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BACKGROUND: Glioma stem cells (GSCs) are glioma cells with stemness and are responsible for a variety of malignant behaviors of glioma. Evidence has shown that signals from tumor microenvironment (TME) enhance stemness of glioma cells. However, identification of the signaling molecules and underlying mechanisms has not been completely elucidated. METHODS: Human samples and glioma cell lines were cultured in vitro to determine the effects of adenovirus (ADV) infection by sphere formation, RT-qPCR, western blotting, FACS and immunofluorescence. For in vivo analysis, mouse intracranial tumor model was applied. Bioinformatics analysis, gene knockdown by siRNA, RT-qPCR and western blotting were applied for further mechanistic studies. RESULTS: Infection of patient-derived glioma cells with ADV increases the formation of tumor spheres. ADV infection upregulated stem cell markers and in turn promoted the capacities of self-renewal and multi-lineage differentiation of the infected tumor spheres. These ADV infected tumor spheres had stronger potential to form xenograft tumors in immune-compromised mice. GSCs formation could be promoted by ADV infection via TLR9, because TLR9 was upregulated after ADV infection, and knockdown of TLR9 reduced ADV-induced GSCs. Consistently, MYD88, as well as total STAT3 and phosphorylated (p-)STAT3, were also upregulated in ADV-induced GSCs. Knockdown of MYD88 or pharmaceutical inhibition of STAT3 attenuated stemness of ADV-induced GSCs. Moreover, we found that ADV infection upregulated lncRNA NEAT1. Knockdown of NEAT1 impaired stemness of ADV-induced GSCs. Lastly, HMGB1, a damage associated molecular pattern (DAMP) that triggers TLR signaling, also upregulated stemness markers in glioma cells. CONCLUSION: ADV, which has been developed as vectors for gene therapy and oncolytic virus, promotes the formation of GSCs via TLR9/NEAT1/STAT3 signaling. Video abstract.
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Infecções por Adenoviridae/complicações , Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Células-Tronco Neoplásicas/patologia , RNA Longo não Codificante/metabolismo , Fator de Transcrição STAT3/metabolismo , Receptor Toll-Like 9/metabolismo , Animais , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/patologia , Regulação Neoplásica da Expressão Gênica , Proteína HMGB1/metabolismo , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fator 88 de Diferenciação Mieloide/metabolismo , Células-Tronco Neoplásicas/metabolismo , RNA Longo não Codificante/genética , Transdução de Sinais , Esferoides Celulares/metabolismo , Esferoides Celulares/patologiaRESUMO
Mammalian neural stem cells (NSCs) are not only responsible for normal development of the central nervous system (CNS), but also participate in brain homeostasis and repair, thus hold promising clinical potentials in the treatment of neurodegenerative diseases and trauma. However the molecular networks regulating the stemness and differentiation of NSCs have not been fully understood. In this study, we show that Tweety-homolog 1 (Ttyh1), a five-pass transmembrane protein specifically expressed in mouse brain, is involved in maintaining stemness of murine NSCs. Blocking or activating Notch signal led to downregulation and upregulation of Ttyh1 in cultured NSCs, respectively, suggesting that Ttyh1 is under the control of Notch signaling. Knockdown of Ttyh1 in cultured NSCs resulted in a transient increase in the number and size of neurospheres, followed by a decrease of stemness as manifested by compromised neurosphere formation, downregulated stem cell markers, and increased neuronal differentiation. We generated Ttyh1 knockout mice by deleting its exon 4 using the CRISPR-Cas9 technology. Surprisingly, in contrast to a previous report, Ttyh1 knockout did not result in embryonic lethality. NSCs derived from Ttyh1 knockout mice phenocopied NSCs transfected with Ttyh1 siRNA. Immunofluorescence showed that loss of Ttyh1 leads to the increase of neurogenesis in adult mice. Taken together, these findings indicate that Ttyh1, which is likely downstream to Notch signaling, plays an important role in regulating NSCs.
