Ischemic stroke is a secondary cause of mortality worldwide, imposing considerable medical and economic burdens on society. Extracellular vesicles, serving as natural nano-carriers for drug delivery, exhibit excellent biocompatibility in vivo and have significant advantages in the management of ischemic stroke. However, the uncertain distribution and rapid clearance of extracellular vesicles impede their delivery efficiency. By utilizing membrane decoration or by encapsulating therapeutic cargo within extracellular vesicles, their delivery efficacy may be greatly improved. Furthermore, previous studies have indicated that microvesicles, a subset of large-sized extracellular vesicles, can transport mitochondria to neighboring cells, thereby aiding in the restoration of mitochondrial function post-ischemic stroke. Small extracellular vesicles have also demonstrated the capability to transfer mitochondrial components, such as proteins or deoxyribonucleic acid, or their sub-components, for extracellular vesicle-based ischemic stroke therapy. In this review, we undertake a comparative analysis of the isolation techniques employed for extracellular vesicles and present an overview of the current dominant extracellular vesicle modification methodologies. Given the complex facets of treating ischemic stroke, we also delineate various extracellular vesicle modification approaches which are suited to different facets of the treatment process. Moreover, given the burgeoning interest in mitochondrial delivery, we delved into the feasibility and existing research findings on the transportation of mitochondrial fractions or intact mitochondria through small extracellular vesicles and microvesicles to offer a fresh perspective on ischemic stroke therapy.

With the discovery of intercellular mitochondrial transfer, the intricate mitochondrial regulatory networks on stem cell fate have aroused intense academic interest. Apart from capturing freely released mitochondria from donor cells, stem cells are able to receive mitochondria through tunneling nanotubes (TNTs), gap junctional channels (GJCs) and extracellular vesicles (EVs), especially when undergoing stressful conditions such as inflammation, hypoxia, chemotherapy drug exposure, and irradiation. Stem cells that are potentiated by exogenous mitochondria show enhanced potential for proliferation, differentiation, and immunomodulation. The well-tolerated nature of either autogenous or allogenous mitochondria when locally injected in the human ischemic heart has validated the safety and therapeutic potential of mitochondrial transplantation. In children diagnosed with mitochondrial DNA deletion syndrome, functional improvements have been observed when empowering their hematopoietic stem cells with maternally derived mitochondria. Apart from the widely investigated applications of mitochondrial transfer in ischemia-reperfusion injury, neurodegenerative diseases and mitochondrial diseases etc., therapeutic potentials of mitochondrial transfer in tissue repair and regeneration are equally noteworthy, though there has been no systematic summary in this regard.This review analyzed the research and development trends of mitochondrial transfer in stem cells and regenerative medicine over the past decade from a bibliometric perspective, introduced the concept and associated mechanisms of mitochondrial transfer, summarized the regulations of intercellular mitochondrial transfer on stem cell fate. Finally, the therapeutic application of mitochondrial transplantation in diseases and tissue regeneration has been reviewed, including recent clinical studies related to mitochondrial transplantation.Mitochondrial transfer shows promise in modifying and reshaping the cellular properties of stem cells, making them more conducive to regeneration. Mesenchymal stem cells (MSCs)-derived mitochondria have shown multifaceted potential in promoting the revitalization and regeneration of cardiac, cutaneous, muscular, neuronal tissue. This review integrates novel research findings on mitochondrial transfer in stem cell biology and regenerative medicine, emphasizing the crucial translational value of mitochondrial transfer in regeneration. It serves to underscore the significant impact of mitochondrial transfer and provides a valuable reference for further exploration in this field.

Mitochondrial metabolism in neurons is necessary for energetically costly processes like synaptic transmission and plasticity. As post-mitotic cells, neurons are therefore faced with the challenge of maintaining healthy functioning mitochondria throughout lifetime. The precise mechanisms of mitochondrial maintenance in neurons, and particularly in morphologically complex dendrites and axons, are not fully understood. Evidence from several biological systems suggests the regulation of cellular metabolism by extracellular vesicles (EVs), secretory lipid-enclosed vesicles that have emerged as important mediators of cell communication. In the nervous system, neuronal and glial EVs were shown to regulate neuronal circuit development and function, at least in part via the transfer of protein and RNA cargo. Interestingly, EVs have been implicated in diseases characterized by altered metabolism, such as cancer and neurodegenerative diseases. Furthermore, nervous system EVs were shown to contain proteins related to metabolic processes, mitochondrial proteins and even intact mitochondria. Here, we present the current knowledge of the mechanisms underlying neuronal mitochondrial maintenance, and highlight recent evidence suggesting the regulation of synaptic mitochondria by neuronal and glial cell EVs. We further discuss the potential implications of EV-mediated regulation of mitochondrial maintenance and function in neuronal circuit development and synaptic plasticity.

Demyelination is a common feature of neuroinflammatory and degenerative diseases of the central nervous system (CNS), such as multiple sclerosis (MS). It is often linked to disruptions in intercellular communication, bioenergetics and metabolic balance accompanied by mitochondrial dysfunction in cells such as oligodendrocytes, neurons, astrocytes, and microglia. Although current MS treatments focus on immunomodulation, they fail to stop or reverse demyelination's progression. Recent advancements highlight intercellular mitochondrial exchange as a promising therapeutic target, with potential to restore metabolic homeostasis, enhance immunomodulation, and promote myelin repair. With this review we will provide insights into the CNS intercellular metabolic decoupling, focusing on the role of mitochondrial dysfunction in neuroinflammatory demyelinating conditions. We will then discuss emerging cell-free biotherapies exploring the therapeutic potential of transferring mitochondria via biogenic carriers like extracellular vesicles (EVs) or synthetic liposomes, aimed at enhancing mitochondrial function and metabolic support for CNS and myelin repair. Lastly, we address the key challenges for the clinical application of these strategies and discuss future directions to optimize mitochondrial biotherapies. The advancements in this field hold promise for restoring metabolic homeostasis, and enhancing myelin repair, potentially transforming the therapeutic landscape for neuroinflammatory and demyelinating diseases.

Extracellular vesicles (EVs) convey complex signals between cells that can be used to promote neuronal plasticity and neurological recovery in brain disease models. These EV signals are multimodal and context-dependent, making them unique therapeutic principles. This review analyzes how EVs released from various cell sources control neuronal metabolic function, neuronal survival and plasticity. Preferential sites of EV communication in the brain are interfaces between pre- and postsynaptic neurons at synapses, between astrocytes and neurons at plasma membranes or tripartite synapses, between oligodendrocytes and neurons at axons, between microglial cells/macrophages and neurons, and between cerebral microvascular cells and neurons. At each of these interfaces, EVs support mitochondrial function and cell metabolism under physiological conditions and orchestrate neuronal survival and plasticity in response to brain injury. In the injured brain, the promotion of neuronal survival and plasticity by EVs is tightly linked with EV actions on mitochondrial function, cell metabolism, oxidative stress and immune responses. Via the stabilization of cell metabolism and immune balance, neuronal plasticity responses are activated and functional neurological recovery is induced. As such, EV lay the ground for neuronal plasticity.

