MCPIP-1 knockdown enhances endothelial colony-forming cell angiogenesis via the TFRC/AKT/mTOR signaling pathway in the ischemic penumbra of MCAO mice.

Cerebral ischemia is a serious disease characterized by brain tissue ischemia and hypoxic necrosis caused by the blockage of blood vessels within the central nervous system. Although stem cell therapy is a promising approach for treating ischemic stroke, the inflammatory, oxidative, and hypoxic environment generated by cerebral ischemia greatly reduces the survival and therapeutic effects of transplanted stem cells. Endothelial colony-forming cells (ECFCs) are a class of precursor cells with strong proliferative potential that can migrate and differentiate directly into mature vascular endothelial cells. Consequently, ECFCs can exert significant therapeutic and reparative effects in diseases associated with vascular injury. Monocyte chemoattractant protein-induced protein 1 (MCPIP-1) exerts multiple biological effects; however, no studies have yet reported its role in the angiogenic function of ECFCs. In this study, we performed Proteome Profiler™ Human Angiogenesis Antibody arrays and tandem mass tag protein profiling to investigate the effect of MCPIP-1 on ECFCs. We demonstrated that MCPIP-1 knockdown enhanced the proliferation, migration, and in vivo and in vitro angiogenic capacity of ECFCs by upregulating the transferrin receptor-activated AKT/m-TOR signaling pathway to promote cellular trophic factor secretion. Furthermore, we found that the lateral ventricular transplantation of ECFCs with lentiviral MCPIP-1 knockdown into mice with middle cerebral artery occlusion increased serum vacular endothelial growth factor(VEGF), angiopoietin-1, and HIF-1a levels, enhanced neovascularization and neurogenesis in the ischemic penumbra, reduced the size of cerebral infarcts, and promoted neurological recovery. Together, these findings suggest new avenues for enhancing the therapeutic efficacy of ECFCs.

Hydroxysafflor yellow a confers neuroprotection against acute traumatic brain injury by modulating neuronal autophagy to inhibit NLRP3 inflammasomes.

ETHNOPHARMACOLOGICAL RELEVANCE: Hydroxysafflor yellow A (HSYA) is the principal bioactive compound isolated from the plant Carthamus tinctorius L. and has been reported to exert neuroprotective effects against various neurological diseases, including traumatic brain injury (TBI). However, the specific molecular and cellular mechanisms underlying HSYA-mediated neuroprotection against TBI are unclear. AIM OF THE STUDY: This study explored the effects of HSYA on autophagy and the NLRP3 inflammasome in mice with TBI and the related mechanisms. MATERIALS AND METHODS: Mice were subjected to TBI and treated with or without HSYA. Neurological severity scoring, LDH assays and apoptosis detection were first performed to assess the effects of HSYA in mice with TBI. RNA-seq was then conducted to explore the mechanisms that contributed to HSYA-mediated neuroprotection. ELISA, western blotting, and immunofluorescence were performed to further investigate the mechanisms of neuroinflammation and autophagy. Moreover, 3-methyladenine (3-MA), an autophagy inhibitor, was applied to determine the connection between autophagy and the NLRP3 inflammasome. RESULTS: HSYA significantly decreased the neurological severity score, serum LDH levels and apoptosis in mice with TBI. A total of 921 differentially expressed genes were identified in the cortices of HSYA-treated mice with TBI and were significantly enriched in the inflammatory response and autophagy. Furthermore, HSYA treatment markedly reduced inflammatory cytokine levels and astrocyte activation. Importantly, HSYA suppressed neuronal NLRP3 inflammasome activation, as indicated by decreased levels of NLRP3, ASC and cleaved caspase-1 and a reduced NLRP3+ neuron number. It increased autophagy and ameliorated autophagic flux dysfunction, as evidenced by increased LC3 II/LC3 I levels and decreased P62 levels. The effects of HSYA on the NLRP3 inflammasome were abolished by 3-MA. Mechanistically, HSYA may enhance autophagy through AMPK/mTOR signalling. CONCLUSION: HSYA enhanced neuronal autophagy by triggering the AMPK/mTOR signalling pathway, leading to inhibition of the NLRP3 inflammasome to improve neurological recovery after TBI.