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Diferenciação Celular , Proteínas de Membrana/deficiência , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Perda do Embrião/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , NeurogêneseRESUMO
The neural stem cell (NSC) niche in subventricular zone (SVZ) of adult mammalian brain contains dense vascular plexus, where endothelial cells (ECs) regulate NSCs by releasing plenty of angiocrine factors. However, the role of ECs-derived exosomes, a novel type of mediators of intercellular communications, in the regulation of NSCs remains unclear. In the current study, primary NSCs isolated from embryonic mouse brains form more neurospheres when cultured in the presence of human umbilical vein endothelial cells (HUVECs). The supportive role of ECs in the coculture was significantly attenuated when GW4869, a blocker of exosome formation, was included, suggesting that HUVECs-derived exosomes played a significant role in supporting NSCs. In order to investigate the role of ECs-derived exosomes on NSCs, we collected exosomes from HUVECs. We found that HUVECs-derived exosomes could significantly promote the formation of neurospheres by primary murine NSCs. EdU incorporation and TUNEL assays indicated that the proliferation of NSCs increased while apoptosis decreased when cultured in the presence of HUVECs-derived exosomes. NSCs incubated with the HUVECs-derived exosomes maintained their potential of multi-lineage differentiation potentials. The expression of stemness-related genes was up-regulated. These data suggested that ECs-derived exosomes could play an importantly role in NSC niche, and they might be used as a reagent for ex vivo NSC amplification for medical application.
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Diferenciação Celular/fisiologia , Exossomos/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/fisiologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Nicho de Células-Tronco/fisiologia , Células Cultivadas , HumanosRESUMO
Ttyh1 is a murine homolog of the Drosophila Tweety and is predicted as a five-pass transmembrane protein. The Ttyh1 mRNA is expressed in mouse brain tissues with a restricted pattern and in human glioma cells. Ttyh1 protein may function as a large-conductance chloride channel, however, the role of Ttyh1 in normal neural development and tumorigenesis has been largely unknown, at least partially due to the lack of effective antibodies. Here we report the expression in E. coli and purification of two recombinant Ttyh1 protein fragments corresponding to one of the predicted extracellular domains and the carboxyl terminus of the mouse Ttyh1. With these Ttyh1 protein products, a set of monoclonal antibodies (mAbs) against the mouse Ttyh1 protein was established by using conventional hybridoma techniques. The specificity of the anti-Ttyh1 mAbs was determined based on their activities in Western blotting and immunofluorescent analysis using embryonic brain tissues and cultured mouse neural stem cells (NSCs). We also show that the mouse Ttyh1 protein was expressed in cultured NSCs, most likely in membrane and cytoplasm. In mouse embryonic brains, it appeared that the Ttyh1 protein was specifically expressed in the apical edge of the ventricular zone as puncta-like structures, as determined by using immunofluorescence. Taken together, our study provided a useful tool for further exploration of the biological functions and pathological significance of Ttyh1 in mice.
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Anticorpos Monoclonais Murinos , Escherichia coli , Expressão Gênica , Proteínas de Membrana , Animais , Anticorpos Monoclonais Murinos/química , Anticorpos Monoclonais Murinos/imunologia , Feminino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Introduction: Macrophages are an important component of innate immunity and involved in the immune regulation of multiple diseases. The functional diversity and plasticity make macrophages to exhibit different polarization phenotypes after different stimuli. During tumor progression, the M2-like polarized tumor-associated macrophages (TAMs) promote tumor progression by assisting immune escape, facilitating tumor cell metastasis, and switching tumor angiogenesis. Our previous studies demonstrated that functional remodeling of TAMs through engineered-modifying or gene-editing provides the potential immunotherapy for tumor. However, lack of proliferation capacity and maintained immune memory of infused macrophages restricts the application of macrophage-based therapeutic strategies in the repressive tumor immune microenvironment (TIME). Although J2 retrovirus infection enabled immortalization of bone marrow-derived macrophages (iBMDMs) and facilitated the mechanisms exploration and application, little is known about the phenotypic and functional differences among multi kinds of macrophages. Methods: HE staining was used to detect the biosafety of iBMDMs, and real-time quantitative PCR, immunofluorescence staining, and ELISA were used to detect the polarization response and expression of chemokines in iBMDMs. Flow cytometry, scratch assay, real-time quantitative PCR, and crystal violet staining were used to analyze its phagocytic function, as well as its impact on tumor cell migration, proliferation, and apoptosis. Not only that, the inhibitory effect of iBMDMs on tumor growth was detected through subcutaneous tumor loading, while the tumor tissue was paraffin sectioned and flow cytometry was used to detect its impact on the tumor microenvironment. Results: In this study, we demonstrated iBMDMs exhibited the features of rapid proliferation and long-term survival. We also compared iBMDMs with RAW264.7 cell line and mouse primary BMDMs with in vitro and in vivo experiments, indicating that the iBMDMs could undergo the same polarization response as normal macrophages with no obvious cellular morphology changes after polarization. What's more, iBMDMs owned stronger phagocytosis and pro-apoptosis functions on tumor cells. In addition, M1-polarized iBMDMs could maintain the anti-tumor phenotypes and domesticated the recruited macrophages of receptor mice, which further improved the TIME and repressed tumor growth. Discussion: iBMDMs can serve as a good object for the function and mechanism study of macrophages and the optional source of macrophage immunotherapy.
