Evaluating the Mechanisms of Action of Mesenchymal Stem Cell-Derived Extracellular Vesicles in Ischemic Stroke

Abstract

The utilization of cell-free therapies derived from extracellular vesicles (EVs) has garnered mounting interest as a promising approach to address the myriad challenges associated with ischemic stroke. These Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) possess considerable therapeutic potential due to their inherent properties, including low immunogenicity, efficient cargo transportation, and the ability to cross the blood-brain barrier. This review examines the mechanisms underlying mesenchymal stem cell-derived EVs in the treatment of ischemic stroke. Future research should aim to identify optimal strategies for EV-based interventions, including combination therapy and preconditioning strategies.

Graphical abstract

Keywords

ischemic stroke / stem cells / extracellular vesicles / exosomes / mesenchymal stem cells

1. Introduction

Globally recognized as the second leading cause of cardiovascular mortality after ischemic heart disease [1], stroke imposes a dual burden of devastating neurological sequelae and substantial socioeconomic strain on healthcare system [2]. Ischemic stroke (IS) is the predominant subtype accounting for approximately 70% of all stroke cases. Consequently, it remains the primary therapeutic target in most stroke trials [3]. The pathophysiology of IS originates from cerebrovascular occlusion, which instigates a series of metabolic perturbations. These perturbations are characterized by hypoxic stress, nutrient deprivation (glucose/lipid), impaired ATP generation, and subsequent energy crisis. Ultimately, these perturbations disrupt ionic gradients and acid-base regulation.

These biochemical alterations culminate in complex pathophysiological cascades encompassing cerebral edema formation, neuroinflammatory activation, redox imbalance, neuronal apoptosis, and synaptic signaling abnormalities [4, 5, 6]. While the majority of patients do survive the initial year following a stroke, over 10% encounter prolonged disability [7]. Rehabilitation constitutes a pivotal component of post-stroke therapeutic interventions.

Current therapeutic paradigms rely on the administration of intravenous rt-PA as the sole FDA-approved thrombolytic intervention. The TRACE-III studies have investigated the efficacy of thrombolytic therapy in patients with acute large vessel occlusion and ischemic penumbra within 4.5 to 24 hours of onset, yielding favorable outcomes. These studies have successfully extended the intravenous thrombolysis time window to 24 hours. [8] Consequently, the development of innovative therapeutic interventions that encompass a wide time window, exhibit high efficacy, and are adaptable to individualized treatment regimens is imperative to enhance outcomes for patients with IS.

Stem cells are undifferentiated, pluripotent cells that have the capacity to develop into various specialized cell types through a process of mitosis and differentiation. These cells possess the capacity to regenerate multiple tissues and organs. A plethora of stem cell types have been investigated in both animal models and clinical studies. Among these, embryonic stem cell therapies give rise to ethical concerns; neural progenitor cells are challenging to isolate and exhibit limited proliferative capacity; and engineered cells, such as induced pluripotent stem cells (iPSCs), face technological limitations [9].

However, mesenchymal stem cells (MSCs) exhibit several advantageous characteristics, including their capacity for straightforward in vitro expansion, minimal immunogenicity, the secretion of a variety of active cytokines, and the ability to elicit potent immunomodulatory effects [10]. Consequently, MSC transplantation for IS treatment remains a prominent research focus in recent years.

According to the Stroke Treatment Academic Industry Roundtable (STAIR), cell therapy has been identified as a leading candidate for enhancing the efficacy of stroke treatment strategies [11]. Specifically, stem cell therapy has demonstrated considerable promise in promoting endogenous neuroprotection and brain repair processes, including immunomodulation, neuronal regeneration, and vascular remodeling [12], rendering it a highly compelling option for enhancing neurological function in IS patients [13]. A meta-analysis of clinical trials investigating the use of stem cell therapy for IS revealed that some statistically significant results showed a favorable trend [14]. However, there are several challenges that must be overcome if this therapy is to be translated into routine clinical practice. These challenges include safety concerns, such as immune reactions, tumorigenicity, and embolism risks. There are also practical barriers, such as stringent regulatory requirements, high production costs, and logistical complexities associated with cell preservation and transfer [15].

Consequently, there has been a growing focus on the paracrine effects of stem cells, particularly the therapeutic potential of extracellular vesicles (EVs). Stem cell-derived EVs are enriched with bioactive molecules that facilitate intercellular communication and exhibit immunomodulatory and tissue repair properties similar to those of their parental cells. Extracellular vesicles (EVs) exhibit substantial structural and compositional heterogeneity and can be classified into three categories: exosome, microvesicle (MV), and apoptotic body (ApoBD). The categorization of EVs is based on their size, density, and biogenesis mechanisms [16]. Their distinctive properties, including immunological inertness, minimal toxicity, and the capacity to traverse biological barriers [17], position EVs as a potentially groundbreaking and more secure alternative to conventional stem cell transplantation for IS treatment [18].

