1. miR-21 was the most abundant miRNA in the uMSC-Exos and was shuttled directly from uMSCs to HUVECs via exosomes.
Accumulating evidence demonstrates that exosomes can fuse with the target cell membrane to deliver various messengers, such as miRNA, proteins or lipids, thereby facilitating signal cross talk between different kinds of cell types to control multiple target genes. Although the miRNA profiles of some typical cancer-derived exosomes have been thoroughly illustrated, the exosome miRNA signatures in uMSCs and other cells have not been fully elucidated and need to be investigated further. To characterize the uMSC-derived exosomal miRNAs, we analysed the expression levels of microRNAs in uMSC-Exos via high-throughput sequencing patterns with a HEK293-Exos as controls. The microRNA expression patterns in uMSCs were then analysed using a GEO data set (GSE46989, http://www.ncbi.nih.gov/geo/). We demonstrated that the specific miRNA signature in uMSC-Exos that was completely different from that of HEK293-Exos and uMSCs. The miR-21 was expressed at the highest level among the total miRNAs in the uMSC-Exos (Figure 1A, B). Based on the top 10 expression of miRNAs in the uMSCs and uMSC-Exos, we found that miR-21-5p, miR-100-5p, miR-125b-5p, and let-7f-5p were also highly expressed in the uMSCs, with miR-21 accounting for the highest proportion in both the maternal cells and the exosomes (Figure 1C, D).
To verify our conjecture, qRT-PCR was used to evaluate the 10 most abundant miRNAs in uMSC-Exos and their pre-miRNAs expression levels in the HUVECs treated with uMSC-Exos or HEK293-Exos for 48 h. Only the expression of miR-21-5p in the uMSC-Exo group was significantly increased compared with that of the other miRNAs, while the pre-miRNAs were not affected (Figure 1E, F). This result confirmed that an amount of mature miR-21 sufficient to perform biological functions was transported into the HUVECs by the uMSC-Exos. Exosomes have been shown to perform biological roles similar to that of their maternal cells. Moreover, the paracrine function of uMSCs is the most critical in the field of tissue regeneration. Therefore, we hypothesized that the role of uMSCs may be associated with the secretion of exosomes that deliver miR-21 to target cells.
To investigate whether exosomes mediate miR transfer, the expression of miR-21 was measured after MSCs were treated with 10 μM GW4869 (an exosome release inhibitor) for 48 h in conditioned medium (CM). GW4869 is a cell-permeable symmetrical dihydroimidazole-amide compound that acts as a potent, specific, non-competitive inhibitor of membrane neutral sphingomyelinase (nSMase) which has been reported to markedly reduce exosome release[21, 22]. As shown in Figure 1G, the levels of miR-21 in the CM collected from MSCs treated with GW4869 were significantly decreased compared with those in the CM obtained from control uMSCs. In addition, the expression of miR-21 in HUVECs treated with GW4869-CM for 48 h was also significantly decreased compared to that of HUVECs treated with uMSC-CM (Figure 1H), indicating that exosomes mediated the miRNA transport between the uMSCs and HUVECs. To improve the visibility of the results, uMSC-Exos fluorescently stained with PKH67 were cultured with HUVECs for 48 h. In addition, FAM was conjugated to miR-21 (miR-21-FAM) in the uMSC-Exos to trace miR-21 by in situ hybridization. The immunofluorescence showed that the green exosomes were concentrated mainly near the nuclei of the HUVECs and the location of the ectogenic miR-21 (red spots) was consistent with that of the exosomes. However, exosome and miRNA components were not detected in the uMSC-Exo-free supernatant (UEFS) (Figure 1I).
All these results indicated that miR-21 might be contained in the exosomes excreted by the uMSCs, a finding that supports our hypothesis that uMSCs deliver miR-21 directly to the target cell for biological function.
2. Preparation and identification of uMSC-Exos with inhibited miR-21
To validate the critical roles of miRNAs, inhibition experiments are more illustrative than overexpression experiments. We developed a strategy to stably inhibit miRNAs inside the uMSC-Exos. The uMSCs were transfected with antagomir RNA-21 to block miR-21 (uMSC-Exo-anti-miR-21) or a scrambled antagomir as a negative control (uMSC-Exo-anti-miR-NC). The exosomes were then extracted using a kit, and the qRT-PCR results demonstrated that the uMSC-Exo-anti-miR-21 group had a significantly downregulated level of miR-21 and the level was not changed in the control groups (Figure 2A).
