Exosome-mediated amplification of endogenous brain repair mechanisms and brain and systemic organ interaction in modulating neurological outcome after stroke

Abstract

Ischemic stroke is caused by a regional interruption of cerebral blood flow to the brain. Rigorous pre-clinical and clinical research has made landmark progress in stroke treatment using thrombolytics and endovascular thrombectomy. Although numerous successful neuroprotective therapeutic agents for ischemic stroke have been reported in pre-clinical studies, most of them failed in clinical testing. Persistent pre-clinical research has demonstrated that the ischemic brain is not only passively dying but is also actively recovering. Within the neurovascular niche in the peri-infarct tissue, repair mechanisms thrive on the interactions between the neural and vascular compartments. In this review, we discuss exogenous therapy using mesenchymal stromal cell-derived exosomes to amplify endogenous brain repair mechanisms and to induce neurorestorative effects after stroke. Emerging evidence indicates that multiple communication axes between the various organs such as the brain, heart, kidney and gut, and whole body immune response mediated by the spleen can also affect stroke outcome. Therefore, in this review, we summarize this evidence and initiate a discussion on the potential to improve stroke outcome by amplifying multiple brain repair mechanisms after stroke, and by targeting peripheral organs and downstream events to enhance recovery in the injured brain and promote over all well being.

Keywords

Exosome microRNA neurorestoration neurovascular niche stroke

Introduction

Despite a declining trend in stroke-related deaths, stroke remains the second leading cause of death behind heart disease, and is a major cause of long-term disability worldwide.¹ Our understanding of the pathophysiological cascade following ischemic injury to the brain has greatly improved over the past few decades. Ischemic brain damage can be divided into two primary zones – the ischemic core and the ischemic penumbra. Failure to meet energy demands is the primary cause of cell death within the ischemic core. Ischemia decreases cerebral blood flow (CBF), impairs energy synthesis, increases extracellular adenosine triphosphate (ATP) as a result of neuronal and glial depolarization, which leads to metabolic and ionic imbalance, and excitotoxicity.^2–4 Therefore, neuronal loss of transmembrane ionic gradients, neuronal depolarization, excessive release of excitatory neurotransmitters and pro-inflammatory molecules, and intracellular ionic imbalance evoke primary neuronal loss via necrosis and apoptosis. The ischemic penumbra is sandwiched between the poorly perfused ischemic core and normally perfused healthy tissue. The penumbra remains viable for hours due to collateral blood flow, presenting a window of opportunity to reperfuse and salvage this region. Reperfusion is a primary goal for thrombolytic and neuroprotective therapies, and is a predictor of improved functional outcome.⁵ In addition to cell death mechanisms which dominate the ischemic core, stroke also stimulates survival and repair mechanisms especially in the penumbra. Activation of these endogenous brain repair mechanisms stimulates spontaneous functional improvement after stroke. Several clinical studies have shown that approximately 50% functional recovery occurs within the first two to six months after stroke onset.^6–8 Initial stroke severity, age of patient, co-morbidities and other factors influence the extent and rate of such spontaneous recovery from stroke.^6–8 Hypoxia and inflammation stimulate tissue repair mechanisms such as angiogenesis, neurogenesis, immunomodulation, neuronal functional reorganization, and rewiring of neuronal networks in the ipsilateral and contralateral brain hemispheres.⁹ The primary aim of neurorestorative therapies after stroke is to enhance these endogenous restorative mechanisms and improve long-term neurological functional outcome.^9,10

The knowledge that the ischemic penumbra is not only passively dying but also is actively recovering, has spurred research to amplify endogenous repair mechanisms to improve recovery after stroke. Following ischemic stroke, there is a graded transition from damage to endogenous repair mechanisms. These repair mechanisms and their signaling pathways are coupled and interdependent, particularly within the neurovascular niche.^10,11 Treatments that can amplify multiple restorative and repair processes are most likely to have maximum therapeutic efficacy. In this review, we will focus our discussion of restorative and repair therapeutics of stroke on the therapeutic effects of mesenchymal stromal cell-derived exosomes (MSC-exosomes).

Remarkable progress has been made in pre-clinical research in elucidating various signaling pathways, and numerous novel therapeutics for stroke prevention, pre-treatment, thrombolytics, neuroprotection, neurorestoration and rehabilitation have emerged.^10,12,13 Unfortunately, clinical translation of investigational drugs remains problematic. Various factors have been implicated in hindering bench to bedside translation of stroke treatments including age and gender bias resulting in widespread use of young male animals that are typically healthy without co-morbidities such as diabetes and hypertension, poor experimental design with insufficient sample size, and reporting and publishing bias.^12,14,15 In this review article, we draw attention to another confounding factor based on emerging preclinical and clinical evidence that indicate multiple communication axes between various organs such as the brain, heart, kidney and gut, and whole body immune response mediated by the spleen that may also contribute to poor outcome after stroke. This new understanding calls for novel treatment approaches that target these peripheral organs to enhance recovery in the injured brain and improve neurological function after stroke.

Endogenous brain repair after stroke

Neurovascular coupling

Coupling of angiogenesis, arteriogenesis and neurogenesis enhances brain plasticity and improves neurological recovery after stroke.¹⁰ The intrinsic ability of the central nervous system to undergo functional reorganization in response to ischemia involves rewiring of brain and spinal cord circuitry, including synaptogenesis to compensate for lost connections and function due to ischemic lesion.^9,10,16,17 Angiogenic signaling is activated as early as an hour after stroke, and persists for several days to weeks.¹⁸ Angiogenesis is a therapeutic target for post stroke recovery; however, there is an associated risk of vascular endothelial growth factor (VEGF) induction of immature and leaky blood vessels that can evoke adverse effects such as edema and vascular regression over time, particularly in diabetic stroke which is associated with extensive vascular damage and poor outcome.^19,20 In addition to angiogenesis, promoting arteriogenesis to form new capillaries and remodel pre-existing collateral blood vessels also improves functional outcome after stroke.²¹ Angiogenesis enhances post stroke synaptogenesis.¹⁷ Increasing synaptophysin, a pre-synaptic vesicle protein, using cell therapy has been correlated with post stroke functional improvement in rodent models of stroke.^22,23 Stroke induces white matter damage with loss of axons, myelin and oligodendrocytes.¹⁰ Regeneration and rewiring of lost neuronal circuitry via axonal outgrowth, dendritic sprouting and remyelination are important for long-term functional recovery.¹⁰ In the weeks following stroke, there is gradual and substantial increase in numbers of myelinated axons and apical dendrite spine density in the peri-infarct brain tissue of rodents.²⁴ Stroke induces differentiation of non-myelinating oligodendrocyte progenitor cells into mature and myelinating oligodendrocytes.²⁴ Post ischemic oligodendrogenesis is required to form myelin sheaths on the newly sprouting axons because mature oligodendrocytes are unable to proliferate and injured oligodendrocytes are unable to form myelin. Magnetic resonance imaging (MRI) of ischemic rodent brain demonstrated that stroke induces increased axon density with axonal projections oriented parallel to the rim of the ischemic boundary zone.²⁵ The temporal and spatial profiles of angiogenesis and axonal remodeling along the ischemic boundary zone coincide after stroke.²⁵ Interestingly, the intact cortical neurons of the contralateral hemisphere also undergo remodeling in response to ischemic injury to form axon collaterals and synaptic-like structures that extend to the denervated side of the spinal cord and improve sensorimotor function in experimental stroke.^9,16 Such intercortical and intracortical axonal connections facilitated by corticospinal neuronal sprouting from the intact cortex to peri-infarct regions can be stimulated and amplified by cell therapies and pharmacological agents.^16,26

Remodeling within the neurovascular niche

Post ischemic angiogenesis regulates several repair mechanisms, including secretion of growth factors that enhance cell survival in the ischemic penumbra, macrophage surge that facilitates debris removal, and scaffolding and guidance for neuroblast migration to the ischemic boundary region.^27,28 Neuroblasts migrate from the subventricular zone in chains or individually to reach a neurovascular niche in the ischemic boundary zone.^11,29–32 The neurovascular niche is a specialized microenvironment in the peri-infarct tissue, where interactions between neural progenitor cells (NPCs) and the vasculature promote adult neurogenesis.¹¹ When NPCs isolated from the subventricular zone of non-ischemic adult rats were co-cultured with cerebral endothelial cells isolated from the stroke boundary, there was a significant increase in NPC proliferation and differentiation into neurons.³³ Similarly, conditioned medium harvested from the SVZ of stroke rats promoted capillary tube formation of normal cerebral endothelial cells.³³ Inhibiting post stroke angiogenesis in the peri-infarct cortex decreased vascular density and resulted in a dramatic decrease in neuroblasts in the peri-infarct cortex indicating that angiogenesis and neurogenesis are causally linked at least within the neurovascular niche after stroke.¹¹ These studies demonstrate that post ischemic angiogenesis and neurogenesis are coupled. While post-stroke neurogenesis has been primarily studied using experimental stroke models in rodents, there is evidence of newborn neurons preferentially localized adjacent to blood vessels in the peri-infarct cortex of stroke patients.³⁴ Therefore, stroke initiates neuronal proliferation and expansion of neuroblasts in the subventricular zone which is followed by their migration to peri-infarct tissue where they differentiate into a region-appropriate phenotype reflecting phenotype of most degenerated neurons and replace neurons destroyed by ischemia.^29,31,32 The innate ability of the brain to replace damaged neurons via adult neurogenesis can be amplified by trophic and growth factors¹¹ and presents a therapeutic target.

