Secondary Release of Exosomes From Astrocytes Contributes to the Increase in Neural Plasticity and Improvement of Functional Recovery After Stroke in Rats Treated With Exosomes Harvested From MicroRNA 133b-Overexpressing Multipotent Mesenchymal Stromal Cells

Abstract

We previously demonstrated that multipotent mesenchymal stromal cells (MSCs) that overexpress microRNA 133b (miR-133b) significantly improve functional recovery in rats subjected to middle cerebral artery occlusion (MCAO) compared with naive MSCs and that exosomes generated from naive MSCs mediate the therapeutic benefits of MSC therapy for stroke. Here we investigated whether exosomes isolated from miR-133b-overexpressing MSCs (Ex-miR-133b+) exert amplified therapeutic effects. Rats subjected to 2 h of MCAO were intra-arterially injected with Ex-miR-133b+, exosomes from MSCs infected by blank vector (Ex-Con), or phosphate-buffered saline (PBS) and were sacrificed 28 days after MCAO. Compared with the PBS treatment, both exosome treatment groups exhibited significant improvement of functional recovery. Ex-miR-133b+ treatment significantly increased functional improvement and neurite remodeling/brain plasticity in the ischemic boundary area compared with the Ex-Con treatment. Treatment with Ex-miR-133b+ also significantly increased brain exosome content compared with Ex-Con treatment. To elucidate mechanisms underlying the enhanced therapeutic effects of Ex-miR-133b+, astrocytes cultured under oxygen- and glucose-deprived (OGD) conditions were incubated with exosomes harvested from naive MSCs (Ex-Naive), miR-133b downregulated MSCs (Ex-miR-133b-), and Ex-miR-133b+. Compared with the Ex-Naive treatment, Ex-miR-133b+ significantly increased exosomes released by OGD astrocytes, whereas Ex-miR-133b- significantly decreased the release. Also, exosomes harvested from OGD astrocytes treated with Ex-miR-133b+ significantly increased neurite branching and elongation of cultured cortical embryonic rat neurons compared with the exosomes from OGD astrocytes subjected to Ex-Con. Our data suggest that exosomes harvested from miR-133b-overexpressing MSCs improve neural plasticity and functional recovery after stroke with a contribution from a stimulated secondary release of neurite-promoting exosomes from astrocytes.

Figure 1.

MicroRNA 133b (miR-133b)-enriched exosome treatment enhances neurological outcome improvement. Foot fault test (A) and modified neurologic severity score (mNSS) (B) data show that compared with the phosphate-buffered saline (PBS) treatment, both multipotent mesenchymal stromal cell (MSC) exosome treatment groups exhibited significant improvement of functional recovery (p < 0.05 after day 14, respectively). Compared with the MSCs infected with blank vector (Ex-Con), exosomes isolated from miR-133b-overexpressing MSCs (Ex-miR-133b⁺) significantly increased functional improvement (p < 0.05 after day 21, respectively). PBS, middle cerebral artery occlusion (MCAO) rats treated with PBS; CON⁺, MCAO rats treated with Ex-Con; 133b⁺, MCAO rats treated with Ex-miR-133b⁺; EXO, exosomes given. ∗p < 0.05, compared to PBS; #p < 0.05 compared to CON⁺, respectively. Mean ± standard error (SE), n = 6/group.

Figure 2.

MicroRNA 133b (miR-133b)-enriched exosomes further increase neurite remodeling and synaptic plasticity in the ischemic boundary zone (IBZ) compared to control multipotent mesenchymal stromal cell (MSC) exosomes. Bielschowsky silver and Luxol fast blue double staining (A row), SMI 31 immunostaining (B row), and synaptophysin immunostaining (C row) show that based on the increase of neurite remodeling and synaptic plasticity in the IBZ by the MSCs infected with blank vector (Ex-Con) treatment, the exosomes isolated from miR-133b-overexpressing MSC (Ex-miR-133b⁺) treatment further increased neurite remodeling and synaptic plasticity in the IBZ. PBS, middle cerebral artery occlusion (MCAO) rats treated with phosphate-buffered saline (PBS); CON⁺, MCAO rats treated with Ex-Con; 133b⁺, MCAO rats treated with Ex-miR-133b⁺. ∗p < 0.05, compared to PBS; #p < 0.05 compared to CON⁺, respectively. Mean ± standard error (SE), n = 6/group. Scale bars: 100 μm.

Figure 3.