Astrocytes, the predominant glial cell type in the mammalian brain, influence a wide variety of brain parameters including neuronal energy metabolism. Exciting recent studies have shown that obesity and diabetes can impact on astrocyte function. We review evidence that dysregulation of astrocytic lipid metabolism and glucose sensing contributes to dysregulation of whole-body energy balance, thermoregulation, and insulin sensitivity. In addition, we consider the overlooked topic of the sex-specific roles of astrocytes and their response to hormonal fluctuations that provide insights into sex differences in metabolic regulation. Finally, we provide an update on potential ways to manipulate astrocyte function, including genetic targeting, optogenetic and chemogenetic techniques, transplantation, and tailored exosome-based therapies, which may lead to improved treatments for metabolic disease.

This study aimed to investigate the mechanisms by which large fibrous connective (LFC) tissue enhances fat graft survival in fat transplantation. A block fat graft model demonstrated that intact fat containing LFC showed significantly higher survival rates compared with liposuctioned fat. In the center of intact grafts, viable fat cells surrounded the LFC, forming a mesh-like living tissue structure. Proteomics of the extracellular matrix (ECM) adjacent to LFC (ALFC) and distant to LFC (DLFC) revealed significant differences in mitochondrial aspects. Staining of LFC tissue showed that it contains a large number of blood vessels and mitochondria, and exhibits stronger antioxidant capacity (p < 0.05) compared with adipose tissue. By mixing LFC with liposuctioned fat and transplanting into nude mice, histological sections showed that LFC promotes SOD1 expression, enhances respiratory chain RNA expression, and reduces ROS and inflammation. Pure mitochondrial-assisted fat transplantation only reduced short-term graft inflammation without improving long-term survival rates. In conclusion, LFC enhances long-term survival rates by reducing oxidative stress in fat grafts and forming a center for fat cell survival, thereby overcoming distance limitations. This represents a novel mechanism distinct from classical fat survival models and provides a reference for clinical practice.

Cardiovascular diseases (CVDs) are the leading cause of mortality among individuals with noncommunicable diseases worldwide. Obesity is associated with an increased risk of developing cardiovascular disease (CVD). Mitochondria are integral to the cardiovascular system, and it has been reported that mitochondrial transfer is associated with the pathogenesis of multiple CVDs and obesity. This review offers a comprehensive examination of the relevance of mitochondrial transfer to cardiovascular health and disease, emphasizing the critical functions of mitochondria in energy metabolism and signal transduction within the cardiovascular system. This highlights how disruptions in mitochondrial transfer contribute to various CVDs, such as myocardial infarction, cardiomyopathies, and hypertension. Additionally, we provide an overview of the molecular mechanisms governing mitochondrial transfer and its potential implications for CVD treatment. This finding underscores the therapeutic potential of mitochondrial transfer and addresses the various mechanisms and challenges in its implementation. By delving into mitochondrial transfer and its targeted modulation, this review aims to advance our understanding of cardiovascular disease treatment, presenting new insights and potential therapeutic strategies in this evolving field.

Intercellular mitochondrial transfer (IMT) is an intriguing biological phenomenon where mitochondria are transferred between different cells and notably, cell types. IMT is physiological, occurring in normal conditions, but also is utilized to deliver healthy mitochondria to cells in distress. Transferred mitochondria can be integrated to improve cellular metabolism, and mitochondrial function. Research on the mitochondrial transfer axis between astrocytes and brain capillaries in vivo is limited by the cellular heterogeneity of the neurovascular unit. To this end, we developed an inducible mouse model that expresses mitochondrial Dendra2 only in astrocytes and then isolated brain capillaries to remove all intact astrocytes. This method allows the visualization of in vivo astrocyte- endothelial cell (EC) and astrocyte-pericyte IMT. We demonstrate evidence of astrocyte-EC and astrocyte-pericyte mitochondrial transfer within brain capillaries. We also show that healthy aging enhances mitochondrial transfer from astrocytes to brain capillaries, revealing a potential link between brain aging and cellular mitochondrial dynamics. Finally, we observe that astrocyte-derived extracellular vesicles transfer mitochondria to brain microvascular endothelial cells, showing the potential route of in vivo IMT. These results represent a breakthrough in our understanding of IMT in the brain and a new target in brain aging and neurovascular metabolism.

As most traditional drugs used to treat central nervous system (CNS) diseases have a single therapeutic target, many of them cannot treat complex diseases or diseases whose mechanism is unknown and cannot effectively reverse the root changes underlying CNS diseases. This raises the question of whether multiple functional components are involved in the complex pathological processes of CNS diseases. Organelles are the core functional units of cells, and the replacement of damaged organelles with healthy organelles allows the multitargeted and integrated modulation of cellular functions. The development of therapies that target independent functional units in the cell, specifically, organelle-based therapies, is rapidly progressing. This article comprehensively discusses the pathogenesis of mitochondrial homeostasis disorders, which involve mitochondria, one of the most important organelles in CNS diseases, and the machanisms of mitochondrion-based therapies, as well as current preclinical and clinical studies on the efficacy of therapies targeting mitochondrial to treat CNS diseases, to provide evidence for use of organelle-based treatment strategies in the future.

Mitochondria transfer is a recently described phenomenon in which donor cells deliver mitochondria to acceptor cells1-3. One possible consequence of mitochondria transfer is energetic support of neighbouring cells; for example, exogenous healthy mitochondria can rescue cell-intrinsic defects in mitochondrial metabolism in cultured ρ0 cells or Ndufs4-/- peritoneal macrophages4-7. Exposing haematopoietic stem cells to purified mitochondria before autologous haematopoietic stem cell transplantation allowed for treatment of anaemia in patients with large-scale mitochondrial DNA mutations8,9, and mitochondria transplantation was shown to minimize ischaemic damage to the heart10-12, brain13-15 and limbs16. However, the therapeutic potential of using mitochondria transfer-based therapies to treat inherited mitochondrial diseases is unclear. Here we demonstrate improved morbidity and mortality of the Ndufs4-/- mouse model of Leigh syndrome (LS) in multiple treatment paradigms associated with mitochondria transfer. Transplantation of bone marrow from wild-type mice, which is associated with release of haematopoietic cell-derived extracellular mitochondria into circulation and transfer of mitochondria to host cells in multiple organs, ameliorates LS in mice. Furthermore, administering isolated mitochondria from wild-type mice extends lifespan, improves neurological function and increases energy expenditure of Ndufs4-/- mice, whereas mitochondria from Ndufs4-/- mice did not improve neurological function. Finally, we demonstrate that cross-species administration of human mitochondria to Ndufs4-/- mice also improves LS. These data suggest that mitochondria transfer-related approaches can be harnessed to treat mitochondrial diseases, such as LS.

