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BACKGROUND: Signaling pathways mediated by microRNAs (miRNAs) have been identified as one of the mechanisms that regulate stroke progression and recovery. Recent investigations using stroke patient blood and cerebrospinal fluid (CSF) demonstrated disease-specific alterations in miRNA expression. In this study, for the first time, we investigated miRNA expression signatures in freshly removed human stroke brain tissue. METHODS: Human brain samples were obtained during craniectomy and brain tissue resection in severe stroke patients with life-threatening brain swelling. The tissue samples were subjected to histopathological and immunofluorescence microscopy evaluation, next generation miRNA sequencing (NGS), and bioinformatic analysis. RESULTS: miRNA NGS analysis detected 34 miRNAs with significantly aberrant expression in stroke tissue, as compared to non-stroke samples. Of these miRNAs, 19 were previously identified in stroke patient blood and CSF, while dysregulation of 15 miRNAs was newly detected in this study. miRNA direct target gene analysis and bioinformatics approach demonstrated a strong association of the identified miRNAs with stroke-related biological processes and signaling pathways. CONCLUSIONS: Dysregulated miRNAs detected in our study could be regarded as potential candidates for biomarkers and/or targets for therapeutic intervention. The results described herein further our understanding of the molecular basis of stroke and provide valuable information for the future functional studies in the experimental models of stroke.
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Encéfalo/metabolismo , MicroRNAs/metabolismo , Acidente Vascular Cerebral/metabolismo , Encéfalo/cirurgia , Biologia Computacional/métodos , Craniectomia Descompressiva/métodos , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Transdução de Sinais/genética , Acidente Vascular Cerebral/cirurgiaRESUMO
microRNAs (miRNAs) are recently identified small RNA molecules that regulate gene expression and significantly influence the essential cellular processes associated with CNS repair after trauma and neuropathological conditions including stroke and neurodegenerative disorders. A number of specific miRNAs are implicated in regulating the development and propagation of CNS injury, as well as its subsequent regeneration. The review focuses on the functions of the miRNAs and their role in brain recovery following CNS damage. The article introduces a brief description of miRNA biogenesis and mechanisms of miRNA-induced gene suppression, followed by an overview of miRNAs involved in the processes associated with CNS repair, including neuroprotection, neuronal plasticity and axonal regeneration, vascular reorganization, neuroinflammation, and endogenous stem cell activation. Specific emphasis is placed on the role of multifunctional miRNA miR-155, as it appears to be involved in multiple neurorestorative processes during different CNS pathologies. In association with our own studies on miR-155, I introduce a new and unexplored approach to cerebral regeneration: regulation of brain tissue repair through a direct modulation of specific miRNA activity. The review concludes with discussion on the challenges and the future potential of miRNA-based therapeutic approaches to CNS repair.
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Doenças do Sistema Nervoso Central/metabolismo , Modelos Animais de Doenças , MicroRNAs/biossíntese , Regeneração Nervosa/fisiologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Doenças do Sistema Nervoso Central/genética , Doenças do Sistema Nervoso Central/terapia , Humanos , MicroRNAs/genéticaRESUMO
BACKGROUND: Brain microvascular endothelial cells form a highly selective blood brain barrier regulated by the endothelial tight junctions. Cerebral ischemia selectively targets tight junction protein complexes, which leads to significant damage to cerebral microvasculature. Short noncoding molecules called microRNAs are implicated in the regulation of various pathological states, including endothelial barrier dysfunction. In the present study, we investigated the influence of microRNA-155 (miR-155) on the barrier characteristics of human primary brain microvascular endothelial cells (HBMECs). METHODS AND RESULTS: Oxygen-glucose deprivation was used as an in vitro model of ischemic stroke. HBMECs were subjected to 3 hours of oxygen-glucose deprivation, followed by transfections with miR-155 inhibitor, mimic, or appropriate control oligonucleotides. Intact normoxia control HBMECs and 4 oxygen-glucose deprivation-treated groups of cells transfected with appropriate nucleotide were subjected to endothelial monolayer electrical resistance and permeability assays, cell viability assay, assessment of NO and human cytokine/chemokine release, immunofluorescence microscopy, Western blot, and polymerase chain reaction analyses. Assessment of endothelial resistance and permeability demonstrated that miR-155 inhibition improved HBMECs monolayer integrity. In addition, miR-155 inhibition significantly increased the levels of major tight junction proteins claudin-1 and zonula occludens protein-1, while its overexpression reduced these levels. Immunoprecipitation and colocalization analyses detected that miR-155 inhibition supported the association between zonula occludens protein-1 and claudin-1 and their stabilization at the HBMEC membrane. Luciferase reporter assay verified that claudin-1 is directly targeted by miR-155. CONCLUSIONS: Based on these results, we conclude that miR-155 inhibition-induced strengthening of endothelial tight junctions after oxygen-glucose deprivation is mediated via its direct target protein claudin-1.
