Acute traumatic spinal cord injury is a devastating event without effective therapeutic approach. The feeble plasticity of spinal cord microvascular endothelial cells (SCMECs) after trauma is one of the major causes for the exacerbation of spinal cord injury. Therefore, improving the plasticity and regeneration of SCMECs is crucial to promote recovery after spinal cord injury. For the present study, we explored the influence of exosomes derived from neural stem cells (NSCs-Exos) on the spinal cord microvascular regeneration after spinal cord injury and determined the underlying mechanisms. After the primary NSCs and SCMECs were extracted, exosomes were isolated from NSCs conditioned medium and used to co-incubated with the SCMECs in vitro, and then the effect of exosomes on the angiogenic activities of SCMECs was measured. The candidate molecules involved in the NSCs-Exos-mediated angiogenesis were screened using Western blotting. The effect of NSCs-Exos on angiogenesis and spinal cord functional recovery after injury in vivo was analyzed. The results demonstrated that NSCs-Exos could enhance the angiogenic activities of SCMECs, and were highly enriched in VEGF-A. The level of VEGF-A was downregulated in NSCs^shVEGF-A-Exos and the pro-angiogenic effects on cocultured SCMECs were inhibited. Furthermore, NSCs-Exos significantly accelerated the microvascular regeneration, reduced the spinal cord cavity, and improved the Basso mouse scale scores in spinal cord injury mice. This work provides the evidence of the underlying mechanism of NSCs-Exos-mediated angiogenesis and suggests a novel therapeutic target for spinal cord injury.Impact statement: The feeble plasticity of SCMECs after trauma is one of the major causes for the exacerbation of SCI. Therefore, improving the regeneration ability of SCMECs is crucial to promote spinal cord functional recovery after injury. Our current study uncovered that NSCs-Exos could promote SCMECs migration, tube formation and proliferation in vitro, and further identified that exosomal VEGF-A mediated the pro-angiogenic effect. Furthermore, we observed a remarkable microvascular density increase, spinal cord cavity shrinkage, and motor function recovery in SCI mice treated with NSCs-Exos, which confirmed the therapeutic effects of NSCs-Exos to alleviate SCI. Downregulating VEGF-A partially abolished these effects of NSCs-Exos. This is the first study to reveal that NSCs-Exos has the pro-angiogenic effect on SCMECs by transferring VEGF-A and promote microvascular regeneration and tissue healing, indicating that NSCs-Exos can become a promising therapeutic bioagent for facilitating the functional recovery of SCI.

Multiple cellular, molecular, and biochemical changes contribute to the etiology and treatment outcome of contusion spinal cord injury (SCI). Dysregulation of microRNAs (miRNAs) has been found following SCI in recent studies. However, little is known about the functional significance of the unique role of miRNAs in SCI. We analyzed the miRNA expression patterns 1 and 3 days following rat SCI using miRNA microarray. Microarray data revealed that nine miRNAs were upregulated and five miRNAs were downregulated 1 day post-injury, and that three miRNAs were upregulated and five miRNAs were downregulated 3 days post-injury, in the sites of contused when compared with sham rat spinal cords. Because miR-21 was one of the miRNAs being most significantly upregulated, we investigated its function. Knockdown of miR-21 by antagomir-21 led to attenuated recovery in hindlimb motor function, increased lesion size, and decreased tissue sparing in rats. Compared with the negative control group, treatment with antagomir-21 significantly increased apoptosis following SCI. Pro-apoptosis genes Fas ligand (FasL), phosphatase and tensin homolog (PTEN), and programmed cell death protein 4 (PDCD4) were proved to be direct targets of miR-21 in many diseases and cell types. In vivo treatment with antagomir-21 increased the expression of FasL and PTEN, but did not affect PDCD4. These results suggested that miR-21 played an important role in limiting secondary cell death following SCI, and that the protective effects of miR-21 might have been the result of its regulation on pro-apoptotic genes. Thus, miR-21 may play an important role in the pathophysiology of SCI.

MicroRNAs are a class of small RNAs that regulate the expression of target mRNAs by inhibiting translation or destabilizing target mRNAs. miR-126 is a microRNA that is highly enriched in endothelial cells. miR-126 has been found to promote angiogenesis and inhibit vascular inflammation in endothelial cells by repressing three target genes Sprouty-related EVH1 domain-containing protein 1 (SPRED1), phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2), and vascular cell adhesion molecule 1 (VCAM1). Our previous study showed that the expression of miR-126 was downregulated after spinal cord injury (SCI). Therefore, we wanted to examine whether upregulation of miR-126 could promote angiogenesis, inhibit inflammation, and exert a positive effect on recovery after contusion SCI. In this study, we found that increased levels of miR-126 promoted angiogenesis, and inhibited leukocyte extravasation into the injured spinal cord, which was concurrent with downregulation of mRNA and protein expression of three validated miR-126 target genes, SPRED1, PIK3R2, and VCAM1. Moreover, a dose-dependent effect of miR-126 was observed in rescuing tissue damage and improving the functional deficit after SCI. Thus, the present study indicated that miR-126 played an important role in angiogenesis and inflammation after SCI.