Angiogenesis is crucial for successful bone defect repair. Co-transplanting Bone Marrow Stromal Cells (BMSCs) and Endothelial Cells (ECs) has shown promise for vascular augmentation, but it face challenges in hostile tissue microenvironments, including poor cell survival and limited efficacy. In this study, the mitochondria of human BMSCs are isolated and transplanted to BMSCs from the same batch and passage number (BMSCsmito). The transplanted mitochondria significantly boosted the ability of BMSCsmito-ECs to promote angiogenesis, as assessed by in vitro tube formation and spheroid sprouting assays, as well as in vivo transplantation experiments in balb/c mouse and SD rat models. The Dll4-Notch1 signaling pathway is found to play a key role in BMSCsmito-induced endothelial tube formation. Co-transplanting BMSCsmito with ECs in a rat cranial bone defect significantly improves functional vascular network formation, and improve bone repair outcomes. These findings thus highlight that mitochondrial transplantation, by acting through the DLL4-Notch1 signaling pathway, represents a promising therapeutic strategy for enhancing angiogenesis and improving bone repair. Hence, mitochondrial transplantation to BMSCS as a therapeutic approach for promoting angiogenesis offers valuable insights and holds much promise for innovative regenerative medicine therapies.

Ischemic stroke-induced mitochondrial dysfunction in the blood-brain barrier-forming brain endothelial cells (BECs) results in long-term neurological dysfunction post-stroke. We previously reported data from a pilot study where intravenous administration of human BEC (hBEC)-derived mitochondria-containing extracellular vesicles (EVs) showed a potential efficacy signal in a mouse middle cerebral artery occlusion (MCAo) model of stroke. We hypothesized that EVs harvested from donor species homologous to the recipient species (e.g., mouse) may improve therapeutic efficacy, and therefore, use of mouse BEC (mBEC)-derived EVs may improve post-stroke outcomes in MCAo mice. We investigated potential differences in the mitochondria transfer of EVs derived from the same species as the recipient cell (mBEC-EVs and recipient mBECs or hBECs-EVs and recipient hBECs) vs. cross-species EVs and recipient cells (mBEC-EVs and recipient hBECs or vice versa). Our results showed that while both hBEC- and mBEC-EVs transferred EV mitochondria, mBEC-EVs outperformed hBEC-EVs in increasing ATP levels and improved recipient mBEC mitochondrial function via increasing oxygen consumption rates. mBEC-EVs significantly reduced brain infarct volume and neurological deficit scores compared to vehicle-injected MCAo mice. The superior therapeutic efficacy of mBEC-EVs in MCAo mice support the continued use of mBEC-EVs to optimize the therapeutic potential of mitochondria-containing EVs in preclinical mouse models.

Cell-based therapeutic strategies have been proposed as an alternative for brain and blood vessels repair after stroke, but their clinical application is hampered by potential adverse effects. We therefore tested the hypothesis that secretome of these cells might be used instead to still focus on cell-based therapeutic strategies. We therefore characterized the composition and the effect of the secretome of brain microvascular endothelial cells (BMECs) on primary in vitro human models of angiogenesis and vascular barrier. Two different secretome batches produced in high scale (scHSP) were analysed by mass spectrometry. Human primary CD34+-derived endothelial cells (CD34+-ECs) were used as well as in vitro models of EC monolayer (CMECs) and blood-brain barrier (BBB). Cells were also exposed to oxygen-glucose deprivation (OGD) conditions and treated with scHSP during reoxygenation. Protein yield and composition of scHSP batches showed good reproducibility. scHSP increased CD34+-EC proliferation, tubulogenesis, and migration. Proteomic analysis of scHSP revealed the presence of growth factors and proteins modulating cell metabolism and inflammatory pathways. scHSP improved the integrity of CMECs, and upregulated the expression of junctional proteins. Such effects were mediated through the activation of the interferon pathway and downregulation of Wnt signalling. Furthermore, OGD altered the permeability of both CMECs and BBB, while scHSP prevented the OGD-induced vascular leakage in both models. These effects were mediated through upregulation of junctional proteins and regulation of MAPK/VEGFR2. Finally, our results highlight the possibility of using secretome from BMECs as a therapeutic alternative to promote brain angiogenesis and to protect from ischemia-induced vascular leakage.

Ischemic stroke-induced mitochondrial dysfunction in the blood-brain barrier-forming brain endothelial cells ( BECs ) results in long-term neurological dysfunction post-stroke. We previously data from a pilot study where intravenous administration of human BEC ( hBEC )-derived mitochondria-containing extracellular vesicles ( EVs ) showed a potential efficacy signal in a mouse middle cerebral artery occlusion ( MCAo ) model of stroke. We hypothesized that EVs harvested from donor species homologous to the recipient species ( e.g., mouse) may improve therapeutic efficacy, and therefore, use of mouse BEC ( mBEC )-derived EVs may improve post-stroke outcomes in MCAo mice. We investigated potential differences in the mitochondria transfer of EVs derived from the same species as the recipient cell (mBEC-EVs and recipient mBECs or hBECs-EVs and recipient hBECs) vs . cross-species EVs and recipient cells (mBEC-EVs and recipient hBECs or vice versa ). Our results showed that while both hBEC- and mBEC-EVs transferred EV mitochondria, mBEC-EVs outperformed hBEC-EVs in increasing ATP levels and improved recipient mBEC mitochondrial function via increasing oxygen consumption rates. mBEC-EVs significantly reduced brain infarct volume and neurological deficit scores compared to vehicle-injected MCAo mice. The superior therapeutic efficacy of mBEC-EVs in a mouse MCAo stroke support the continued use of mBEC-EVs to optimize the therapeutic potential of mitochondria-containing EVs in preclinical mouse models.

