Abstract
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.Graphical
Highlights
Spinal cord injury (SCI) affects 12,000 individuals annually in the U.S, and current medicamentous and therapeutic possibilities after SCI are very limited [1]
Isolation and characterization of Human umbilical cord derived mesenchymal stem cell (hucMSC)‐extracellular vesicles (EVs) and CD73+hucMSC‐EVs Under light microscope, hucMSCs derived from umbilical cord tissue and the modified CD73+hucMSCs by lentiviral transduction appeared similar growth characteristics (Fig. 1A)
A and B BV2 cells are treated with 1 μg/ml LPS in the presence or absence of 30 μg/ml CD73+hucMSC-EVs, and together with 1 μM MRS1706 (adenosine 2b receptor (A2bR) inhibitor) or 1 μM SCH58261 (Adenosine 2a receptor (A2aR) inhibitor)
Summary
Spinal cord injury (SCI) affects 12,000 individuals annually in the U.S, and current medicamentous and therapeutic possibilities after SCI are very limited [1]. The infiltrating macrophages and in situ microglia reach their peaks on 3–7 days after SCI, and polarize to proinflammatory M1 subtype, which further secrete proinflammatory cytokines and lead to the secondary injury. This microglia might trigger A1 phenotype astrocyte proliferation and astrocyte scar formation, which is harmful for the surrounding microenvironment. Extracellular vesicles have been designed as nano drug carriers in many diseases. 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
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