are recognized by pattern recognition receptors, so mitochondrial DAMPs are an important part of regulating inflammation.

Among trauma patients, severe ischemia, hemorrhagic shock, and infection are predispositions to systemic inflammatory response syndrome (SIRS). Conditional pathogens colonized in the gut are transferred to the blood during ischemia, and then cause SIRS. mtDNA levels in trauma patients' plasma were thousands of folds higher than those in patients with no open wounds or gastrointestinal injuries (Simmons et al., 2013). TLR-9 as the inducer for synthesis of proinflammatory cytokines interacted with mtDNA to activate p38 mitogen-activated protein kinase pathway at clinical plasma concentrations (Garcia-Martinez et al., 2016). Increases of blood serum extracellular mtDNA were reported in arterial hypertension individuals (Veiko et al., 2010). mtDNA were detected in the synovial fluids of rheumatoid arthritis patients as well as plasma of femur fracture patients (Gan et al., 2015; Du et al., 2020). These data suggest that mtDNA has the ability to induce inflammatory responses in the form of mitochondria DAMPs. However, extracellular DNA can act as extracellular trap to kill pathogens efficiently. Ejection of mtDNA dependent on ROS was responded to pathogen-associated molecular patterns, and trapped microbial pathogens to kill them by antimicrobial peptides in the extracellular space (Yousefi et al., 2008). The question comes what the exact role of mitochondria DAMPs is since they contribute to inflammation for one side and assist efficient clearance of harmful cellular components for the other side. The comprehensive mechanism of mtDNA activating antigen-presenting cells and other immune cells may help deliver the answer.

Extracellular Mitochondria in Cardiovascular Diseases

Atherosclerosis

Cardiovascular disease includes various diseases such as coronary artery disease, hypertension, stroke, and peripheral neurovascular disease. Up to 2017, the population suffering from cardiovascular disease increased beyond 20% (Decandia, 2018). Atherosclerosis is the main basic disease that attributes to cardiovascular disease morbidity and mortality. It is a chronic disease characterized by endothelial injury, inflammatory cell infiltration, cell proliferation, and lipid deposition. Platelets are the first cells to the site of endothelial injury and express glycoproteins Ib and IIb/IIIa, which may contribute to the activation of endothelial cells. When subjected to irritating stimuli (such as dyslipidemia, hypertension, or proinflammatory mediators), endothelial cells will be activated and express adhesion molecules. Chemokines mediate the migration of leukocytes to the innermost layer of the artery and promote the entry and retention of cholesterol-containing low-density lipoprotein particles in the artery wall, which finally progress to atherosclerotic plaque (Hansson, 2005). The activation of vascular inflammation can promote acute coronary syndrome caused by vulnerable plaques, leading to thrombosis formation, myocardial infarction, pulmonary embolism, and stroke.

Since the activation of vascular endothelium is the main part of the inflammatory response, there is no doubt that intercellular communication between vascular cells represents a key aspect of this process. In blood circulation, platelets appear to be the main structure that carries extracellular mitochondria. They are small anucleate cell fragments in a discoid form released by megakaryocytes in the bone marrow. Each platelet has about four mitochondria located near its outer membrane (Thon and Italiano, 2012). Platelets can be activated by a variety of danger signals and participate in inflammatory reactions by releasing the contents of platelet cytoplasmic granules into the extracellular environment. Activated platelets also release thrombotic and proinflammatory microvesicles. Boudreau et al. (2014) observed that activated platelets not only released mitochondria containing microvesicles into the extracellular space but also free mitochondria with respiratory function. Although free mitochondria are smaller than intact platelets and have submicron size and membrane fraction, they still meet the current structural definition of conventional microvesicles, which provides a new explanation for the recognized heterogeneity in platelet-derived microvesicles (Dean et al., 2009). More importantly, activated platelets released free mitochondria by relying on PLA 2 -IIA hydrolysis to produce bioactive mediators (fatty acids, lysophospholipids, and mtDNA) and promoted the proinflammatory response of neutrophils (Fig. 2).

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FIG. 2.

Cell-to-cell communication mediated by extracellular mitochondria. Activation of platelets leads to mitochondria release into extracellular space. These mitochondria are liberated in the form of mitoMVs and freeMito. mtDNA acting as mtDAMPs can be also released from dysfunctional mitochondria. After fusion, extracellular mitochondria induce inflammatory cytokines and ultimately cause inflammation in neutrophils. mtDAMP, mitochondria associated damage-related molecular pattern; mitoMV, mitochondria-microvesicles; freeMito, free mitochondria. Color images are available online.

