Exosomes derived from M2 Macrophages Improve Angiogenesis and Functional Recovery after Spinal Cord Injury through HIF-1α/VEGF Axis.

Exosomes are nano-sized vesicles that contain a variety of mRNAs, miRNAs, and proteins. They are capable of being released by a variety of cells and are essential for cell-cell communication. The exosomes produced by cells have shown protective benefits against spinal cord damage (SCI). Recently, it was discovered that M2 macrophages aid in the angiogenesis of numerous illnesses. However, the functional role of M2 macrophage-derived exosomes on SCI is unclear. Here, we investigate the pro-angiogenesis of M2 macrophage-derived exosomes on SCI. We founded that M2 macrophage exosomes alleviated tissue damage and enhanced functional recovery post-SCI. We discovered that M2 macrophage exosome administration increased angiogenesis after SCI in vivo using immunohistochemistry, immunofluorescence labeling, and Western blot analysis. Additionally, the expression of the pro-angiogenesis factors, HIF-1α and VEGF, were enhanced with the treatment of the M2 macrophage exosomes. Furthermore, we found that M2 macrophage exosomes enhanced neurogenesis after SCI in vivo. In vitro, we found that M2 macrophage exosomes can be taken up by the brain endothelial cell line (bEnd.3) and that they enhanced the tube formation, migration, and proliferation of bEnd.3 cells. Furthermore, by using special siRNA to inhibit HIF-1α expression, we observed that the expression of VEGF decreased, and the tube formation, migration, and proliferation of bEnd.3 cells were attenuated with the treatment of HIF-1α-siRNA. In conclusion, our findings reveal that M2 macrophage exosomes improve neurological functional recovery and angiogenesis post-SCI, and this process is partially associated with the activation of the HIF-1/VEGF signaling pathway.

Schwann cells-derived exosomes promote functional recovery after spinal cord injury by promoting angiogenesis.

Exosomes are small vesicles that contain diverse miRNA, mRNA, and proteins that are secreted by multiple cells, and play a vital function in cell-cell communication. Numerous exosomes produced by cells have been demonstrated to be protective against spinal cord injury (SCI). This study aims to investigate the neuroprotective effect of Schwann cells-derived exosomes (SCs-Exos) on spinal cord injury. We found that SCs-Exos can be taken directly by brain-derived endothelial cells.3 (bEnd.3 cells) and promoted to proliferate, migrate, and form bEnd.3 tube. Additionally, our results showed that the pro-angiogenesis molecules, Integrin-ß1, were highly expressed in SCs-Exos. Moreover, we used special shRNA technology to investigate the role of Integrin-ß1 in mediating the effect of SCs-Exos-induced angiogenesis on bEnd.3 cells. We observed that the pro-angiogenic effect of SCs-Exos on bEnd.3 cells was suppressed by inhibiting the expression of integrin-ß1 in SCs-Exos. In the SCI model, we found that SCs-Exos attenuated tissue damage and improved functional recovery after SCI. Using immunofluorescence staining, we observed that SCs-Exos treatment promoted angiogenesis in SCI, and integrin-ß1 was required to promote angiogenesis. In conclusion, our results indicate that SCs-Exos promote angiogenesis by delivering integrin-ß1 and may serve as a promising novel therapeutic agent for enhancing neurological functional recovery after SCI.

The inhibition of microRNA-326 by SP1/HDAC1 contributes to proliferation and metastasis of osteosarcoma through promoting SMO expression.

Osteosarcoma (OS) is a malignant bone cancer lacking of effective treatment target when the metastasis occurred. This study investigated the implication of MicroRNA-326 in OS proliferation and metastasis to provide the clue for the treatment of metastatic OS. This study knocked down SP1 in MG63 and 143B cells and then performed Microarray assay to find the expression of miRNAs that were influenced by SP1. MTT, EdU, wound-healing and cell invasion assays were performed to evaluated cell proliferation and invasion. OS metastasis to lung was detected in a nude mice model. ChIP assay and DAPA were applied to determine the regulatory effect of SP1 and histone deacetylase 1 (HDAC) complex on miR-326 expression. Human OS tissues showed lowly expressed miR-326 but highly expressed Sp1 and HDAC. Sp1 recruited HDAC1 to miR-326 gene promoter, which caused the histone deacetylation and subsequent transcriptional inhibition of miR-326 gene. miR-326 deficiency induced the stimulation of SMO/Hedgehog pathway and promoted the proliferation and invasion of 143B and MG63 cells as well as the growth and metastasis in nude mice. SP1/HDAC1 caused the transcriptional inhibition of miR-326 gene by promoting histone deacetylation; miR-326 deficiency conversely stimulated SMO/Hedgehog pathway that was responsible for the proliferation and metastasis of OS.

