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Hypoxic preconditioned bone mesenchymal stem cells ameliorate spinal cord injury in rats via improved survival and migration.

ABSTRACT: The unique hypoxic inflammatory microenvironment observed in the spinal cord following spinal cord injury (SCI) limits the survival and efficacy of transplanted bone mesenchymal stem cells (BMSCs). The aim of the present study was to determine whether hypoxic preconditioning (HP) increased the therapeutic effects of BMSC on SCI. BMSCs were pretreated with cobalt chloride (CoCl2) in vitro, and the proliferative apoptotic and migratory abilities of these hypoxic BMSCs (H?BMSCs) were assessed. BMSCs and H?BMSCs derived from green fluorescent protein (GFP) rats were transplanted into SCI rats in vivo. The neurological function, histopathology, inflammation, and number and migration of transplanted cells were examined. HP significantly enhanced BMSC migration (increased hypoxia inducible factor 1? and C?X?C motif chemokine receptor 4 expression) and tolerance to apoptotic conditions (decreased caspase?3 and increased B?cell lymphoma 2 expression) in vitro. In vivo, H?BMSC transplantation significantly improved neurological function, decreased spinal cord damage and suppressed the inflammatory response associated with microglial activation. The number of GFP?positive cells in the SCI core and peripheral region of H?BMSC animals was increased compared with that in those of BMSC animals, suggesting that HP may increase the survival and migratory abilities of BMSCs and highlights their therapeutic potential for SCI.

SUBMITTER: Wang W

PROVIDER: S-EPMC6192716 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Hypoxic preconditioned bone mesenchymal stem cells ameliorate spinal cord injury in rats via improved survival and migration.

Wang Weiheng W Huang Xiaodong X Lin Wenbo W Qiu Yuanyuan Y He Yunfei Y Yu Jiangming J Xi Yanhai Y Ye Xiaojian X

International journal of molecular medicine 20180807 5

The unique hypoxic inflammatory microenvironment observed in the spinal cord following spinal cord injury (SCI) limits the survival and efficacy of transplanted bone mesenchymal stem cells (BMSCs). The aim of the present study was to determine whether hypoxic preconditioning (HP) increased the therapeutic effects of BMSC on SCI. BMSCs were pretreated with cobalt chloride (CoCl2) in vitro, and the proliferative apoptotic and migratory abilities of these hypoxic BMSCs (H‑BMSCs) were assessed. BMSC ...[more]

PMID: 30106084

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Project description:BACKGROUND:Spinal cord injury (SCI) can lead to severe motor and sensory dysfunction with high disability and mortality. In recent years, mesenchymal stem cell (MSC)-secreted nano-sized exosomes have shown great potential for promoting functional behavioral recovery following SCI. However, MSCs are usually exposed to normoxia in vitro, which differs greatly from the hypoxic micro-environment in vivo. Thus, the main purpose of this study was to determine whether exosomes derived from MSCs under hypoxia (HExos) exhibit greater effects on functional behavioral recovery than those under normoxia (Exos) following SCI in mice and to seek the underlying mechanism. METHODS:Electron microscope, nanoparticle tracking analysis (NTA), and western blot were applied to characterize differences between Exos and HExos group. A SCI model in vivo and a series of in vitro experiments were performed to compare the therapeutic effects between the two groups. Next, a miRNA microarray analysis was performed and a series of rescue experiments were conducted to verify the role of hypoxic exosomal miRNA in SCI. Western blot, luciferase activity, and RNA-ChIP were used to investigate the underlying mechanisms. RESULTS:Our results indicate that HExos promote functional behavioral recovery by shifting microglial polarization from M1 to M2 phenotype in vivo and in vitro. A miRNA array showed miR-216a-5p to be the most enriched in HExos and potentially involved in HExos-mediated microglial polarization. TLR4 was identified as the target downstream gene of miR-216a-5p and the miR-216a-5p/TLR4 axis was confirmed by a series of gain- and loss-of-function experiments. Finally, we found that TLR4/NF-κB/PI3K/AKT signaling cascades may be involved in the modulation of microglial polarization by hypoxic exosomal miR-216a-5p. CONCLUSION:Hypoxia preconditioning represents a promising and effective approach to optimize the therapeutic actions of MSC-derived exosomes and a combination of MSC-derived exosomes and miRNAs may present a minimally invasive method for treating SCI.