The presentation of encouraging preclinical data has generated a positive outlook on the potential of EVs as a broadly applicable therapeutic modality for IS patients. This review highlights the pivotal role of MSCs-derived EVs in treating IS, and systematically explores the mechanisms by which MSCs and their derived vesicles modulate cell fate (inhibition of autophagy, apoptosis, and pyroptosis), exert neuroprotective and immunomodulatory effects, promote angiogenesis and blood-brain barrier repair, and exhibit antioxidant and anti-inflammatory properties. The objective of this study is to facilitate the clinical translation of cell-free therapies for stroke by providing a comprehensive theoretical framework.

2. A Brief Overview of Stem Cell-Derived Extracellular Vesicle

MSCs are a heterogeneous subset of multipotent adult stromal cells characterized by self-renewal capacity and multilineage differentiation potential [19]. The therapeutic appeal of these cells is attributable to several factors, including their rapid replicative kinetics, immunomodulatory properties, low immunogenicity, and fewer ethical constraints when compared with embryonic stem cells [20].

Two phase II trials by Jaillard et al. [21] and Law et al. [22] validated the safety and feasibility of MSC transplantation in subacute IS, while parallel studies in chronic stroke demonstrated comparable outcomes [23]. To date, clinical trials have collectively established the safety profile of MSC-based therapies in stroke patients. However, the efficacy of these therapies remains to be definitively assessed. Large-scale, placebo-controlled trials are necessary to make this assessment [24].

The challenges associated with the translation of cell-based therapies include, but are not limited to, the poor survival of transplanted mesenchymal stromal cells (MSCs) in the ischemic penumbra, where the survival rate of cells is less than 1% beyond 72 hours post-transplantation [25, 26]. Additional limitations as drug delivery vehicles encompass unpredictable differentiation, risk of microvascular embolization, infection susceptibility, and logistical hurdles in manufacturing, storage, and transport [27]. EVs are lipid bilayer structures secreted by somatic cells, which engage in processes including tissue repair, immunomodulation, and cell proliferation under pathological conditions of the organism [28, 29].

Extracellular vesicles (EVs) are a heterogeneous population of particles that are typically categorized into different subtypes, including exosome (30–150 nanometers), microvesicle (MV, 150 nanometers–1 micrometer), and ApoBD (1–5 micrometers) based on their subcellular origin, biogenesis, particle size, and molecular composition [30].

The process of exosome biogenesis is initiated by the inward budding of endosomal limiting membranes, which gives rise to the formation of multivesicular bodies (MVBs). These MVBs subsequently undergo fusion with the plasma membrane, resulting in the release of exosome into the extracellular environment. Following its release, the exosome has the capacity to interact with the extracellular matrix, elicit responses in cells within the local microenvironment, or act on cells located at a distance [31, 32].

The process of microvesicle formation involves direct budding from the plasma membrane, yielding a population of EVs that exhibit significant heterogeneity in size. Conversely, ApoBDs are produced at the cell surface and are exclusively released by apoptotic cells during programmed cell death [33].

It is noteworthy that in bone marrow mesenchymal stromal cell (BM-MSC)-derived EVs, exosome and microvesicles exhibit distinct lipid profiles. Exosomes feature an outer leaflet enriched in phosphatidylserine lyso-derivatives and free fatty acids, with cardiolipin localized to the inner leaflet. In contrast, microvesicles exhibit a lipid composition analogous to that of the donor cell plasma membrane, thereby emphasizing the intrinsic structural diversity among EV subtypes [34].

Most significantly, EVs serve as natural carriers for diverse biomolecules, including proteins, lipids, DNA, and various RNA species [35], positioning them as promising candidates for therapeutic interventions and drug delivery systems.

MSC-derived EVs and exosome paracrine effectors encapsulate the therapeutic cargo of parental MSCs while mitigating risks associated with live cell administration. These nanovesicles inherit the immunomodulatory, pro-angiogenic, and neuroprotective properties of MSCs, offering a cell-free alternative that circumvents issues of cell viability, embolization, and ethical complexity [36, 37, 38, 39]. A critical analysis reveals that, in comparison with MSCs, exosome production exhibits several advantageous characteristics. Firstly, it is more streamlined, cost-effective, and scalable. Secondly, its nanoscale dimensions (approximately 1/1,000,000 the volume of MSCs) facilitate simplified manufacturing and storage [40].