Circulating cell-free mitochondrial DNA (ccf-mtDNA) is an indicator of cell death, inflammation, and oxidative stress. ccf-mtDNA in pregnancies with placental dysfunction differs from that in healthy pregnancies, and the direction of this difference depends on gestational age and method of mtDNA quantification. Reactive oxygen species (ROS) trigger release of mtDNA, yet it is unknown whether trophoblast cells release mtDNA in response to oxidative stress, a common feature of pregnancies with placental pathology. We hypothesized that oxidative stress would induce cell death and release of mtDNA from trophoblast cells. BeWo cells were treated with antimycin A (10-320 µM) or rotenone (0.2-50 µM) to induce oxidative stress. A multiplex real-time quantitative PCR (qPCR) assay was used to quantify mtDNA and nuclear DNA in membrane-bound, non-membrane-bound, and vesicle-bound forms in cell culture supernatants and cell lysates. Treatment with antimycin A increased ROS (P < 0.0001), induced cell necrosis (P = 0.0004) but not apoptosis (P = 0.6471), and was positively associated with release of membrane-bound and non-membrane-bound mtDNA (P < 0.0001). Antimycin A increased mtDNA content in exosome-like extracellular vesicles (vesicle-bound form; P = 0.0019) and reduced autophagy marker expression (LC3A/B, P = 0.0002; p62, P < 0.001). Rotenone treatment did not influence mtDNA release or cell death (P > 0.05). Oxidative stress induces release of mtDNA into the extracellular space and causes nonapoptotic cell death and a reduction in autophagy markers in BeWo cells, an established in vitro model of human trophoblast cells. Intersection between autophagy and necrosis may mediate the release of mtDNA from the placenta in pregnancies exposed to oxidative stress.NEW & NOTEWORTHY This is the first study to test whether trophoblast cells release mitochondrial (mt)DNA in response to oxidative stress and to identify mechanisms of release and biological forms of mtDNA from this cellular type. This research identifies potential cellular mechanisms that can be used in future investigations to establish the source and biomarker potential of circulating mtDNA in preclinical experimental models and humans.

Intercellular mitochondrial transfer (MT) is a newly discovered form of cell-to-cell signalling involving the active incorporation of healthy mitochondria into stressed/injured recipient cells, contributing to the restoration of bioenergetic profile and cell viability, reduction of inflammatory processes and normalisation of calcium dynamics. Recent evidence has shown that MT can occur through multiple cellular structures and mechanisms: tunneling nanotubes (TNTs), via gap junctions (GJs), mediated by extracellular vesicles (EVs) and other mechanisms (cell fusion, mitochondrial extrusion and migrasome-mediated mitocytosis) and in different contexts, such as under physiological (tissue homeostasis and stemness maintenance) and pathological conditions (hypoxia, inflammation and cancer). As Mesenchimal Stromal/ Stem Cells (MSC)-mediated MT has emerged as a critical regulatory and restorative mechanism for cell and tissue regeneration and damage repair in recent years, its potential in stem cell therapy has received increasing attention. In particular, the potential therapeutic role of MSCs has been reported in several articles, suggesting that MSCs can enhance tissue repair after injury via MT and membrane vesicle release. For these reasons, in this review, we will discuss the different mechanisms of MSCs-mediated MT and therapeutic effects on different diseases such as neuronal, ischaemic, vascular and pulmonary diseases. Therefore, understanding the molecular and cellular mechanisms of MT and demonstrating its efficacy could be an important milestone that lays the foundation for future clinical trials.

Mitochondrial transfer occurs between cells, and it is important for damaged cells to receive healthy mitochondria to maintain their normal function and protect against cell death. Accumulating evidence suggests that the functional mitochondria of astrocytes are released and transferred to oxygen-glucose deprivation/reoxygenation (OGD/R)-injured neurons. Mild hypothermia (33 °C) is capable of promoting this process, which partially restores the function of damaged neurons. However, the pathways and mechanisms by which mild hypothermia facilitates mitochondrial transfer remain unclear. We are committed to studying the role of mild hypothermia in neuroprotection to provide reliable evidences and insights for the clinical application of mild hypothermia in brain protection. Tunneling nanotubes (TNTs) are considered to be one of the routes through which mitochondria are transferred between cells. In this study, an OGD/R-injured neuronal model was successfully established, and TNTs, mitochondria, neurons and astrocytes were double labeled using immunofluorescent probes. Our results showed that TNTs were present and involved in the transfer of mitochondria between cells in the mixed-culture system of neurons and astrocytes. When neurons were subjected to OGD/R exposure, TNT formation and mitochondrial transportation from astrocytes to injured neurons were facilitated. Further analysis revealed that mild hypothermia increased the quantity of astrocytic mitochondria transferred into damaged neurons through TNTs, raised the mitochondrial membrane potential (MMP), and decreased the neuronal damage and death during OGD/R. Altogether, our data indicate that TNTs play an important role in the endogenous neuroprotection of astrocytic mitochondrial transfer. Furthermore, mild hypothermia enhances astrocytic mitochondrial transfer into OGD/R-injured neurons via TNTs, thereby promoting neuroprotection and neuronal recovery.

The dramatic increase in intracranial pressure after subarachnoid hemorrhage leads to a decrease in cerebral perfusion pressure and a reduction in cerebral blood flow. Mitochondria are directly affected by direct factors such as ischemia, hypoxia, excitotoxicity, and toxicity of free hemoglobin and its degradation products, which trigger mitochondrial dysfunction. Dysfunctional mitochondria release large amounts of reactive oxygen species, inflammatory mediators, and apoptotic proteins that activate apoptotic pathways, further damaging cells. In response to this array of damage, cells have adopted multiple mitochondrial quality control mechanisms through evolution, including mitochondrial protein quality control, mitochondrial dynamics, mitophagy, mitochondrial biogenesis, and intercellular mitochondrial transfer, to maintain mitochondrial homeostasis under pathological conditions. Specific interventions targeting mitochondrial quality control mechanisms have emerged as promising therapeutic strategies for subarachnoid hemorrhage. This review provides an overview of recent research advances in mitochondrial pathophysiological processes after subarachnoid hemorrhage, particularly mitochondrial quality control mechanisms. It also presents potential therapeutic strategies to target mitochondrial quality control in subarachnoid hemorrhage.

Tumour necrosis factor alpha-induced protein 2 (TNFAIP2) is a gene induced by tumor necrosis factor in endothelial cells. TNFAIP2 has important functions in physiological and pathological processes, including cell proliferation, adhesion, migration, angiogenesis, inflammation, tunneling nanotube (TNT) formation and tumorigenesis. Moreover, TNFAIP2 is the key factor in the formation of TNTs. TNTs are related to signal transduction between different cell types and are considered a novel means of cell-to-cell communication. Mesenchymal stem cells (MSCs) are pluripotent cells that exhibit self-renewal, multidirectional differentiation, paracrine function and immune-regulating ability. MSCs can transfer mitochondria through TNTs to improve the functions of target cells. This review revealed that TNFAIP2 promotes the formation of TNTs and that MSCs rely on TNTs for mitochondrial transfer to ameliorate cell dysfunction.