Assuntos
Barreira Hematoencefálica/metabolismo , Permeabilidade Capilar , Células Endoteliais/metabolismo , Glucose/deficiência , MicroRNAs/metabolismo , Junções Íntimas/metabolismo , Barreira Hematoencefálica/patologia , Hipóxia Celular , Células Cultivadas , Claudina-1/genética , Claudina-1/metabolismo , Impedância Elétrica , Células Endoteliais/patologia , Humanos , MicroRNAs/genética , Junções Íntimas/genética , Junções Íntimas/patologia , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Stroke-induced endothelial cell injury leads to destruction of cerebral microvasculature and significant damage to the brain tissue. A subacute phase of cerebral ischemia is associated with regeneration involving the activation of vascular remodeling, neuroplasticity, neurogenesis, and neuroinflammation processes. Effective restoration and improvement of blood supply to the damaged brain tissue offers a potential therapy for stroke. microRNAs (miRNAs) are recently identified small RNA molecules that regulate gene expression and significantly influence the essential cellular processes associated with brain repair following stroke. A number of specific miRNAs are implicated in regulating the development and propagation of the ischemic tissue damage as well as in mediating post-stroke regeneration. In this review, I discuss the functions of the miRNA miR-155 and the effect of its in vivo inhibition on brain recovery following experimental cerebral ischemia. The article introduces new and unexplored approach to cerebral regeneration: regulation of brain tissue repair through a direct modulation of specific miRNA activity.
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BACKGROUND: MicroRNA miR-155 is implicated in modulation of the inflammatory processes in various pathological conditions. In our previous studies, we demonstrated that in vivo inhibition of miR-155 promotes functional recovery after mouse experimental stroke. In the present study, we explored if this beneficial effect is associated with miR-155 inhibition-induced alterations in post-stroke inflammatory response. METHODS: Intravenous injections of a specific miR-155 inhibitor were initiated at 48 h after mouse distal middle cerebral artery occlusion (dMCAO). Temporal changes in the expression of cytokines and key molecules associated with cytokine signaling were assessed at 7, 14, and 21 days after dMCAO, using mouse cytokine gene and protein arrays and Western blot analyses. Electron and immunofluorescence confocal microscopy techniques were used to evaluate the ultrastructural changes, as well as altered expression of specific phenotypic markers, at different time points after dMCAO. RESULTS: In the inhibitor-injected mice (inhibitor group), there was a significant decrease in CCL12 and CXCL3 cytokine expression at 7 days and significantly increased levels of major cytokines IL-10, IL-4, IL-6, MIP-1α, IL-5, and IL-17 at 14 days after dMCAO. These temporal changes correlated with altered expression of miR-155 target proteins SOCS-1, SHIP-1, and C/EBP-ß and phosphorylation levels of cytokine signaling regulator STAT-3. Electron microscopy showed decreased number of phagocytically active peri-vascular microglia/macrophages in the inhibitor samples. Immunofluorescence and Western blot of these samples demonstrated that expression of leukocyte/ macrophage marker CD45 and phagocytosis marker CD68 was reduced at 7 days, and in contrast, significantly increased at 14 days after dMCAO, as compared to controls. CONCLUSIONS: Based on our findings, we propose that in vivo miR-155 inhibition following mouse stroke significantly alters the time course of the expression of major cytokines and inflammation-associated molecules, which could influence inflammation process and tissue repair after experimental cerebral ischemia.