The MicroRNA-106a/20b Strongly Enhances the Antitumour Immune Responses of Dendritic Cells Pulsed with Glioma Stem Cells by Targeting STAT3.

Background: Evaluate the effect of the miRNA-106a/20b on the efficacy of DCs pulsed with GSCs in activating GSC-specific T cell responses. Methods: We cultured GSCs and prepared GSC antigen lysates by apoptosis. Then, immature DCs were pulsed with GSC antigen lysates in vitro. STAT3 levels in DCs were assessed by Western blotting, and the expression of CD80, CD86, and MHC-II was tested by fluorescence-activated cell sorting. The production and secretion of the cytokines IL-6, IL-12, TNF-α, and IL-10 in DCs induced by GSCs were determined by enzyme-linked immunosorbent assay. Finally, the cytotoxic functions of T cells stimulated by GSC-DC fusion cells transfected with a miR-106a/20b mimic in vitro and the antitumour activity in vivo were detected. Results: We found that the levels of miR-106a/20b were downregulated, but the expression of STAT3 was significantly upregulated. Simultaneously, the inhibition of STAT3 in the fusion cells by STAT3-specific siRNA caused significant upregulation of the expression of CD80, CD86, and MHC-II, and the secretion of the cytokines IL-6 and IL-12 was substantially increased, IL-10 was markedly decreased. These findings revealed that STAT3 is an important regulator of DC maturation. Furthermore, the interactional binding sites between the 3'-untranslated region (3'-UTR) of STAT3 mRNA and miR-106a/20b were predicted by bioinformatics and verified by a dual-luciferase assay. Moreover, the reduction in STAT3 levels in GSC-DCs enhanced the generation of CD8+ T cells and reduced the generation of Foxp3+ regulatory T cells. Meanwhile, the secretion of the T cell cytokine IFN-γ was significantly increased. Further research showed that DCs after miR-106a/20b-mimics transfection could promote the inhibition of GSC proliferation by T cells in vitro and suppress tumour growth in vivo. Conclusions: This study indicted that the miR-106a/20b activation could be one of the important molecular mechanisms leading to enhance antitumour immune responses of GSC-mediated DCs, which downregulated the expression of STAT3 to alleviate its the inhibitory effect.

Mesenchymal stem cells protect against TBI-induced pyroptosis in vivo and in vitro through TSG-6.

BACKGROUND: Pyroptosis, especially microglial pyroptosis, may play an important role in central nervous system pathologies, including traumatic brain injury (TBI). Transplantation of mesenchymal stem cells (MSCs), such as human umbilical cord MSCs (hUMSCs), has been a focus of brain injury treatment. Recently, MSCs have been found to play a role in many diseases by regulating the pyroptosis pathway. However, the effect of MSC transplantation on pyroptosis following TBI remains unknown. Tumor necrosis factor α stimulated gene 6/protein (TSG-6), a potent anti-inflammatory factor expressed in many cell types including MSCs, plays an anti-inflammatory role in many diseases; however, the effect of TSG-6 secreted by MSCs on pyroptosis remains unclear. METHODS: Mice were subjected to controlled cortical impact injury in vivo. To assess the time course of pyroptosis after TBI, brains of TBI mice were collected at different time points. To study the effect of TSG-6 secreted by hUMSCs in regulating pyroptosis, normal hUMSCs, sh-TSG-6 hUMSCs, or different concentrations of rmTSG-6 were injected intracerebroventricularly into mice 4 h after TBI. Neurological deficits, double immunofluorescence staining, presence of inflammatory factors, cell apoptosis, and pyroptosis were assessed. In vitro, we investigated the anti-pyroptosis effects of hUMSCs and TSG-6 in a lipopolysaccharide/ATP-induced BV2 microglial pyroptosis model. RESULTS: In TBI mice, the co-localization of Iba-1 (marking microglia/macrophages) with NLRP3/Caspase-1 p20/GSDMD was distinctly observed at 48 h. In vivo, hUMSC transplantation or treatment with rmTSG-6 in TBI mice significantly improved neurological deficits, reduced inflammatory cytokine expression, and inhibited both NLRP3/Caspase-1 p20/GSDMD expression and microglial pyroptosis in the cerebral cortices of TBI mice. However, the therapeutic effect of hUMSCs on TBI mice was reduced by the inhibition of TSG-6 expression in hUMSCs. In vitro, lipopolysaccharide/ATP-induced BV2 microglial pyroptosis was inhibited by co-culture with hUMSCs or with rmTSG-6. However, the inhibitory effect of hUMSCs on BV2 microglial pyroptosis was significantly reduced by TSG-6-shRNA transfection. CONCLUSION: In TBI mice, microglial pyroptosis was observed. Both in vivo and in vitro, hUMSCs inhibited pyroptosis, particularly microglial pyroptosis, by regulating the NLRP3/Caspase-1/GSDMD signaling pathway via TSG-6. Video Abstract.