Circulating cell-free mitochondrial DNA (ccf-mtDNA) is an indicator of cell death, inflammation, and oxidative stress. ccf-mtDNA differs in pregnancies with placental dysfunction from healthy pregnancies and the direction of this difference depends on gestational age and method of mtDNA quantification. Reactive oxygen species (ROS) trigger release of mtDNA from non-placental cells; yet it is unknown whether trophoblast cells release mtDNA in response to oxidative stress, a common feature of pregnancies with placental pathology. We hypothesized that oxidative stress would induce cell death and release of mtDNA from trophoblast cells. BeWo cells were treated with antimycin A (10-320 μM) or rotenone (0.2-50 μM) to induce oxidative stress. A multiplex real-time quantitative PCR (qPCR) assay was used to quantify mtDNA and nuclear DNA in membrane bound, non-membrane bound, and vesicular-bound forms in cell culture supernatants and cell lysates. Treatment with antimycin A increased ROS (p<0.0001), induced cell necrosis (p=0.0004) but not apoptosis (p=0.6471) and was positively associated with release of membrane-bound and non-membrane bound mtDNA (p<0.0001). Antimycin A increased mtDNA content in exosome-like extracellular vesicles (vesicular-bound form; p=0.0019) and reduced autophagy marker expression (LC3A/B, p=0.0002; p62, p<0.001). Rotenone treatment did not influence mtDNA release or cell death (p>0.05). Oxidative stress induces release of mtDNA into the extracellular space and causes non-apoptotic cell death and a reduction in autophagy markers in BeWo cells, an established in vitro model of human trophoblast cells. Intersection between autophagy and necrosis may mediate the release of mtDNA from the placenta in pregnancies exposed to oxidative stress.

This is the first study to test whether trophoblast cells release mitochondrial DNA in response to oxidative stress and to identify mechanisms of release and biological forms of mtDNA from this cellular type. This research identifies potential cellular mechanisms that can be used in future investigations to establish the source and biomarker potential of circulating mitochondrial DNA in preclinical experimental models and humans.

Angiogenesis is a complex process that involves the expansion of the pre-existing vascular plexus to enhance oxygen and nutrient delivery and is stimulated by various factors, including hypoxia. Since the process of angiogenesis requires a lot of energy, mitochondria play an important role in regulating and promoting this phenomenon. Besides their roles as an oxidative metabolism base, mitochondria are potential bioenergetics organelles to maintain cellular homeostasis via sensing alteration in oxygen levels. Under hypoxic conditions, mitochondria can regulate angiogenesis through different factors. It has been indicated that unidirectional and bidirectional exchange of mitochondria or their related byproducts between the cells is orchestrated via different intercellular mechanisms such as tunneling nanotubes, extracellular vesicles, and gap junctions to maintain the cell homeostasis. Even though, the transfer of mitochondria is one possible mechanism by which cells can promote and regulate the process of angiogenesis under reperfusion/ischemia injury. Despite the existence of a close relationship between mitochondrial donation and angiogenic response in different cell types, the precise molecular mechanisms associated with this phenomenon remain unclear. Here, we aimed to highlight the possible role of mitochondria concerning angiogenesis, especially the role of mitochondrial transport and the possible relation of this transfer with autophagy, the housekeeping phenomenon of cells, and angiogenesis.

Extracellular vesicles (EVs) are extremely versatile naturally occurring membrane particles that convey complex signals between cells. EVs of different cellular sources are capable of inducing striking therapeutic responses in neurological disease models. Differently from pharmacological compounds that act by modulating defined signalling pathways, EV-based therapeutics possess multiple abilities via a variety of effectors, thus allowing the modulation of complex disease processes that may have very potent effects on brain tissue recovery. When applied in vivo in experimental models of neurological diseases, EV-based therapeutics have revealed remarkable effects on immune responses, cell metabolism and neuronal plasticity. This multimodal modulation of neuroimmune networks by EVs profoundly influences disease processes in a highly synergistic and context-dependent way. Ultimately, the EV-mediated restoration of cellular functions helps to set the stage for neurological recovery. With this review we first outline the current understanding of the mechanisms of action of EVs, describing how EVs released from various cellular sources identify their cellular targets and convey signals to recipient cells. Then, mechanisms of action applicable to key neurological conditions such as stroke, multiple sclerosis and neurodegenerative diseases are presented. Pathways that deserve attention in specific disease contexts are discussed. We subsequently showcase considerations about EV biodistribution and delineate genetic engineering strategies aiming at enhancing brain uptake and signalling. By sketching a broad view of EV-orchestrated brain plasticity and recovery, we finally define possible future clinical EV applications and propose necessary information to be provided ahead of clinical trials. Our goal is to provide a steppingstone that can be used to critically discuss EVs as next generation therapeutics for brain diseases.

Mitochondria have been primarily considered intracellular organelles that are responsible for generating energy for cell survival. However, accumulating evidence suggests that mitochondria are secreted into the extracellular space under physiological and pathological conditions, and these secreted mitochondria play diverse roles by regulating metabolism, the immune response, or the differentiation/maturation in target cells. Furthermore, increasing amount of research shows the therapeutic effects of local or systemic administration of mitochondria in various disease models. These findings have led to growing interest in exploring mitochondria as potential therapeutic agents. Here, we discuss the emerging roles of mitochondria as extracellularly secreted organelles to shed light on their functions beyond energy production. Additionally, we provide information on therapeutic outcomes of mitochondrial transplantation in animal models of diseases and an update on ongoing clinical trials, underscoring the potential of using mitochondria as a novel therapeutic intervention.

Established by brain endothelial cells, the blood-brain barrier (BBB) regulates the trafficking of molecules, restricts immune cell entry into the CNS, and has an active role in neurovascular coupling (the regulation of cerebral blood flow to support neuronal activity). In the early stages of multiple sclerosis, around the time of symptom onset, inflammatory BBB damage is accompanied by pathogenic immune cell infiltration into the CNS. In the later stages of multiple sclerosis, dysregulation of neurovascular coupling is associated with grey matter atrophy. Genetic and environmental factors associated with multiple sclerosis, including dietary habits, the gut microbiome, and vitamin D concentrations, might contribute directly and indirectly to brain endothelial cell dysfunction. Damage to brain endothelial cells leads to an influx of deleterious molecules into the CNS, accelerating leakage across the BBB. Potential future therapeutic approaches might help to prevent BBB damage (eg, monoclonal antibodies targeting cell adhesion molecules and fibrinogen) and help to repair BBB dysfunction (eg, mesenchymal stromal cells) in people with multiple sclerosis.