What needs to be pointed out is the existence of intact cell-free mitochondria under normal physiological conditions should not be ignored. Al Amir Dache et al. (2020) discovered the presence of particles containing mtDNA in the circulation. Then they confirmed that the blood contained complete cell-free full-length mtDNA with a diameter of more than 0.22 μm. Using cell sorting analysis and transmission methods, these particles were identified as intact cell-free mitochondria with dense and double-layer membrane structure. The latest findings indicate that free mitochondria are also one of the key mediators of vascular inflammation. Monocytes exposed to lipopolysaccharide (LPS) resulted in the release of free mitochondria, which subsequently induced inflammatory responses mediated by TNF-α and type I interferon (IFN) in endothelial cells (Puhm et al., 2019). It is worth mentioning that free and microvesicle-embedded mitochondria seem to affect the function of endothelial cell in the same inflammatory pathways. This phenomenon needs to be thought provoking, in terms of inhibiting inflammation, whether free mitochondria and mitochondria coated with membrane vesicles still have the same effect. These findings have attracted great attention that, in addition to being the energy-providing component of eukaryotic cells, mitochondria are also messengers that play an important role in cell-cell communication, especially in vascular inflammation.

Human antimicrobial peptide LL-37 producing IFN-α has been found in neutrophil and NETs in atherosclerotic lesions. In addition, mtDNA present in NETs induces formation of LL-37-mtDNA complexes. Zhang and colleagues demonstrated not only free mtDNA but also the LL-37-mtDNA complex was higher in plasma of patients with atherosclerosis. The complex triggered activation of TLR9-mediated inflammatory responses through escaping from autophagy and degradation by DNase II. The results were further supported by mouse model that atherosclerotic lesion enlarged ApoE ^-/- mice injected with LL-37-mtDNA complex and plaque size reduced with reduction of serum TNF-α, IL-6, and IFN-α in mice treated with antibody against the complex (Zhang et al., 2015). Oxidative stress is one of the acknowledged formation mechanisms for atherogenesis. Lectin-like oxidized low-density lipoprotein scavenger receptor-1 (LOX-1) is the major receptor for oxidized low-density lipoproteins (ox-LDL) accumulating in the arterial wall, and ox-LDL activation dependent on LOX-1is involved in atherosclerosis (Honjo et al., 2003). In an LDLR knockout mouse model, antibody against LOX-1 attenuated ox-LDL-mediated autophagy, TLR9, and inflammatory signal (CD45 and CD68) expressions (Mehta et al., 2007). LOX-1 deletion also reduced damaged mtDNA in mice aortas. Macrophages overloaded ox-LDL and cholesterol crystals eventually converted into foam cells due to imbalance between lipid uptake and efflux. Foam cells deposited underneath endothelium make arterial wall thickening, leading to obstructive atherosclerotic plaque formation. Macrophage cellular oxidation, 7-hydroperoxide, autophagy protein 5 enhances macrophage oxidative stress and accelerates atherosclerosis progression. Consistent with the previous study, LPS induced mtDNA damage, LOX-1, and NLRP3 inflammasome expression in THP-1 macrophages and primary macrophages. The data showed NLRP3 inflammasome was inhibited with the absence of LOX-1 and enhanced after autophagy was blocked (Ding et al., 2014).

Extracellular Mitochondria Transplantation as Therapeutic Agent for Heart

The heart is an organ composed of a large number of muscle fibers and mitochondria. A steady flow of blood is pumped from the heart to guarantee sufficient oxygen and nutrients (such as water, inorganic salt, glucose, protein, and various kinds of water-soluble vitamins) for other body tissues and organs, meanwhile taking away the end products of metabolism (such as carbon dioxide, urea, and uric acid) from them. Almost all the energy required for the contraction of cardiomyocytes is provided by ATP produced by mitochondria, which accounts for 30% of the volume of cardiomyocytes. In myocardial I/R events, mitochondrial ATP synthesis decreases with hydrolysis increase, and then ROS are formed. Free radicals released from necrotic cells destroy myocardial cell membrane potential, resulting in arrhythmia, decreased myocardial contractility and cardiac output. I/R can damage mitochondria, including (1) rapid restoration of pH; (2) further increase of mitochondrial and intracellular Ca2⁺ overload; and (3) production of ROS (Shin et al., 2017). Therefore, in recent years, clinical trials have explored a new paradigm of mitochondrial therapy based on delivery of mitochondria from autologous or nonautologous sources to treat diseases related to mitochondrial damage and dysfunction. Mitochondrial transplantation can replace injured mitochondria by supplementing exogenous healthy mitochondria to reverse excessive ROS production and restore mitochondrial function.

