Cellular Communication Networks Mediated by Microglia in Ischemic Stroke

Stroke is a leading cause of mortality and long-term disability worldwide, with limited spontaneous repair processes occurring after injury. Immune cells are involved in multiple aspects of ischemic stroke, from early damage processes to late recovery-related events. Compared with the substantial advances that have been made in elucidating how immune cells modulate acute ischemic injury, the understanding of the impact of the immune system on functional recovery is limited. In this review, we summarized the mechanisms of brain repair after ischemic stroke from both the neuronal and non-neuronal perspectives, and we review advances in understanding of the effects on functional recovery after ischemic stroke mediated by infiltrated peripheral innate and adaptive immune cells, immune cell-released cytokines and cell-cell interactions. We also highlight studies that advance our understanding of the mechanisms underlying functional recovery mediated by peripheral immune cells after ischemia. Insights into these processes will shed light on the double-edged role of infiltrated peripheral immune cells in functional recovery after ischemic stroke and provide clues for new therapies for improving neurological function.

Following ischemic stroke, peripheral immune cell infiltration is characterized by myeloid cell predominance in the acute phase and lymphoid cell infiltration in the subacute to chronic phases. Endothelial cells, as a critical interface between the peripheral circulation and the brain, upregulate adhesion molecules to facilitate immune cell infiltration. However, it remains unclear whether endothelial cells exhibit functional differences at different stages after ischemic stroke and how these differences affect immune cell infiltration. We performed single-cell RNA sequencing on peripheral immune and endothelial cells from Sham and middle cerebral artery occlusion (MCAO) mice at 3 and 14 days post-MCAO. Subsequent analysis of the sequencing data, combined with flow cytometry and immunofluorescence staining, was used to investigate the relationship between endothelial cell changes at different stages of stroke and immune cell infiltration. We observed that the infiltration capacity of peripheral immune cells did not significantly increase at different stages after MCAO. However, endothelial cells underwent significant changes. By Day 3 post-MCAO, there was an increased proportion of venous endothelial cells with enhanced angiogenesis and adhesion functions. In this acute phase, newly formed venous endothelial cells with high expression of the adhesion molecule ICAM-1 were observed, promoting the infiltration of myeloid cells and NKT cells. From the acute to chronic phases, endothelial angiogenesis gradually decreased, accompanied by a marked increase in antigen presentation function. At 14 days post-MCAO, an increased proportion of VCAM-1-expressing venous endothelial cells was observed, potentially facilitating the infiltration of T cells and a subset of neutrophils. Furthermore, we discovered that the differential changes in venous endothelial cells at different stages after MCAO may be driven by distinct differentiation and proliferation patterns regulated by different signaling pathways. Our study highlights that the differential expression of adhesion molecules and functional changes in endothelial cells at distinct stages after ischemic stroke may regulate the infiltration patterns of peripheral immune cells.

Patients presenting with stroke symptoms suffer from either ischemic stroke, hemorrhagic stroke, transient ischemic attacks (TIA), or "stroke mimics," which include benign headaches, epilepsy, and vestibular disorders. As ischemic and hemorrhagic stroke patients require different medical treatments, early identification of the underlying cause of symptoms is essential for tailored and urgent medical intervention. This study investigates whether extracellular vesicles (EVs), present in peripheral blood of patients presenting with stroke symptoms, can be used to identify patients with ischemic stroke. Blood was collected from 155 patients presenting with stroke symptoms in the emergency room and analyzed for EVs by flow cytometry (ethics approval number NL72929.018.20). The primary endpoint was to compare platelet EV concentrations between patients with (n = 66) and without (n = 89) ischemic stroke. Concentrations of EVs from both activated platelets and leukocytes were lower in patients presenting with ischemic stroke compared to other patients (p = 0.038 and p = 0.015, respectively). No significant differences in other EV types were observed. In addition, ischemic stroke patients were older and had a higher diastolic blood pressure compared to patients with other diagnoses. In a multivariable analysis, leukocyte EVs and diastolic blood pressure were independent indicators of ischemic stroke. To conclude, this study demonstrates that the plasma concentration of leukocyte EVs can be useful to identify ischemic stroke patients in an emergency setting.