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Fenótipo , Animais , Camundongos , Microambiente Tumoral/imunologia , Macrófagos Associados a Tumor/imunologia , Macrófagos Associados a Tumor/metabolismo , Macrófagos/imunologia , Proliferação de Células , Linhagem Celular Tumoral , Camundongos Endogâmicos C57BL , Apoptose , Fagocitose , Movimento Celular/imunologiaRESUMO
Inherited and age-related retinal degenerations are the commonest causes of blindness without effective treatments. Retinal progenitor cells (RPCs), which have the multipotency to differentiate into various retinal cell types, are regarded as a promising source of cell transplantation therapy for retinal degenerative diseases. However, the self-limited expansion of RPCs causes difficulty in cell source supply and restrict its clinical treatment. In this work, we found that inhibition of microRNA-449a (miR-449a) in RPCs can promote proliferation and inhibit apoptosis of RPCs, partially through upregulating Notch signaling. Further optimization of transduction miR-449a inhibitor into RPCs by endothelial cell-derived exosomes can promote the survival of RPCs transplanted in vivo and reduce cell apoptosis in retinal degeneration mouse models. In summary, these studies have shown that exosome-miR-449a inhibitor can effectively promote the expansion of RPCs in vitro and enhance transplanted RPCs survival in vivo, which might provide a novel intervention strategy for retinal degenerations in the future.
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OBJECTIVE: To analyze the clinical characteristics of hemophagocytic syndrome (HLH) children with different EB virus (EBV) DNA loads, and to explore the relationship between differential indicators and prognosis. METHODS: Clinical data of 73 children with HLH treated in our hospital from January 2015 to April 2022 were collected. According to EBV DNA loads, the children were divided into negative group (≤5×102 copies/ml), low load group (>5×102-<5×105 copies/ml) and high load group (≥5×105copies/mlï¼. The clinical symptoms and laboratory indexes of the three groups were compared, and the ROC curve was used to determine the best cut-off value of the different indexes. Cox regression model was used to analyze the independent risk factors affecting the prognosis of children, and to analyze the survival of children in each group. RESULTS: The proportion of female children, the swelling rate of liver and spleen lymph nodes and the involvement rate of blood, liver, circulation and central nervous system in the high load group were higher than those in the negative group. The incidence of disseminated intravascular coagulation(DIC) and central nervous system(CNS) involvement in the high load group were higher than those in the low load group. The liver swelling rate and circulatory system involvement rate in the low load group were higher than those in the negative group(P<0.05). PLT counts in the high load group were significantly lower than those in the negative group, and the levels of GGT, TBIL, CK-MB, LDH, TG, SF, and organ involvement were significantly higher than those in the negative group. The levels of CK, LDH, SF and the number of organ involvement in the high load group were significantly higher than those in the low load group. The levels of GGT and TBIL in low load group were significantly higher than those in negative group. In terms of treatment, the proportion of blood purification therapy in the high and low load group was significantly higher than that in the negative group(P<0.01). ROC curve analysis showed that the best cut-off values of PLT, LDH, TG and SF were 49.5, 1139, 3.12 and 1812, respectively. The appellate laboratory indicators were dichotomized according to the cut-off value, and the differential clinical symptoms were included in the Cox regression model. Univariate analysis showed that LDH>1139 U/L, SF>1812 µg/L, dysfunction of central nervous system, number of organ damage, DIC and no blood purification therapy were the risk factors affecting the prognosis of children (P<0.05); Multivariate analysis shows that PLT≤49.5×109/L and dysfunction of central nervous system were risk factors affecting the prognosis of children (P<0.05). Survival analysis showed that there was no significant difference in the survival rate among the three groups. CONCLUSION: The incidence of adverse prognostic factors in children with HLH in the EBV-DNA high load group is higher, and there is no significant difference in the survival rate of the three groups after blood purification therapy. Therefore, early identification and application of blood purification therapy is of great significance for children with HLH in the high load group.