In a 2009 study, for the first time, respiratory active mitochondria isolated from healthy donors were injected into the ischemic area of rabbit hearts after 30 “/120” (I/R). The injected mitochondria survived under the endocardium, decreased levels of creatine kinase MB, cardiac troponin I, and caspase-3, and exerted cardioprotective effect by increasing ATP content, reducing infarct size, preventing myocardial cell loss, and improving myocardial function after ischemia (McCully et al., 2009). Similar results were observed in the transplantation of autogenous mitochondria. After injecting autologous mitochondria into regional ischemia, the heart showed recovery for 4 weeks and the infarct size was significantly reduced. The transplanted mitochondria were internalized by cardiomyocytes in 2–8 h, which restored normal oxygen consumption and high energy phosphate synthesis, and enhanced myocardial contraction (Masuzawa et al., 2013). Transplantation of mitochondria immediately after myocardial ischemia and just before reperfusion reduced the ratio of infarct size to risk area. Magnetic resonance image showed that this effect could last for 4 weeks (Kaza et al., 2017). Prophylactic administration of single or multiple doses of mitochondria in vitro before ischemia rescued myocardial function. Coronary blood flow could be temporarily restored during reperfusion rather than before (Guariento et al., 2020). These results suggest that transplantation of healthy extracellular mitochondria exerts therapeutic effect at different stages of I/R.

Exogenous mitochondria include two different delivery modes: direct myocardial injection and intracoronal artery infusion. By tracing 18 F-rhodamine 6G and iron oxide nanoparticle-labeled mitochondria, it was found that compared with direct injection, intracoronary delivery of mitochondria led to rapid and wider distribution of the whole myocardium, but both had similar protective effects on the heart. Transplanted mitochondria appeared in the interstitial space around cardiomyocytes 10 min after injection. Within 1–2 h postdelivery, transplanted mitochondria were gradually detected around the sarcolemma, nucleus, and endogenous damaged mitochondria. Such time-dependent changes were consistent with the time of myocardial function enhancement (Shin et al., 2019). Observed through three-dimensional super resolution microscopy and transmission electron microscopy, exogenous mitochondria from pluripotent stem cell-derived cardiomyocytes and human cardiac fibroblasts were integrated into cardiomyocytes within a few minutes, and then transported to endosomes and lysosomes. Most of these mitochondria were fused with the endogenous mitochondrial network, which represents that the fusion of extracellular mitochondria and recipient mitochondria is an evolutionarily conserved and universal biological process (Ali Pour et al., 2020). Other studies have shown that Cytochalasin D, a specific inhibitor of actin polymerization, reduced mitochondrial internalization to cardiomyocytes and ATP content; furthermore, mitochondrial intercellular transfer was also involved in tunnel nanotubes within cardiomyocyte syncytial structures (Pacak et al., 2015).

Controlled myocardial ischemia results in diastolic cardiac arrest by placement of aortic cross-forceps to block coronary blood flow. If extracorporeal membrane oxygenation (ECMO) is supported for more than 72 h, myocardial damage will be induced. Therefore, children undergoing congenital heart disease surgery treated with ECMO are the most suitable population for mitochondrial transplantation therapy. In the five patients who received the initial mitochondrial transplantation, ∼1 × 10⁹ mitochondria were injected into each patient through epicardial injection, and four patients recovered cardiac function and successfully separated from ECMO (Emani and McCully , 2017). The mean cold ischemia time after heart transplantation is 4–6 h, and the survival rate is inversely proportional to cold ischemia time due to I/R injury. In a mouse transplantation model with mitochondrial injection concentration of 5 × 10 ⁸ per gram wet weight, mitochondrial transplantation prolonged cold ischemia time to 29 h, enhanced calculated ejection fraction and shortening fraction in recipient heart, and reduced graft tissue injury (Moskowitzova et al., 2019).

Although mitochondrial transplantation compensates for the deficiency of myocardial cell viability caused by mitochondrial dysfunction, which cannot be corrected by drugs, and is even considered a possible lifesaving measure, the long-term effects of mitochondrial transplantation have declined. A 28-day observation of biological energy showed that basic respiratory function and ATP production were improved significantly in the short term, but these biological advantages gradually disappeared over time (McCully et al., 2017). However, this does not mean that mitochondrial transplantation has little clinical significance, because the production of mitochondrial superoxide in transplanted cells does not increase, nor does transplantation itself produce obvious post-transplant adverse effects. Therefore, mitochondrial transplantation may be more suitable to save acute stress injuries.

Isolation and Storage of Mitochondria

To ensure the viability of transplanted mitochondria, the isolation and purification of functional mitochondria must be obtained within a short time. Standard mitochondrial isolation techniques take up to 90 min, while most cardiac surgeries require around 60 min for optimal results, making it unreliable to use the mitochondria separated by standard method to rescue myocardial injury. McCully and his team (2017) have developed a new method of mitochondrial isolation that can be obtained within 30 min. They used standardized tissue dissociator and differential filtration technique to dissociate 0.1 g skeletal muscle surgical sample into homogenate. The homogenate was digested with subtilisin A, and then filtered by a sterile mesh to obtain mitochondria that can be directly used or concentrated by centrifugation. The purity and activity of mitochondria isolated from this method were verified by oxygen consumption rate, ATP analysis, fluorescent probe analysis, light microscopy with fluorescent mitochondrial labeling (MitoTracker CMXros), and transmission electron microscope, which proved the functionality and clinical application of isolated mitochondria.

