MiR-133b promotes neural plasticity and functional recovery after treatment of stroke with multipotent mesenchymal stromal cells in rats via transfer of exosome-enriched extracellular particles

Abstract

To test, in vivo, the hypothesis that exosomes from multipotent mesenchymal stromal cells (MSCs) mediate microRNA 133b (miR-133b) transfer which promotes neurological recovery from stroke, we used knockin and knockdown technologies to upregulate or downregulate the miR-133b level in MSCs (miR-133b(+) MSCs or miR-133b(-) MSCs) and their corresponding exosomes, respectively. Rats were subjected to middle cerebral artery occlusion (MCAo) and were treated with naïve MSCs, miR-133b(+) MSCs, or miR-133b(-) MSC at 1 day after MCAo. Compared with controls, rats receiving naïve MSC treatment significantly improved functional recovery and exhibited increased axonal plasticity and neurite remodeling in the ischemic boundary zone (IBZ) at day 14 after MCAo. The outcomes were significantly enhanced with miR-133b(+) MSC treatment, and were significantly decreased with miR-133b(-) MSC treatment, compared to naïve MSC treatment. The miR-133b level in exosomes collected from the cerebral spinal fluid was significantly increased after miR-133b(+) MSC treatment, and was significantly decreased after miR-133b(-) MSC treatment at day 14 after MCAo, compared to naïve MSC treatment. Tagging exosomes with green fluorescent protein demonstrated that exosomes-enriched extracellular particles were released from MSCs and transferred to adjacent astrocytes and neurons. The expression of selective targets for miR-133b, connective tissue growth factor and ras homolog gene family member A, was significantly decreased in the IBZ after miR-133b(+) MSC treatment, while their expression remained at similar elevated levels after miR-133b(-) MSC treatment, compared to naïve MSC treatment. Collectively, our data suggest that exosomes from MSCs mediate the miR-133b transfer to astrocytes and neurons, which regulate gene expression, subsequently benefit neurite remodeling and functional recovery after stroke.

Keywords: Exosomes; Multipotent mesenchymal stromal cells; Neurite remodeling; Stroke; microRNA 133b.

Figure 1

Specific lentiviruses modify MSC miR-133b expression. Puromycin selected stable MSC cell lines infected with LentimiRa-GFP-hsa-mir-133b lentivirus (miR-133b⁺MSC), GFP Blank miRNA/microRNA lentivirus (miR-133b^+CONMSC), miRZip-133b anti-miR-133b microRNA lentivirus (miR-133bMSC) and pGreenPuro Scramble Hairpin Control lentivirus (miR-133b^−CONMSC), respectively. Green fluorescent protein (GFP) was used to monitor the infection efficiency (A). RT-PCR data show the miR-133b expression level in miR-133b⁺MSCs and their exosomes were significantly increased compared to those in miR-133b^+CONMSCs; however, miR-133bMSCs exhibited significantly decreased the miR-133b expression level in cells and exosomes compared to those in miR-133b^−CONMSCs (B). Growth curve analysis shows that miR-133b⁺MSCs and miR-133bMSCs and their corresponding control MSCs exhibit similar cell proliferation character(C). The data of counting GFP tag positive cells show that miR-133b⁺MSCs and miR-133bMSCs have similar characters on survival and trafficking to brain (D,E). 133b+con: miR-133b^+CONMSCs, 133b+: miR-133b⁺MSCs, 133b-con: miR-133b^−CONMSCs, 133b-: miR-133bMSCs. *P<0.01 compared with miR-133b^+CONMSCs, respectively; #P<0.01 compared with miR-133b^−CONMSC, respectively. Scale bar = 50 µm. Mean ± SE, n=3/group (B,C) and n=6/group (D).

Figure 2

MiR-133b mediates MSC induced functional recovery. The adhesive-removal test (A) and Foot-fault test (B) were performed prior to the treatment after MCAo (baseline), at day 3 and 7, and at day 14 before sacrifice after MCAo. Compared with PBS treatment, naive MSCs, miR-133b^+CONMSCs and miR-133b^−CONMSCs significantly improved functional recovery at day 14 after MCAo, while miR-133b⁺MSCs significantly improved functional recovery at both day 7 (adhesive-removal test only) and 14 after MCAo, and miR-133bMSCs had no obvious beneficial effects at these two time points. Compared with their corresponding control MSC treatment, miR-133b⁺MSCs significantly improved, while miR-133bMSCs treatment significantly decreased, functional recovery at day 14 after MCAo. MCAo: MCAo rats administered with PBS, MSCs: MCAo rats administered with naive MSCs, 133b+: MCAo rats administered with miR-133b⁺MSCs, 133b+con: MCAo rats administered with miR-133b^+CONMSCs, 133b-: MCAo rats administered with miR-133bMSCs, 133b-con: MCAo rats administered with miR-133b^−CONMSCs. Respective color * in A and B indicates P<0.05, compared with PBS treatment; respective color # in A and B indicates P<0.05, compared with their corresponding control MSC treatment. Mean ± SE, n=6/group.