Mechanisms of neurovascular coupling

The neurovascular unit (NVU) comprises structural and functional interactions between neuronal, glial (astrocytes, microglia, and oligodendrocytes), and vascular (endothelial cells, pericytes, and smooth muscle cells) compartments, and the extracellular matrix of the brain. The molecular mechanisms underlying coupling of angiogenesis and neurogenesis after stroke remain unknown. Stroke increases ischemic brain expression of several growth and trophic factors and chemokines such as VEGF, insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2), brain-derived neurotrophic factor (BDNF), stromal cell-derived factor 1 (SDF1), monocyte chemoattractant protein-1 (MCP-1), endothelial nitric oxide synthase (eNOS), and angiopoietin-1/Tie2 (Ang1/Tie2) some of which are multi-functional and promote neurogenesis, angiogenesis, oligodendrogenesis, and may potentially mediate their coupling.³⁵ Neuroblast proliferation, migration, and differentiation are coupled to post ischemic oligodendrogenesis.^{11,24,36–38} In addition to the proliferation of oligodendrocyte progenitor cells in the corpus callosum, stroke stimulates NSCs in the SVZ to generate oligodendrocyte progenitor cells which then migrate to the white matter tracts of the corpus callosum and striatum as well as ischemic boundary zone and differentiate into mature myelin producing oligodendrocytes in the vicinity of sprouting axons.²⁴ In the neurovascular niche, secretion of SDF-1, VEGF and Ang1 by activated endothelial cells, angiogenic blood vessels, and astrocytes guide neuroblast and oligodendrocyte progenitor cell migration, and the inhibition of SDF-1 and Ang1 in remodeling cerebral vessels attenuates neuroblast migration toward the peri-infarct cortex.^11,36–38 Exosomes derived from ischemic endothelial cells and NSCs can modulate neurogenesis and angiogenesis, respectively, via transfer of exosomal cargo microRNAs (miRs).³⁹ Ischemic endothelial cell-derived exosomes rich in cargo miR-146 a and miR-125 a were taken up by non-ischemic NSCs and promoted neuronal differentiation.³⁹ Similarly, ischemic NSC-derived exosomes exhibiting increased miR-106 b and miR-125 b were taken up by non-ischemic endothelial cells and significantly increased their capillary tube formation.³⁹ In-vitro, ischemia-activated NPCs were found to stimulate angiogenesis in cultured endothelial cells via secretion of VEGF.³³ MiR-9 has been implicated to link developmental neurogenesis and angiogenesis through the formation of neurons expressing VEGF-A.⁴⁰ Ischemia increased expression of serum response factor in oligodendrocyte progenitor cells and oligodendrocytes which coincided with decreased expression of miR-9 and miR-200 family in the injured white matter tracts of corpus callosum and striatum.⁴¹ Additionally, inhibition of serum response factor or over expression of miR-9 and miR-200 in oligodendrocyte progenitor cells was found to inhibit oligodendrocyte progenitor cells differentiation into oligodendrocytes.⁴¹ Therefore, exosomes and their cargo miRs may play an important role in facilitating neurovascular coupling. Treatments that regulate multiple brain repair mechanisms may improve long-term functional recovery after stroke.¹⁰

Inflammatory responses after stroke

Disruptions of the NVU and blood–brain barrier (BBB) are key pathological steps that initiate secondary tissue damage after stroke. Platelets and endothelial cells are activated immediately after vascular occlusion, and the adhesion molecule P-selectin translocates to the cell surface membrane initiating leukocyte recruitment to the sites of brain injury.⁴² BBB disruption facilitates the infiltration of peripheral/circulating neutrophils, macrophages, monocytes, and lymphocytes from the blood into ischemic brain.^43,44 Inflammatory responses begin within a few hours after stroke and evolve over the following several weeks contributing to secondary brain damage, infarct expansion and secondary organ dysfunction after stroke. Neuronal, glial and transmembrane depolarization leads to an increase in extracellular ATP which can activate resident microglia.⁴ Stroke-induced rapid local inflammatory responses in the brain parenchyma include microgliosis, astrogliosis, and cytokine and chemokine secretion. Exosomes secreted by microglia and astrocytes can store and release inflammatory cytokines such as IL-1β.^45,46 Microglia-derived exosomes can also stimulate neuronal activity and enhance propagation of inflammatory signals.⁴⁷ Exosomes secreted by injured brain cells in response to ischemic stimuli can influence bystander cells by the transfer of dysregulated miRs that suppress the expression of essential genes in the recipient cells.⁴⁸ In their resting state, resident microglia continuously monitor the microenvironment using their motile processes and protrusions and aid in metabolic byproduct and debris removal.⁴⁹ Upon ischemic injury to the brain, microglia are rapidly activated, undergo structural and functional alterations to a phagocytic phenotype, accumulate near the injury site and secrete inflammatory mediators.⁴⁹ Following neutrophil invasion and microglial activation, macrophages derived from local microglia and hematogenous macrophages invade the ischemic tissue.⁴² In their degenerative M1 phenotype, microglia and macrophages promote inflammation by increasing proinflammatory cytokines (interleukins (IL-6, IL-1β), tumor necrosis factor-α (TNF-α) etc.), integrins, adhesion molecules, chemokines and their receptor molecules and reactive oxygen species (ROS).^4,50,51 During this sub-acute phase of stroke, the various pro-inflammatory cytokines, chemokines, ROS, and activated matrix metalloproteinases (MMPs) increase glial activation and peripheral immune cell infiltration into the brain which worsen BBB leakage, brain edema, neuronal death, and hemorrhagic transformation.^51,52 During the recovery phase of stroke, microglia and macrophages assume an anti-inflammatory M2 phenotype and facilitate debris removal from the ischemic brain.⁴ M2 phenotype microglia and macrophages decrease the expression of inflammatory factors, protect neurons, and improve cell survival in the ischemic environment.⁵³ Therefore, extending the M2 phase of these macrophages and microglia and inducing macrophage polarization from M1 to M2 phase are desirable neurorestorative effects. However, the distinction of M1 and M2 phenotypes is not clearly defined and the simple M1 and M2 classifications are likely inadequate to explain the complex role of macrophage polarization in mediating inflammatory responses.⁵⁴

Exosome therapy to amplify brain repair

Exosomes are nanosized vesicles (∼30–100 nm in diameter) that are generated by nearly all cells, and constitute major vehicles for intercellular communication.⁵⁵ Exosomes have multiple roles in regulating physiological and pathological processes.⁵⁵ Based on their parent cell and functional cargo, exosomes derived from various cell types have been reported to induce neuroprotective and neurorestorative effects by modulating gene, protein and miR expression in their target cells and tissues.^55,56 MiRs are small non-coding RNA sequences approximately 22 nucleotides in length that can regulate multiple genes, pathways, and complicated cellular networks by acting either alone or in concert with other miRs.¹⁰ MiRs regulate post-transcriptional gene regulation by binding to complementary sites in the 3′-untranslated region of messenger RNAs.⁵⁷ The miR signature in ischemic stroke is being unraveled and has potential use as diagnostic and prognostic markers as well as novel therapeutic targets.⁵⁸ Exosomes are transported by biological fluids and provide an effective means of communication between neighboring as well as remote cells.⁵⁹ Exosomes contain and deliver proteins, lipids, and nucleic acids that are characteristic of its parent cell to recipient cells.⁵⁹ Exosomes enable a graft-free delivery of therapeutic molecules and offer several advantages over cell-therapy. For instance, exosomes have low immunogenicity, low risk of microvascular thrombosis and can easily cross the BBB and be internalized by endogenous brain cells.^56,60–62 Exosome content can be readily modified by manipulating contents of the parent cell and miR expression. Therefore, exosomes can be programmed to maximize therapeutic efficacy by targeting specific neuroprotective and neurorestorative pathways, thereby enabling the development of personalized targeted drug delivery vehicles to deliver designer genetic instructions as well as the active components of cell-based therapy to treat ischemic stroke.^63–65 Exosomes derived from a variety of cell types such as embryonic stem cells, induced pluripotent stem cells (iPSCs), and adult stem cells such as mesenchymal stromal cells (MSCs), human umbilical cord blood cells (HUCBCs), and neural stem cells (NSCs) have been employed to treat ischemic stroke and other cerebrovascular diseases in pre-clinical studies.^66,67 In this review, we will discuss the therapeutic effects of a multipotent restorative therapy for stroke, MSC-exosomes.