Assuntos
Antagomirs/uso terapêutico , Citocinas/metabolismo , Encefalite/etiologia , Encefalite/metabolismo , Infarto da Artéria Cerebral Média/complicações , MicroRNAs/metabolismo , Animais , Antagomirs/farmacologia , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Citocinas/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Macrófagos/patologia , Macrófagos/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microglia/patologia , Microglia/ultraestrutura , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Transdução de Sinais/fisiologia , Proteína 1 Supressora da Sinalização de Citocina/genética , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Fatores de TempoRESUMO
A multifunctional microRNA, miR-155, has been recently recognized as an important modulator of numerous biological processes. In our previous in vitro studies, miR-155 was identified as a potential regulator of the endothelial morphogenesis. The present study demonstrates that in vivo inhibition of miR-155 supports cerebral vasculature after experimental stroke. Intravenous injections of a specific miR-155 inhibitor were initiated at 48 h after mouse distal middle cerebral artery occlusion (dMCAO). Microvasculature in peri-infarct area, infarct size, and animal functional recovery were assessed at 1, 2, and 3 weeks after dMCAO. Using in vivo two-photon microscopy, we detected improved blood flow and microvascular integrity in the peri-infarct area of miR-155 inhibitor-injected mice. Electron microscopy revealed that, in contrast to the control group, these animals demonstrated well preserved capillary tight junctions (TJs). Western blot analysis data indicate that improved TJ integrity in the inhibitor-injected animals could be associated with stabilization of the TJ protein ZO-1 and mediated by the miR-155 target protein Rheb. MRI analysis showed significant (34%) reduction of infarct size in miR-155 inhibitor-injected animals at 21 d after dMCAO. Reduced brain injury was confirmed by electron microscopy demonstrating decreased neuronal damage in the peri-infarct area of stroke. Preservation of brain tissue was reflected in efficient functional recovery of inhibitor-injected animals. Based on our findings, we propose that in vivo miR-155 inhibition after ischemia supports brain microvasculature, reduces brain tissue damage, and improves the animal functional recovery. Significance statement: In the present study, we investigated an effect of the in vivo inhibition of a microRNA, miR-155, on brain recovery after experimental cerebral ischemia. To our knowledge, this is the first report describing the efficiency of intravenous anti-miRNA injections in a mouse model of ischemic stroke. The role of miRNAs in poststroke revascularization has been unexplored and in vivo regulation of miRNAs during the subacute phase of stroke has not yet been proposed. Our investigation introduces a new and unexplored approach to cerebral regeneration: regulation of poststroke angiogenesis and recovery through direct modulation of specific miRNA activity. We expect that our findings will lead to the development of novel strategies for regulating neurorestorative processes in the postischemic brain.
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Infarto da Artéria Cerebral Média/terapia , MicroRNAs/genética , Terapêutica com RNAi , Animais , Injeções Intravenosas , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/metabolismo , Microvasos/patologia , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/uso terapêutico , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/metabolismo , Junções Íntimas/ultraestruturaRESUMO
Hypertensive small vessel disease is a major cause of vascular cognitive impairment (VCI). Spontaneously hypertensive/stroke prone rats (SHR/SP) with unilateral carotid artery occlusion (UCAO) and a Japanese permissive diet (JPD) have white-matter (WM) damage similar to that seen in VCI. We hypothesized that WM injury was due to hypoxia-mediated, blood-brain barrier (BBB) disruption. Twelve-week-old SHR/SP had UCAO/JPD and were studied with immunohistochemistry, biochemistry, multimodal magnetic resonance imaging (MRI), and Morris water maze (MWM) testing. One week after UCAO/JPD, WM showed a significant increase in hypoxia inducible factor-1α (HIF-1α), which increased further by 3 weeks. Prolyl hydroxylase-2 (PHD2) expression decreased at 1 and 3 weeks. Infiltrating T cells and neutrophils appeared around endothelial cells from 1 to 3 weeks after UCAO/JPD, and matrix metalloproteinase-9 (MMP-9) colocalized with inflammatory cells. At 3 weeks, WM immunostained for IgG, indicating BBB leakage. Minocycline (50 mg/kg intraperitoneally) was given every other day from weeks 12 to 20. Multimodal MRI showed that treatment with minocycline significantly reduced lesion size and improved cerebral blood flow. Minocycline improved performance in the MWM and prolonged survival. We propose that BBB disruption occurred secondary to hypoxia, which induced an MMP-9-mediated infiltration of leukocytes. Minocycline significantly reduced WM damage, improved behavior, and prolonged life.