The exosomes in the three groups were successfully precipitated by an ExoQuick-TC kit (ExoQuick-TC, System Biosciences) according to the manufacturer’s instructions. The morphology of the purified exosomes was observed by using transmission electron microscopy (TEM). Whether exposed to the inhibitor or not, all exosomes had a saucer-like shape with a diameter ranging from 40 to 100 nm (Figure 2B). The diameter of the exosomes was determined with a NanoSight LM10 instrument (NanoSight, Amesbury, U.K., http://www.nanosight.com) (Figure 2C). The markers of the exosomes, namely, CD9, CD63 and CD81, were also detected by Western blotting, and the results showed that compared with the amounts in the UEFS, the CD9, CD63 and CD81 levels were enriched in the exosome samples (Figure 2D). This finding indicates that the remodelling and extraction of the uMSC-Exos were effective and reliable.
3. Effects of exosomal miR-21 on the cellular functions of HUVECs
We evaluated the role of miR-21 in uMSC-Exo by in vitro and in vivo experiments. All phenotypic experiments were conducted with four groups: The uMSC-Exo-anti-miR-NC, uMSC-Exo-anti-miR-21, uMSC-Exo groups and a blank group (HUVECs with PBS). Previous reports demonstrated that miR-21 has an effect on cancer cell proliferation. However, the mechanism of uMSC exosomal miR-21 in HUVEC proliferation remains unknown. The CCK-8 analysis showed that exosome stimulation resulted in a significant increase in HUVEC proliferation, although the effect was reduced by the miR-21 inhibitor (Figure 3A).
To determine whether exosomal miR-21 modulates cell migration, HUVECs were incubated with PBS, uMSC-Exo-anti-miR-NC, uMSC-Exo-anti-miR-21 or uMSC-Exo and the monolayer was disrupted with a straight scratch. After 12 h and 24 h, the wound width in the culture of each group was photographed (Figure 3B). We observed that at the same time point (12 h or 24 h), the migration rate of HUVECs in the uMSC-Exo-anti-miR-21 group was significantly lower than that of the other two groups (uMSC-Exo and uMSC-Exo-anti-miR-NC) and similar to that of the blank control group. Furthermore, we conducted a Transwell assay, and the results showed that the number of migrating HUVECs was reduced by approximately one-half after the treatment with exosomal miR-21 inhibitor (Figure 3C). These results indicated that the loss of miR21 uMSC-Exo no longer enhanced the migration capacity of HUVECs.
The tube formation assay was performed to obtain direct evidence of the angiogenic function of miR-21 in HUVECs. The results showed that there were newly growing branch points and tube lengths in the uMSC-Exo group and uMSC-Exo-anti-miR-NC group that formed in a time-dependent manner while the tube formation in the group with inhibited miR-21 was weak (Figure 4A). Thus, these results directly demonstrated the angiogenic role of uMSC-Exos in HUVECs, which could be suppressed by the miR-21 inhibitor.
The role of miR-21 derived from uMSC-Exos in promoting angiogenesis was verified by an in ovo angiogenesis assay. Eight-day-old embryonated chicken eggs were used for different treatments once a day. Two days after treatment, the CAM was assessed for changes in the number and length of blood vessels. The data showed that uMSC-Exo-anti-miR-21 was able to impair in ovo neovascularization, and the value was two-fold less than that of the other two control groups as assessed on the tenth day. In addition, on the 12th day, the density of the vessels increased compared to that observed in previous days, although the inhibition effect was most obvious in the experimental group (Figure 4B). This finding is consistent with our in vitro test results showing that exosomes with inhibited miR-21 have a significantly reduced effect on angiogenesis.
4. Exosomal miR-21 derived from uMSCs activates PI3K/AKT signalling by targeting SPRY1 in HUVECs
VEGFA has been reported to have an essential role in HUVEC-mediated angiogenesis. Various types of evidence verified that VEGF increases the vascular density through SPRY1 and that SPRY1 negatively regulates angiogenesis. SPRY1 was predicted to be a potential target gene of miR-21 by the microRNA (http://www.microrna.org/) and TargetScan (http://www.targetscan.org/) databases. However, whether uMSC-Exo-miR-21 plays a critical role through SPRY1 in HUVEC-mediated angiogenesis has not been reported, and the signalling pathways are also unknown. In this study, the luciferase reporter assay results showed that miR-21-5p can bind to the 3’-UTR of SPRY1 and suppress the transcription of SPRY1 when the miR-21 overexpression vector was transfected into HEK293 cells (Figure 5A, B).