Extracellular Vesicles Derived from Epidural Fat-Mesenchymal Stem Cells Attenuate NLRP3 Inflammasome Activation and Improve Functional Recovery After Spinal Cord Injury.

Spinal cord injury (SCI) is a devastating event which caused high mortality and morbidity. Recently, nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome has been showed to act a critical t role in the secondly injury phase of SCI. In current study, we aimed to investigate the effect and underlying molecular mechanisms of extracellular vesicles derived from epidural fat (EF)- mesenchymal stem cells (MSCs) for the treatment of SCI. Ninety-six Sprague-Dawley rats were used for current study and randomly divided into four groups: sham group, SCI group, SCI + Saline group, SCI + Extracellular vesicles group. Basso-Beattie-Bresnahan (BBB) scores was applied to evaluate the neurological functional recovery. Cresyl violet-stained was conducted evaluate the protective effect of EF-MSCs-Extracellular vesicles on lesion volume after SCI. ELISA, immunohistochemistry assay, TUNEL assay and western blotting were conducted to investigate the underlying molecular mechanisms. Our results demonstrated that the administration of EF-MSCs-Extracellular vesicles via tail vein injection improved neurological functional recovery and reduced the lesion volume after SCI. And systemic administration of EF-MSCs-Extracellular vesicles significantly inhibited NLRP3 inflammasome activation and reduced the expression of inflammatory cytokines. Additionally, the expression levels of proapoptotic protein Bax was decreased and antiapoptotic Bcl-2 was upregulated with the treatment of EF-MSCs-Extracellular vesicles after SCI. In summary, in current study, we demonstrated for the first time that the EF-MSCs-Extracellular vesicles can improve neurological functional recovery after SCI, and the underlying molecular mechanisms may partly through the inhibition of NLRP3 inflammasome activation.

Exosomes Derived from miR-126-modified MSCs Promote Angiogenesis and Neurogenesis and Attenuate Apoptosis after Spinal Cord Injury in Rats.

Spinal cord injury (SCI) is a devastating neurological event that results in incomplete or complete loss of voluntary motor and sensory function. Until recently, there has been no effective curative strategy for SCI. Our previous study showed that microRNA (miR)-126 promoted angiogenesis and attenuated inflammation after SCI; however, the effect of miR-126-based treatment is limited because of the low efficiency of miR delivery in vivo. Recently, accumulating evidence has indicated that exosomes can serve as a valuable therapeutic vehicle for miR delivery to the central nervous system (CNS). Thus, the present study aimed to investigate whether exosomes derived from mesenchymal stem cells (MSCs) can be used to deliver miR-126 to treat SCI. In this study, we found that MSCs can load miR-126 into secreted exosomes. In a rat model of SCI, exosomes transferred miR-126 to the injured site of the spinal cord, reduced the lesion volume and improved functional recovery after SCI. Additionally, miR-126-loaded exosomes promoted angiogenesis post-SCI. Moreover, the administration of miR-126 exosomes promoted neurogenesis and reduced cell apoptosis after SCI. In vitro, we observed that exosomes derived from miR-126-modified MSCs promoted the angiogenesis and migration of human umbilical venous endothelial cells (HUVECs) by inhibiting the expression of Sprouty-related EVH1 domain-containing protein 1 (SPRED1) and phosphoinositide-3-kinase regulatory subunit 2 (PIK3R2). In conclusion, our study demonstrated that exosomes derived from MSCs transfected with miR-126 may promote angiogenesis and neurogenesis, inhibit apoptosis and promote functional recovery after SCI. These findings suggest that exosomes derived from miR-126-modified MSCs may serve as a novel potential therapeutic strategy for treating SCI.

Systemic Administration of Exosomes Released from Mesenchymal Stromal Cells Attenuates Apoptosis, Inflammation, and Promotes Angiogenesis after Spinal Cord Injury in Rats.