Stroke treatment using MSC-exosomes

MSCs are a prolific source of exosomes⁶⁸ and MSCs as well as MSC-derived exosomes have been widely studied for the treatment of stroke and other neurological diseases.⁶⁹ Treatment of stroke with MSCs was found to improve neurological recovery by secreting a plethora of growth and trophic factors and promoting endogenous brain repair mechanisms, primarily via the secretion of exosomes.^63,70–76 The secreted exosomes were found to carry and transfer their cargo miRs (such as miR-133 b) to astrocytes and neurons in the brain and regulate target genes which stimulate restorative mechanisms such as neurite outgrowth and improve neurological functional outcome.⁷⁵ Pre-clinical studies using rodent models of ischemic stroke have demonstrated therapeutic efficacy, ability to promote endogenous brain repair mechanisms, and improvement of long-term neurological functional outcome after stroke when MSC-exosomes were administered intravenously at 24 h after stroke onset.^63,73–76 When mice subjected to stroke were intravenously treated with MSCs at 24 h after stroke or with MSC-derived exosomes at one, three, five days after stroke, similar functional recovery was observed.⁷⁶ In addition to therapeutic efficacy which is at least equivalent to parent cell therapy,⁷⁶ treatment with exosomes has become popular due to advantages over cell therapy such as low risk of tumor formation and reduced immunogenicity, and their ability to be readily expanded in-vitro and stored until use. Figure 1 presents a simplified schematic summarizing MSC-exosome-derived therapeutic effect after stroke.

Figure 1.

Summary of MSC-exosome-derived therapeutic effects after stroke. MSC-derived exosomes transfer microRNA to the various cells of the neurovascular unit as well as stimulate parenchymal brain cells to secrete growth and trophic factors which promote neurorestorative mechanisms such as angiogenesis, neurogenesis, synaptogenesis, oligodendrogenesis, and anti-inflammatory responses which contribute to neurological functional recovery after stroke.

Apart from their therapeutic potential, plasma exosomal miRs can also serve as biomarkers for prognosis of stroke.⁵⁸ Compared to a healthy control patient population, serum exosomal content of brain-specific miRs (miR-9 and miR-124) was significantly increased in acute ischemic stroke patients, and the expression level correlated with stroke severity.⁷⁷ Another study compared plasma exosome levels in healthy and ischemic stroke patients and found that employing the combination of miR-422 a and miR-125 b-2-3 p may serve as blood-based biomarkers for monitoring and classifying stroke patients into acute (decreased expression) and sub-acute (increased expression) stages.⁵⁸

MSC-exosome induced neurovascular remodeling after stroke

MSC-exosome therapy favors neurovascular remodeling.^63,76 Systemic administration of MSC-exosomes at 24 h after ischemic stroke was found to significantly increase axon density, myelin density, synaptic plasticity and angiogenesis in the ischemic border zone as well as promote neuroblast migration from the subventricular zone to ischemic border zone.^63,76 In a model of sub-cortical stroke, intravenous administration of MSC-exosomes at 24 h after stroke was found to improve long-term functional outcome, axonal sprouting from the intact cortical tissue to injured striatum leading to increased axon density in the injured striatum, and increased oligodendrocyte progenitor cell number as well mature oligodendrocyte counts resulting in increased number of myelinated axons compared to control stroke animals.⁷⁸ The mechanisms underlying MSC-exosome-induced brain plasticity after stroke and other cerebrovascular diseases are under investigation.^79,80 The characteristics of the parent cell and cargo of exosomes particularly miR content appear to influence therapeutic efficacy. MSC-exosomes are internalized by endothelial cells, neurons, oligodendrocytes and microglia in the ischemic brain.⁷⁸ MSC-exosomes are internalized by both neuronal cell bodies and axons resulting in increased axonal outgrowth in cultured primary cortical neurons.⁸¹ The miR content of exosomes appears to mediate neuronal plasticity as inhibition of argonaute 2 protein (a primary miR machinery proteins) in MSC-exosomes blocked MSC-exosome effects on axonal outgrowth.⁸¹ In addition, modified MSC-exosomes over expressing miR-17-92 cluster amplified axonal growth by activating the PTEN/mTOR signaling pathway in recipient neurons.⁸¹ Therefore, MSC-exosomes can directly transfer their cargo miR to recipient neurons and thereby influence neuronal remodeling. Glial cells and glial cell-derived exosomes likely modulate neurovascular coupling and brain plasticity by mediating communication between neuronal, glial, and vascular compartments.^82,83 Exosomes derived from miR-133 b-overexpressing MSCs were found to amplify therapeutic effects of MSC-exosome in rats subjected to stroke.⁸⁴ Interestingly, the amplification of neural plasticity following miR-133 b+MSC-exosomes was attributed to a stimulated secondary release of neurite-promoting exosomes from astrocytes.⁸⁴ Therefore, it is likely that MSC-exosomes exert their therapeutic potential by acting directly on parenchymal and vascular cells and also via secondary secretion of exosomes by astrocytes and other parenchymal cells and promote remodeling of the ischemic brain.

Effect of MSC-exosome therapy on inflammatory and immune response

Intravenous transplantation of MSCs at a chronic time point of 60 days after stroke in rats showed that MSCs preferentially home to the spleen over the ischemic brain, regulate inflammatory responses and decrease ischemic lesion volume compared to control rats.⁸⁵ MSC-exosomes are less immunogenic compared to MSCs due to the reduction in membrane-bound protein content.⁸⁶ MSC-exosomes are immunologically active and promote anti-inflammatory effects.^87,88 Exosomes derived from normal MSCs or hypoxia-preconditioned MSCs were found to exhibit anti-inflammatory properties and induce M2 polarization of bone-marrow-derived macrophages in-vitro and in-vivo in a model of skeletal muscle injury.⁸⁸ MSC-exosome treatment in stroke mice did not alter the infiltration of immune cells such as neutrophils, dendritic cells and macrophages.⁷⁶ However, at six days after stroke, post ischemic immunosuppression characterized by substantial reduction of total white blood cells counts (∼50%), monocyte (∼75%) and lymphocyte subsets (∼60%) in the peripheral blood of saline-treated stroke mice was significantly attenuated in MSC-exosome-treated stroke mice which had significantly increased numbers of B cells, NK cells, and T cells.⁷⁶ Therefore, MSC-exosomes can potentially attenuate post stroke immunosuppression which in addition to extending protection against infection also creates a hospitable environment favoring brain remodeling.