Exosomes exhibit minimal membrane-bound immunogenic epitopes, thereby significantly reducing the risk of allogeneic immune rejection. These nanovesicles have been shown to facilitate intercellular communication by delivering bioactive cargo, including microRNAs (miRNAs), proteins, and lipids, to recipient cells. The membranes of these structures have been found to contain adhesion molecules and ligand-specific receptors. These molecules and receptors have the capacity to facilitate targeted accumulation in injured tissues or specific microenvironments. This property can be further enhanced through synthetic modifications, which serve to augment cell/tissue tropism [41].

Collectively, EVs-based therapies offer distinct advantages over traditional stem cell approaches for stroke treatment, including improved safety, logistical feasibility, and programmable targeting capabilities.

3. Potential of Mesenchymal Stem Cell-Derived Extracellular Vesicles in Treating Ischemic Stroke: Administration Methods and Efficacy

In the context of research on stem cell and secretome therapy for ischemic stroke, a critical examination of the four administration routes—intravenous, intra-arterial, intranasal, and intracerebral—has revealed a series of distinguishing characteristics (Table 1, Ref. [39, 42, 43]; Table 2, Ref. [44, 45, 46, 47, 48, 49, 50, 51]; Table 3, Ref. [52, 53, 54]; Table 4, Ref. [55, 56, 57, 58, 59, 60, 61]), accompanied by notable disparities in efficacy, operational complexity, and clinical translation potential. Intra-arterial administration has been the subject of comparatively less research. Despite its invasive nature, this approach offers the significant advantage of expeditious drug delivery to the ischemic area, thereby reducing circulatory loss. Intravenous administration is more frequently reported in models of middle cerebral artery occlusion (MCAO), encompassing both transient and permanent models. Intranasal administration has been utilized in rat and mouse MCAO stroke models; however, studies demonstrating its therapeutic efficacy in stroke models remain limited. As a non-invasive method that is in close proximity to the target organ, intranasal administration may be more acceptable to patients. Intracerebral administration has been demonstrated to offer several advantages, including its capacity to reach the target organ directly, its need for a reduced quantity of mesenchymal stem cells and secretions, and its ability to minimize systemic effects. However, it demands extremely high operational precision, necessitating the accurate positioning of the injection site. The intricacies of the operation may impede its extensive clinical implementation.

In future applications, standardization of dosages and procedures, in addition to the selection of the optimal treatment route based on the unique characteristics of each patient, will be essential.

To date, however, there has been a paucity of studies that have directly compared the efficacy of exosome therapeutic strategies derived from different stem cell sources. Absent direct comparative evidence, a network meta-analysis was employed to establish an indirect comparison, utilizing cerebral infarct volume percentage and modified neurological severity score (mNSS) as outcome indicators [62].

Among the adipose mesenchymal stem cell-derived Exosomes (ADSC-Exos), bone marrow mesenchymal stem cell-derived Exosomes (BMSC-Exos), and dental pulp stem cell-derived Exosomes (DPSC-Exos), BMSC-Exos were identified as the optimal type for reducing cerebral infarct volume (%), and mNSS.

Nevertheless, the procurement of bone marrow mesenchymal stem cells (BMSCs), the primary source of BMSC-exosomes, necessitates an invasive and painful procedure, which represents a substantial drawback when juxtaposed with more readily obtainable mesenchymal stem cell sources [63].

BMSC-Exos demonstrate a high degree of vulnerability to age-related changes in their parental cells. As donors age, their regenerative capacity and therapeutic efficacy may decline more significantly than those of mesenchymal stem cells from other sources [64].

Human umbilical cord mesenchymal stem cells (hUCMSCs) can be obtained through a minimally invasive procedure that involves the extraction of blood from the umbilical cord. This collection method is significantly less complex than the methods employed for most other MSC sources. Another type of mesenchymal stem cell, adipose-derived stem cells (ADSCs), exhibits a shorter population doubling time and stronger anti-apoptotic potential [65].

Consequently, there is a necessity for a greater number of high-quality randomized controlled animal experiments, especially direct comparative evidence, to ascertain the most efficacious administration strategy for stem cell-derived exosome utilization in the treatment of ischemic stroke.

4. MSC-derived EVs Can Regulate Cell Function and Fate

MSC-EVs and interoception (intero) have been demonstrated to play a crucial role in regulating brain cell function and fate, especially in the context of programmed cell death pathways (e.g., autophagy, apoptosis, and thermoapoptosis). The regulation in question is modulated by bioactive cargoes, including, but not limited to, microRNAs (miRNAs) and proteins. These bioactive cargoes selectively modulate spatiotemporal signaling networks (Fig. 1).