Indole-3-propionic acid alleviates ischemic brain injury in a mouse middle cerebral artery occlusion model.

Increasing evidence highlights the importance of gut microbiota and its metabolites as an environmental factor affecting ischemic stroke. However, the role of microbial indole metabolites in ischemic stroke remains largely unknown. Here, we evaluated the effects and the underlying mechanism of indole-3-propionic acid (IPA) in a mouse model of acute middle cerebral artery occlusion (MCAO) and the mechanisms underlying these effects. We collected blood samples and evaluated serum indole derivatives levels using ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS) in 8-10-week-old male C57 mice undergoing MCAO or sham. Intragastric IPA administration (400 µg/20 g/d) was performed in mice with MCAO, and its effects and mechanisms were assessed. We found that the serum IPA levels were significantly lower in mice with MCAO than in sham-treated subjects. 16S rRNA gene sequencing revealed that IPA treatment ameliorated the MCAO-induced alterations of the gut microbiome structure, specifically reshaping the microbial community composition in mice with MCAO to resemble that in the mice from the control group, with an increase in the abundance of probiotics and a decrease in the abundance of harmful bacteria. IPA repaired the integrity of the intestinal barrier and regulated the activities of regulatory T cells (Tregs) and Th17 cells in the gut-associated lymphoid tissue. Intragastric IPA administration effectively alleviated neuroinflammation, neurological impairment and brain infarction. Of note, Tregs in the IPA treatment group inhibited A1 reactive astrogliosis in vitro. The beneficial effects of IPA are thus mediated by the gut microbiota, which could enable the development of prebiotics for microbiome-based treatments for ischemic stroke.

Human umbilical cord mesenchymal stem cell-derived exosomal miR-146a-5p reduces microglial-mediated neuroinflammation via suppression of the IRAK1/TRAF6 signaling pathway after ischemic stroke.

To investigate the therapeutic mechanism of action of transplanted stem cells and develop exosome-based nanotherapeutics for ischemic stroke, we assessed the effect of exosomes (Exos) produced by human umbilical cord mesenchymal stem cells (hUMSCs) on microglia-mediated neuroinflammation after ischemic stroke. Our results found that injected hUMSC-Exos were able to access the site of ischemic damage and could be internalized by cells both in vivo and in vitro. In vitro, treatment with hUMSC-Exos attenuated microglia-mediated inflammation after oxygen-glucose deprivation (OGD). In vivo results demonstrated that treatment with hUMSC-Exos significantly reduced infarct volume, attenuated behavioral deficits, and ameliorated microglia activation, as measured three days post-transient brain ischemia. Furthermore, miR-146a-5p knockdown (miR-146a-5p k/d Exos) partially reversed the neuroprotective effect of hUMSC-Exos. Our mechanistic study demonstrated that miR-146a-5p in hUMSC-Exos reduces microglial-mediated neuroinflammatory response through IRAK1/TRAF6 pathway. We conclude that miR-146a-5p derived from hUMSC-Exos can attenuate microglia-mediated neuroinflammation and consequent neural deficits following ischemic stroke. These results elucidate a potential therapeutic mechanism of action of mesenchymal stem cells and provide evidence that hUMSC-Exos represent a potential cell-free therapeutic option for ischemic stroke.

LncRNA GAS5 regulates the proliferation, migration, invasion and apoptosis of brain glioma cells through targeting GSTM3 expression. The effect of LncRNA GAS5 on glioma cells.