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Linfo-Histiocitose Hemofagocítica , Humanos , Criança , Feminino , Estudos Retrospectivos , Fatores de Risco , DNA , PrognósticoRESUMO
Epigenetic regulations on the maintenance of neural stem cells (NSCs) are complicated and far from been fully understood. Our previous findings have shown that after blocking Notch signaling in NSCs in vivo, the stemness of NSCs decreases, accompanied by the downregulated expression of miR-582-5p. In the current study, we further investigated the function and mechanism of miR-582-5p in the maintenance of NSCs in vitro and in vivo. After transfecting a mimic of miR-582-5p, the formation of neurospheres and proliferation of NSCs and intermediate progenitor cells (NS/PCs) were enhanced, and the expression of stemness markers such as Sox2, Nestin, and Pax6 also increased. The results were reversed after transfection of an inhibitor of miR-582-5p. We further generated miR-582 knock-out (KO) mice to investigate its function in vivo, and we found that the number of NSCs in the subventricular zone (SVZ) region decreased and the number of neuroblasts increased in miR-582 deficient mice, indicating reduced stemness and enhanced neurogenesis of NSCs. Moreover, RNA-sequencing and molecular biological analysis revealed that miR-582-5p regulates the stemness and proliferation of NSCs by inhibiting secretory protein FAM19A1. In summary, our research uncovered a new epigenetic mechanism that regulates the maintenance of NSCs, therefore providing novel targets to amplify NSCs in vitro and to promote neurogenesis in vivo during brain pathology and aging.
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During neurodevelopment, differentiation of neural stem/progenitor cells (NSPCs) into neurons are regulated by many factors including Notch signaling pathway. Herein, we report the effect of a Notch signaling blocker, i.e. γ -secretase inhibitor (GSI), on this differentiating process, especially on the morphological development. NSPCs were cultured and induced to differentiate with or without GSI. The neurite outgrowth was impeded by GSI application and the expression of a Notch signaling downstream effector miR-342-5p increased with the downregulated expression of Notch effectors Hes1 and Hes5. Upregulated expression of miR-342-5p in differentiating NSPCs could shorten the neurite length of progeny neurons, which was similar to the effect of GSI. To avoid the possible influence from astrocytes into neurons, we directly applied cultured neurons, on which GSI could shorten the processes and RBP-J knockdown could also reduce the neurite length. Similarly, transfection of miR-342-5p mimics or inhibitors into PC12 cells led to shorter or longer processes of cells compared with control ones. Furthermore, in differentiating NSPCs, GSI-induced shorter neurites could be partially rescued by miR-342-5p inhibitors, and STAT3 was one of the possible targets of miR-342-5p during this differentiating process as indicated by results of Western Blot test, luciferase reporter assay and GFP reporter assay. To further demonstrate the role of STAT3, it was introduced into GSI-treated neurons and the GSI-affected neurites could also be partially rescued. In conclusion, GSI could influence the morphological development of neurons and the possible mechanism involved Notch/miR-342-5p and STAT3. These results would be informative for future therapeutic research.
