C-C chemokine receptor type 2-overexpressing exosomes alleviated experimental post-stroke cognitive impairment by enhancing microglia/macrophage M2 polarization

BACKGROUND Human-derived mesenchymal stromal cells have been shown to improve cognitive function following experimental stroke.The activity of exosomes has been verified to be comparable to the therapeutic effects of mesenchymal stromal cells.However,the effects of exosomes derived from human umbilical cord mesenchymal stem cells(HUC-MSCs)(ExoCtrl)on post-stroke cognitive impairment(PSCI)have rarely been reported.Moreover,whether exosomes derived from C-C chemokine receptor type 2(CCR2)-overexpressing HUC-MSCs(ExoCCR2)can enhance the therapeutic effects on PSCI and the possible underlying mechanisms have not been studied.AIM To investigate the effects of ExoCtrl on PSCI and whether ExoCCR2 can enhance therapeutic effects on PSCI.METHODS Transmission electron microscopy,qNano®particles analyzer,and Western blotting were employed to determine the morphology and CCR2 expression of ExoCtrl or ExoCCR2.ELISA was used to study the binding capacity of exosomes to CC chemokine ligand 2(CCL2)in vivo.After the intravenous injection of ExoCtrl or ExoCCR2 into experimental rats,the effect of ExoCtrl and ExoCCR2 on PSCI was assessed by Morris water maze.Remyelination and oligodendrogenesis were analyzed by Western blotting and immunofluorescence microscopy.QRT-PCR and immunofluorescence microscopy were conducted to compare the microglia/macrophage polarization.The infiltration and activation of hematogenous macrophages were analyzed by Western blotting and transwell migration analysis.RESULTS CCR2-overexpressing HUC-MSCs loaded the CCR2 receptor into their exosomes.The morphology and diameter distribution between ExoCtrl and ExoCCR2 showed no significant difference.ExoCCR2 bound significantly to CCL2 but ExoCtrl showed little CCL2 binding.Although both ExoCCR2 and ExoCtrl showed beneficial effects on PSCI,oligodendrogenesis,remyelination,and microglia/macrophage polarization,ExoCCR2 exhibited a significantly superior beneficial effect.We also found that ExoCCR2 could suppress the CCL2-induced macrophage migration and activation in vivo and in vitro,compared with ExoCtrl treated group.CONCLUSION CCR2 over-expression enhanced the therapeutic effects of exosomes on the experimental PSCI by promoting M2 microglia/macrophage polarization,enhancing oligodendrogenesis and remyelination.These therapeutic effects are likely through suppressing the CCL2-induced hematogenous macrophage migration and activation.Key words:Cognitive impairment;Stroke;Exosomes;C-C chemokine receptor type 2;Microglia/macrophage polarization;Remyelination.

BACKGROUND Human-derived mesenchymal stromal cells have been shown to improve cognitive function following experimental stroke. The activity of exosomes has been verified to be comparable to the therapeutic effects of mesenchymal stromal cells. However, the effects of exosomes derived from human umbilical cord mesenchymal stem cells（HUC-MSCs）（Exo Ctrl） on post-stroke cognitive impairment（PSCI） have rarely been reported. Moreover, whether exosomes derived from C-C chemokine receptor type 2（CCR2）-overexpressing HUC-MSCs(ExoCCR2) can enhance the therapeutic effects on PSCI and the possible underlying mechanisms have not been studied.AIM To investigate the effects of Exo Ctrl on PSCI and whether ExoCCR2 can enhance therapeutic effects on PSCI.METHODS Transmission electron microscopy, q Nano? particles analyzer, and Western blotting were employed to determine the morphology and CCR2 expression of Exo Ctrl or ExoCCR2. ELISA was used to study the binding capacity of exosomes to CC chemokine ligand 2（CCL2） in vivo. After the intravenous injection of Exo Ctrl or ExoCCR2 into experimental rats, the effect of Exo Ctrl and ExoCCR2 on PSCI was assessed by Morris water maze. Remyelination and oligodendrogenesis were analyzed by Western blotting and immunofluorescence microscopy. QRT-PCR and immunofluorescence microscopy were conducted to compare the microglia/macrophage polarization. The infiltration and activation of hematogenous macrophages were analyzed by Western blotting and transwellmigration analysis.RESULTS CCR2-overexpressing HUC-MSCs loaded the CCR2 receptor into their exosomes.The morphology and diameter distribution between Exo Ctrl and ExoCCR2 showed no significant difference. ExoCCR2 bound significantly to CCL2 but Exo Ctrl showed little CCL2 binding. Although both ExoCCR2 and Exo Ctrl showed beneficial effects on PSCI, oligodendrogenesis, remyelination, and microglia/macrophage polarization, ExoCCR2 exhibited a significantly superior beneficial effect. We also found that ExoCCR2 could suppress the CCL2-induced macrophage migration and activation in vivo and in vitro, compared with Exo Ctrl treated group.CONCLUSION CCR2 over-expression enhanced the therapeutic effects of exosomes on the experimental PSCI by promoting M2 microglia/macrophage polarization,enhancing oligodendrogenesis and remyelination. These therapeutic effects are likely through suppressing the CCL2-induced hematogenous macrophage migration and activation.