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Mesenchymal stromal cell-derived extracellular vesicles afford neuroprotection by modulating PI3K/AKT pathway and calcium oscillations.

ABSTRACT: Mesenchymal stromal cells (MSC) are widely recognized as potential effectors in neuroprotective therapy. The protective properties of MSC were considered to be associated with the secretion of extracellular vesicles (MSC-EV). We explored the effects of MSC-EV in vivo on models of traumatic and hypoxia-ischemia (HI) brain injury. Neuroprotective mechanisms triggered by MSC-EV were also studied in vitro using a primary neuroglial culture. Intranasal administration of MSC-EV reduced the volume of traumatic brain damage, correlating with a recovery of sensorimotor functions. Neonatal HI-induced brain damage was mitigated by the MSC-EV administration. This therapy also promoted the recovery of sensorimotor functions, implying enhanced neuroplasticity, and MSC-EV-induced growth of neurites in vitro supports this. In the in vitro ischemic model, MSC-EV prevented cell calcium (Ca²⁺) overload and subsequent cell death. In mixed neuroglial culture, MSC-EV induced inositol trisphosphate (IP3) receptor-related Ca²⁺ oscillations in astrocytes were associated with resistance to calcium overload not only in astrocytes but also in co-cultured neurons, demonstrating intercellular positive crosstalk between neural cells. This implies that phosphatidylinositol 3-Kinase/AKT signaling is one of the main pathways in MSC-EV-mediated protection of neural cells exposed to ischemic challenge. Components of this pathway were identified among the most enriched categories in the MSC-EV proteome.

SUBMITTER: Turovsky EA

PROVIDER: S-EPMC9461662 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Mesenchymal stromal cell-derived extracellular vesicles afford neuroprotection by modulating PI3K/AKT pathway and calcium oscillations.

Turovsky Egor A EA Golovicheva Victoria V VV Varlamova Elena G EG Danilina Tatyana I TI Goryunov Kirill V KV Shevtsova Yulia A YA Pevzner Irina B IB Zorova Ljubava D LD Babenko Valentina A VA Evtushenko Ekaterina A EA Zharikova Anastasia A AA Khutornenko Anastasia A AA Kovalchuk Sergey I SI Plotnikov Egor Y EY Zorov Dmitry B DB Sukhikh Gennady T GT Silachev Denis N DN

International journal of biological sciences 20220821 14

Mesenchymal stromal cells (MSC) are widely recognized as potential effectors in neuroprotective therapy. The protective properties of MSC were considered to be associated with the secretion of extracellular vesicles (MSC-EV). We explored the effects of MSC-EV <i>in vivo</i> on models of traumatic and hypoxia-ischemia (HI) brain injury. Neuroprotective mechanisms triggered by MSC-EV were also studied <i>in vitro</i> using a primary neuroglial culture. Intranasal administration of MSC-EV reduced t ...[more]

PMID: 36147480

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Project description:BackgroundMesenchymal stromal cell derived extracellular vesicles (MSC-EVs) are a promising therapeutic for neuroinflammation. MSC-EVs can interact with microglia, the resident immune cells of the brain, to exert their immunomodulatory effects. In response to inflammatory cues, such as cytokines, microglia undergo phenotypic changes indicative of their function e.g. morphology and secretion. However, these changes in response to MSC-EVs are not well understood. Additionally, no disease-relevant screening tools to assess MSC-EV bioactivity exist, which has further impeded clinical translation. Here, we developed a quantitative, high throughput morphological profiling approach to assess the response of microglia to neuroinflammation-relevant signals and whether this morphological response can be used to indicate the bioactivity of MSC-EVs.ResultsUsing an immortalized human microglia cell-line, we observed increased size (perimeter, major axis length) and complexity (form factor) upon stimulation with interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Upon treatment with MSC-EVs, the overall morphological score (determined using principal component analysis) shifted towards the unstimulated morphology, indicating that MSC-EVs are bioactive and modulate microglia. The morphological effects of MSC-EVs in TNF-γ/IFN-α stimulated cells were concomitant with reduced secretion of 14 chemokines/cytokines (e.g. CXCL6, CXCL9) and increased secretion of 12 chemokines/cytokines (e.g. CXCL8, CXCL10). Proteomic analysis of cell lysates revealed significant increases in 192 proteins (e.g. HIBADH, MEAK7, LAMC1) and decreases in 257 proteins (e.g. PTEN, TOM1, MFF) with MSC-EV treatment. Of note, many of these proteins are involved in regulation of cell morphology and migration. Gene Set Variation Analysis revealed upregulation of pathways associated with immune response, such as regulation of cytokine production, immune cell infiltration (e.g. T cells, NK cells) and morphological changes (e.g. Semaphorin, RHO/Rac signaling). Additionally, changes in microglia mitochondrial morphology were measured suggesting that MSC-EV modulate mitochondrial metabolism.ConclusionThis study comprehensively demonstrates the effects of MSC-EVs on human microglial morphology, cytokine secretion, cellular proteome, and mitochondrial content. Our high-throughput, rapid, low-cost morphological approach enables screening of MSC-EV batches and manufacturing conditions to enhance EV function and mitigate EV functional heterogeneity in a disease relevant manner. This approach is highly generalizable and can be further adapted and refined based on selection of the disease-relevant signal, target cell, and therapeutic product.