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Inibidores e Moduladores de Secretases gama , MicroRNAs , Células-Tronco Neurais , Receptores Notch , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Inibidores e Moduladores de Secretases gama/farmacologia , MicroRNAs/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Ratos , Receptores Notch/metabolismo , Transdução de SinaisRESUMO
The quiescence, activation, and subsequent neurogenesis of neural stem cells (NSCs) play essential roles in the physiological homeostasis and pathological repair of the central nervous system. Previous studies indicate that transmembrane protein Ttyh1 is required for the stemness of NSCs, whereas the exact functions in vivo and precise mechanisms are still waiting to be elucidated. By constructing Ttyh1-promoter driven reporter mice, we determined the specific expression of Ttyh1 in quiescent NSCs and niche astrocytes. Further evaluations on Ttyh1 knockout mice revealed that Ttyh1 ablation leads to activated neurogenesis and enhanced spatial learning and memory in adult mice (6-8 weeks). Correspondingly, Ttyh1 deficiency results in accelerated exhaustion of NSC pool and impaired neurogenesis in aged mice (12 months). By RNA-sequencing, bioinformatics and molecular biological analysis, we found that Ttyh1 is involved in the regulation of calcium signaling in NSCs, and transcription factor NFATc3 is a critical effector in quiescence versus cell cycle entry regulated by Ttyh1. Our research uncovered new endogenous mechanisms that regulate quiescence versus activation of NSCs, therefore provide novel targets for the intervention to activate quiescent NSCs to participate in injury repair during pathology and aging.
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Formation of glioma stem cells (GSCs) is considered as one of the main reasons of temozolomide (TMZ) resistance in glioma patients. Recent studies have shown that tumor microenvironment-derived signals could promote GSCs formation. But the critical molecule and underlying mechanism for GSCs formation after TMZ treatment is not entirely identified. Our study showed that TMZ treatment promoted GSCs formation by glioma cells; TMZ treatment of biopsy-derived glioblastoma multiforme cells upregulated HMGB1; HMGB1 altered gene expression profile of glioma cells with respect to mRNA, lncRNA and miRNA. Furthermore, our results showed that TMZ-induced HMGB1 increased the formation of GSCs and when HMGB1 was downregulated, TMZ-mediated GSCs formation was attenuated. Finally, we showed that the effect of HMGB1 on glioma cells was mediated by TLR2, which activated Wnt/ß-catenin signaling to promote GSCs. Mechanistically, we found that HMGB1 upregulated NEAT1, which was responsible for Wnt/ß-catenin activation. In conclusion, TMZ treatment upregulates HMGB1, which promotes the formation of GSCs via the TLR2/NEAT1/Wnt pathway. Blocking HMGB1-mediated GSCs formation could serve as a potential therapeutic target for preventing TMZ resistance in GBM patients.
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Tweety-homolog 1 (Ttyh1) is expressed in neural tissue and has been implicated in the generation of several brain diseases. However, its functional significance in pain processing is not understood. By disrupting the gene encoding Ttyh1, we found a loss of Ttyh1 in nociceptors and their central terminals in Ttyh1-deficient mice, along with a reduction in nociceptor excitability and synaptic transmission at identified synapses between nociceptors and spinal neurons projecting to the periaqueductal grey (PAG) in the basal state. More importantly, the peripheral inflammation-evoked nociceptor hyperexcitability and spinal synaptic potentiation recorded in spinal-PAG projection neurons were compromised in Ttyh1-deficient mice. Analysis of the paired-pulse ratio and miniature excitatory postsynaptic currents indicated a role of presynaptic Ttyh1 from spinal nociceptor terminals in the regulation of neurotransmitter release. Interfering with Ttyh1 specifically in nociceptors produces a comparable pain relief. Thus, in this study we demonstrated that Ttyh1 is a critical determinant of acute nociception and pain sensitization caused by peripheral inflammation.