It is well known that appropriate mechanical stimulation benefits tendon-bone (T-B) healing, however, the mechanisms behind this are still uncovered completely. Here, we aimed to explore whether the IL-4/JAK/STAT signaling pathway mediated macrophage polarization was involved in mechanical stimulation induced T-B healing. C57BL/6 mice rotator cuff (RC) repair model was established, and the mice were randomly allocated to the following group. 1. Mice were allowed for free cage activities after surgery (FC group); 2. Mice received treadmill running initiated on postoperative day 7 (TR group); 3. Mice only received a local injection of hydrogel containing IL-4 neutralizing antibody without postoperative intervention (FC ​+ ​AF-404-SP group); 4. Mice received a local injection of hydrogel containing IL-4 neutralizing antibody and postoperative treadmill running (TR ​+ ​AF-404-SP group). The expression of IL-4 within supraspinatus tendon (SST) enthesis was measured by Enzyme-linked immunosorbent assay (ELISA). In addition, the activation of JAK/STAT signaling pathway in macrophages and identification of macrophage phenotype at the RC insertion site was detected by Flow cytometry and qRT-PCR. T-B healing quality in this RC repair model was evaluated by histological staining, Micro-computed tomography (Micro-CT) scanning, and biomechanical testing. In this study, using the RC repair model, we confirmed that generation of IL-4, activation of the JAK/STAT signaling pathway in macrophages, the ability of macrophages to polarize towards M2 subtype, and T-B healing quality were significantly enhanced in TR group compared to FC group. When comparing FC ​+ ​AF-404-SP group with TR ​+ ​AF-404-SP group, it was found that the mechanical stimulation induced this effect was depleted following the blockade of the IL-4/JAK/STAT signaling pathway. Our finding suggested that mechanical stimulation could accelerate T-B healing via activating the IL-4/JAK/STAT signaling pathway that modulates macrophages to polarize towards M2 subtype. This is the first study to reveal a significant role of mechanical stimulation in the IL-4/JAK/STAT signaling pathway activation and macrophage polarization during RC T-B healing, which highlights the IL-4/JAK/STAT signaling pathway as a potential target to mediate macrophage M2 polarization and improves T-B healing for RC repair.

MicroRNAs are a novel set of small, non-protein-coding nucleotide RNAs that negatively regulate the expression of target mRNAs. miRNA-21 is a microRNA that is highly enriched in endothelial cells. miRNA-21 has been shown to be a potential pro-angiogenic factor in some biological systems. Our previous study showed that the expression of miRNA-21 was up-regulated after spinal cord injury. However, the effect of miRNA-21 on angiogenesis in the spinal cord was unclear. In this study, to understand the role of miRNA-21 on injured endothelial cells exclusively, an oxygen and glucose deprivation model of endothelial cells was constructed, and the up-regulation of miRNA-21 was discovered in this model. An increased level of miRNA-21 by mimics promoted the survival, migration and tube formation of endothelial cells, which simultaneously inhibited tissue inhibitor of metalloproteinase-3 (TIMP3) expression and promoted matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9) expression and secretion. A decreased level of miRNA-21 by antagomir exerted an opposite effect. As is well known, survival, migration and tube formation of endothelial cells are necessary prerequisites for angiogenesis after injury. TIMP3 was validated as a direct target of miRNA-21 by dual-luciferase reporter assay. Silencing with small interfering RNA against TIMP3 promoted tube formation and increased MMP2 and MMP9 expression at the protein level. In vivo, we found that decreased levels of miRNA-21 inhibited angiogenesis after spinal cord injury in rats using synchrotron radiation micro-computed tomography. In summary, these findings suggest that miRNA-21 has a protective effect on angiogenesis by reducing cell death and promoting cell survival, migration and tube formation via partially targeting the TIMP3 by potentially regulating MMP2 and MMP9. TIMP3 is a functional target gene. Identifying the role of miRNA-21 in the protection of angiogenesis might offer a novel therapeutic target for secondary spinal cord injury, in which angiogenesis is indispensable.