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Anti-Inflamatórios/uso terapêutico , Hipóxia/complicações , Inflamação/tratamento farmacológico , Minociclina/uso terapêutico , Substância Branca/efeitos dos fármacos , Substância Branca/patologia , Animais , Antibacterianos/uso terapêutico , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/imunologia , Barreira Hematoencefálica/patologia , Circulação Cerebrovascular/efeitos dos fármacos , Hipóxia/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/análise , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Inflamação/complicações , Inflamação/imunologia , Inflamação/patologia , Masculino , Metaloproteinase 2 da Matriz/análise , Metaloproteinase 2 da Matriz/imunologia , Metaloproteinase 9 da Matriz/análise , Metaloproteinase 9 da Matriz/imunologia , Ratos , Ratos Endogâmicos SHR , Substância Branca/imunologia , Substância Branca/lesõesRESUMO
Pulsed electromagnetic fields (PEMF) have been demonstrated to have anti-inflammatory and pro-regenerative effects in animals and humans. We used the FDA-approved Sofpulse (Ivivi Health Sciences, LLC) to study effect of PEMF on infarct size and poststroke inflammation following distal middle cerebral artery occlusion (dMCAO) in mice. Electromagnetic field was applied within 30-45 min after ischemic brain damage and utilized twice a day for 21 consecutive days. Ischemic infarct size was assessed using MRI and histological analysis. At 21 days after dMCAO, the infarct size was significantly (by 26%) smaller in PEMF-treated animals as compared to controls. Neuroinflammation in these animals was evaluated using specialized cytokine/chemokine PCR array. We demonstrate that PEMF significantly influenced expression profile of pro- and anti-inflammatory factors in the hemisphere ipsilateral to ischemic damage. Importantly, expression of gene encoding major pro-inflammatory cytokine IL-1α was significantly reduced, while expression of major anti-inflammatory IL-10 was significantly increased. PEMF application significantly downregulated genes encoding members of the major pro-apoptotic tumor necrosis factor (TNF) superfamily indicating that the treatment could have both anti-inflammatory and anti-apoptotic effects. Both reduction of infarct size and influence on neuroinflammation could have a potentially important positive impact on the poststroke recovery process, implicating PEMF as a possible adjunctive therapy for stroke patients.
Assuntos
Isquemia Encefálica/terapia , Encéfalo/metabolismo , Encéfalo/patologia , Infarto da Artéria Cerebral Média/terapia , Magnetoterapia , Acidente Vascular Cerebral/terapia , Animais , Citocinas/metabolismo , Inflamação/terapia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Functional signaling between neural stem/progenitor cells (NSPCs) and brain endothelial cells (ECs) is essential to the coordination of organized responses during initial embryonic development and also during tissue repair, which occurs following brain injury. In this study, we investigated the molecular mechanisms underlying this functional signaling, using primary mouse brain ECs and NSPCs from embryonic mouse brain. EC/NSPC co-culture experiments have revealed that neural progenitors secrete factors supporting angiogenesis, which induce noticeable changes in endothelial morphology. We demonstrate that NSPCs influence the expression of mTOR and TGF-ß signaling pathway components implicated in the regulation of angiogenesis. Endothelial morphogenesis, an essential component of vascular development, is a complex process involving gene activation and the upregulation of specific cell signaling pathways. Recently identified small molecules, called microRNAs (miRNAs), regulate the expression of genes and proteins in many tissues, including brain and vasculature. We found that NSPCs induced considerable changes in the expression of at least 24 miRNAs and 13 genes in ECs. Three NSPC-regulated EC miRNAs were identified as the potential primary mediators of this NSPC/EC interaction. We found that the specific inhibition, or overexpression, of miRNAs miR-155, miR-100, and miR-let-7i subsequently altered the expression of major components of the mTOR, TGF-ß and IGF-1R signaling pathways in ECs. Overexpression of these miRNAs in ECs suppressed, while inhibition activated, the in vitro formation of capillary-like structures, a process representative of EC morphogenesis. In addition, we demonstrate that inhibition of FGF, VEGF, and TGF-ß receptor signaling abolished NSPC-promoted changes in the endothelial miRNA profiles. Our findings demonstrate that NSPCs induce changes in the miRNA expression of ECs, which are capable of activating angiogenesis by modulating distinct cell signaling pathways.
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Gestational alcohol exposure leads to a spectrum of neurological symptoms which range from severe mental retardation caused by high dose exposure, to subtle cognitive and neuropsychiatric symptoms caused by low-to-moderate doses. We and other investigators have demonstrated that exposure to moderate levels of alcohol throughout gestation leads to impaired neurogenesis in the adult hippocampus, although the mechanisms by which this occurs are not known. To begin to distinguish cell-intrinsic from microenvironmental contributions to impaired adult neurogenesis, we isolated neural stem progenitor cells (NSPCs) from the adult SVZ of mice exposed to moderate levels of alcohol throughout gestation. We found that NSPCs isolated from fetal alcohol exposed (FAE) mice displayed reduced neurosphere formation in culture, as assessed by a serial passage neurosphere assay, and reduced neuronal differentiation upon growth factor withdrawal. These studies suggest that gestational alcohol exposure leads to long-lasting NSPC-intrinsic dysregulation, which may underlie in vivo neurogenic deficits.