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Nociceptores , Transmissão Sináptica , Animais , Proteínas de Membrana/metabolismo , Camundongos , Neurônios/metabolismo , Dor , Substância Cinzenta PeriaquedutalRESUMO
Abnormal metabolism serves a critical role in the development and progression of different types of malignancies including glioblastoma (GBM), and may therefore serve as a promising target for treatment of cancer. Preclinical studies have indicated that a ketogenic diet (KD) may exhibit beneficial effects in patients with GBM; however, the underlying mechanisms remain incompletely understood. The aim of the present study was to evaluate the effects of a KD on glioma stemlike cells (GSCs), by culturing patientderived primary GSCs as well as a GSC cell line in glucoserestricted, ßhydroxybutyratecontaining medium (BHBGlow) which was used to mimic clinical KD treatment. GSCs cultured in BHBGlow medium exhibited reduced proliferation and increased apoptosis compared with cells grown in the control medium. Furthermore, decreased expression of stem cell markers, diminished selfrenewal in vitro, and reduced tumorigenic capacity in vivo, providing evidence that the stemness of GSCs was compromised. Mechanistically, culturing in BHBGlow medium reduced glucose uptake and inhibited glycolysis in GSCs. Furthermore, culturing in the BHBGlow medium resulted in morphological and functional disturbances to the mitochondria of GSCs. These metabolic changes may have reduced ATP production, promoted lactic acid accumulation, and thus, increased the production of reactive oxygen species (ROS) in GSCs. The expression levels and activation of mammalian target of rapamycin, hypoxiainducible factor 1 and Bcell lymphoma 2 were decreased, consistent with the reduced proliferation of GSCs in BHBGlow medium. ROS scavenging reversed the inhibitory effects of a KD on GSCs. Taken together, the results demonstrate that treatment with KD inhibited proliferation of GSCs, increased apoptosis and attenuated the stemness in GSCs by increasing ROS production.
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Ácido 3-Hidroxibutírico/farmacologia , Neoplasias Encefálicas/dietoterapia , Dieta Cetogênica , Glioblastoma/dietoterapia , Células-Tronco Neoplásicas/patologia , Adolescente , Adulto , Idoso , Animais , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/cirurgia , Proliferação de Células/efeitos dos fármacos , Meios de Cultura/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/patologia , Glioblastoma/cirurgia , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/metabolismo , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais Cultivadas , Adulto JovemRESUMO
Extracellular vesicles (EVs) including exosomes can serve as mediators of cell-cell communication under physiological and pathological conditions. However, cargo molecules carried by EVs to exert their functions, as well as mechanisms for their regulated release and intake, have been poorly understood. In this study, we examined the effects of endothelial cells-derived EVs on neurons suffering from oxygen-glucose deprivation (OGD), which mimics neuronal ischemia-reperfusion injury in human diseases. In a human umbilical endothelial cell (HUVEC)-neuron coculture assay, we found that HUVECs reduced apoptosis of neurons under OGD, and this effect was compromised by GW4869, a blocker of exosome release. Purified EVs could be internalized by neurons and alleviate neuronal apoptosis under OGD. A miRNA, miR-1290, was highly enriched in HUVECs-derived EVs and was responsible for EV-mediated neuronal protection under OGD. Interestingly, we found that OGD enhanced intake of EVs by neurons cultured in vitro. We examined the expression of several potential receptors for EV intake and found that caveolin-1 (Cav-1) was upregulated in OGD-treated neurons and mice suffering from middle cerebral artery occlusion (MCAO). Knock-down of Cav-1 in neurons reduced EV intake, and canceled EV-mediated neuronal protection under OGD. HUVEC-derived EVs alleviated MCAO-induced neuronal apoptosis in vivo. These findings suggested that ischemia likely upregulates Cav-1 expression in neurons to increase EV intake, which protects neurons by attenuating apoptosis via miR-1290.
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Caveolina 1/metabolismo , Células Endoteliais/metabolismo , Vesículas Extracelulares/metabolismo , MicroRNAs/metabolismo , Neurônios/metabolismo , Animais , Apoptose , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regulação para CimaRESUMO
Neurologic impairments are usually irreversible as a result of limited regeneration in the central nervous system. Therefore, based on the regenerative capacity of stem cells, transplantation therapies of various stem cells have been tested in basic research and preclinical trials, and some have shown great prospects. This manuscript overviews the cellular and molecular characteristics of embryonic stem cells, induced pluripotent stem cells, neural stem cells, retinal stem/progenitor cells, mesenchymal stem/stromal cells, and their derivatives in vivo and in vitro as sources for regenerative therapy. These cells have all been considered as candidates to treat several major neurological disorders and diseases, owing to their self-renewal capacity, multi-directional differentiation, neurotrophic properties, and immune modulation effects. We also review representative basic research and recent clinical trials using stem cells for neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, and age-related macular degeneration, as well as traumatic brain injury and glioblastoma. In spite of a few unsuccessful cases, risks of tumorigenicity, and ethical concerns, most results of animal experiments and clinical trials demonstrate efficacious therapeutic effects of stem cells in the treatment of nervous system disease. In summary, these emerging findings in regenerative medicine are likely to contribute to breakthroughs in the treatment of neurological disorders. Thus, stem cells are a promising candidate for the treatment of nervous system diseases.