Project description:BackgroundHypoxia preconditioning has been proven to be an effective method to enhance the therapeutic action of mesenchymal stem cells (MSCs). However, the beneficial effects of hypoxic MSCs in ischemia/reperfusion (I/R) lung injury have yet to be investigated. In this study, we hypothesized that the administration of hypoxic MSCs would have a positive therapeutic impact on I/R lung injury at molecular, cellular, and functional levels.MethodsI/R lung injury was induced in isolated and perfused rat lungs. Hypoxic MSCs were administered in perfusate at a low (2.5×105 cells) and high (1×106 cells) dose. Rats ventilated with a low tidal volume of 6 ml/kg served as controls. Hemodynamics, lung injury indices, inflammatory responses and activation of apoptotic pathways were determined.ResultsI/R induced permeability pulmonary edema with capillary leakage and increased levels of reactive oxygen species (ROS), pro-inflammatory cytokines, adhesion molecules, cytosolic cytochrome C, and activated MAPK, NF-κB, and apoptotic pathways. The administration of a low dose of hypoxic MSCs effectively attenuated I/R pathologic lung injury score by inhibiting inflammatory responses associated with the generation of ROS and anti-apoptosis effect, however this effect was not observed with a high dose of hypoxic MSCs. Mechanistically, a low dose of hypoxic MSCs down-regulated P38 MAPK and NF-κB signaling but upregulated glutathione, prostaglandin E2, IL-10, mitochondrial cytochrome C and Bcl-2. MSCs infused at a low dose migrated into interstitial and alveolar spaces and bronchial trees, while MSCs infused at a high dose aggregated in the microcirculation and induced pulmonary embolism.ConclusionsHypoxic MSCs can quickly migrate into extravascular lung tissue and adhere to other inflammatory or structure cells and attenuate I/R lung injury through anti-oxidant, anti-inflammatory and anti-apoptotic mechanisms. However, the dose of MSCs needs to be optimized to prevent pulmonary embolism and thrombosis.

Project description:BackgroundSpinal cord injury (SCI) is an inflammatory condition, and excessive adenosine triphosphate (ATP) is released into the extracellular space, which can be catabolized into adenosine by CD73. Extracellular vesicles have been designed as nano drug carriers in many diseases. However, their impacts on delivery of CD73 after SCI are not yet known. We aimed to construct CD73 modified extracellular vesicles and explore the anti-inflammatory effects after SCI.MethodsCD73 engineered extracellular vesicles (CD73+ hucMSC-EVs) were firstly established, which were derived from human umbilical cord mesenchymal stem cells (hucMSCs) transduced by lentiviral vectors to upregulate the expression of CD73. Effects of CD73+ hucMSC-EVs on hydrolyzing ATP into adenosine were detected. The polarization of M2/M1 was verified by immunofluorescence. Furthermore, A2aR and A2bR inhibitors and A2bR knockdown cells were used to investigate the activated adenosine receptor. Biomarkers of microglia and levels of cAMP/PKA were also detected. Repetitively in vivo study, morphology staining, flow cytometry, cytokine analysis, and ELISA assay, were also applied for verifications.ResultsCD73+ hucMSC-EVs reduced concentration of ATP and promoted the level of adenosine. In vitro experiments, CD73+ hucMSC-EVs increased macrophages/microglia M2:M1 polarization, activated adenosine 2b receptor (A2bR), and then promoted cAMP/PKA signaling pathway. In mice using model of thoracic spinal cord contusion injury, CD73+ hucMSC-EVs improved the functional recovery after SCI through decreasing the content of ATP in cerebrospinal fluid and improving the polarization from M1 to M2 phenotype. Thus, the cascaded pro-inflammatory cytokines were downregulated, such as TNF-α, IL-1β, and IL-6, while the anti-inflammatory cytokines were upregulated, such as IL-10 and IL-4.ConclusionsCD73+ hucMSC-EVs ameliorated inflammation after spinal cord injury by reducing extracellular ATP, promoting A2bR/cAMP/PKA pathway and M2/M1 polarization. CD73+ hucMSC-EVs might be promising nano drugs for clinical application in SCI therapy.