Peripheral organ dysfunction after stroke

Secondary organ dysfunction is present after stroke and treatment strategies should expand beyond treating the injured brain to also target secondary organ recovery.^89–95 In recent years, there has been a focus on elucidating the mechanisms underlying peripheral organ dysfunction after stroke and the signaling pathways that may mediate inter organ communication, that are discussed in this section. Almost all the cell types constituting the NVU can produce extracellular vesicles such as exosomes and microvesicles which may mediate intercellular communication within the NVU as well as with distant target cells. Extracellular vesicles are emerging as intrinsic communication mediators between the brain and immune system.⁹⁶ Stroke-induced BBB disruption facilitates brain-derived antigens, and extracellular vesicles derived from injured brain cells to enter the blood stream alter peripheral immune response and potentially induce dysfunction of peripheral organs.⁹⁶ For instance, activated endothelial cells and astrocytes at the site of inflammation secrete extracellular vesicles that can cross the BBB, enter the peripheral circulation, and regulate acute cytokine response and mediate trafficking of peripheral immune cells to injured brain tissue.⁹⁶ On the other hand, systemic inflammation and circulating exosomes in response to infectious stimuli are potential neuroinflammatory mediators that can stimulate microglial and astrocytic activation and increase the expression of inflammatory cytokines in the brain.⁹⁷ High doses of endotoxins and severe peripheral inflammation can potentially damage the BBB and aggravate injury to the brain.⁹⁷ Therefore, it is important to understand the various communication axes between the brain and other organs and the role played by systemic inflammatory and immune responses in mediating secondary organ dysfunction after stroke. Figure 2 represents simplified schematic summarizing peripheral organ injury after stroke.

Figure 2.

Summary of stroke-induced peripheral organ injury. Stroke induces systemic inflammation and activates peripheral immune response which aggaravates ischemic brain injury as well as contributes to stroke-induced peripheral organ injury such as gut dysbiosis, cardiac dysfunction and renal disease.

Brain–immune axis

Systemic inflammatory responses after stroke are mainly driven by cytokines, chemokines and stress hormones.⁹⁸ Exosomes can cross the BBB and potentially transfer brain-derived antigens to the periphery and activate/aggravate peripheral immune response after stroke.^99,100 Exosomes can also transfer pro-inflammatory messages from the periphery to recipient brain cells.¹⁰¹ The spleen is a major regulator of peripheral immune response after ischemic stroke.^98,102–104 Large artery occlusion resulting in large lesions affecting cortical and sub-cortical areas stimulates systemic inflammatory responses irrespective of left- or right-sided stroke.¹⁰² Rats that underwent splenectomy two weeks prior to stroke induction were found to have significantly lower lesion volume and decreased local inflammation in the ischemic brain tissue characterized by reduction in number of activated microglia, macrophages, and neutrophils, indicating that peripheral inflammation mediated by the spleen plays an important role in neurodegeneration after stroke.¹⁰⁵ Stroke significantly alters spleen function, increases production of circulating proinflammatory cytokines, induces systemic activation of T and B lymphocytes, aids invasion of peripheral immune cells and inflammatory mediators to the brain, and exacerbates local inflammatory responses.^98,102 Lymphocyte infiltration in the ischemic brain begins about 48 h after acute ischemic stroke, and the invading T lymphocytes promote detrimental inflammatory cascades and contribute to secondary brain injury.^43,50 Cytotoxic CD8+ T-cells and CD4+ T-helper 1 (TH1) cells aggravate ischemic injury via secretion of proinflammatory cytokines such as TNF-α, Interferon-γ, interleukins (IL-2, IL-12), while CD4+TH2 cells secrete anti-inflammatory cytokines such as IL-4, IL-5, IL-10, and IL-13 which may exert neuroprotection.¹⁰⁶ In pre-clinical studies using rodent models of ischemic stroke, early after stroke (6–24 h after stroke) activated spleen cells were found to increase proinflammatory cytokines, while at later time points (96 h after stroke), thymus and splenic atrophy (gross spleen size reduction increased apoptosis and decreased splenocyte numbers) decreased CD8+T-cell counts, reduced inflammatory cytokine secretion and increased Tregs, resulting in a state of profound immunosuppression.^98,103,104 Post stroke immunosuppression increases risk of infection and contributes to post-stroke mortality.¹⁰⁷ Infection complicates acute stroke outcome in approximately 30% of patients.¹⁰⁸ Advancing age, history of alcohol abuse, hyperglycemia, hypertension, and cardiac abnormalities such as atrial fibrillation increased susceptibility to post stroke pneumonia.¹⁰⁹ Stroke survivors often encounter respiratory complications and abnormal breathing patterns.¹¹⁰ Stroke-induced immune suppression, altered stress responses and environmental factors/hygiene status (urinary catheterization, feeding tubes) are some of the factors that have been implicated in post stroke lung infection.¹⁰⁹ Immunomodulation after stroke has been found to improve neurological outcome after stroke and is a promising therapeutic strategy.^111,112

Brain–heart axis

Cardiac abnormalities are common in patients with neurological injuries, even in the absence of primary cardiac disease.^89,90 Stroke patients often present with symptoms of myocardial injury, ischemia-like electrocardiographic (ECG) changes and arrhythmias.^89,90,113 Cardiac complications such as heart attack, congestive heart failure, cardiac arrest and abnormal heart rhythms-like atrial fibrillation can contribute towards mortality and morbidity in stroke patients.^114,115 The risk of cardiac complications after stroke increases with stroke severity, neurological deficits and co-morbidities such as hypertension, diabetes, high cholesterol, and age^116,117 which are also predictors of worse functional outcome and secondary complications such as vasospasm, delayed cerebral ischemia and pulmonary edema.¹¹⁸ Ischemic stroke affecting the right insular cortex decreases sympathetic tone and causes parasympathetic over activity.¹¹⁹ Compared to left hemispheric stroke, right insular lesions are associated with higher mortality and cardiac dysfunction.^120–122 Inflammation and immune response may play an important role in mediating brain–heart interaction in the context of stroke.¹²³ Mice subjected to stroke exhibit cardiac dysfunction with evidence of cardiomyocyte hypertrophy, interstitial fibrosis and increased inflammatory factors, macrophages and oxidative stress in the heart compared to non-stroke control mice.¹²⁴ Concomitant with cell death in the ischemic brain, cardiomyocyte death in the heart tissue was also observed in rats subject to ischemic stroke.¹²⁵ Ischemia-damaged brain cells can secrete soluble factors that can transmit cell death signals to cardiomyocytes and induce cardiac dysfunction after stroke.^125,126 Proinflammatory cytokines secreted by damaged neuronal and glial cells can stimulate the posterior hypothalamus to increase sympathetic output and circulating catecholamine level.^127–129 Catecholamine surge is known to cause cardiac hypertrophy or myocardial ischemia.^130,131 Preclinical studies have demonstrated that elevated plasma catecholamine levels following ischemic stroke are directly proportional to the incidence of myocardial lesions and cardiac damage.¹³² Detailed mechanisms of brain–heart interaction after stroke have been discussed elsewhere.¹²³

The role of exosomes, miRs and immune response in mediating cardiac dysfunction after stroke and other cerebrovascular diseases is emerging.^123,124,133 Since stroke alters miR profile in the brain as well as in the circulation and some of these miRs may play critical roles in regulating the functioning of other organs such as heart and kidney, it is likely that alteration of exosomes and miR content influences secondary organ damage. For example, miR-126 has been well documented to play critical roles in maintaining vascular integrity and regulating angiogenesis.¹³⁴ Stroke significantly decreases circulating miR-126 level and miR-126 deficiency has been associated with several cardiac abnormalities and diseases including heart failure, atrial fibrillation and coronary artery disease.^{124,134–136} Employing a rodent model of stroke, ischemic stroke in rodents significantly decreases serum and heart miR-126 expression, and induces post stroke cardiac dysfunction.¹²⁴ In addition, endothelial cell miR-126 knockout mice exhibited cardiac dysfunction and increased cardiomyocyte hypertrophy, fibrosis, and inflammatory factor expression after ischemic stroke compared to control stroke mice.¹²⁴ On one hand, employing miRs as biomarkers to predict and diagnose such cardiac dysfunction may be clinically useful, and on the other, targeting these miRs might be necessary and/or amplify the therapeutic benefits of exosome therapy. Thus, improving cardiac function after stroke may provide a means to improve stroke recovery.

Brain–kidney axis

Stroke patients often present with symptoms of kidney dysfunction depending on severity and stroke subtype.^91,92 Patients with large artery atherosclerotic stroke often have low estimated glomerular filtration rate that is associated with increased stroke severity and six-month mortality rate, and worse neurological functional outcome.¹³⁷ Ischemic stroke patients can also develop acute kidney injury or chronic kidney disease.^138–140 Post ischemic systemic inflammation likely plays an important role in mediating kidney dysfunction after stroke. Stroke-induced elevation of inflammatory mediators such as C-reactive protein, IL-6 and ROS can also affect endothelial cell permeability in the kidney and enable entry of high-molecular-weight compounds, inflammatory mediators, immune cells and immune complexes, into the endothelium of glomerular filtration membrane.¹⁴¹ ROS can degrade the glomerular basement membrane, impair glomerular and tubular cell functions and promote renal fibrosis.^141–143 Interestingly, systemic administration of microvesicles derived from MSCs has been employed successfully to treat acute kidney injury in rats subject to renal ischemic reperfusion injury as well as extended protection against subsequent chronic kidney disease.¹⁴⁴ Future studies are needed to understand brain–kidney axis and how injury to one organ affects the other.