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OBJECTIVE: To investigate the effects of adiponectin (APN) on anxiety and memory impairment of 9-month-old triple transgenic Alzheimer's disease (3xTg-AD) model mice. METHODS: The 9-month-old 3xTg-AD mice and C57BL/6J mice were randomly divided into four groups (n=8 for each group):Wild type(WT)+Saline, 3xTg-AD +Saline, WT+APN and 3xTg-AD +APN group. All mice were implanted cannula in lateral ventricle and each mouse was intracerebroventricular injected with adiponectin or saline under free moving condition after 7 days recovery. The anxiety and memory ability of each mouse were observed by using open field test, object recognition task and Y-maze test. RESULTS: â In the open field test, compared to WT+Saline group, the time of 3xTg-AD +Saline mice spent in center was significantly decreased, and the time spent in periphery was obviously increased. However, APN treatment effectively reversed the phenomenon appeared in 3xTg-AD mice, indicating that APN could alleviate the anxiety observed in 3xTg-AD mice. â¡In novel object recognition task, the discrimination index of 3xTg-AD+Saline group was (-16.7±10.1)%, significantly lower than (18.0±8.2)% in WT+Saline group (P<0.01) and (15.7±8.8)% in 3xTg-AD+APN group (P<0.01), which indicated that APN could effectively prevent the recognition memory impairment in 3xTg mice. â¢In Y-maze test, the spontaneous alternation rate of 3xTg-AD +Saline group was (40.0±1.7)%,significantly lower than (56.6±4.6)% in WT+Saline group and (53.9±5.6)% in 3xTg-AD +APN group (P<0.01), which indicated that APN could prevent working memory impairment in 3xTg-AD mice. CONCLUSIONS: Adiponectin could effectively alleviate the anxiety and reverse the impairment of recognition memory and working memory of 9-month-old 3xTg-AD mice, and might play an important role in the prevention and treatment of AD.
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Adiponectina/farmacologia , Doença de Alzheimer/tratamento farmacológico , Ansiedade/tratamento farmacológico , Memória de Curto Prazo , Animais , Modelos Animais de Doenças , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Distribuição AleatóriaRESUMO
Notch signaling is critically involved in neural development, but the downstream effectors remain incompletely understood. In this study, we cultured neurospheres from Nestin-Cre-mediated conditional Rbp-j knockout (Rbp-j cKO) and control embryos and compared their miRNA expression profiles using microarray. Among differentially expressed miRNAs, miR-342-5p showed upregulated expression as Notch signaling was genetically or pharmaceutically interrupted. Consistently, the promoter of the miR-342-5p host gene, the Ena-vasodilator stimulated phosphoprotein-like (Evl), was negatively regulated by Notch signaling, probably through HES5. Transfection of miR-342-5p promoted the differentiation of neural stem cells (NSCs) into intermediate neural progenitors (INPs) in vitro and reduced the stemness of NSCs in vivo. Furthermore, miR-342-5p inhibited the differentiation of neural stem/intermediate progenitor cells into astrocytes, likely mediated by targeting GFAP directly. Our results indicated that miR-342-5p could function as a downstream effector of Notch signaling to regulate the differentiation of NSCs into INPs and astrocytes commitment.
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Proliferação de Células , MicroRNAs/genética , Células-Tronco Neurais/citologia , Neurogênese , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Regulação da Expressão Gênica , Proteína Glial Fibrilar Ácida/genética , Camundongos , Células-Tronco Neurais/metabolismoRESUMO
UNLABELLED: This study aimed to elucidate the associations between interleukin-4 (IL-4) single nucleotide polymorphisms (SNPs), 590C/T and 589C/T, serum IL-4 levels, and atopic dermatitis (AD) in children. METHODS: A total of 82 children with AD were randomly selected as the case group and divided into mild group (15 cases), moderate group (46 cases), and severe group (21 cases). Additionally, 100 healthy children were selected as the control group. Genotype frequencies of IL-4 SNPs were detected by PCR-RFLP. Serum IL-4 levels were measured by ELISA. RESULTS: Significant differences were shown in genotype distributions and allele frequencies of 589C/T and allele frequencies of 590C/T (all P < 0.05). Serum IL-4 levels in the mild, moderate, and severe groups were significantly higher than those in the control group; significant differences were found among these three groups with increased severity of AD. Serum IL-4 levels of heterozygote and mutant homozygote carriers in the mild, moderate, and severe groups were higher than wild homozygote carriers in those three groups and the control group (all P < 0.05). CONCLUSION: 590T and 589T alleles of IL-4 gene may be associated with high levels of serum IL-4, which may increase the risk of AD in children.