Project description:BackgroundStem cell transplantation therapy is a potential approach for the repair of spinal cord injuries and other neurodegenerative diseases, but its effectiveness is hampered by the low rate of targeted migration of cells to the area of injury. The aim of this study was to investigate the effects of miR-31 on the migration of bone marrow mesenchymal stem cells (BMSCs) and the regulation of MMP-2 and CXCR4 expression in vitro and in vivo.MethodseGFP-expressing BMSCs were isolated and cultured for subsequent experiments. The experiments were divided into three groups: control group, miR-31agomir group, and miR-31antagomir group. Proliferation was analyzed using CCK-8 and flow cytometry; cell migration in vitro was analyzed using wound-healing and transwell assays. The mouse SCI model was prepared by the impact method, and cells were transplanted (3 groups, 12 per group). Relevant inflammatory factors were detected by ELISA. The BMS score was used to evaluate the functional recovery of the mouse spinal cord and the frozen section was used to analyze the cell migration ability in vivo. The in vitro and in vivo expression levels of MMP-2 and CXCR4 were evaluated by Western blot and immunohistochemical staining.ResultsIn vitro experiments showed that cells in the miR-31agomir group exhibited enhanced cell proliferation (P<0.05, P<0.001) and migration (P<0.001) and upregulated protein expression levels of CXCR4 (P<0.01) and MMP-2 (P<0.001) compared with cells in the control group. The results of in vivo experiments showed that the expression of pro-inflammatory factors was reduced after cell transplantation treatment. Cells in the miR-31agomir group showed enhanced cell-targeted migration ability (P<0.001), improved the function of damaged tissues (P<0.001), and upregulated CXCR4 and MMP-2 expression compared to the control group (P<0.001).ConclusionOur experiment demonstrated that miR-31 could promote the migration of BMSCs and miR-31 could repair and improve the function of damaged tissues in SCI.

Project description:BackgroundSecondary injury after spinal cord injury (SCI) is a major obstacle to their neurological recovery. Among them, changes in astrocyte phenotype regulate secondary injury dominated by neuroinflammation. Hypoxia-preconditioned mesenchymal stem cells (MSCs)-derived extracellular vesicle (H-EV) plays a multifaceted role in secondary injury by interacting with cellular components and signaling pathways. They possess anti-inflammatory properties, regulate oxidative stress, and modulate apoptotic pathways, promoting cell survival and reducing neuronal loss. Given the unique aspects of secondary injury, H-EV shows promise as a therapeutic approach to mitigate its devastating consequences. Our study aimed to determine whether H-EV could promote SCI repair by altering the phenotype of astrocytes.MethodsRat bone marrow MSCs (BMSCs) and EVs secreted by them were extracted and characterized. After the SCI model was successfully constructed, EV and H-EV were administered into the tail vein of the rats, respectively, and then their motor function was evaluated by the Basso-Beattie-Bresnahan (BBB) score, Catwalk footprint analysis, and electrophysiological monitoring. The lesion size of the spinal cord was evaluated by hematoxylin-eosin (HE) staining. The key point was to use glial fibrillary acidic protein (GFAP) as a marker of reactive astrocytes to co-localize with A1-type marker complement C3 and A2-type marker S100A10, respectively, to observe phenotypic changes in astrocytes within tissues. The western blot (WB) of the spinal cord was also used to verify the results. We also compared the efficacy differences in apoptosis and inflammatory responses using terminal deoxynucleotidyl transferase dUTP terminal labeling (TUNEL) assay, WB, and enzyme-linked immunosorbent assay (ELISA). Experiments in vitro were also performed to verify the results. Subsequently, we performed microRNA (miRNA) sequencing analysis of EV and H-EV and carried out a series of knockdown and overexpression experiments to further validate the mechanism by which miRNA in H-EV plays a role in promoting astrocyte phenotypic changes, as well as the regulated signaling pathways, using WB both in vivo and in vitro.ResultsOur findings suggest that H-EV is more effective than EV in the recovery of motor function, anti-apoptosis, and anti-inflammatory effects after SCI, both in vivo and in vitro. More importantly, H-EV promoted the conversion of A1 astrocytes into A2 astrocytes more than EV. Moreover, miR-21, which was found to be highly expressed in H-EV by miRNA sequencing results, was also demonstrated to influence changes in astrocyte phenotype through a series of knockdown and overexpression experiments. At the same time, we also found that H-EV might affect astrocyte phenotypic alterations by delivering miR-21 targeting the JAK2/STAT3 signaling pathway.ConclusionH-EV exerts neuroprotective effects by delivering miR-21 to promote astrocyte transformation from the A1 phenotype to the A2 phenotype, providing new targets and ideas for the treatment of SCI.

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Hypoxic preconditioned bone mesenchymal stem cells ameliorate spinal cord injury in rats via improved survival and migration.

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Hypoxic preconditioned bone mesenchymal stem cells ameliorate spinal cord injury in rats via improved survival and migration.

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