Extracellular vesicles derived from CD73 modified human umbilical cord mesenchymal stem cells ameliorate inflammation after spinal cord injury

Abstract

Background Spinal 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. Methods CD73 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. Results CD73+ 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. Conclusions CD73+ 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 Abstract

Enhancing Functional Recovery Through Intralesional Application of Extracellular Vesicles in a Rat Model of Traumatic Spinal Cord Injury

“…Adenosine signaling may contribute to the immunomodulatory activity of EVs via the conversion of AMP to adenosine by the GPI-anchored 5′-ecto-nucleotidase CD73 present on enriched UC-MSC EVs (Priglinger et al, 2021 ). A recent report by Zhai et al ( 2021 ) demonstrated that the use of EVs produced by virus-transformed hUC-MSC engineered to increase the levels of CD73 alleviated inflammation after SCI in a mouse model, and regulated macrophage polarization in vitro . Along the same line, Xu et al ( 2018 ) demonstrated that CD73-deficient mice displayed overwhelming immune responses and poor locomotor recovery after SCI.…”

Preconditioning and Engineering Strategies for Improving the Efficacy of Mesenchymal Stem Cell-Derived Exosomes in Cell-Free Therapy

“…The overexpression of HIF in MSCs-exosomes exerted a better proangiogenic effect in cranial defects [ 138 ], myocardial infarction [ 139 ], and early ischemic necrosis of the femoral head [ 140 ]. A recent study found that CD73-modified MSC-derived EVs could break down overreleased adenosine triphosphate (ATP) and improve the M1 to M2 polarization phenotype of microglia, thereby improving locomotion in mice with spinal cord contusion [ 141 ].…”

Regulatory Role of Mesenchymal Stem Cells on Secondary Inflammation in Spinal Cord Injury

“… 140 To date, the accumulated evidence confirms that MSCs can skew the balance between M1/M2 macrophages towards the M2 phenotype, thereby facilitating functional neurological improvement post-SCI. 141 , 142 …”

“…140 To date, the accumulated evidence confirms that MSCs can skew the balance between M1/M2 macrophages towards the M2 phenotype, thereby facilitating functional neurological improvement post-SCI. 141,142 Study investigating the mechanism involved found that this effect was associated with some soluble factors secreted by MSCs. 134 Stimulated MSCs can significantly secrete numerous cytokines including TGF-β, indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), IL-4, and IL-6 following stimulation by inflammatory mediators.…”