Freshly isolated mitochondria during operation are undoubtedly the best choice for mitochondrial transplantation, but cryopreserved mitochondria may be more suitable for bedside patients. The inner and outer membrane of mitochondria will be impaired when they are stored in cold or frozen state. When the inner membrane of mitochondria is damaged, its energy production, pH gradient, and membrane potential decrease. When the outer membrane of the mitochondria is damaged, its integrity is lost and membrane permeability increases, leading to cytochrome C release from the gap between the mitochondrial membranes. Therefore, it is necessary to refrigerate and store mitochondria under the condition of maintaining the stability of mitochondria inner and outer membrane. A study has shown that the structure of mitochondria inner and outer membrane was intact and their oxidative phosphorylation ability was preserved at −80°C when 10% (V/V) dimethyleneimine was used for cryopreservation (Nukala et al., 2006). Another study showed that freezing mitochondria with trehalose prevented the release of cytochrome C, retaining the ability of mitochondria to induce apoptosis (Yamaguchi et al., 2007). Cold 4-(2-HYDROXyethyl) -1-piperazineethanesulfonic acid (HEPES) sucrose-based buffer maintained ∼80% mitochondrial respiratory capacity after 24 h of mitochondrial refrigeration. Adding cytochrome C to the above cache solution enhanced the respiratory capacity to around 100% for 24 h after refrigeration started (Gnaiger et al., 2000). The University of Wisconsin solution belongs to phosphate buffer, which restored the content of cytochrome C and the activity of complex II in rat liver mitochondria. The addition of antioxidants and colloid also effectively protected complex III and IV activity and mitochondrial structure (Yamada et al., 2020).

Clinical Drug Delivery Trials for Mitochondrial Transplantation

Pep-1 is an amphiphilic peptide consisting of three domains: hydrophobic point, hydrophilic site, and septal region. The main role of PEP-1 is to improve the uptake efficiency of cells, thus improving mitochondrial function. After co-culture of fibroblasts with Pep-1-modified mitochondria, the mitochondrial morphology of fibroblasts was prolonged, ROS production was reduced, and mitochondrial membrane potential was increased (Chang et al., 2013, 2017). Mitochondrial drug delivery system can transport therapeutic molecules to the mitochondria of the targeted diseased cells, which may help to accelerate the mitochondrial transplantation. A versatile mitochondrial targeting liposomal-based nanodevice, namely a Mito-Porter system, is established to ensure that therapeutic molecules are absorbed by target cells, and to localize and function within the mitochondria. Mito-porter first gained access to cells through macropinocytosis, then combined with mitochondria through electrostatic interaction of negative membrane potential, and finally transferred the cargos to mitochondria through membrane fusion (Yamada and Harashima, 2008; Yamada et al., 2019). With the advantage of not limiting the physical nature or size of the cargos, Mito-Porter is expected to be a basis for developing nanomaterials for cellular therapy and ischemic disease therapy.

Conclusion

Mitochondrial transcellular transfer has been considered a new mechanism of mitochondrial movement. Depending on the type and state of the mitochondria, extracellular mitochondria play a role in promoting tissue repair or inducing inflammation in health and diseases. Complete mitochondria or fc-mtDNA wrapped with vectors (such as microvesicls or exosomes) are released into tissue fluid and circulation as the active components of intercellular signaling, for playing immunomodulatory functions through intercellular interactions. Extracellular mitochondria are a new medium for intercellular communication between inflammatory vascular cells; moreover, fc-mtDNA can even be used as a biomarker for the prognosis of cardiovascular diseases (Table 1).

In injured cells, especially ischemic cardiomyocytes, the damaged mitochondria are replaced by isolated viable and respirable mitochondria, which can improve the severity of myocardial infarction, cell viability, and ventricular function. The success rate of mitochondrial transplantation for the purpose of saving the recipient cell function needs to be carefully considered, because whether the transferred mitochondria remain functional is still the focus of debate. Mitochondria are highly dynamic organelles and mtDNA has high immunogenicity, so intercellular signaling undergoes changes according to the nature of mitochondria, leading to the survival or death of recipient cells. The existence form, transmission mode, and internalization mechanism of these mitochondria still need to be explored. In addition, how to quickly extract high-purity mitochondria has inspired experimental innovations for future researches. Extracellular mitochondria enrich the content of vascular intercellular communication and have great therapeutic potential in the treatment of cardiovascular diseases.