Autophagy displays a dual role in cerebral ischemia: it is neuroprotective in the initial stages of ischemia (\le24 hours), but during prolonged activation, it becomes neurotoxic. Furthermore, induced mesenchymal stem cells-EV (iMSC-EV) have been shown to activate the STAT3 pathway, thereby significantly inhibiting excessive autophagy in the delayed ischemic phase (48–72 hours) when administered in combination with the autophagy inhibitor 3-methyladenine (3-MA) [66].

In vitro and in vivo models of hypoxia and middle cerebral artery occlusion (MCAO), respectively, demonstrate that adipose-derived MSCs (adMSCs) can regulate neuronal autophagy-related genes through the delivery of miRNA-25 [67]. MSC-EVs derived from olfactory mucosa have been shown to maintain physiological autophagy levels by alleviating oxygen-glucose deprivation/reoxygenation (OGD/R)-induced endoplasmic reticulum stress through enhanced PI3K/Akt/mTOR phosphorylation [68].

MSC-EVs have been shown to exert multidimensional anti-apoptotic effects by synergizing endogenous/exogenous apoptotic pathways and mitochondrial homeostasis. Neurotrophic factors (e.g., GM-CSF, NGF) in MSC-EVs support neuronal survival in the peri-infarct region [69, 70]. For instance, the bone marrow-derived MSC-exosome miR-133a-3p has been shown to modulate DAPK2 and Akt/mTOR, thus counteracting the process of apoptosis in SH-SY5Y cells following OGD/R. Bone marrow MSC-derived exosome miR-138-5p enhances STAT3 phosphorylation, thereby inhibiting LCN2-dependent apoptosis in astrocytes [71]. The MSC exosome miR-26a-5p has been shown to target CDK6 and attenuate microglia apoptosis in ischemia/reperfusion (I/R) injury [72]. MSC-EVs have been shown to protect microglia from I/R-induced injury by enhancing FOXO3a-mediated mitophagy, thereby reducing subsequent neuronal damage [73].

Furthermore, MSC-EV has been observed to impede cellular pyroptosis by modulating inflammatory vesicle activity and mitochondrial dynamics. Co-culture with MSC-EVs has been demonstrated to restore AMPK-dependent autophagy flux in OGD-treated PC12 cells, leading to the clearance of NLRP3 inflammasomes and the prevention of cellular pyroptosis [74]. A growing body of research has demonstrated that dong quai methylin, an active ingredient in the MSC-EVs treatment model, has the capacity to attenuate caspase-9-driven neuronal focal death and reduce neurological damage in stroke mice by inhibiting RIPK3-dependent mitochondrial autophagy [75].

5. Neuroprotective Effects of MSC-Derived EVs

The centrality of neuronal injury, neurological deficits, and impaired neuroplasticity to the pathogenesis of IS is well-documented. The process of neural repair subsequent to ischemia is contingent upon the proliferation, migration, and differentiation of endogenous neural precursor cells (NPCs). Furthermore, human MSCs have been shown to upregulate neural-specific and synapse-associated proteins within 30 days post-transplantation [76], suggesting that transplanted MSCs successfully differentiate and contribute to endogenous repair processes. From a mechanistic perspective, the therapeutic efficacy of MSCs (mesenchymal stem cells) is attributed to their ability to enhance the migration and differentiation of NPCs (neural progenitor cells) through the activation of the PI3K-Akt signaling pathway [77]. Furthermore, Shiota et al. [78] demonstrated that MSCs promote NPC proliferation/migration in ischemic brain injury by up-regulating the expression of chemokines and sialyltransferases. Secreted factors such as SDF-1 and NRG1 have also been demonstrated to promote NPC proliferation [79], which, in turn, facilitates the replenishment of new neurons in damaged tissues and enhances neurological function.