Mitochondrial transfer (MT) is a biological process that allows a donor cell to horizontally share its own mitochondria with a recipient cell. Mitochondria are highly dynamic membrane-bound sub-cellular organelles prominently involved in the regulation of the cell energy balance, calcium homeostasis, and apoptotic machinery activation. They physiologically undergo fusion and fission processes in response to the cell requirement, with a continuous morphological re-arrangement. This structural and functional plasticity is at the basis of the MT, described in tissue regeneration, cardiac and neurological diseases, as well as in cancer. Here, the MT has been observed in the tumor micro-environment (TME) from the adipose-derived stem cells (ASCs) to the cancer cells, eventually reverting the lack of the mitochondria respiration function, or enhancing their motility and drug resistance. In this chapter, we outline some key protocols for evaluating this exciting phenomenon of MT. These methodological and technical approaches are very important, considering all the limitations that scientists constantly face, especially in this field of the research.

Mitochondria serve as a vital energy source for nerve cells. The mitochondrial network also acts as a defense mechanism against external stressors that can threaten the stability of the nervous system. However, excessive accumulation of damaged mitochondria can lead to neuronal death. Mitophagy is an essential pathway in the mitochondrial quality control system and can protect neurons by selectively removing damaged mitochondria. In most neurological disorders, dysfunctional mitochondria are a common feature, and drugs that target mitophagy can improve symptoms. Here, we reviewed the role of mitophagy in Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, stroke, and traumatic brain injuries. We also summarized drug and non-drug approaches to promote mitophagy and described their therapeutic role in neurological disorders in order to provide valuable insight into the potential therapeutic agents available for neurological disease treatment. However, most studies on mitophagy regulation are based on preclinical research using cell and animal models, which may not accurately reflect the effects in humans. This poses a challenge to the clinical application of drugs targeting mitophagy. Additionally, these drugs may carry the risk of intolerable side effects and toxicity. Future research should focus on the development of safer and more targeted drugs for mitophagy.

Mitochondria are believed to have originated through an ancient endosymbiotic process in which proteobacteria were captured and co-opted for energy production and cellular metabolism. Mitochondria segregate during cell division and differentiation, with vertical inheritance of mitochondria and the mitochondrial DNA genome from parent to daughter cells. However, an emerging body of literature indicates that some cell types export their mitochondria for delivery to developmentally unrelated cell types, a process called intercellular mitochondria transfer. In this Review, we describe the mechanisms by which mitochondria are transferred between cells and discuss how intercellular mitochondria transfer regulates the physiology and function of various organ systems in health and disease. In particular, we discuss the role of mitochondria transfer in regulating cellular metabolism, cancer, the immune system, maintenance of tissue homeostasis, mitochondrial quality control, wound healing and adipose tissue function. We also highlight the potential of targeting intercellular mitochondria transfer as a therapeutic strategy to treat human diseases and augment cellular therapies.

Epithelial transport is a multifaceted process crucial for maintaining normal physiological functions in the human body. This comprehensive review delves into the pathophysiological mechanisms underlying epithelial transport and its significance in disease pathogenesis. Beginning with an introduction to epithelial transport, it covers various forms, including ion, water, and nutrient transfer, followed by an exploration of the processes governing ion transport and hormonal regulation. The review then addresses genetic disorders, like cystic fibrosis and Bartter syndrome, that affect epithelial transport. Furthermore, it investigates the involvement of epithelial transport in the pathophysiology of conditions such as diarrhea, hypertension, and edema. Finally, the review analyzes the impact of renal disease on epithelial transport and highlights the potential for future research to uncover novel therapeutic interventions for conditions like cystic fibrosis, hypertension, and renal failure.

Mitochondria control cellular functions through their metabolic role. Recent research that has gained considerable attention is their ability to transfer between cells. This has the potential of improving cellular functions in pathological or energy-deficit conditions, but little is known about the role of mitochondrial transfer in sustaining cellular homeostasis. Few studies have investigated the potential of skeletal muscle as a source of healthy mitochondria that can be transferred to other cell types. Thus, we isolated intermyofibrillar mitochondria from murine skeletal muscle and incubated them with host cells. We observed dose- and time-dependent increases in mitochondrial incorporation into myoblasts. This resulted in elongated mitochondrial networks and an enhancement of bioenergetic profile of the host cells. Mitochondrial donation also rejuvenated the functional capacities of the myoblasts when respiration efficiency and lysosomal function were inhibited by complex I inhibitor rotenone and bafilomycin A, respectively. Mitochondrial transfer was accomplished via tunneling nanotubes, extracellular vesicles, gap junctions, and by macropinocytosis internalization. Murine muscle mitochondria were also effectively transferred to human fibroblast cells having mitochondrial DNA mutations, resulting in augmented mitochondrial dynamics and metabolic functions. This improved cell function by diminishing reactive oxygen species (ROS) emission in the diseased cells. Our findings suggest that mitochondria from donor skeletal muscle can be integrated in both healthy and functionally compromised host cells leading to mitochondrial structural refinement and respiratory boost. This mitochondrial trafficking and bioenergetic reprogramming to maintain and revitalize tissue homeostasis could be a useful therapeutic strategy in treating diseases.NEW & NOTEWORTHY In our study, we have shown the potential of mouse skeletal muscle intermyofibrillar mitochondria to be transplanted in myoblasts and human fibroblast cells having mitochondrial DNA mutations. This resulted in an augmentation of mitochondrial dynamics and enhancement of bioenergetic profile in the host cells. Our findings suggest that mitochondria from donor skeletal muscle can be integrated into both healthy and functionally compromised host cells leading to mitochondrial structural refinement and respiratory boost.

Bone marrow-derived mononuclear cells (BMMNCs) have been used for decades in preclinical and clinical studies to treat various neurological diseases. However, there is still a knowledge gap in the understanding of the underlying mechanisms of BMMNCs in the treatment of neurological diseases. In addition, prerequisite factors for the efficacy of BMMNC administration, such as the optimal route, dose, and number of administrations, remain unclear. In this review, we discuss known and unknown aspects of BMMNCs, including the cell harvesting, administration route and dose; mechanisms of action; and their applications in neurological diseases, including stroke, cerebral palsy, spinal cord injury, traumatic brain injury, amyotrophic lateral sclerosis, autism spectrum disorder, and epilepsy. Furthermore, recommendations on indications for BMMNC administration and the advantages and limitations of BMMNC applications for neurological diseases are discussed. BMMNCs in the treatment of neurological diseases. BMMNCs have been applied in several neurological diseases. Proposed mechanisms for the action of BMMNCs include homing, differentiation and paracrine effects (angiogenesis, neuroprotection, and anti-inflammation). Further studies should be performed to determine the optimal cell dose and administration route, the roles of BMMNC subtypes, and the indications for the use of BMMNCs in neurological conditions with and without genetic abnormalities.