Project description:BackgroundThe cerebrospinal fluid (CSF), which surrounds the brain and spinal cord, is predominantly produced by the choroid plexus of the ventricle. Although CSF-derived extracellular vesicles (CSF-EVs) may be utilized as diagnostic and prognostic indicators for illnesses of the central nervous system (CNS), it is uncertain if CSF-EVs may have an impact on neurological function after spinal cord injury (SCI).MethodsHere, we isolated EVs using ultracentrifugation after extracting CSF from Bama miniature pigs. We then combined CSF-EVs with hydrogel and put it on the spinal cord's surface. To determine if CSF-EVs had an impact on mice's neurofunctional recovery, behavioral evaluations were employed. Both in vitro and in vivo, the effect of CSF-EVs on angiogenesis was assessed. We investigated whether CSF-EVs stimulated the PI3K/AKT pathway to alter angiogenesis using the PI3K inhibitor LY294002.ResultsCSF-EVs were successfully isolated and identified by transmission electron microscope (TEM), nano-tracking analysis (NTA), and western blot. CSF-EVs could be ingested by vascular endothelial cells as proved by in vivo imaging and immunofluorescence. We demonstrated that CSF-EVs derived from pigs with SCI (SCI-EVs) showed a better effect on promoting vascular regeneration as compared to CSF-EVs isolated from pigs receiving laminectomy (Sham-EVs). Behavioral assessments demonstrated that SCI-EVs could dramatically enhance motor and sensory function in mice with SCI. Western blot analysis suggested that SCI-EVs promote angiogenesis by activating PI3K/AKT signaling pathway, and the pro-angiogenetic effect of SCI-EVs was attenuated by the application of the LY294002 (PI3K inhibitor).ConclusionOur study revealed that CSF-EVs could enhance vascular regeneration by activating the PI3K/AKT pathway, hence improving motor function recovery after SCI, which may offer potential novel therapeutic options for acute SCI.The translational potential of this articleThis study demonstrated the promotion of vascular regeneration and neurological function of CSF-derived exosomes, which may provide a potential therapeutic approach for the treatment of spinal cord injury.

Project description:BackgroundGlioblastomas are lethal brain tumors under the current combinatorial therapeutic strategy that includes surgery, chemo- and radio-therapies. Extensive changes in the tumor microenvironment is a key reason for resistance to chemo- or radio-therapy and frequent tumor recurrences. Understanding the tumor-nontumor cell interaction in TME is critical for developing new therapy. Glioblastomas are known to recruit normal cells in their environs to sustain growth and encroachment into other regions. Neural progenitor cells (NPCs) have been noted to migrate towards the site of glioblastomas, however, the detailed mechanisms underlying glioblastoma-mediated NPCs' alteration remain unkown.MethodsWe collected EVs in the culture medium of three classic glioblastoma cell lines, U87 and A172 (male cell lines), and LN229 (female cell line). U87, A172, and LN229 were co-cultured with their corresponding EVs, respectively. Mouse NPCs (mNPCs) were co-cultured with glioblastoma-derived EVs. The proliferation and migration of tumor cells and mNPCs after EVs treatment were examined. Proteomic analysis and western blotting were utilized to identify the underlying mechanisms of glioblastoma-derived EVs-induced alterations in mNPCs.ResultsWe first show that glioblastoma cell lines U87-, A172-, and LN229-derived EVs were essential for glioblastoma cell prolifeartion and migration. We then demonstrated that glioblastoma-derived EVs dramatically promoted NPC proliferation and migration. Mechanistic studies identify that glioblastoma-derived EVs achieve their functions via activating PI3K-Akt-mTOR pathway in mNPCs. Inhibiting PI3K-Akt pathway reversed the elevated prolfieration and migration of glioblastoma-derived EVs-treated mNPCs.ConclusionOur findings demonstrate that EVs play a key role in intercellular communication in tumor microenvironment. Inhibition of the tumorgenic EVs-mediated PI3K-Akt-mTOR pathway activation might be a novel strategy to shed light on glioblastoma therapy. Video Abstract.

Dataset Information

Mesenchymal stromal cell-derived extracellular vesicles afford neuroprotection by modulating PI3K/AKT pathway and calcium oscillations.

Publications

Mesenchymal stromal cell-derived extracellular vesicles afford neuroprotection by modulating PI3K/AKT pathway and calcium oscillations.

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