In IS, the occurrence of white matter demyelination occurs prior to axonal damage, thereby indicating that myelin regeneration serves as a pivotal mechanism in the prevention of axonal damage [80]. However, the process of axonal regeneration is impeded by glial cell scarring, which is characterized by the deposition of extracellular matrix and the proliferation of reactive astrocytes. The expression of Connective Tissue Growth Factor (CTGF), which is elevated in glial scarring, is repressed by MicroRNA-133 [81]. The BMSC-derived exosome, miR-133b, has been demonstrated to induce secondary exosome secretion from astrocytes, reduce glial scar thickness, and enhance neuroplasticity [82]. Furthermore, studies have shown that miR-17-92 clusters promote oligodendrocytogenesis and stroke-induced neurogenesis [83]. In addition, bone marrow MSCs have been observed to activate PI3K/AKT/GSK-3\beta signaling to promote axon growth after MCAO [84]. Finally, recent research has revealed that axonal growth after MCAO [85] is also enhanced by BMSC-derived exosome signaling. The MSC-derived exosome miR-146-5p has been shown to promote myelin repair and protect axons [82], and the study of miR-17-92-enriched exosome has demonstrated their capacity to enhance axonal myelination and synaptic plasticity through PTEN/PI3K/Akt/mTOR/GSK-3\beta signaling [86]. The inactivation of GSK-3\beta, a critical element in axonal regeneration, has been demonstrated to expedite CNS repair [87].

MiR-124 has been identified as the most prevalent microRNA (miRNA) in the brain, with a pivotal role in determining the fate of neurons within the subventricular zone (SVZ). It has been demonstrated that this protein plays a pivotal role in the mediation of stroke-induced neurogenesis in the adult subventricular zone and striatum. Moreover, the promotion of NPC-to-neuron differentiation and ischemic resistance through enhanced neurogenesis has been observed to be a consequence of cortical overexpression [88]. Adipose MSC-derived miR-22-3p has been shown to attenuate ischemic injury by inhibiting the KDM6B-mediated BMP2/BMF axis [89], thus underscoring the role of EV-miRNA as a pivotal regulator of neurogenesis.

Neuroapoptosis and excitotoxicity have been identified as significant contributors to neuronal death following ischemia. MiR-345-3p and MiR-124-3p, derived from BMSC-derived exosome, have been shown to inhibit OGD/R-induced apoptosis by down-regulating TRAF6 [90] and other pro-apoptotic targets [91], respectively. The secretion of factors by mesenchymal stem cells (MSCs) has been shown to attenuate glutamate excitotoxicity by reducing the expression of NMDAR subunit proteins, the influx of calcium ions, and surface GluR1 levels [92].

Neuronal metabolic function and mitochondrial health are critical for neuronal survival. the BMSC-derived exosome KLF4 reduces N6-methyladenosine modification of Drp1 by targeting lncRNA-ZFAS1, which improves mitochondrial dynamics and thereby attenuates ischemic neuronal injury [93].

It is evident that stem cell-derived EVs and Exosomes have the potential to serve as effective candidates for promoting neurogenesis and neuroplasticity following cerebral ischaemia (Fig. 2).

6. Immunomodulatory Effects of MSC-derived EVs

MSC-EVs have been shown to play a pivotal role in the remodelling of the inflammatory microenvironment after IS (Fig. 3). This is thought to be attributed to their own constituent complex and delicate paracrine signaling network, which dynamically and precisely regulates both the innate and adaptive immune systems.

6.1 Regulation of Microglia/Macrophage Activation

Subsequent to IS, microglia undergo rapid activation, resulting in a significant disruption to the dynamic equilibrium of their M1/M2 phenotype. MSC plays a pivotal regulatory role in modulating microglia. Inhibition of TLR4 expression has been demonstrated to result in a reduction in microglia activation. It has been demonstrated that, in the presence of activated microglia, MSCs have the capacity to inhibit proliferation and migration, as well as to reduce phagocytosis [94]. A study revealed that bone marrow MSCs overexpressing miR-182-5p hindered microglia M1 polarization, diminished inflammatory cytokines, and repressed TLR4/NF-\kappaB signaling in a mouse model of cerebral ischemia [95]. MSC exosome miR-223-3p has been demonstrated to attenuate cerebral ischemia-reperfusion injury by inhibiting microglia M1 polarization-mediated pro-inflammatory responses through the inhibitory effect on CysLT2R [50]. In this study, the research team investigated the modulatory effects of bone marrow mesenchymal stem cells (BMSCs)-derived exosome H19 on lipopolysaccharide (LPS)-stimulated microglia M1/M2 polarization and inflammation-mediated neurotoxicity. The study found that exosome H19 can attenuate these effects through the adsorption of miR-29b-3p [96].

Overexpression of miR-22-3p has been demonstrated to promote M2 polarization of macrophages, inhibit inflammation, and alleviate ischemia/reperfusion (I/R) injury by downregulating interferon regulatory factor 5 (IRF5). Furthermore, the promotion of M2 phenotypic polarization through targeted negative regulation of IRF5 is another notable property of miR-125a derived from BMSC-Exos [97]. ADSC-Exos have been observed to modulate the expression levels of various markers, including CD163, Arg1, CD206, TGF-\beta1, and IL-10, while concomitantly decreasing the expression levels of TNF-\alpha, IL-6, and IL-8. This regulatory effect is attributed to the targeting of the Rho-associated coiled-coil containing protein kinase 1/PTEN pathway.