INTRODUCTION: To investigate the effects of lncRNA GAS5 on the proliferation, migration, invasion and apoptosis of brain glioma cells. METHODS: The expression levels of lncRNA GAS5 and GSTM3 in normal glial cells (HEB) and glioma cells (U251 and U87) were detected by RT-qPCR and western blot, respectively. Glioma cells were transfected with ctrl vector, pcDNA-GAS5, siRNA ctrl (siNC) or GSTM3 siRNA and the effects of lncRNA GAS5 and GSTM3 on the proliferation, migration, invasion and apoptosis of glioma cells were detected by CCK-8 assay, transwell assay and Caspase 3/7 activity assay, respectively. RESULTS: The expression of lncRNA GAS5 was significantly decreased in glioma cell lines U251 and U87 compared with normal glial cells HEB (p < 0.01). In addition, overexpression of lncRNA GAS5 inhibited the proliferation, migration and invasion of U251 and U87 cells, and promoted cell apoptosis as demonstrated by the increased activity of Caspase 3/7. Furthermore, GSTM3 was predicted as a target gene of lncRNA GAS5 by bioinformatics analysis and its expression was increased in glioma cells compared with the normal cells as indicated by western blotting and RT-qPCR experimental results. Silencing of GSTM3 with GSTM3 siRNA decreased the proliferation, migration and invasion but increased the apoptosis of glioma cell lines U251 and U87, which was similar to that the effect lncRNA GAS5 over-expression. CONCLUSION: lncRNA GAS5 can effectively inhibit the proliferation, migration and invasion of glioma cells and promote cell apoptosis through targeting GSTM3 expression.

Meta-Analysis of the Safety and Efficacy of Stem Cell Therapies for Ischemic Stroke in Preclinical and Clinical Studies.

Recent studies have indicated that stem cell transplantation may be effective in the treatment of ischemic stroke. Therefore, we performed a meta-analysis to evaluate the safety and efficacy of stem cell therapy for ischemic stroke in preclinical and clinical studies. In accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, we searched the PubMed, Cochrane Library, Embase, Web of science, and Ovid databases from inception through May 2018. A total of 11 preclinical studies-18 independent interventions were ultimately included. Similarly, 11 clinical studies were finally included. Two authors independently screened trials. Lesion volume and modified neurological severity scores (mNSSs) were regarded as outcome measures for preclinical studies. The composite weighted mean [95% confidence interval (CI)] effect sizes for lesion volume, percentage of lesion volume, and mNSSs were -46.59 (-62.04 to -31.15; P < 0.001), -13.18 (-25.62 to -0.73; P = 0.04), and -1.85 (-2.17 to -1.53; P < 0.001), respectively. Our analysis revealed that all three outcomes were significantly more favorable in the stem cell group than in the control group. Barthel index (BI) values, modified Rankin scale (mRS) scores, National Institutes of Health Stroke Scale (NIHSS) scores, and Fugl-Meyer assessment (FMA) scores were regarded as outcome measures for human studies. Our results were as follows: NIHSS [mean differences, MDs = -2.57, 95% CI (-3.45 to -1.68), I2 = 51%, P < 0.001]; BI [MD = 7.93, 95% CI (3.11 to 12.75), I2 = 59%, P = 0.001]; mRS [MD = -0.53, 95% CI (-0.73 to -0.28), I2 = 0%, P < 0.001]; FMA [MD = 5.50, 95% CI (2.05 to 8.95), I2 = 15%, P = 0.002]. These results suggest that stem cell transplantation was associated with significantly better outcomes than control treatment. Adverse reactions such as mild headache and fever resolved shortly after treatment. Stem cell transplantation can significantly improve neurological deficits and quality of life in patients with ischemic stroke, without severe adverse reactions. Our results also suggest that such treatment is most effective when provided earlier and through the intravenous route.

Circ-U2AF1 promotes human glioma via derepressing neuro-oncological ventral antigen 2 by sponging hsa-miR-7-5p.