Brain–gut axis

It is increasingly evident that there exists a gut–brain axis which affects normal brain functioning as well as influences disease outcome under pathological states.¹⁴⁵ Gut dysbiosis after stroke is proportional to stroke severity and is characterized by an increase in opportunistic bacterial count and diversity often with decline in beneficial bacterial diversity numbers, and can adversely affect stroke outcome via several intersecting mechanisms.^93–95 Gut dysbiosis after stroke can affect neuroinflammatory and immune responses as the brain–gut axis consists of neural and humoral pathways that can regulate peripheral immune response and lymphocyte population and worsen neurological function.^145–147 Bacterial metabolites can also alter immune responses in favor of proinflammatory signaling.¹⁴⁸ In rats subjected to stroke, significant decrease in gastrointestinal motility and intestinal mucosal injury including necrosis and shedding of the intestinal epithelium was observed at 6 h after stroke which worsened by 24 h after stroke.^93,95 Intestinal lymphocytes infiltrate into the brain and aggravate ischemic injury.¹⁴⁶ Using a mouse stroke model, antibiotics were shown to induce alterations in the intestinal microbiota composition, and reduce ischemic lesion volume and neurological deficits.¹⁴⁶ Systemic inflammation can increase gut permeability.¹⁴⁹ In mice subjected to stroke, increased gut permeability facilitated bacterial translocation and leads to infection, sepsis and increase mortality.¹⁵⁰ While stroke increases gut permeability and bacterial translocation in both young and aged mice, aged animals were found to be particularly more susceptible to sepsis and immune dysfunction.¹⁵⁰ Bacterial translocation from the gut to mesenteric lymph nodes and peripheral organs such as spleen, liver and lungs triggers adaptive and innate immune responses.¹⁵⁰ Gut dysbiosis-mediated inflammatory responses can also affect cardiac and kidney function after stroke.^123,151,152 Interestingly, MSCs are known to have anti-microbial effects and can attenuate bacterial infection-induced sepsis, sepsis-induced inflammatory responses and sepsis-related mortality while improving bacterial clearance in sepsis rodent models.^153,154 Thus, there is promising therapeutic potential in amplifying the anti-microbial effects of MSCs-exosomes via cell conditioning and/or drug loading.¹⁵⁵

Conclusions and future directions

The interaction between various cell types within the NVU and neurovascular niche dictates the injury and repair processes after ischemic stroke, respectively. Understanding the sequential course of pathological events and transition from injury to repair mechanisms within the brain will enable neuroprotective and restorative interventions to amplify endogenous brain repair mechanisms to reach therapeutic levels. A rigorous understanding of the interdependence and coupling between various restorative events after stroke await fruition and it is crucial to develop therapeutics that targets multiple signaling pathways to maximize therapeutic efficacy. MSC-derived exosomes offer a powerful therapeutic tool to promote endogenous brain repair mechanisms after stroke while overcoming limitations of cell-based therapy. Current and future research focusing on optimization of MSC isolation and culture conditions, collection and characterization of exosomes, and importantly technology for expansion of MSC-derived exosomes to obtain large quantity of clinically effective exosomes are warranted. Optimizations of MSC-exosomes including manipulation of key miRs that promote neurorestorative effects to amplify therapeutic effects of MSC-exosomes are needed. The effects of MSC-exosome therapy on systemic inflammatory and peripheral immune response are yet to be fully understood. Local and systemic inflammatory and immune responses mediate delayed brain injury after stroke and likely regulate peripheral organ dysfunction after stroke. Recent and emerging literature delineating the effect of stroke on other organs such as the heart, gut, spleen, lungs, and kidney stress the importance of treating secondary injury to peripheral organs after stroke to improve overall recovery. It is likely that there are several such communication axes between the various organs which dictate over all brain recovery after stroke. When employing systemic administration of MSC-exosomes to treat the ischemic brain, it is likely that there is some level of direct/indirect protection rendered to peripheral organs such as heart, kidney, spleen and gut, and further studies are warranted.

Footnotes

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by National Institute of Neurological Disorders and Stroke R01NS083078 (JC), R01NS099030 (JC), R01NS097747 (JC) and R01NS088656 (MC), National Institutes of Health R01HL143432 (JC), and American Heart Association award 17POST33410580 (PV).

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

References

Writing Group

Benjamin

Blaha

et al.

Heart disease and stroke statistics – 2017 update: a report from the American Heart Association. Circulation 2017; 135: e146–e603.

Lai

Zhang

Wang

. Excitotoxicity and stroke: identifying novel targets for neuroprotection. Prog Neurobiol 2014; 115: 157–188.

Deng

et al.

MicroRNAs regulate mitochondrial function in cerebral ischemia-reperfusion injury. Int J Mol Sci 2015; 16: 24895–24917.

Iadecola

Anrather

. The immunology of stroke: from mechanisms to translation. Nat Med 2011; 17: 796–808.

National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group. Tissue plasminogen activator for acute ischemic stroke. N Engl J Med 1995; 333: 1581–1587.

Wade

Wood

Hewer

. Recovery after stroke – the first 3 months. J Neurol Neurosurg Psychiatry 1985; 48: 7–13.

Duncan

Lai

Keighley

. Defining post-stroke recovery: implications for design and interpretation of drug trials. Neuropharmacology 2000; 39: 835–841.

Jorgensen

Nakayama

Raaschou

et al.

Outcome and time course of recovery in stroke. Part II: Time course of recovery. The Copenhagen Stroke Study. Arch Phys Med Rehabil 1995; 76: 406–412.

Liu

Zhang

et al.

Contralesional axonal remodeling of the corticospinal system in adult rats after stroke and bone marrow stromal cell treatment. Stroke 2008; 39: 2571–2577.

10.

Chen

Venkat

Zacharek

et al.

Neurorestorative therapy for stroke. Front Hum Neurosci 2014; 8: 382.

11.

Ohab

Fleming

Blesch

et al.

A neurovascular niche for neurogenesis after stroke. J Neurosci 2006; 26: 13007–13016.

12.

Minnerup

Sutherland

Buchan

et al.

Neuroprotection for stroke: current status and future perspectives. Int J Mol Sci 2012; 13: 11753–11772.

13.

Powers

Derdeyn

Biller

et al.

2015 American Heart Association/American Stroke Association focused update of the 2013 guidelines for the early management of patients with acute ischemic stroke regarding endovascular treatment: a guideline for healthcare professionals from the American Heart Association/American Stroke Association. Stroke 2015; 46: 3020–3035.

14.

Jolkkonen

Kwakkel

. Translational hurdles in stroke recovery studies. Transl Stroke Res 2016; 7: 331–342.

15.

Pennypacker

Bix

Fraser

. Correcting the trajectory of stroke therapeutic research. Transl Stroke Res 2017; 8: 65–66.

16.

Zai

Ferrari

Subbaiah

et al.

Inosine alters gene expression and axonal projections in neurons contralateral to a cortical infarct and improves skilled use of the impaired limb. J Neurosci 2009; 29: 8187–8197.

17.

Zhang

Chopp

. Neurorestorative therapies for stroke: underlying mechanisms and translation to the clinic. Lancet Neurol 2009; 8: 491–500.

18.

Hayashi

Noshita

Sugawara

et al.

Temporal profile of angiogenesis and expression of related genes in the brain after ischemia. J Cereb Blood Flow Metab 2003; 23: 166–180.

19.

Yan

Venkat

Chopp

et al.

Neurorestorative responses to delayed hMSC treatment of stroke in type 2 diabetic rats. Stroke 2016; 47: 2850–2858.

20.

Venkat

Chopp

Chen

. Blood–brain barrier disruption, vascular impairment, and ischemia/reperfusion damage in diabetic stroke. J Am Heart Assoc 2017; 6: e005819.

21.

Wei

Erinjeri

Rovainen

et al.

Collateral growth and angiogenesis around cortical stroke. Stroke 2001; 32: 2179–2184.

22.

Gutierrez-Fernandez

Rodriguez-Frutos

Ramos-Cejudo

et al.