Mitochondrial dysfunction is reiteratively involved in the pathogenesis of diverse neurodegenerative diseases. Current in vitro and in vivo approaches support that mitochondrial dysfunction is branded by several molecular and cellular defects, whose impact at different levels including the calcium and iron homeostasis, energetic balance and/or oxidative stress, makes it difficult to resolve them collectively given their multifactorial nature. Mitochondrial transfer offers an overall solution since it contains the replacement of damage mitochondria by healthy units. Therefore, this review provides an introducing view on the structure and energy-related functions of mitochondria as well as their dynamics. In turn, we summarize current knowledge on how these features are deregulated in different neurodegenerative diseases, including frontotemporal dementia, multiple sclerosis, amyotrophic lateral sclerosis, Friedreich ataxia, Alzheimer´s disease, Parkinson´s disease, and Huntington's disease. Finally, we analyzed current advances in mitochondrial transfer between diverse cell types that actively participate in neurodegenerative processes, and how they might be projected toward developing novel therapeutic strategies.

As the powerhouse and core of cellular metabolism and survival, mitochondria are the essential organelle in mammalian cells and maintain cellular homeostasis by changing their content and morphology to meet demands through mitochondrial quality control. It has been observed that mitochondria can move between cells under physiological and pathophysiological conditions, which provides a novel strategy for preserving mitochondrial homeostasis and also a therapeutic target for applications in clinical settings. Therefore, in this review, we will summarize currently known mechanisms of intercellular mitochondrial transfer, including modes, triggers, and functions. Due to the highly demanded energy and indispensable intercellular linkages of the central nervous system (CNS), we highlight the mitochondrial transfer in CNS. We also discuss future application possibilities and difficulties that need to be addressed in the treatment of CNS injury and diseases. This clarification should shed light on its potential clinical applications as a promising therapeutic target in neurological diseases. Intercellular mitochondrial transfer maintains the homeostasis of central nervous system (CNS), and its alteration is related to several neurological diseases. Supplementing exogenous mitochondrial donor cells and mitochondria, or utilizing some medications to regulate the process of transfer might mitigate the disease and injury.

Mitochondrial dysfunction is a central event in the pathogenesis of several degenerative brain disorders. It entails fission and fusion dynamics disruption, progressive decline in mitochondrial clearance, and uncontrolled oxidative stress. Many therapeutic strategies have been formulated to reverse these alterations, including replacing damaged mitochondria with healthy ones. Spontaneous mitochondrial transfer is a naturally occurring process with different biological functions. It comprises mitochondrial donation from one cell to another, carried out through different pathways, such as the formation and stabilization of tunneling nanotubules and Gap junctions and the release of extracellular vesicles with mitochondrial cargoes. Even though many aspects of regulating these mechanisms still need to be discovered, some key enzymatic regulators have been identified. This review summarizes the current knowledge on mitochondrial dysfunction in different neurodegenerative disorders. Besides, we analyzed the usage of mitochondrial transfer as an endogenous revitalization tool, emphasizing the enzyme regulators that govern this mechanism. Going deeper into this matter would be helpful to take advantage of the therapeutic potential of mitochondrial transfer.

Endothelial cells (ECs) angiogenesis is the process of sprouting new vessels from the existing ones, playing critical roles in physiological and pathological processes such as wound healing, placentation, ischemia/reperfusion, cardiovascular diseases and cancer metastasis. Although mitochondria are not the major sites of energy source in ECs, they function as important biosynthetic and signaling hubs to regulate ECs metabolism and adaptations to local environment, thus affecting ECs migration, proliferation and angiogenic process. The understanding of the importance and potential mechanisms of mitochondria in regulating ECs metabolism, function and the process of angiogenesis has developed in the past decades. Thus, in this review, we discuss the current understanding of mitochondrial proteins and signaling molecules in ECs metabolism, function and angiogeneic signaling, to provide new and therapeutic targets for treatment of diverse cardiovascular and angiogenesis-dependent diseases.

Background: The transfer of mitochondria from healthy mesenchymal stem cells (MSCs) to injured MSCs has been shown to have potential therapeutic benefits for neural cell post-ischemic stroke. Specifically, functional mitochondria can perform their normal functions after being internalized by stressed cells, leading to host cell survival. However, while this approach shows promise, there is still a lack of understanding regarding which neural cells can internalize functional mitochondria and the regulatory mechanisms involved. To address this gap, we investigated the ability of different neural cells to internalize exogenous functional mitochondria extracted from MSCs. Methods: Functional mitochondria (F-Mito) isolated from umbilical cord derived-MSCs (UCMSCs) were labeled with lentivirus of HBLV-mito-dsred-Null-PURO vector. The ability of stressed cells to internalize F-Mito was analyzed using a mouse (C57BL/6 J) middle cerebral artery occlusion (MCAO) model and an oxygen-glucose deprivation/reoxygenation (OGD/R) cell model. The cell viability was measured by CCK-8 kit. Time-course of intracellular ROS levels in stressed cells were analyzed by DCFH-DA staining after OGD/R and F-Mito treatment. MitoSOX, Mitotracker and WGA labeling were used to assess the relationship between ROS levels and the uptake of F-Mito at the single-cell level. Pharmacological modulation of ROS was performed using acetylcysteine (ROS inhibitor). Results: Our findings demonstrate that neurons and endothelial cells are more effective at internalizing mitochondria than astrocytes, both in vitro and in vivo, using an ischemia-reperfusion model. Additionally, internalized F-Mito decreases host cell reactive oxygen species (ROS) levels and rescues survival. Importantly, we found that the ROS response in stressed cells after ischemia is a crucial determinant in positively mediating the internalization of F-Mito by host cells, and inhibiting the generation of ROS chemicals in host cells may decrease the internalization of F-Mito. These results offer insight into how exogenous mitochondria rescue neural cells via ROS response in an ischemic stroke model. Overall, our study provides solid evidence for the translational application of MSC-derived mitochondria as a promising treatment for ischemic stroke.

In addition to critical roles in bioenergetics, mitochondria are key contributors to the regulation of many other functions in cells, ranging from steroidogenesis to apoptosis. Numerous studies further demonstrate that cell type-specific differences exist in mitochondria, with cells of a given lineage tailoring their endogenous mitochondrial population to suit specific functional needs. These findings, coupled with studies of the therapeutic potential of mitochondrial transplantation, provide a strong impetus to better understand how mitochondria can influence cell function or fate. Here an inducible mitochondrial depletion modelis used to study how cells lacking endogenous mitochondria respond, on a global protein expression level, to transplantation with lineage-mismatched (LM) mitochondria. It is shown that LM mitochondrial transplantation does not alter the proteomic profile in nonmitochondria-depleted recipient cells; however, enforced depletion of endogenous mitochondria results in dramatic changes in the proteomic landscape, which returns to the predepletion state following internalization of LM mitochondria. These data, derived from a cell system that can be rendered free of influence by endogenous mitochondria, indicate that transplantation of mitochondria-even from a source that differs significantly from the recipient cell population, effectively restores a normal proteomic landscape to cells lacking their own mitochondria.