The prognosis for human glioma, a malignant tumor of the central nervous system, is poor due to its rapid growth, genetic heterogeneity, and inadequate understanding of its underlying molecular mechanisms. Circular RNAs composed of exonic sequences, represent an understudied form of noncoding RNAs (ncRNAs) that was discovered more than a decade ago, function as microRNA sponges. We aimed to assess the relationship between circ-U2AF1 (CircRNA ID: hsa_circ_0061868) and hsa-mir-7-5p and examine their effects on proliferation, apoptosis, and the metastatic phenotype of glioma cells regulated by neuro-oncological ventral antigen 2 (NOVA2). We found that the expression levels of circ-U2AF1 and NOVA2 were upregulated, while hsa-miR-7-5p was downregulated in human glioma tissues and glioma cell lines. Our data and bioinformatic analysis indicated the association of these molecules with glioma grade, a positive correlation between circ-U2AF1 and NOVA2 expression levels and a negative correlation of hsa-miR-7-5p with both circ-U2AF1 and NOVA2, respectively. In addition, silencing of circ-U2AF1 expression resulted in increased hsa-miR-7-5p expression and decreased NOVA2 expression both in vitro and in vivo. Luciferase assay confirmed hsa-miR-7-5p as a direct target of circ-U2AF1 and NOVA2 as a direct target of hsa-miR-7-5p. Functionally, silencing of circ-U2AF1 inhibits glioma development by repressing NOVA2 via upregulating hsa-miR-7-5p both in vitro and in vivo. Thus, we assumed that circ-U2AF1 promotes glioma malignancy via derepressing NOVA2 by sponging hsa-miR-7-5p. Taken together, we suggest that circ-U2AF1 can be a prognostic biomarker and the circ-U2AF1/hsa-miR-7-5p/NOVA2 regulatory pathway may be a novel therapeutic target for treating gliomas.

Oncostatin M-induced upregulation of SDF-1 improves Bone marrow stromal cell migration in a rat middle cerebral artery occlusion stroke model.

Bone marrow-derived mesenchymal stem cells (BMSCs) exhibit potential regenerative effects on the injured brain. However, these effects are constrained by their limited ability to migrate to the injured site. Oncostatin M (OSM) has been shown to affect the proliferation and migration of mesenchymal stem cells. Therefore, in the present study, we explored whether OSM improves BMSC migration and secretion of growth factors and cytokines in a rat middle cerebral artery occlusion (MCAO) stroke model. The effect of OSM on the proliferation and apoptosis of rat BMSCs was first assessed in vitro, and the gene and secretion levels of factors related to cell nutrition and migration, such as SDF-1 and VEGF, were detected. To further explore underlying pathways triggered by OSM, BMSCs were treated with OSM in the presence or absence of inhibitors of the STAT3 and ERK pathways. Effects of OSM on SDF-1 expression in astrocytes and BMSC migration were also evaluated. In the rat MCAO model, OSM secretion levels were detected in the brain for up to 72 h after model establishment. Ventricle injection of OSM alone or OSM combined with caudal vein graft of BMSCs was then performed in MCAO stroke rats. After 72 h, production of SDF-1 and grafted BMSCs was detected in the lesion areas of the brain, and the nerve function score was evaluated. We found that the production of OSM continually increased in the brains of MCAO rats from 12 h to 72 h. OSM significantly upregulated SDF-1 in BMSCs via the STAT3 and ERK pathways and significantly promoted the expression of VEGF and MMP-2. OSM also promoted the secretion of SDF-1 in astrocytes through the STAT3 and ERK pathways to in turn enhance BMSC migration. Combination treatment with OSM and BMSCs in MCAO rats increased the migration efficiency of BMSCs in the brain, which significantly improved neurofunctional recovery while reducing the expression of inflammatory mediators and promoting the secretion of nutrition factors. Overall, these results show that OSM is highly expressed in the brains of MCAO stroke rats and can upregulate SDF-1 to promote BMSC migration. Thus, combination treatment with OSM and BMSCs improves the graft efficiency of BMSCs and neurofunctional recovery.

miR­141 inhibits glioma vasculogenic mimicry by controlling EphA2 expression.