Effects of intravenous administration of allogenic bone marrow- and adipose tissue-derived mesenchymal stem cells on functional recovery and brain repair markers in experimental ischemic stroke. Stem Cell Res Ther 2013; 4: 11.

23.

Zhang

Chopp

et al.

Intravenous administration of human umbilical tissue-derived cells improves neurological function in aged rats after embolic stroke. Cell Transplant 2013; 22: 1569–1576.

24.

Ueno

Chopp

Zhang

et al.

Axonal outgrowth and dendritic plasticity in the cortical peri-infarct area after experimental stroke. Stroke 2012; 43: 2221–2228.

25.

Guangliang

Quan

Lian

et al.

Magnetic resonance imaging investigation of axonal remodeling and angiogenesis after embolic stroke in sildenafil-treated rats. J Cereb Blood Flow Metab 2008; 28: 1440–1448.

26.

Liu

Zhang

et al.

Bone marrow stromal cells enhance inter- and intracortical axonal connections after ischemic stroke in adult rats. J Cereb Blood Flow Metab 2010; 30: 1288–1295.

27.

Zhang

Chopp

Roberts

et al.

Stroke increases neural stem cells and angiogenesis in the neurogenic niche of the adult mouse. PLoS One 2014; 9: e113972.

28.

Manoonkitiwongsa

Jackson-Friedman

McMillan

et al.

Angiogenesis after stroke is correlated with increased numbers of macrophages: the clean-up hypothesis. J Cereb Blood Flow Metab 2001; 21: 1223–1231.

29.

Zhang

Chopp

Gregg

et al.

Patterns and dynamics of subventricular zone neuroblast migration in the ischemic striatum of the adult mouse. J Cereb Blood Flow Metab 2009; 29: 1240–1250.

30.

Zhang

Wang

et al.

Activated neural stem cells contribute to stroke-induced neurogenesis and neuroblast migration toward the infarct boundary in adult rats. J Cereb Blood Flow Metab 2004; 24: 441–448.

31.

Arvidsson

Collin

Kirik

et al.

Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat Med 2002; 8: 963–970.

32.

Parent

Vexler

Gong

et al.

Rat forebrain neurogenesis and striatal neuron replacement after focal stroke. Ann Neurol 2002; 52: 802–813.

33.

Hua

Zheng Gang

Lei

et al.

Coupling of angiogenesis and neurogenesis in cultured endothelial cells and neural progenitor cells after stroke. J Cereb Blood Flow Metab 2007; 28: 764–771.

34.

Jin

Wang

Xie

et al.

Evidence for stroke-induced neurogenesis in the human brain. Proc Natl Acad Sci U S A 2006; 103: 13198–13202.

35.

Yoshimura

Takagi

Harada

et al.

FGF-2 regulation of neurogenesis in adult hippocampus after brain injury. Proc Natl Acad Sci U S A 2001; 98: 5874–5879.

36.

Robin

Zhang

Wang

et al.

Stromal cell-derived factor 1alpha mediates neural progenitor cell motility after focal cerebral ischemia. J Cereb Blood Flow Metab 2006; 26: 125–134.

37.

Hayakawa

Seo

Pham

et al.

Cerebral endothelial derived vascular endothelial growth factor promotes the migration but not the proliferation of oligodendrocyte precursor cells in vitro. Neurosci Lett 2012; 513: 42–46.

38.

Imitola

Raddassi

Park

et al.

Directed migration of neural stem cells to sites of CNS injury by the stromal cell-derived factor 1alpha/CXC chemokine receptor 4 pathway. Proc Natl Acad Sci U S A 2004; 101: 18117–18122.

39.

Liu

Pan

et al.

Abstract 177: exosomal micrornas from ischemic cerebral endothelial cells and neural stem cells regulate coupling of neurogenesis and angiogenesis. Stroke 2018; 49: A177.

40.

Madelaine

Sloan

Huber

et al.

microRNA-9 couples brain neurogenesis and angiogenesis. Cell Rep 2017; 20: 1533–1542.

41.

Buller

Chopp

Ueno

et al.

Regulation of serum response factor by miRNA-200 and miRNA-9 modulates oligodendrocyte progenitor cell differentiation. Glia 2012; 60: 1906–1914.

42.

Okada

Copeland

Mori

et al.

P-selectin and intercellular adhesion molecule-1 expression after focal brain ischemia and reperfusion. Stroke 1994; 25: 202–211.

43.

Emsley

Smith

Gavin

et al.

An early and sustained peripheral inflammatory response in acute ischaemic stroke: relationships with infection and atherosclerosis. J Neuroimmunol 2003; 139: 93–101.

44.

Gelderblom

Leypoldt

Steinbach

et al.

Temporal and spatial dynamics of cerebral immune cell accumulation in stroke. Stroke 2009; 40: 1849–1857.

45.

Bianco

Pravettoni

Colombo

et al.

Astrocyte-derived ATP induces vesicle shedding and IL-1 beta release from microglia. J Immunol 2005; 174: 7268–7277.

46.

Bianco

Perrotta

Novellino

et al.

Acid sphingomyelinase activity triggers microparticle release from glial cells. EMBO J 2009; 28: 1043–1054.

47.

Turola

Furlan

Bianco

et al.

Microglial microvesicle secretion and intercellular signaling. Front Physiol 2012; 3: 149.

48.

Gupta

Pulliam

. Exosomes as mediators of neuroinflammation. J Neuroinflammation 2014; 11: 68.

49.

Brennan

Suh

Won

et al.

NADPH oxidase is the primary source of superoxide induced by NMDA receptor activation. Nat Neurosci 2009; 12: 857–863.

50.

Liesz

Zhou

Mracsko

et al.

Inhibition of lymphocyte trafficking shields the brain against deleterious neuroinflammation after stroke. Brain 2011; 134: 704–720.

51.

Yilmaz

Granger

. Leukocyte recruitment and ischemic brain injury. Neuromol Med 2010; 12: 193–204.

52.

Jin

Yang

. Inflammatory mechanisms in ischemic stroke: role of inflammatory cells. J Leukoc Biol 2010; 87: 779–789.

53.

Guo

et al.

Microglia/macrophage polarization dynamics reveal novel mechanism of injury expansion after focal cerebral ischemia. Stroke 2012; 43: 3063–3070.

54.

Murray

. Macrophage Polarization. Annu Rev Physiol 2017; 79: 541–566.

55.

Ailawadi

Wang

et al.

Pathologic function and therapeutic potential of exosomes in cardiovascular disease. Biochim Biophys Acta 2015; 1852: 1–11.

56.

Xin

Chopp

. Exosomes/miRNAs as mediating cell-based therapy of stroke. Front Cell Neurosci 2014; 8: 377.

57.

Koutsis

Siasos

Spengos

. The emerging role of microRNA in stroke. Curr Topics Med Chem 2013; 13: 1573–1588.

58.

Liu

Wang

et al.

Plasma exosomal miR-422a and miR-125b-2-3p serve as biomarkers for ischemic stroke. Curr Neurovasc Res 2017; 14: 330–337.

59.

Tamkovich

Tutanov

Laktionov

. Exosomes: generation, structure, transport, biological activity, and diagnostic application. Biochem (Moscow) Supplt Ser A 2016; 10: 163–173.

60.

ELA

Mager

Breakefield

et al.

Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov 2013; 12: 347–357.

61.

Otero-Ortega

Gomez de Frutos

Laso-Garcia

et al.

Exosomes promote restoration after an experimental animal model of intracerebral hemorrhage. J Cereb Blood Flow Metab 2018; 38: 767–779.

62.

Chen

Liu

et al.

Elucidation of exosome migration across the blood-brain barrier model in vitro. Cell Mol Bioeng 2016; 9: 509–529.

63.

Xin

Cui

et al.

Systemic administration of exosomes released from mesenchymal stromal cells promote functional recovery and neurovascular plasticity after stroke in rats. J Cereb Blood Flow Metab 2013; 33: 1711–1715.

64.

Chopp

Zhang

. Emerging potential of exosomes and noncoding microRNAs for the treatment of neurological injury/diseases. Expert Opin Emerg Drugs 2015; 20: 523–526.

65.

Luarte

Batiz

Wyneken

et al.

Potential therapies by stem cell-derived exosomes in CNS diseases: focusing on the neurogenic niche. Stem Cells Int 2016; 2016: 5736059.

66.

Liu

Yan

et al.