Ischemic stroke is a leading cause of mortality and long-term disability globally. One of its aspects is the breakdown of the blood-brain barrier (BBB). The disruption of BBB's integrity during stroke exacerbates neurological damage and hampers therapeutic intervention. Recent advances in regenerative medicine suggest that mesenchymal stem cells (MSCs) derived extracellular vesicles (EVs) show promise for restoring BBB integrity. This review explores the potential of MSC-derived EVs in mediating neuroprotective and reparative effects on the BBB after ischemic stroke. We highlight the molecular cargo of MSC-derived EVs, including miRNAs, and their role in enhancing angiogenesis, promoting the BBB and neural repair, and mitigating apoptosis. Furthermore, we discuss the challenges associated with the clinical translation of MSC-derived EV therapies and the possibilities of further enhancing EVs' innate protective qualities. Our findings underscore the need for further research to optimize the therapeutic potential of EVs and establish their efficacy and safety in clinical settings.

Engineered extracellular vesicles for ischemic stroke treatment

Artificial mesenchymal stem cell extracellular vesicles enhanced ischemic stroke treatment through targeted remodeling brain microvascular endothelial cells

Stem cell‐based therapies and extracellular vesicle (EV) treatment have demonstrated significant potential for neuroprotection against ischemic stroke. Although the neuroprotective mechanisms are not yet fully understood, targeting microglia is central to promoting neuroprotection. Microglia are the resident immune cells of the central nervous system. These cells are crucial in the pathogenesis of ischemic stroke. They respond rapidly to the site of injury by releasing pro‐inflammatory cytokines, phagocytizing dead cells and debris, and recruiting peripheral immune cells to the ischemic area. Although these responses are essential for clearing damage and initiating tissue repair, excessive or prolonged microglial activation can exacerbate brain injury, leading to secondary neuroinflammation and neurodegeneration. Moreover, microglia exhibit a dynamic range of activation states with the so‐called M1 pro‐inflammatory and M2 anti‐inflammatory phenotypes, representing the two ends of the spectrum. The delivery of both EVs and stem cells modulates microglial activation, suppressing pro‐inflammatory genes, influencing the expression of transcription factors, and altering receptor expression, ultimately contributing to neuroprotection. These findings underscore the importance of understanding the complex and dynamic role of microglia in the development of effective neuroprotective strategies to reduce the effects of ischemic stroke. In this review, we examine the current state of knowledge regarding the role of microglia in ischemic stroke, including their molecular and cellular mechanisms, activation states, and interactions with other cells. We also discuss the multifaceted contributions of microglia to stem cell‐ and EV‐based neuroprotection during an ischemic stroke to provide a comprehensive understanding of microglial functions and their potential implications in stroke therapies.

Neuroinflammation is a fundamental feature of many chronic neurodegenerative diseases, where it contributes to disease onset, progression, and severity. This persistent inflammatory state arises from the activation of innate and adaptive immune responses within the central nervous system (CNS), orchestrated by a complex interplay of resident immune cells, infiltrating peripheral immune cells, and an array of molecular mediators such as cytokines, chemokines, and extracellular vesicles. Among CNS-resident cells, microglia play a central role, exhibiting a dynamic spectrum of phenotypes ranging from neuroprotective to neurotoxic. In chronic neurodegenerative diseases, sustained microglial activation often leads to the amplification of inflammatory cascades, reinforcing a pathogenic cycle of immune-mediated damage. Intercellular communication within the inflamed CNS is central to the persistence and progression of neuroinflammation. Microglia engage in extensive crosstalk with astrocytes, neurons, oligodendrocytes, and infiltrating immune cells, shaping both local and systemic inflammatory responses. These interactions influence key processes such as synaptic pruning, phagocytosis, blood–brain barrier integrity, and cytokine-mediated signaling. Understanding the mechanisms of cell–cell signaling in this context is critical for identifying therapeutic strategies to modulate the immune response and restore homeostasis. This review explores the key players in CNS neuroinflammation, with a focus on the role of microglia, the molecular pathways underlying intercellular communication, and potential therapeutic approaches to mitigate neuroinflammatory damage in chronic neurodegenerative diseases.