6.2 Regulation of Innate Immune Cells

IS has been demonstrated to cause the disruption of the blood-brain barrier (BBB), thus allowing the infiltration of peripheral innate immune cells into the brain tissue. This phenomenon has been demonstrated to compound neuroinflammation. MSC-EVs have been observed to modulate innate immune cells, including neutrophils, natural killer (NK) cells, and dendritic cells (DCs).

Neutrophils are the first immune cells to infiltrate ischemic tissues via the compromised BBB. The present study investigates the effects of MSC small extracellular vesicles (sEVs) on brain neutrophils in a rodent model of focal cerebral ischemia. The results demonstrate that sEVs have the capacity to antagonize the deleterious effects of brain neutrophils, thereby reducing neutrophil adhesion and activation without interfering with the peripheral immune response [44]. In addition, the use of MSC-exosome has been demonstrated to enhance immunity by increasing neutrophilic phagocytosis and survival [98].

MSC-Exos have been shown to reduce neutrophil infiltration and inhibit neutrophil respiratory burst, thereby decreasing the expression of inflammatory mediators (including IL-1\beta, IL-6, and TNF-\alpha) and suppressing the production of ROS in neutrophils [99]. As demonstrated by Soni et al. [100], the administration of bone marrow-derived mesenchymal stem cell-derived exosome has been shown to be more effective in prolonging the lifespan of neutrophils.

MSCs have been observed to down-regulate the expression of the NKG2D receptor through the secretion of active molecules, including PGE2, sHLA-G5, and TGF-\beta. These molecules have been shown to inhibit the activity of NK cells. Inhibition of NK cell toxicity by hypoxic MSCs has been demonstrated to enhance the engraftment of allogeneic recipients and reduce the accumulation of host-derived NK cells during in vivo transplantation [101].

DCs are a target of MSC-EVs immunosuppression. The secretion of interleukin-10 (IL-10) and direct contact with DCs by MSC-EVs have been demonstrated to inhibit the expression of co-stimulatory molecules, including CD80 and CD86, among others [102].

6.3 Regulation of the Adaptive Immune System

MSC-EVs have been demonstrated to exhibit a remarkable homeostatic capacity in the regulation of T cell subsets. On the one hand, it has been demonstrated to promote the differentiation of Treg cells through the induction of TGF-\beta, thus inhibiting the expansion of helper T cells (Th1) and Th17 cells [103, 104, 105, 106].

Furthermore, memory CD4+ T cells undergo a process of reprogramming in response to MSC-EVs, and IL-17-mediated neutrophil phagocytosis is enhanced [107].

Furthermore, MSC-EVs have been demonstrated to induce functional remodeling of B cells. It has been demonstrated that modulation of the CXCR4/CXCR5 signaling pathway, it has been demonstrated that they can inhibit proliferation and chemotaxis [108]. Concurrently, the remodeling of PI3K-AKT signaling induced by EVs results in the differentiation of B cells towards a CD24highCD38high regulatory B cell (Breg) phenotype [109, 110]. Furthermore, the activation of the PD1-PDL1 pathway has been shown to enhance immunoglobulin M production and antigen presentation by B cells, thereby effectively suppressing the associated immune response [111, 112, 113].

7. MSC-derived EVs Can Promote Angiogenesis and Blood-brain Barrier Repair

7.1 Neovascularization (Angiogenesis)

MSC-EVs have been demonstrated to elicit a substantial augmentation in the number of BrdU+/vWF+ cells and the expression of angiogenic markers (VEGF, VEGFR2, Ang-1, Tie-2) [114]. These “signaling molecules” such as vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) are delivered to the ischemic region, thereby laying a solid foundation for neovascularization [115, 116, 117, 118, 119].

Exosomes from BMSCs have been demonstrated to induce endothelial tube formation in OGD-exposed bEnd.3 cells by facilitating the delivery of Egr2. This, in turn, has been shown to enhance SIRT6 expression, thereby inhibiting Notch1 hyperactivation [120].

Induced MSC-EVs have been demonstrated to activate the STAT3 pathway, thereby inhibiting autophagy and promoting angiogenesis [66]. Furthermore, MSC-derived extracellular vesicles have been observed to reduce pericyte detachment from capillaries by inactivating the nuclear factor-\kappaB (NF-\kappaB) pathway [121].