Cell based therapies for ischemic stroke: from basic science to bedside. Prog Neurobiol 2014; 115: 92–115.

67.

Venkat

Shen

Chopp

et al.

Cell-based and pharmacological neurorestorative therapies for ischemic stroke. Neuropharmacology 2018; 134(Pt B): 310–322.

68.

Yeo

Lai

Zhang

et al.

Mesenchymal stem cell: an efficient mass producer of exosomes for drug delivery. Adv Drug Deliv Rev 2013; 65: 336–341.

69.

Phinney

Pittenger

. Concise review: MSC-derived exosomes for cell-free therapy. Stem cells 2017; 35: 851–858.

70.

Chen

Wang

et al.

Ischemic rat brain extracts induce human marrow stromal cell growth factor production. Neuropathology 2002; 22: 275–279.

71.

Zacharek

Chen

Cui

et al.

Angiopoietin1/Tie2 and VEGF/Flk1 induced by MSC treatment amplifies angiogenesis and vascular stabilization after stroke. J Cereb Blood Flow Metab 2007; 27: 1684–1691.

72.

Chen

Zhang

et al.

Intravenous administration of human bone marrow stromal cells induces angiogenesis in the ischemic boundary zone after stroke in rats. Circ Res 2003; 92: 692–699.

73.

Xin

Liu

et al.

MiR-133b promotes neural plasticity and functional recovery after treatment of stroke with multipotent mesenchymal stromal cells in rats via transfer of exosome-enriched extracellular particles. Stem Cells 2013; 31: 2737–2746.

74.

Xin

Katakowski

Wang

et al.

MicroRNA cluster miR-17-92 cluster in exosomes enhance neuroplasticity and functional recovery after stroke in rats. Stroke 2017; 48: 747–753.

75.

Xin

Buller

et al.

Exosome-mediated transfer of miR-133b from multipotent mesenchymal stromal cells to neural cells contributes to neurite outgrowth. Stem cells 2012; 30: 1556–1564.

76.

Doeppner

Herz

Görgens

et al.

Extracellular vesicles improve post-stroke neuroregeneration and prevent postischemic immunosuppression. Stem Cells Transl Med 2015; 4: 1131–1143.

77.

Peng

et al.

Increased brain-specific MiR-9 and MiR-124 in the serum exosomes of acute ischemic stroke patients. PLoS One 2016; 11: e0163645.

78.

Otero-Ortega

Laso-García

Gómez-de Frutos

MdC

et al.

White matter repair after extracellular vesicles administration in an experimental animal model of subcortical stroke. Sci Rep 2017; 7: 44433.

79.

Marote

Teixeira

Mendes-Pinheiro

et al.

MSCs-derived exosomes: cell-secreted nanovesicles with regenerative potential. Front Pharmacol 2016; 7: 231.

80.

Börger

Bremer

Ferrer-Tur

et al.

Mesenchymal Stem/stromal cell-derived extracellular vesicles and their potential as novel immunomodulatory therapeutic agents. Int J Mol Sci 2017; 18: 1450.

81.

Zhang

Chopp

Liu

et al.

Exosomes derived from mesenchymal stromal cells promote axonal growth of cortical neurons. Mol Neurobiol 2017; 54: 2659–2673.

82.

Fruhbeis

Frohlich

Kuo

et al.

Extracellular vesicles as mediators of neuron-glia communication. Front Cell Neurosci 2013; 7: 182.

83.

Venkat

Chopp

Chen

. New insights into coupling and uncoupling of cerebral blood flow and metabolism in the brain. Croat Med J 2016; 57: 223–228.

84.

Xin

Wang

et al.

Secondary release of exosomes from astrocytes contributes to the increase in neural plasticity and improvement of functional recovery after stroke in rats treated with exosomes harvested from MicroRNA 133b-overexpressing multipotent mesenchymal stromal cells. Cell Transplant 2017; 26: 243–257.

85.

Acosta

Tajiri

Hoover

et al.

Intravenous bone marrow stem cell grafts preferentially migrate to spleen and abrogate chronic inflammation in stroke. Stroke 2015; 46: 2616–2627.

86.

Lou

Chen

Zheng

et al.

Mesenchymal stem cell-derived exosomes as a new therapeutic strategy for liver diseases. Exp Mol Med 2017; 49: e346.

87.

Zhang

Yin

Lai

et al.

Mesenchymal stem cells secrete immunologically active exosomes. Stem Cells Dev 2014; 23: 1233–1244.

88.

Sicco

Reverberi

Balbi

et al.

Mesenchymal stem cell-derived extracellular vesicles as mediators of anti-inflammatory effects: endorsement of macrophage polarization. Stem Cells Transl Med 2017; 6: 1018–1028.

89.

Koroshetz

Benner

et al.

Neuroanatomic correlates of stroke-related myocardial injury. Neurology 2006; 66: 1325–1329.

90.

Tokgozoglu

Batur

Topcuoglu

et al.

Effects of stroke localization on cardiac autonomic balance and sudden death. Stroke 1999; 30: 1307–1311.

91.

Kudo

Konta

Degawa

et al.

Relationship between kidney damage and stroke types in Japanese patients. Clin Exp Nephrol 2012; 16: 564–569.

92.

Wang

Luo

Wang

et al.

Comparison of associations of outcomes after stroke with estimated GFR using Chinese modifications of the MDRD study and CKD-EPI creatinine equations: results from the China National Stroke Registry. Am J Kidney Dis 2014; 63: 59–67.

93.

Zhu

Chuai

. Changes in serum ghrelin and small intestinal motility in rats with ischemic stroke. Anat Rec 2012; 295: 307–312.

94.

Winek

Engel

Koduah

et al.

Depletion of cultivatable gut microbiota by broad-spectrum antibiotic pretreatment worsens outcome after murine stroke. Stroke 2016; 47: 1354–1363.

95.

Liu

Luo

Kou

et al.

Ischemic stroke damages the intestinal mucosa and induces alteration of the intestinal lymphocytes and CCL19 mRNA in rats. Neurosci Lett 2017; 658: 165–170.

96.

Dickens

Tovar

YRLB

Yoo

et al.

Astrocyte-shed extracellular vesicles regulate the peripheral leukocyte response to inflammatory brain lesions. Sci Signal 2017; 10(473): eaai7696.

97.

Wang

Kodali

et al.

In vivo evidence for the contribution of peripheral circulating inflammatory exosomes to neuroinflammation. J Neuroinflamm 2018; 15: 8.

98.

Offner

Subramanian

Parker

et al.

Experimental stroke induces massive, rapid activation of the peripheral immune system. J Cereb Blood Flow Metab 2006; 26: 654–665.

99.

Selmaj

Mycko

Raine

et al.

The role of exosomes in CNS inflammation and their involvement in multiple sclerosis. J Neuroimmunol 2017; 306: 1–10.

100.

de Rivero Vaccari

Brand

3rd Adamczak

et al.

Exosome-mediated inflammasome signaling after central nervous system injury. J Neurochem 2016; 136(Suppl 1): 39–48.

101.

Balusu

Van Wonterghem

De Rycke

et al.

Identification of a novel mechanism of blood-brain communication during peripheral inflammation via choroid plexus-derived extracellular vesicles. EMBO Mol Med 2016; 8: 1162–1183.

102.

Gendron

Teitelbaum

Cossette

et al.

Temporal effects of left versus right middle cerebral artery occlusion on spleen lymphocyte subsets and mitogenic response in Wistar rats. Brain Res 2002; 955: 85–97.

103.

Offner

Subramanian

Parker

et al.

Splenic atrophy in experimental stroke is accompanied by increased regulatory T cells and circulating macrophages. J Immunol 2006; 176: 6523–6531.

104.

Vendrame

Gemma

Pennypacker

et al.

Cord blood rescues stroke-induced changes in splenocyte phenotype and function. Exp Neurol 2006; 199: 191–200.

105.

Ajmo

Vernon

DOL

Collier

et al.

The spleen contributes to stroke-induced neurodegeneration. J Neurosci Res 2008; 86: 2227–2234.

106.

Arumugam

Granger

Mattson

. Stroke and T-cells. Neuromol Med 2005; 7: 229–242.

107.

Prass

Meisel

Hoflich

et al.

Stroke-induced immunodeficiency promotes spontaneous bacterial infections and is mediated by sympathetic activation reversal by poststroke T helper cell type 1-like immunostimulation. J Exp Med 2003; 198: 725–736.