Human glioma is a pernicious tumor from the central nervous system; it has been reported that microRNAs (miRs) may have carcinogenic or tumor suppressor effects on human glioma. The aim of the present study was to assess miR­141 expression and functional role in human primary glioma, as well as in tumor­derived cell lines. The expression of miR­141 in primary human glioma tissues and cell lines was assessed by employing reverse transcription­quantitative polymerase chain reaction. Next, its role in cellular growth, migration, invasion and vasculogenic mimicry (VM) regulation was determined using various in vitro and in vivo assays, and on the identification its target gene(s) using luciferase assays. The results demonstrated that miR­141 expression was downregulated, and Ephrin type­A receptor 2 (EphA2) was upregulated in the primary human gliomas and human glioma­derived cell lines tested. In addition, a negative correlation existed between miR­141 and EphA2 expression levels in glioma grades II, III and IV. Furthermore, exogenous miR­141 expression resulted in decreased proliferation, migration and invasion, as well as in apoptosis and cell cycle arrest in vitro. It was also revealed that exogenous miR­141 expression resulted in in vivo inhibition of tumor growth and inhibition of the development of VM. Finally, the present study successfully confirmed that EphA2 was a direct target of miR­141 in glioma­derived cells using luciferase assays. Based on these results, it was concluded that miR­141 may regulate cell proliferation, migration, invasion and VM formation by controlling EphA2 expression; also, its target EphA2 may be a novel diagnostic/prognostic biomarker and a potential anti­VM therapeutic target.

Blocking the CD47-SIRPα axis by delivery of anti-CD47 antibody induces antitumor effects in glioma and glioma stem cells.

Tumor initiating cells or cancer stem cells (CSCs) play an important role in the initiation, development, metastasis, and recurrence of tumors. However, traditional therapies have limited effects against CSCs and targeting these cells is crucial when developing new therapeutic strategies against cancer. One potentially targetable factor is CD47, a member of the immunoglobulin superfamily. This protein acts as an anti-phagocytic "don't eat me" signal and is often found expressed by cancer cells, particularly CSCs. CD47 functions by activating signal regulatory protein-α (SIRP-α) expressed on macrophages, preventing phagocytosis. However, the role of CD47 in glioma stem cells (GSCs) has been not been thoroughly investigated. Our study therefore examined the expression and function of this protein in glioma cells and GSCs. We found that CD47 was highly expressed on glioma cells, especially GSCs, and that expression associated with worse clinical outcomes. We also found that CD47+ glioma cells possessed stem/progenitor cell-like characteristics and knocking down CD47 expression resulted in a reduction in these characteristics. Treatment with anti-CD47 antibody led to increased phagocytosis of glioma cells and GSCs by macrophages. We next examined the effects of anti-CD47 antibody on glioma cells/GSCs in an immune competent mouse glioma model, revealing significant inhibition of tumor growth and prolonged survival times. Importantly, there were no apparent side effects in the animal model. In summary, we have shown that CD47 is a potentially safe and effective therapeutic target for glioma.

Anti-inflammatory Effect of Mesenchymal Stromal Cell Transplantation and Quercetin Treatment in a Rat Model of Experimental Cerebral Ischemia.

Here, we have investigated the synergistic effect of quercetin administration and transplantation of human umbilical cord mesenchymal stromal cells (HUMSCs) following middle cerebral artery occlusion in rat. Combining quercetin treatment with delayed transplantation of HUMSCs after local cerebral ischemia significantly (i) improved neurological functional recovery; (ii) reduced proinflammatory cytokines (interleukin(IL)-1ß and IL-6), increased anti-inflammatory cytokines (IL-4, IL-10, and transforming growth factor-ß1), and reduced ED-1 positive areas; (iii) inhibited cell apoptosis (caspase-3 expression); and (iv) improved the survival rate of HUMSCs in the injury site. Altogether, our results demonstrate that combined HUMSC transplantation and quercetin treatment is a potential strategy for reducing secondary damage and promoting functional recovery following cerebral ischemia.

Krüppel-like factor 9 inhibits glioma cell proliferation and tumorigenicity via downregulation of miR-21.

Krüppel-like factors (KLFs) are zinc finger-containing transcription factors that play key roles in the regulation of differentiation and development as well as biological processes central to the development of malignancies. Increasing evidence indicates that Krüppel-like factor 9 (KLF9) plays a critical role in regulating tumorigenesis. However, the biological role and molecular mechanism of KLF9 in glioma progression remain unclear. Herein, we found that KLF9 expression was strongly reduced in gliomas. Reduced KLF9 expression promoted glioma cell proliferation. Importantly, re-constitution of KLF9 expression inhibited glioma cell proliferation and tumor growth in vivo. Furthermore, we determined that KLF9 interacted with the miR-21 promoter, leading to suppression of miR-21 expression and cell cycle arrest. Taken together, our findings indicate a novel mechanism for KLF function in tumorigenesis and may also suggest new targets for clinical intervention in human cancer.