MicroRNA 133b (miR-133b)-enriched exosomes increased exosomes in brain ischemic boundary zone (IBZ). Western blot shows that the common exosomal proteins CD63 and CD81 are present in the exosome extraction from the IBZ (A). The transmission electron microscopic (TEM) images show the morphology of exosomes presented in the brain extracellular (B), within a size range of 30–100 nm. qNano quantification data show that compared to the multipotent mesenchymal stromal cells (MSCs) infected with blank vector (Ex-Con) treatment and phosphate-buffered saline (PBS) control, the extracellular exosomes present in the IBZ significantly increased after miR-133b-overexpressing MSC (Ex-miR-133b⁺) treatment (C). PBS, middle cerebral artery occlusion (MCAO) rats treated with PBS; CON⁺, MCAO rats treated with Ex-Con; 133b⁺, MCAO rats treated with Ex-miR-133b⁺. ∗p <0.05, compared to Ex-Con. Mean ± standard error (SE), n = 6/group. Scale bar: 100 nm.

Figure 4.

MicroRNA 133b (miR-133b)-enriched exosomes enhance the release of exosomes from primary cultured oxygen-and glucose-deprived (OGD) astrocytes. MSC exosomes significantly increased the exosomes released by OGD astrocytes, and there was no obvious difference on the release from OGD astrocytes, after treatment with harvested from naive MSCs (Ex-Naive) and two control exosomes (A). Compared with the exosome Ex-Naive treatment, exosomes released by OGD astrocytes treated with miR-133b-overexpressing MSCs (Ex-miR-133b⁺) were significantly increased (A) and significantly decreased (B) by treatment with Ex-miR-133b⁻, respectively. Correlated with the miR-133b levels in the multipotent mesenchymal stromal cells (MSCs) (Table 1), the number of exosomes released by MSCs has no difference between naive MSCs and the two control MSCs (miR-CON⁺MSCs and miR-CON⁻MSCs), but the exosomal release was significantly increased from the miR-133b⁺MSCs and decreased from the miR-133b⁻MSCs, compared with that from naive MSCs (B). NivM, naive MSCs; NivA, naive astrocytes; NON, nontreated OGD astrocytes; MSC, OGD astrocytes treated with naive MSC exosomes; CON⁻, miR-CON⁻MSC or OGD astrocytes treated with miR-CON⁻MSC exosomes; 133b⁻, miR-133b⁻MSC or OGD astrocytes treated with miR-133b⁻MSC exosomes; CON⁺, miR-CON⁺MSC or OGD astrocytes treated with miR-CON⁺MSC exosomes; 133b⁺, miR-133b⁺MSC or OGD astrocytes treated with miR-133b⁺MSC exosomes. ∗p < 0.05, mean ± SD, n = 3/group.

Figure 5.

Exosomes harvested from oxygen- and glucose-deprived (OGD) astrocytes after treatment with miR-133b-overexpressing MSCs (Ex-miR-133b⁺) increase neurite outgrowth. (A) The morphology of cultured neurons in this assay. Exosomes harvested from OGD astrocytes significantly increased the neurite number and total neurite length per neuron compared with the non-exosome-treated primary cultured cortical neurons. The neurite number and total neurite length substantially increased after the neurons were treated with exosomes harvested from OGD astrocytes that were pretreated by the MSCs infected with blank vector (Ex-Con) and Ex-miR-133b⁺, and Ex-miR-133b⁺-pretreated OGD astrocytic exosomes have the enhanced effect compared with the Ex-Con pretreatment (p < 0.05, respectively; B–D). Con, non-exosome-treated neurons; OGD, neurons treated with exosomes harvested from OGD astrocytes; Con⁺, neurons treated with exosomes harvested from OGD astrocytes pretreated with Ex-Con; 133b⁺, neurons treated with exosomes harvested from OGD astrocytes pretreated with Ex-miR-133b⁺. ∗p < 0.05, mean ± standard deviation (SD), n = 3/group. Scale bar: 50 μm.

Figure 6.

MicroRNA 133b (miR-133b)-enriched exosomes downregulate the protein level of Rab9 effector protein with kelch motifs (RABEPK). Western blot data show that the MSCs infected with blank vector (Ex-Con) treatment significantly downregulated the RABEPK level increased in the ischemic boundary zone (IBZ) after middle cerebral artery occlusion (MCAO), and miR-133b-overexpressing MSC (Ex-miR-133b⁺) treatment further reduces the protein level (A, B) (p < 0.05, respectively). Double immunostaining shows the RABEPK largely expressed in the astrocytes (E–H). Ex-Con treatment significantly reduced the RABEPK level increase in the astrocytes subjected to oxygen and glucose deprivation (OGD), and Ex-miR-133b⁺ treatment further reduces the protein level (C, D) (p < 0.05, respectively). Sham, tissue from sham surgery rats; PBS, MCAO rats treated with phosphate-buffered saline (PBS); Naive, naive astrocytes; OGD, astrocytes subjected to OGD; CON⁺, MCAO rats or OGD astrocytes treated with Ex-Con; 133b⁺, MCAO rats or OGD astrocytes treated with Ex-miR-133b⁺. ∗p < 0.05, mean ± standard deviation (SD), n = 3/group. Scale bar: 50 μm.