Ischemic stroke is the third cause of death in the developed countries and the main reason of severe disability. Brain ischemia leads to the production of damage-associated molecular patterns (DAMPs) by neurons and glial cells which results in astrocyte and microglia activation, pro-inflammatory cytokines and chemokines production, blood-brain barrier (BBB) disruption, infiltration of leukocytes from the peripheral blood into the infarcted area, and further exacerbation of tissue damage. However, some immune cells such as microglia or monocytes are capable to change their phenotype to anti-inflammatory, produce anti-inflammatory cytokines, and protect injured nervous tissue. In this situation, therapies, which will modulate the immune response after brain ischemia, such as transplantation of mesenchymal stem cells (MSCs) are catching interest. Many experimental studies of ischemic stroke revealed that MSCs are able to modulate immune response and act neuroprotective, through stimulation of neurogenesis, oligodendrogenesis, astrogenesis, and angiogenesis. MSCs may also have an ability to replace injured cells, but the release of paracrine factors directly into the environment or via extracellular vesicles (EVs) seems to play the most pronounced role. EVs are membrane structures containing proteins, lipids, and nucleic acids, and they express similar properties as the cells from which they are derived. However, EVs have lower immunogenicity, do not express the risk of vessel blockage, and have the capacity to cross the blood-brain barrier. Experimental studies of ischemic stroke showed that EVs have immunomodulatory and neuroprotective properties; therefore, they can stimulate neurogenesis and angiogenesis. Up to now, 20 clinical trials with MSC transplantation into patients after stroke were performed, from which two concerned on only hemorrhagic stroke and 13 studied only on ischemic stroke. There is no clinical trial with EV injection into patients after brain ischemia so far, but the case with miR-124-enriched EVs administration is planned and probably there will be more clinical studies with EV transplantation in the near future.

Ischaemic stroke is a leading cause of death and disability. Circulating extracellular vesicles (EVs) post‐stroke may help brain endothelial cells (BECs) counter ischaemic injury. However data on how EVs from ischaemic stroke patients, considering injury severity, affect these cells are limited. The aims were to characterize the inflammatory and angiogenic components of circulating EVs in acute ischaemic stroke patients, considering stroke severity, and to investigate whether these circulating EVs differentially influence the proangiogenic properties and blood–brain barrier (BBB) integrity of human BECs. Eighteen ischaemic stroke patients (acute phase: 24–48 h) and nine controls matched by age, sex, and blood pressure were studied. Stroke severity was classified as severe (n = 9) or mild (n = 9). Plasma EVs were analysed for size, concentration, and protein markers (CD63, Alix, CD81, TSG101, HSP70), as well as proinflammatory and angiogenic proteins. EV uptake, cell viability, proangiogenic capacity, electrical resistance [TEER (transendothelial electrical resistance)], and dextran‐70 kD permeability were assessed using human brain microvascular endothelial cells (hCMEC/D3). Stroke patients had lower EV concentrations than controls (p = 0.075), with mild‐stroke patients having the smallest EVs. Stroke‐derived EVs had higher levels of interleukin 6 (IL‐6), tumour necrosis factor α (TNF‐α), nitrotyrosine, and vascular endothelial growth factor (VEGF) but lower placental growth factor (PLGF) compared to controls. IL‐6 was higher in mild strokes (p = 0.0025), and VEGF was higher in severe strokes (p = 0.048). EVs from severe‐stroke cases enhanced proangiogenic capacity and minimally disrupted the BBB. Stroke severity influences EV number, size, and composition. EVs from severe strokes may promote BBB restoration and cerebral angiogenesis, suggesting their role in intercellular communication and homeostasis in ischaemic tissue. imageKey points Ischaemic stroke is one of the leading causes of death worldwide. After an ischaemic stroke several physiological processes are triggered to recover the injured tissue. Increasing evidence has suggested that extracellular vesicles (EVs) present in the bloodstream could play a role in brain recovery, but their specific impact, especially concerning stroke severity, was unclear. This study demonstrates that plasma‐derived EVs from first‐ever ischaemic stroke patients have distinctive characteristics and effects over brain angiogenesis and blood–brain barrier (BBB) integrity. Our study proposes that circulating EVs from patients with severe stroke may carry protective factors to initiate brain endothelial cell recovery after acute episodes. These findings underscore the role of EVs as potential effectors of BBB recovery and biomarkers in severe ischaemic stroke.