Decreased percentages of regulatory T cells are necessary to activate Th1-Th17-Th22 responses during acute rejection of the peripheral nerve xenotransplantation in mice.

BACKGROUND: T cells have major functions in the initiation and perpetuation of nerve graft rejection. Our study aimed to investigate the function of regulatory T cells (Treg)-Th1-Th17-Th22 cells in the rejection of peripheral nerve xenotransplantation. METHODS: Adult male C57 BL/6 mice were used as the recipient for nerve xenotransplantation, and Sprague-Dawley rats were used as the donor. These nerve xenotransplanted mice were used as the experimental groups, and those that received autograft transplant were chosen as the control group. All of the animals were pretreated with interferon (IFN)-γ, interleukin (IL)-17, and IL-22 before the experiment was conducted. The percentages of spleen Treg-Th1-Th17-Th22 cells were evaluated by flow cytometry 1, 3, 7, 14, and 28 days after transplantation. Serum levels of IFN-γ, IL-17, and IL-22 were assessed by enzyme-linked immunosorbent assay. Statistical analysis was performed by Wilcoxon rank sum and Spearman correlation test. RESULTS: During acute rejection, the percentages of Th1-Th17-Th22 cells in the spleen and serum IFN-γ, IL-17, and IL-22 levels in the experimental group increased compared with those in the control group. By contrast, CD4CD25Foxp3 T cell level decreased. The rejection of xenograft was significantly prevented after the mice were treated with IL-17-neutralizing, IL-22-neutralizing, and IFN-γ-neutralizing antibodies. Moreover, the percentage of CD4CD25Foxp3 Treg was negatively correlated with the percentages of Th1-Th17-Th22 cells and levels of IL-17, IL-22, and IFN-γ. CONCLUSION: These results suggested that the Treg-Th1-Th17-Th22 cells involved in xenotransplant rejection and imbalance between Tregs and Th1-Th17-Th22 cells contribute to the acute rejection of peripheral nerve xenotransplant.

Nitric oxide-mediated immunosuppressive effect of human amniotic membrane-derived mesenchymal stem cells on the viability and migration of microglia.

Human amniotic membrane-derived mesenchymal stem cells (AMSCs) are considered a novel and promising source of stem cells for cell replacement-based therapy. Current research is mostly limited to investigating the cellular differentiation potential of AMSCs, while few have focused on their immunosuppressive properties. This study is aimed at exploring and evaluating the immunosuppressive effect of human AMSCs on the viability and migratory properties of microglia. We found, from results of cell viability assays, that AMSCs can reduce the activity of inflammatory cells by secreting nitric oxide (NO). Also, based on results from wound healing and transwell migration assays, we show that AMSCs can inhibit the migration of human microglia as well as the mouse microglial cell line BV2, suggesting that they have the ability to inhibit the recruitment of certain immune cells to injury sites. Furthermore, we found that NO contributes significantly to this inhibitory effect. Our study provides evidence that human AMSCs can have detrimental effects on the viability and migration of microglia, through secretion of NO. This mechanism may contribute to anti-inflammatory processes in the central nervous system.

Blood-brain barrier disruption induced by hemoglobin in vivo: Involvement of up-regulation of nitric oxide synthase and peroxynitrite formation.

Accumulating evidence has demonstrated that up-regulation of nitric oxide synthase (NOS) and subsequent peroxynitrite (ONOO(-)) formation exert a devastating effect on the damage of BBB in multiple diseases. However, considerably less attention has been focused on the role of NOS/ONOO(-) in BBB disruption after intracerebral hemorrhage (ICH). Using an experimental stroke model by injecting hemoglobin (Hb) into the caudate nucleus of male Sprague Dawley rats, we explored the role of NOS/ONOO(-) in BBB disruption after ICH. Brain edema content, behavioral changes, alterations of TJ proteins (claudin-5 and ZO-1), expression of neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS), formation of 3-nitrotyrosine (3-NT), as well as NO production were investigated. Hb in the rat brain led to a significant brain edema production and neurological deficits. Overexpressed NOS was concomitant with large quantities of 3-NT formation. Moreover, sites of enhanced nNOS, iNOS, eNOS and 3-NT immunoreactivity were colocalized with diminished or discontinuous ZO-1 and/or claudin-5 staining as evidenced by Western blot and immunofluorescence, indicating the involvement of NOS and ONOO(-) in the BBB disruption. Meaningfully, levels of 3-NT in serum, which had a similar tendency with that of in brain tissues (r=0.934, P<0.001), had a marked correlation with brain edema content (r=0.782, P<0.001) and neurological deficits (r=0.851, P<0.001). We concluded that ONOO(-) formation by the upregulation of NOS may play a central role in promoting the BBB damage following ICH. Moreover, ONOO(-) may be a promising biomarker for the judgment or prediction of brain injury and clinical prognosis after ICH.