Figure 3

MiR-133b increases axonal plasticity and neurite remodeling in the IBZ. A representative image shows the BDA-positive labeling in a rat vobratome coronal brain section (A). The BDA labeled axons in the CFA which ipsilateral to the lesion were enlarged in B. Analysis data (C) show that intracortical axonal density was significantly increased after naive MSC treatment compared with PBS treatment at day 14 after MCAo. MiR-133b⁺MSC treatment significantly increased while miR-133bMSC treatment significantly decreased the cortical axonal density at day 14 after MCAo compared with naive MSC treatment (P<0.05, C). Bielshowsky silver staining, NF-200 and synaptophysin immunostaining performed on adjacent frozen coronal brain sections to detect the neurite remodeling. Arrows indicate Bielshowsky silver (D), NF-200 (G) and synaptophysin (J) staining in the IBZ, respectively, of the ipsilateral cortex (E, H and K are enlarged from D, G, and J, respectively). Compared with PBS treatment, the positive staining of Bielshowsky silver, NF-200 and synaptophysin significantly increased at day 14 after MCAo along the IBZ after naive MSC treatment. MiR-133b⁺MSC treatment significantly increased, while miR-133bMSC treatment significantly decreased the positive staining area of Bielshowsky silver, NF-200 and synaptophysin at day 14 after MCAo compared with naive MSC treatment. MCAo: MCAo rats administered with PBS, MSCs: MCAo rats administered with naive MSCs, 133b+: MCAo rats administered with miR-133b⁺MSCs, 133b-: MCAo rats administered with miR-133bMSCs. *P<0.05 compared with MCAo control; #P<0.05 compared with naive MSCs. Scale bar = 1mm in A and 250 µm in B, 50 µm of the rest, Mean ± SE, n=3/group (C). n=6/group (F, I and L).

Figure 4

MiR-133b levels in CSF exosomes and MSC exosomes communicated with astrocytes and neurons. RT-PCR was employed to measure the miR-133b level in exosomes collected from the CSF at day 14 after MCAo. Data show that MCAo decreased the miR-133b level in the CSF exosomes. The miR-133b level in the CSF exosomes was significantly increased compared to the MCAo control after treatment with naive MSCs. MiR-133b⁺MSC treatment significantly increased, while miR-133bMSC treatment significantly decreased miR-133b levels in the CSF exosomes, respectively, compared with the naive MSC treatment (A). B–C show that exosomes-enriched particles of MSCs (arrows) in the IBZ and taken up by adjacent astrocytes and neurons (arrow heads). NOR: Normal rats, PBS: MCAo rats administered with PBS, MSCs: MCAo rats administered with naive MSCs, 133b+: MCAo rats administered with miR-133b⁺MSCs, 133b-: MCAo rats administered with miR-133bMSCs. *P<0.05 compared with NOR; #P<0.05 compared with PBS; ^P<0.05 compared with MSCs. Mean±SE, n=3/group.

Figure 5

MiR-133b regulates CTGF expression in astrocytes and RhoA expression in the IBZ. Immunostaining shows CTGF expression in the IBZ (A) and colocalized with GFAP in astrocytes (B). Immunostaining analysis data (C) show that CTGF expression significantly increased at day 14 after MCAo compared with normal rats. The administration of naive MSCs significantly decreased the CTGF expression at day 14 after MCAo, compared with MCAo alone rats. MiR-133b⁺MSC treatment further significantly decreased CTFG at day 14 after MCAo compared with naïve MSC treatment, while the CTGF expression in miR-133b⁻MSC treatment was sustained at a significantly elevated level at day 14 after MCAo compared with naïve MSC treatment. Western blot was used to detect the RhoA level in the IBZ (D). The data (E) show that RhoA expression significantly increased at day 14 after MCAo compared with normal rats. Naive MSC treatment significantly decreased the RhoA expression at day 14 after MCAo, compared with MCAo alone rats. MiR-133b⁺MSC treatment further significantly decreased RhoA at day 14 after MCAo compared with that naive MSC treatment, and the RhoA expression after miR-133bMSC treatment was sustained at a similar elevated level at day 14 after MCAo compared with that after naive MSC treatment. NOR: Normal rats, PBS: MCAo rats administered with PBS, MSCs: MCAo rats administered with naive MSCs, 133b+: MCAo rats administered with miR-133b⁺MSCs, 133b-: MCAo rats administered with miR-133bMSCs. *P<0.05 compared with NOR; #P<0.05 compared with PBS; ^P<0.05 compared with MSCs. Scale bar = 50 µm. Mean ± SE, n=6/group.