MSC activate the PI3K/Akt/eNOS signaling pathway by secreting exosome to upregulate the expression of microRNAs and angiogenic factors. As demonstrated in [122, 123], the up-regulation of microRNAs such as miR-126-3p, miR-140-5p, let-7C-5p and miR-486, and the down-regulation of other microRNAs, including miR-186-5p, miR-370-3p and miR-409-3p, have been observed in the relevant context.

In a study of the effects of hiPS-MSC-EV on a mouse model of MCAO, it was found that the treatment led to a reduction in infarct size, an improvement in spontaneous motility, and an enhancement of angiogenesis. This was achieved through the expression of VEGF and CXCR4 proteins in the infarct hemisphere (Fig. 4) [124].

7.2 Blood-brain Barrier (BBB) Protection and Repair

The blood-brain barrier (BBB) is a highly selective permeability barrier that maintains the brain’s microenvironment by separating blood from the brain’s extracellular fluid (Fig. 5).

The BBB is a significant target of cerebral ischemia-reperfusion injury and can be disrupted by up-regulation of the expression of matrix metalloproteinase-2 and matrix metalloproteinase-9. These enzymes have been shown to degrade the basement membrane following ischemia [125], thereby facilitating acute neurovascular and parenchymal destruction [126]. Matrix metalloproteinases (MMPs) have been identified as a key factor in the opening of the BBB, with studies demonstrating their ability to degrade tight junction proteins (TJPs) [127].

The BMSC exosomes derived from QXZG intervention has been shown to repair the blood-brain barrier by reducing the expression levels of MMP-2 and MMP-9 [128].

BMSC-EVs have been demonstrated to attenuate basement membrane degradation by inhibiting the caveolin-1/CD147/VEGFR2/MMP signaling axis [129, 130]. Furthermore, MSC-Exos- miR-132-3p have been demonstrated to be more efficacious in reducing endothelial cell tight junction breaks, upregulating Claudin-5 and ZO-1, and attenuating BBB dysfunction [131]. In addition, BMSCs-Exos have been demonstrated to maintain the integrity of the blood-brain barrier and reduce early brain damage in SAH [132].

MSC-EV-delivered miR-125b-5p has been demonstrated to maintain BBB integrity by targeting Toll-like receptor 4 (TLR4) and inhibiting nuclear transcription factor-\kappaB (NF-\kappaB) signaling in astrocytes [133].

8. Antioxidant and Anti-inflammatory Mechanisms of MSC-derived EVs

MSC-EVs have been demonstrated to exert a pivotal role in the process of remodeling the inflammatory microenvironment subsequent to IS (Fig. 3). This phenomenon is believed to occur through direct cell-to-cell contact and the intricate and nuanced paracrine signaling network, which meticulously and precisely regulates both the innate and adaptive immune systems.

In the aftermath of an ischemic brain injury, MSC-EVs have been observed to deliver superoxide dismutase (SOD) and glutathione peroxidase (GPx) to the ischemic region, thereby directly neutralizing reactive oxygen species (ROS) [134].

The MSC-EVs have been demonstrated to inhibit astrocyte activation and associated inflammation through the miR-125b-5p/TLR4/NF-\kappaB pathway [133]. MSC exosome KLF3-AS1 has been demonstrated to attenuate OGD/R-induced inflammation in SK-N-SH and SH-SY5Y cells by regulating Sphk1. bMC-Exos KLF3-AS1 has also been shown to ameliorate brain I/R-induced inflammatory injury by facilitating Sirt1 deubiquitylation through the KLF3-AS1/miR-206/USP22 network [135]. The study found that the expression of microRNA-146a-5p in human umbilical cord blood-derived mesenchymal stem cells (hUMSC-Exos) led to a reduction in microglia-mediated neuroinflammatory responses through the interleukin-1 receptor-associated kinase 1 (IRAK1)/TNF receptor-associated factor 6 (TRAF6) pathway [136].

In addition, exosomes carrying long non-coding RNA ZFAS1 derived from bone marrow MSCs alleviate oxidative stress and inflammatory responses in IS by inhibiting microRNA-15a-5p (Fig. 6) [137].

9. Discussion

The most recent research by Chen’s team reveals the synergistic effect of combined extracellular vesicle therapy: the co-administration of endothelial progenitor cell-derived and neural progenitor cell-derived extracellular vesicles significantly enhances anti-apoptotic effects and inhibits reactive oxygen species production. In comparison with the therapeutic effect of a single type of extracellular vesicle, this combined therapy demonstrates significantly superior efficacy [138]. This finding not only expands the dimension of extracellular vesicles in IS treatment but also indicates an innovative direction for personalized regenerative medicine models.