MiR-124-5p inhibits the growth of high-grade gliomas through posttranscriptional regulation of LAMB1.

BACKGROUND: Glioblastoma multiforme (GBM) is the most aggressive form of human brain tumor. It was previously shown that high levels of laminin-8 expression were a predictor of tumor recurrence and patient survival. It is thus important to elucidate the mechanism by which laminin-8 expression is regulated and determine how this contributes to glioma progression. This study investigated the mechanism of regulation of LAMB1, which encodes the ß1 chain of laminin-8, in glioma cells lines and in a mouse model of GBM. METHODS: The expression levels of LAMB1 and miR-124-5p were examined in glioma cell lines (U87 and U251) and GBM tissue samples by quantitative PCR and Western blotting. The potential regulation of LAMB1 by miR-124-5p was investigated by assessing the effects of restored miR-124-5p expression on cell proliferation, colony formation, and tumor growth and angiogenesis. The effects of inhibiting LAMB1 on tumor growth and angiogenesis were also assessed. RESULTS: The upregulation of LAMB1 expression was highly correlated with the downregulation of miR-124-5p. LAMB1 protein expression was suppressed by miR-124-5p. The restoration of miR-124-5p expression suppressed glioma growth by inhibiting angiogenesis, effects that were also observed upon LAMB1 knockdown. CONCLUSIONS: The findings indicate that miR-124-5p functions as a tumor suppressor and could serve as a molecular marker for glioma diagnosis and as a potential therapeutic target in GBM treatment.

Dual expression of hTERT and VEGF prolongs life span and enhances angiogenic ability of aged BMSCs.

Previous studies have confirmed the therapeutic effects of bone marrow stromal cells (BMSCs) transplantation on cerebral ischemia. However, the proliferative, differentiative, and homing capacity of BMSC from the elderly are significantly reduced, especially after several passages expansion in vitro. In this study, by introducing lentivirus-mediated hTERT and VEGF genes to modify human BMSCs from aged donors, we observed extended lifespan, promoted angiogenic capacity while less enhanced tumorigenicity of the genetically engineering BMSCs. These results therefore suggest that the modification of aged BMSCs by dual expression of hTERT and VEGF may be used for autologous cell replacement for ischemic cerebrovascular disease in elderly patients.

Comparison of the neural differentiation potential of human mesenchymal stem cells from amniotic fluid and adult bone marrow.

Human mesenchymal stem cells (MSCs) are considered a promising tool for cell-based therapies of nervous system diseases. Bone marrow (BM) has been the traditional source of MSCs (BM-MSCs). However, there are some limitations for their clinical use, such as the decline in cell number and differentiation potential with age. Recently, amniotic fluid (AF)-derived MSCs (AF-MSCs) have been shown to express embryonic and adult stem cell markers, and can differentiate into cells of all three germ layers. In this study, we isolated AF-MSCs from second-trimester AF by limiting dilution and compared their proliferative capacity, multipotency, neural differentiation ability, and secretion of neurotrophins to those of BM-MSCs. AF-MSCs showed a higher proliferative capacity and more rapidly formed and expanded neurospheres compared to those of BM-MSCs. Both immunocytochemical and quantitative real-time PCR analyses demonstrated that AF-MSCs showed higher expression of neural stemness markers than those of BM-MSCs following neural stem cell (NSC) differentiation. Furthermore, the levels of brain-derived growth factor and nerve growth factor secreted by AF-MSCs in the culture medium were higher than those of BM-MSCs. In addition, AF-MSCs maintained a normal karyotype in long-term cultures after NSC differentiation and were not tumorigenic in vivo. Our findings suggest that AF-MSCs are a promising and safe alternative to BM-MSCs for therapy of nervous system diseases.