108.

Westendorp

Nederkoorn

Vermeij

et al.

Post-stroke infection: a systematic review and meta-analysis. BMC Neurol 2011; 11: 110.

109.

Matz

Seyfang

Dachenhausen

et al.

Post-stroke pneumonia at the stroke unit – a registry based analysis of contributing and protective factors. BMC Neurol 2016; 16: 107.

110.

Rochester

Mohsenin

. Respiratory complications of stroke. Semin Respir Crit Care Med 2002; 23: 248–260.

111.

Yang

Hamilton

Valenzuela

et al.

Multipotent adult progenitor cells enhance recovery after stroke by modulating the immune response from the spleen. Stem cells 2017; 35: 1290–1302.

112.

Dames C, Winek K, Beckers Y, et al. Immunomodulatory treatment with systemic GM-CSF augments pulmonary immune responses and improves neurological outcome after experimental stroke. J Neuroimmunol. Epub ahead of print 9 March 2018. DOI: 10.1016/j.jneuroim.2018.03.005.

113.

Cheshire

Jr Saper

. The insular cortex and cardiac response to stroke. Neurology 2006; 66: 1296–1297.

114.

Samuels

. The brain–heart connection. Circulation 2007; 116: 77–84.

115.

Kolin

Norris

. Myocardial damage from acute cerebral lesions. Stroke 1984; 15: 990–993.

116.

Yoshimura

Toyoda

Ohara

et al.

Takotsubo cardiomyopathy in acute ischemic stroke. Ann Neurol 2008; 64: 547–554.

117.

Wolf

Abbott

Kannel

. Atrial fibrillation as an independent risk factor for stroke: the Framingham Study. Stroke 1991; 22: 983–988.

118.

Milionis

Faouzi

Cordier

et al.

Characteristics and early and long-term outcome in patients with acute ischemic stroke and low ejection fraction. Int J Cardiol 2013; 168: 1082–1087.

119.

Soros

Hachinski

. Cardiovascular and neurological causes of sudden death after ischaemic stroke. Lancet Neurol 2012; 11: 179–188.

120.

Laowattana

Zeger

Lima

et al.

Left insular stroke is associated with adverse cardiac outcome. Neurology 2006; 66: 477–483. discussion 463.

121.

Christensen

Boysen

Christensen

et al.

Insular lesions, ECG abnormalities, and outcome in acute stroke. J Neurol Neurosurg Psychiatry 2005; 76: 269–271.

122.

Algra

Gates

Fox

et al.

Side of brain infarction and long-term risk of sudden death in patients with symptomatic carotid disease. Stroke 2003; 34: 2871–2875.

123.

Chen

Venkat

Seyfried

et al.

Brain-heart interaction: cardiac complications after stroke. Circ Res 2017; 121: 451–468.

124.

Chen

Cui

Yang

et al.

MiR-126 affects brain-heart interaction after cerebral ischemic stroke. Transl Stroke Res 2017; 8: 374–385.

125.

Ishikawa

Tajiri

Vasconcellos

et al.

Ischemic stroke brain sends indirect cell death signals to the heart. Stroke 2013; 44: 3175–3182.

126.

Gonzales-Portillo

Ishikawa

Shinozuka

et al.

Stroke and cardiac cell death: two peas in a pod. Clin Neurol Neurosurg 2016; 142: 145–147.

127.

Johnson

Campisi

Sharkey

et al.

Catecholamines mediate stress-induced increases in peripheral and central inflammatory cytokines. Neuroscience 2005; 135: 1295–1307.

128.

Tracey

. The inflammatory reflex. Nature 2002; 420: 853–859.

129.

Borovikova

Ivanova

Zhang

et al.

Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin. Nature 2000; 405: 458–462.

130.

Samuels

. Neurogenic heart disease: a unifying hypothesis. Am J Cardiol 1987; 60: 15j–19j.

131.

Schomig

. Catecholamines in myocardial ischemia. Systemic and cardiac release. Circulation 1990; 82: Ii13–22.

132.

Hachinski

Smith

Silver

et al.

Acute myocardial and plasma catecholamine changes in experimental stroke. Stroke 1986; 17: 387–390.

133.

Yan

Chopp

et al.

Abstract WMP84: immune response mediates brain- heart interaction after subarachnoid hemorrhage. Stroke 2018; 49: AWMP84.

134.

Wang

Aurora

Johnson

et al.

The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis. Dev Cell 2008; 15: 261–271.

135.

Long

Wang

et al.

Circulating miR-30a, miR-126 and let-7b as biomarker for ischemic stroke in humans. BMC Neurol 2013; 13: 178.

136.

Qiang

Hong

Ningfu

et al.

Expression of miR-126 and miR-508-5p in endothelial progenitor cells is associated with the prognosis of chronic heart failure patients. Int J Cardiol 2013; 168: 2082–2088.

137.

Yeh

Jeng

Tang

et al.

Low estimated glomerular filtration rate is associated with poor outcomes in patients who suffered a large artery atherosclerosis stroke. Atherosclerosis 2015; 239: 328–334.

138.

Kumai

Kamouchi

Hata

et al.

Proteinuria and clinical outcomes after ischemic stroke. Neurology 2012; 78: 1909–1915.

139.

Tsagalis

Akrivos

Alevizaki

et al.

Long-term prognosis of acute kidney injury after first acute stroke. Clin J Am Soc Nephrol 2009; 4: 616–622.

140.

Covic

Schiller

Mardare

et al.

The impact of acute kidney injury on short-term survival in an Eastern European population with stroke. Nephrol Dial Transplant 2008; 23: 2228–2234.

141.

Baud

Ardaillou

. Reactive oxygen species: production and role in the kidney. Am J Physiol 1986; 251: F765–776.

142.

Rhyu

Yang

et al.

Role of reactive oxygen species in TGF-beta1-induced mitogen-activated protein kinase activation and epithelial-mesenchymal transition in renal tubular epithelial cells. J Am Soc Nephrol 2005; 16: 667–675.

143.

Jiang

Liu

Dusting

et al.

NADPH oxidase-dependent redox signaling in TGF-beta-mediated fibrotic responses. Redox Biol 2014; 2: 267–272.

144.

Gatti

Bruno

Deregibus

et al.

Microvesicles derived from human adult mesenchymal stem cells protect against ischaemia-reperfusion-induced acute and chronic kidney injury. Nephrol Dial Transplant 2011; 26: 1474–1483.

145.

Bercik

Collins

Verdu

. Microbes and the gut-brain axis. Neurogastroenterol Motil 2012; 24: 405–413.

146.

Benakis

Brea

Caballero

et al.

Commensal microbiota affects ischemic stroke outcome by regulating intestinal γδT cells. Nat Med 2016; 22: 516–523.

147.

Singh

Roth

Llovera

et al.

Microbiota dysbiosis controls the neuroinflammatory response after stroke. J Neurosci 2016; 36: 7428–7440.

148.

Arpaia

Campbell

Fan

et al.

Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation. Nature 2013; 504: 451–455.

149.

Kelly

Kennedy

Cryan

et al.

Breaking down the barriers: the gut microbiome, intestinal permeability and stress-related psychiatric disorders. Front Cell Neurosci 2015; 9: 392.

150.

Crapser

Ritzel

Verma

et al.

Ischemic stroke induces gut permeability and enhances bacterial translocation leading to sepsis in aged mice. Aging 2016; 8: 1049–1060.

151.

Adrie

Parlato

Salmi

et al.

Bacterial translocation and plasma cytokines during transcatheter and open-heart aortic valve implantation. Shock 2015; 43: 62–67.

152.

Nagatomo

Tang

. Intersections between microbiome and heart failure: revisiting the gut hypothesis. J Card Fail 2015; 21: 973–980.

153.

Krasnodembskaya

Song

Fang

et al.

Antibacterial effect of human mesenchymal stem cells is mediated in part from secretion of the antimicrobial peptide LL-37. Stem cells 2010; 28: 2229–2238.

154.

Mei

Haitsma

Dos Santos

et al.

Mesenchymal stem cells reduce inflammation while enhancing bacterial clearance and improving survival in sepsis. Am J Respir Crit Care Med 2010; 182: 1047–1057.

155.

Alcayaga-Miranda

Cuenca

Khoury

. Antimicrobial activity of mesenchymal stem cells: current status and new perspectives of antimicrobial peptide-based therapies. Front Immunol 2017; 8: 339.