Simple SummaryIschemic stroke represents one of the leading causes of death and disability worldwide. The identification of new prognostic factors and biomarkers for patients’ risk stratification could reduce the burden of disease. In this perspective, given the possibility of non-invasively collecting the extracellular vesicles and characterizing them on the basis of parental surface markers, we verified whether extracellular vesicles could represent an interesting prognostic biomarker in ischemic stroke. We found that specific extracellular vesicle subtypes are associated with stroke severity and both short- and long-term outcomes.The possibility of characterizing the extracellular vesicles (EVs) based on parental cell surface markers and their content makes them a new attractive prognostic biomarker. Thus, our study aims to verify the role of EVs as relevant prognostic factors for acute and mid-term outcomes in ischemic stroke. Forty-seven patients with acute ischemic stroke were evaluated at admission (T0), immediately after recanalization treatment or after 2 h in non-treated patients (T1) and after one week (Tw) using the National Institutes of Health Stroke Scale (NIHSS), and after 3 months using the Modified Rankin Scale (mRS). Total count and characterization of EVs were assessed by Nanosight analysis and flow cytometry. The relationships between stroke outcomes and EV count were assessed through multivariable negative binomial regression models. We found that the amount of platelet-derived EVs at admission was positively associated with the severity of ischemic stroke at the onset as well as with the severity of mid-term outcome. Moreover, our study revealed that T-cell-derived EVs at admission were positively related to both early and mid-term ischemic stroke outcomes. Finally, T-cell-derived EVs at T1 were positively related to mid-term ischemic stroke outcome. The present study suggests that specific EV subtypes are associated with stroke severity and both short- and long-term outcomes. EVs could represent a valid tool to improve risk stratification in patients with ischemic stroke and post-recanalization treatment monitoring.

ACE2-enriched extracellular vesicles enhance infectivity of live SARS-CoV-2 virus.

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

Chronic cancer-associated inflammation and immunosuppression are common features in most patients with solid malignancies. The causes of this chronic inflammatory-immunosuppressive state are still largely undefined. We hypothesized that selective RNAs can be secreted by cancer cells in extracellular vesicles (EVs) and may trigger proinflammatory responses in target cells, leading to chronic inflammation linked to immune cell dysfunction and immunosuppression. We found that tumor cell lines from pancreatic ductal adenocarcinoma (PDAC), prostate cancer (PCa) and from pediatric cancer such as Ewing sarcoma (EwS) continuously secrete large numbers of small (40-200 nm) EVs. In contrast to non-transformed fibroblasts, cancer cell-derived EVs are enriched with large subsets of retroelement and pericentromeric transcripts, including LINE, SINE and HERV retroelements, and human satellite 2 and 3 (HSAT2,3) RNAs. These virus-like RNAs were highly elevated in plasma EVs from EwS patients but not in healthy donors. Some of them, including HERV-K and HSAT2, were detected in peripheral blood myeloid cells with CD33+HLA-DR− immunosuppressive phenotypes, and these cell populations were expanded in EwS patients compared to healthy donors. Using mouse xenografts and in vitro models, we also found that at least some of these RNAs, such as HSAT2, are transmitted to stromal fibroblasts and immune cells in the tumor microenvironment. They also accumulated in fibroblasts after treatment with EwS EVs, coincident with the induction of proinflammatory and DNA damage responses. Prolonged exposure of fibroblasts to EwS EVs also led to mitotic defects and senescence. Expression and dissemination of these highly immunogenic virus-like RNAs in EVs may thus be a common feature of multiple human malignancies, potentially affecting host cells in the local and systemic tumor environment. This, in turn, may induce chronic inflammation contributing to an overall immunosuppressed state in patients. Citation Format: Valentina Evdokimova, Peter Ruzanov, Hendrik Gassmann, Lincoln D. Stein, Poul H. Sorensen, Stefan Burdach, Laszlo Radvanyi. Tumor-derived extracellular vesicles transmit retroelement and pericentromeric RNAs to drive proinflammatory and DNA damage responses in stromal fibroblasts and immune cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4468.

Ischemic stroke is a fatal and disabling disease worldwide and imposes a significant burden on society. At present, biological markers that can be conveniently measured in body fluids are lacking for the diagnosis of ischemic stroke, and there are no effective treatment methods to improve neurological function after ischemic stroke. Therefore, new ways of diagnosing and treating ischemic stroke are urgently needed. The neurovascular unit, composed of neurons, astrocytes, microglia, and other components, plays a crucial role in the onset and progression of ischemic stroke. Extracellular vesicles are nanoscale lipid bilayer vesicles secreted by various cells. The key role of extracellular vesicles, which can be released by cells in the neurovascular unit and serve as significant facilitators of cellular communication, in ischemic stroke has been extensively documented in recent literature. Here, we highlight the role of neurovascular unit-derived extracellular vesicles in the diagnosis and treatment of ischemic stroke, the current status of extracellular vesicle engineering for ischemic stroke treatment, and the problems encountered in the clinical translation of extracellular vesicle therapies. Extracellular vesicles derived from the neurovascular unit could provide an important contribution to diagnostic and therapeutic tools in the future, and more studies in this area should be carried out.