An inhibitory effect of SPRY1 on angiogenesis, which is induced by PI3K/AKT activation in cancer cells, has been demonstrated. Thus, we used the method of SPRY1 knockdown to identify the downstream pathway in HUVECs. The qRT-PCR results indicated that the mRNA level of SPRY1 increased dramatically by approximately two-fold when uMSC-Exo-anti-miR-21 was used to interfere with HUVECs, and no difference was observed between the other two groups (Figure 5C). The mRNA level of the downstream gene P13K showed the opposite trend under the same conditions, although the changes in the total AKT levels were not apparent. The miR-21 inhibitor markedly decreased the upregulated mRNA levels of HIF-1α and VEGFA (genes related to vascularization) induced by exosomes T (Figure 5D). These results were consistent with those of the Western blot assay (Figure 5E). Notably, uMSC-Exo and uMSC-Exo-anti-miR21 induced significant increases in the phosphorylation of Akt while the total AKT remained unchanged.. Hence, we suggested that uMSC-Exo-miR-21 suppressed the expression of SPRY1 and promoted the hyperactivation of AKT (p-AKT), leading to increased angiogenesis by regulating the SPRY1/PI3K/AKT signalling axis in the HUVECs. Thus, the activation of SPRY1/PI3K/AKT pathways may be the underlying mechanism by which miR-21 containing uMSC-Exos enhance angiogenesis.
5. SPRY1 knockdown increases PI3K/AKT activation and HUVEC proliferation, migration and angiogenesis.
To confirm the key role of SPRY1 in angiogenesis and assess whether knocking down the expression of SPRY1 can achieve similar effects as uMSCs-Exo on angiogenesis, we used siRNA to inhibit the expression of SPRY1 in HUVECs. First, we examined the inhibitory efficiency of these siRNAs by qRT-PCR and Western blot (Figure 6A, B), and the most effective siRNA (siSPRY1 #1) was used for the following functional assays. Next, HUVECs were transfected with SPRY1 siRNAs#1 in culture for 24 h. PI3K/AKT activation and the target genes (VEGFA and HIF-α) were determined by qRT-PCR and Western blot. As expected, we observed an increased level of PI3k, AKT, VEGFA and HIF-1α in the uMSC-Exo+siRNA-SPRY1#1 group. (Figure 6C, D)
Because SPRY1 was found to decrease PI3K/AKT activation, we then tested whether SPRY1 knockdown truly stimulates HUVEC-induced angiogenesis. To determine the extent of HUVEC migration, the scratch experiment showed that at 12 h and 24 h, the HUVECs in the uMSC-Exo+siRNA-SPRY1#1 group migrated at a faster rate, although the number of cells that migrated increased in both groups (Figure 6E). Similarly, the tube formation data showed that after SPRY1 was silenced, the number of branches and blood vessels more than doubled (Figure 6F).
Taken together, the data from our in vitro functional assays on HUVECs suggested that SPRY1 knockdown increased PI3K/AKT activation and HUVEC proliferation, migration and angiogenesis.
6. Exosomal miR-21 derived from uMSCs enhanced local microvascular network formation and bone regeneration in vivo
To study the influence of exosomal miR-21 on bone formation in vivo, the rat cranial bone defect model was carried out for a duration of 6 weeks. In particular, to evaluate the potential therapeutic role of regulated exosomes in bone healing, we developed an experimental group with exosomes that overexpressed miR-21 (uMSC-Exo-miR-21+) and compared the findings with those of the uMSC-Exo group and the negative control (NC) group.
Then, micro-CT and histological analyses were used to evaluate the extent of calvarial defect repair in the three groups (Figure 7A). Similar to our previous study results in a femur fracture model, the administration of uMSC‐Exos led to an obvious increase in the amount of new bone formation compared with that in the NC group. Moreover, compared to both the uMSC-Exo and the control groups, treatment with uMSC‐Exos with overexpressed miR-21 led to a significant increase in the bone volume (BV), BV/TV and bone mineral density (BMD). Vascular growth within the bone callus was evaluated by imaging of contrast‐perfused, decalcified specimens. The vessel volume was remarkably increased in the uMSC-Exo-miR-21+ group.
H&E staining was used to observe the microscopic bone and soft tissues surrounding the calvarial defect 6 weeks post-operation (Figure 7B). Histological observations at low magnification (40×) revealed that the implanted scaffolds were covered with abundant cells and tissue in both the uMSC-Exo and uMSC-Exo-miR-21+ groups, although a greater amount of callus tissue was observed on the scaffolds in the inner spaces of the uMSC-Exo-miR-21+ group. Moreover, the immunohistochemistry assay results showed that significantly more CD31‐positive blood vessels formed in the uMSC-Exo-miR-21+ group. Compared with that in the control and uMSC-Exo groups, the expression of VEGFA in the new callus tissue was upregulated in the uMSC-Exo-miR-21+ group. Unsurprisingly, the expression of SPRY1 in the uMSC-Exo-miR-21+ group was decreased. These results were consistent with the results from our cell experiments, implying that exosomal miR-21 delivery stimulated greater vessel formation within callus regions and enhanced bone formation in the defect space.