Mitochondrial dysfunction is a hallmark of numerous diseases, including neurodegenerative disorders, metabolic disorders, and cancer. Mitochondrial transfer, the transfer of mitochondria from one cell to another, has recently emerged as a potential therapeutic approach for restoring mitochondrial function in diseased cells. In this review, we summarize the current understanding of mitochondrial transfer, including its mechanisms, potential therapeutic applications, and impact on cell death pathways. We also discuss the future directions and challenges in the field of mitochondrial transfer as a novel therapeutic approach in disease diagnosis and treatment.

Mitochondrial transplantation (MTx) is an emerging but poorly understood technology with the potential to mitigate severe ischemia-reperfusion injuries after cardiac arrest (CA). To address critical gaps in the current knowledge, we test the hypothesis that MTx can improve outcomes after CA resuscitation.

This study consists of both in vitro and in vivo studies. We initially examined the migration of exogenous mitochondria into primary neural cell culture in vitro. Exogenous mitochondria extracted from the brain and muscle tissues of donor rats and endogenous mitochondria in the neural cells were separately labeled before co-culture. After a period of 24 h following co-culture, mitochondrial transfer was observed using microscopy. In vitro adenosine triphosphate (ATP) contents were assessed between freshly isolated and frozen-thawed mitochondria to compare their effects on survival. Our main study was an in vivo rat model of CA in which rats were subjected to 10 min of asphyxial CA followed by resuscitation. At the time of achieving successful resuscitation, rats were randomly assigned into one of three groups of intravenous injections: vehicle, frozen-thawed, or fresh viable mitochondria. During 72 h post-CA, the therapeutic efficacy of MTx was assessed by comparison of survival rates. The persistence of labeled donor mitochondria within critical organs of recipient animals 24 h post-CA was visualized via microscopy.

The donated mitochondria were successfully taken up into cultured neural cells. Transferred exogenous mitochondria co-localized with endogenous mitochondria inside neural cells. ATP content in fresh mitochondria was approximately four times higher than in frozen-thawed mitochondria. In the in vivo survival study, freshly isolated functional mitochondria, but not frozen-thawed mitochondria, significantly increased 72-h survival from 55 to 91% (P = 0.048 vs. vehicle). The beneficial effects on survival were associated with improvements in rapid recovery of arterial lactate and glucose levels, cerebral microcirculation, lung edema, and neurological function. Labeled mitochondria were observed inside the vital organs of the surviving rats 24 h post-CA.

MTx performed immediately after resuscitation improved survival and neurological recovery in post-CA rats. These results provide a foundation for future studies to promote the development of MTx as a novel therapeutic strategy to save lives currently lost after CA.

Neural stem and progenitor cell (NSPC) transplants provide neuroprotection in models of acute brain injury, but the underlying mechanisms are not fully understood. Here, we provide evidence that caspase-dependent apoptotic cell death of NSPCs is required for sending survival signals to the injured brain. The secretome of dying NSPCs contains heat-stable proteins, which protect neurons against glutamate-induced toxicity and trophic factor withdrawal in vitro, and from ischemic brain damage in vivo. Our findings support a new concept suggesting a bystander effect of apoptotic NSPCs, which actively promote neuronal survival through the release of a protective "farewell" secretome. Similar protective effects by the secretome of apoptotic NSPC were also confirmed in human neural progenitor cells and neural stem cells but not in mouse embryonic fibroblasts (MEF) or human dopaminergic neurons, suggesting that the observed effects are cell type specific and exist for neural progenitor/stem cells across species.

Mammalian genes were long thought to be constrained within somatic cells in most cell types. This concept was challenged recently when cellular organelles including mitochondria were shown to move between mammalian cells in culture via cytoplasmic bridges. Recent research in animals indicates transfer of mitochondria in cancer and during lung injury in vivo, with considerable functional consequences. Since these pioneering discoveries, many studies have confirmed horizontal mitochondrial transfer (HMT) in vivo, and its functional characteristics and consequences have been described. Additional support for this phenomenon has come from phylogenetic studies. Apparently, mitochondrial trafficking between cells occurs more frequently than previously thought and contributes to diverse processes including bioenergetic crosstalk and homeostasis, disease treatment and recovery, and development of resistance to cancer therapy. Here we highlight current knowledge of HMT between cells, focusing primarily on in vivo systems, and contend that this process is not only (patho)physiologically relevant, but also can be exploited for the design of novel therapeutic approaches.

Ischemic stroke causes brain endothelial cell (BEC) death and damages tight junction integrity of the blood-brain barrier (BBB). We harnessed the innate mitochondrial load of BEC-derived extracellular vesicles (EVs) and utilized mixtures of EV/exogenous 27 kDa heat shock protein (HSP27) as a one-two punch strategy to increase BEC survival (via EV mitochondria) and preserve their tight junction integrity (via HSP27 effects). We demonstrated that the medium-to-large (m/lEV) but not small EVs (sEV) transferred their mitochondrial load, that subsequently colocalized with the mitochondrial network of the recipient primary human BECs. Recipient BECs treated with m/lEVs showed increased relative ATP levels and mitochondrial function. To determine if the m/lEV-meditated increase in recipient BEC ATP levels was associated with m/lEV mitochondria, we isolated m/lEVs from donor BECs pre-treated with oligomycin A (OGM, mitochondria electron transport complex V inhibitor), referred to as OGM-m/lEVs. BECs treated with naïve m/lEVs showed a significant increase in ATP levels compared to untreated OGD cells, OGM-m/lEVs treated BECs showed a loss of ATP levels suggesting that the m/lEV-mediated increase in ATP levels is likely a function of their innate mitochondrial load. In contrast, sEV-mediated ATP increases were not affected by inhibition of mitochondrial function in the donor BECs. Intravenously administered m/lEVs showed a reduction in brain infarct sizes compared to vehicle-injected mice in a mouse middle cerebral artery occlusion model of ischemic stroke. We formulated binary mixtures of human recombinant HSP27 protein with EVs: EV/HSP27 and ternary mixtures of HSP27 and EVs with a cationic polymer, poly (ethylene glycol)-b-poly (diethyltriamine): (PEG-DET/HSP27)/EV. (PEG-DET/HSP27)/EV and EV/HSP27 mixtures decreased the paracellular permeability of small and large molecular mass fluorescent tracers in oxygen glucose-deprived primary human BECs. This one-two punch approach to increase BEC metabolic function and tight junction integrity may be a promising strategy for BBB protection and prevention of long-term neurological dysfunction post-ischemic stroke.