Spinal cord injury (SCI) is one of the most common devastating injuries, which causes permanent disabilities such as paralysis and loss of movement or sensation. The precise pathogenic mechanisms of the disease remain unclear, and, as of yet, there is no effective cure. Mesenchymal stem cells (MSCs) show promise as an effective therapy in the experimental models of SCI. MSCs secrete various factors that can modulate a hostile environment, which is called the paracrine effect. Among these paracrine molecules, exosome is considered to be the most valuable therapeutic factor. Thus, exosomes from MSCs (MSCs-exosomes) can be a potential candidate of therapeutic effects of stem cells. The present study was designed to investigate the effect of whether systemic administration of exosomes generated from MSCs can promote the function recovery on the rat model of SCI in vivo. In the present study, we observed that systemic administration of MSCs-exosomes significantly attenuated lesion size and improved functional recovery post-SCI. Additionally, MSCs-exosomes treatment attenuated cellular apoptosis and inflammation in the injured spinal cord. Expression levels of proapoptotic protein (Bcl-2-associated X protein) and proinflammatory cytokines (tumor necrosis factor alpha and interleukin [IL]-1ß) were significantly decreased after MSCs-exosomes treatment, whereas expression levels of antiapoptotic (B-cell lymphoma 2) and anti-inflammatory (IL-10) proteins were upregulated. Further, administration of MSCs-exosomes significantly promoted angiogenesis. These results show, for the first time, that systemic administration of MSCs-exosomes attenuated cell apoptosis and inflammation, promoted angiogenesis, and promoted functional recovery post-SCI, suggesting that MSCs-exosomes hold promise as a novel therapeutic strategy for treating SCI.

Tetramethylpyrazine enhances functional recovery after contusion spinal cord injury by modulation of MicroRNA-21, FasL, PDCD4 and PTEN expression.

Our previous study showed Tetramethylpyrazine (TMP) has protective effects against SCI. In this study, we aimed to uncover the mechanism underlying the protective effects of TMP in SCI. SCI was induced in Sprague-Dawley rats with a modified weight-drop device. One group was subjected to SCI in combination with TMP administration at a dose of 200mg/kgd, for 3 days. Concurrently, another group received SCI in combination with an equal volume of 0.9% saline. Locomotor functional recovery was assessed during the 4 weeks post-injury by performing the Basso, Beattie, and Bresnahan (BBB) rating procedure. Lesion size and spared tissue were measured by cresyl violet staining. MicroRNA-21 (miR-21) expression was determined by real-time PCR and in situ hybridization. FasL, PDCD4, and PTEN are direct targets of miR-21 in many diseases and cell types; their levels were analyzed by western blot. Immunohistochemistry was performed to observe the expression of PDCD4 and PTEN. Cell apoptosis was assessed by TUNEL staining and DNA laddering. TMP treatment after contusion SCI significantly improved functional recovery, decreased lesion size, and increased tissue sparing and miR-21 levels; expression of FasL, PDCD4, and PTEN was decreased. TMP treatment also reduced apoptosis after SCI. Thus, TMP administration improved functional recovery and reduced cell apoptosis. Its protective effect may partly based on increasing the expression of miR-21 and decreasing the expression of FasL, PDCD4, and PTEN. These could serve as new exploratory targets for SCI treatment.

Anti-apoptotic effect of microRNA-21 after contusion spinal cord injury in rats.

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.

Tetramethylpyrazine accelerates the function recovery of traumatic spinal cord in rat model by attenuating inflammation.

In the present study, we explored the effects of tetramethylpyrazine (TMP), an alkaloid extracted from the Chinese herbal medicine Ligusticum wallichii Franchat (chuanxiong), on a rat model of contusion spinal cord injury (SCI). The contusion SCI model was induced in rats by a modified Allen's weight-drop method with a severity of 5 g × 50 mm impacting on the T10 segment. In the TMP treatment group, rats were injected intraperitoneally (i.p.) with TMP (200mg/kg), every 24h for 5 days, starting half an hour after contusion SCI. The control group was treated with saline. Compared with the control group, the TMP group significantly ameliorated the recovery of hindlimb function of rats. TMP treatment significantly reduced the expression of macrophage migration inhibitory factor, nuclear factor κappa B, pro-inflammatory cytokine interleukin-18 and neutrophil infiltration. On the other hand, TMP enhanced the expression of inhibitor κappa B and anti-inflammation cytokine interleukin-10. In conclusion, our results demonstrate that TMP inhibits the development of inflammation and tissue injury associated with spinal cord contusion in rats which may improve the rats' hindlimb function.