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Dermatite Atópica/genética , Interleucina-4/sangue , Interleucina-4/genética , Polimorfismo de Nucleotídeo Único , Criança , Pré-Escolar , Dermatite Atópica/metabolismo , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Lactente , MasculinoRESUMO
The growth of solid tumors depends on neovascularization. Several therapies targeting tumor angiogenesis have been developed. However, poor response in some tumors and emerging resistance necessitate further investigations of new drug targets. Notch signal pathway plays a pivotal role in vascular development and tumor angiogenesis. Either blockade or forced activation of this pathway can inhibit angiogenesis. As blocking Notch pathway results in the formation of vascular neoplasm, activation of Notch pathway to prevent tumor angiogenesis might be an alternative choice. However, an in vivo deliverable reagent with highly efficient Notch-activating capacity has not been developed. Here, we generated a polypeptide, hD1R, which consists of the Delta-Serrate-Lag-2 fragment of the human Notch ligand Delta-like 1 and an arginine-glycine-aspartate (RGD) motif targeting endothelial cells (ECs). We showed that hD1R could bind to ECs specifically through its RGD motif and effectively triggered Notch signaling in ECs. We demonstrated both in vitro and in vivo that hD1R inhibited angiogenic sprouting and EC proliferation. In tumor-bearing mice, the injection of hD1R effectively repressed tumor growth, most likely through increasing tumor hypoxia and tissue necrosis. The amount and width of vessels reduced remarkably in tumors of mice treated with hD1R. Moreover, vessels in tumors of mice treated with hD1R recruited more NG2(+) perivascular cells and were better perfused. Combined application of hD1R and chemotherapy with cisplatin and teniposide revealed that these two treatments had additive antitumor effects. Our study provided a new strategy for antiangiogenic tumor therapy.
Assuntos
Células Endoteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica/metabolismo , Domínios e Motivos de Interação entre Proteínas , Animais , Antígenos/metabolismo , Proteínas de Ligação ao Cálcio , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Hipóxia , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Neoplasias/genética , Neovascularização Patológica/genética , Ligação Proteica , Proteoglicanas/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Carga Tumoral/genéticaRESUMO
BACKGROUND: Notch ligands enhance ex vivo expansion of hematopoietic stem cells (HSCs). But to use Notch ligands in HSC therapies of human diseases, efforts are required to improve ex vivo expansion efficiency and in vivo transplant engraftment. DESIGN AND METHODS: We designed and produced an endothelium-targeted soluble Notch ligand, the DSL domain of Delta-like 1 fused with a RGD motif (D1R), and examined the effects of this protein on HSCs ex vivo and in vivo. RESULTS: D1R efficiently promoted ex vivo expansion of both mouse bone marrow (BM) and human umbilical cord blood HSCs. HSCs expanded with D1R up-regulated many of the stemness-related genes, and showed high BM engraftment efficacy with long-term repopulation capacity after transplantation. Moreover, in vivo administration of D1R increased the number of BM HSCs in mice, and facilitated BM recovery of mice after irradiation. Injection of D1R significantly improved HSC engraftment and myeloid recovery after BM transplantation in irradiated mice. D1R enhanced HSC engraftment not only in BM, but also in the liver and spleen after BM transplantation in mice. D1R induced the formation of compact cell clusters containing the transplanted HSCs in close contact with endothelial cells, reminiscent of HSC niches, in the liver and spleen. CONCLUSIONS: D1R might be applied in improving both HSC expansion ex vivo and HSC engraftment in vivo in transplantation.