Furthermore, the employment of preconditioning strategies has emerged as a potentially efficacious approach [139]. Conditioned medium derived from hypoxic BMSCs has demonstrated protective effects in a rat stroke model, and its therapeutic effect may be mediated by stem cell-derived EVs [140]. It has also been recently discovered that the pro-angiogenic capacity of hypoxic preconditioned MSC-derived EVs is not influenced by inflammatory stimuli [141]. The therapeutic effects of EVs are evident in the absence of their elimination by the hostile microenvironment.

Despite the evidence indicating that MSC-derived extracellular vesicles and exosomes possess mechanisms such as regulating cell fate (inhibiting autophagy, apoptosis, and pyroptosis), neuroprotection, immunomodulation, promoting angiogenesis and BBB repair, and antioxidation and anti-inflammation in the treatment of IS, current research still faces significant translational bottlenecks.

The present state of research in this field is encumbered by significant translational obstacles. Firstly, extant evidence is primarily derived from animal models and in vitro experiments, exhibiting a paucity of clinical-grade research data. Furthermore, the clinical safety and delivery efficiency of targeted delivery systems (e.g., nanocarriers or biological scaffolds) have yet to be elucidated. Moreover, the determination of therapeutic time windows, dosage optimization protocols, and other key parameters have not been systematically studied. Consequently, the establishment of a multi-center clinical research network and the development of an integrated research system from basic to translational levels will be pivotal approaches to promote the clinical translation of extracellular vesicles in IS treatment.

10. Conclusions

In summary, IS, a neurological disease with high morbidity and mortality, involves complex cascade reactions in its pathophysiological processes, including energy metabolism disorders, neuroinflammation, oxidative stress, and blood-brain barrier (BBB) damage. The limitations in time window and efficacy of existing treatments underscore the pressing need to develop new therapeutic approaches. Mesenchymal stem cells (MSCs) have demonstrated considerable promise in the treatment of ischemic stroke (IS) due to their low immunogenicity, robust immunomodulatory capacity, and paracrine properties. Nevertheless, the process of cell transplantation is encumbered by considerable challenges, including its limited capacity for survival and the potential risk of embolism.

This review methodically expounds on the fundamental mechanisms and benefits of MSC-derived extracellular vesicles (MSC-EVs), particularly the role of exosome, as a form of acellular therapy.

First, MSC-EVs have been shown to deliver biologically active cargo, such as microRNAs (miRNAs) and proteins, which have been found to inhibit excessive autophagy, apoptosis, and pyroptosis of neurons. This, in turn, has been demonstrated to contribute to the maintenance of cellular homeostasis. Secondly, MSC-EVs have been shown to activate endogenous neurogenesis, regulate axonal regeneration and myelination, and reduce the obstruction of glial scars, thereby achieving neuroprotective effects. MSC-EVs have been demonstrated to target and regulate microglial polarization, neutrophil infiltration, and the balance of T cell subsets, thereby alleviating excessive inflammatory responses and remodeling the immune microenvironment. Furthermore, MSC-EVs have been shown to promote angiogenesis in ischemic regions, repair BBB damage by inhibiting matrix metalloproteinase activity and stabilizing tight junction proteins. Finally, MSC-EVs have been shown to directly deliver antioxidant enzymes, such as SOD and GPx, and to inhibit oxidative stress and inflammatory cascades through signaling pathways, including Nrf2 and NF-\kappaB.

In comparison with conventional stem cell transplantation, MSC-EVs offer several advantages, including their high immune inertness, ease of storage and transportation, and the ability to produce them in bulk. These cells effectively circumvent the safety and logistical challenges associated with cell therapies, positioning them as optimal candidates for IS treatment. However, further clinical translation is required to address issues such as standardized isolation and purification, dosage optimization, targeted delivery efficiency, and long-term safety. Future research should concentrate on the regulation of MSC-EV heterogeneity, in-depth analysis of mechanisms, and large-scale clinical trials to provide theoretical support and practical guidance for promoting the transition of acellular therapies from basic research to clinical application, ultimately improving the neurological prognosis of patients with IS.

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Funding

Chongqing Natural Science Foundation(cstc2021jcyj-msxmx0848)

Bishan Science and Technology Research Project(BSKJ2022006)

National Natural Science Foundation of China (NSFC)(81773954)

National College Student Innovation and Entrepreneurship Program(202310617015)

Chongqing College Students’ Innovation and Entrepreneurship Project(S202310617003)