Assuntos
Etanol/farmacologia , Ventrículos Laterais/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Animais , Células Cultivadas , CamundongosRESUMO
Approximately 60,000 patients in the United States are waiting for a kidney transplant due to genetic, immunologic and environmentally caused kidney failure. Adult human renal stem cells could offer opportunities for autologous transplant and repair of damaged organs. Current data suggest that there are multiple progenitor types in the kidney with distinct localizations. In the present study, we characterize cells derived from human kidney papilla and show their capacity for tubulogenesis. In situ, nestin(+) and CD133/1(+) cells were found extensively intercalated between tubular epithelia in the loops of Henle of renal papilla, but not of the cortex. Populations of primary cells from the renal cortex and renal papilla were isolated by enzymatic digestion from human kidneys unsuited for transplant and immuno-enriched for CD133/1(+) cells. Isolated CD133/1(+) papillary cells were positive for nestin, as well as several human embryonic stem cell markers (SSEA4, Nanog, SOX2, and OCT4/POU5F1) and could be triggered to adopt tubular epithelial and neuronal-like phenotypes. Isolated papillary cells exhibited morphologic plasticity upon modulation of culture conditions and inhibition of asymmetric cell division. Labeled papillary cells readily associated with cortical tubular epithelia in co-culture and 3-dimensional collagen gel cultures. Heterologous organ culture demonstrated that CD133/1(+) progenitors from the papilla and cortex became integrated into developing kidney tubules. Tubular epithelia did not participate in tubulogenesis. Human renal papilla harbor cells with the hallmarks of adult kidney stem/progenitor cells that can be amplified and phenotypically modulated in culture while retaining the capacity to form new kidney tubules. This article is part of a Special Issue entitled: Polycystic Kidney Disease.
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Células-Tronco Adultas/citologia , Células-Tronco Adultas/imunologia , Antígenos CD/metabolismo , Glicoproteínas/metabolismo , Medula Renal/citologia , Túbulos Renais/citologia , Túbulos Renais/crescimento & desenvolvimento , Peptídeos/metabolismo , Antígeno AC133 , Células-Tronco Adultas/transplante , Animais , Diferenciação Celular , Separação Celular , Técnicas de Cocultura , Ensaio de Unidades Formadoras de Colônias , Humanos , Camundongos , Técnicas de Cultura de Órgãos , Rim Policístico Autossômico Dominante/terapiaRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutation of PKD1 and PKD2 that encode polycystin-1 and polycystin-2. Polycystin-1 is tyrosine phosphorylated and modulates multiple signaling pathways including AP-1, and the identity of the phosphatases regulating polycystin-1 are previously uncharacterized. Here we identify members of the LAR protein tyrosine phosphatase (RPTP) superfamily as members of the polycystin-1complex mediated through extra- and intracellular interactions. The first extracellular PKD1 domain of polycystin-1 interacts with the first Ig domain of RPTPσ, while the polycystin-1 C-terminus of polycystin-1 interacts with the regulatory D2 phosphatase domain of RPTPγ. Additional homo- and heterotypic interactions between RPTPs recruit RPTPδ. The multimeric polycystin protein complex is found localised in cilia. RPTPσ and RPTPδ are also part of a polycystin-1/E-cadherin complex known to be important for early events in adherens junction stabilisation. The interaction between polycystin-1 and RPTPγ is disrupted in ADPKD cells, while RPTPσ and RPTPδ remain closely associated with E-cadherin, largely in an intracellular location. The polycystin-1 C-terminus is an in vitro substrate of RPTPγ, which dephosphorylates the c-Src phosphorylated Y4237 residue and activates AP1-mediated transcription. The data identify RPTPs as novel interacting partners of the polycystins both in cilia and at adhesion complexes and demonstrate RPTPγ phosphatase activity is central to the molecular mechanisms governing polycystin-dependent signaling. This article is part of a Special Issue entitled: Polycystic Kidney Disease.
Assuntos
Proteínas Tirosina Fosfatases Semelhantes a Receptores/química , Canais de Cátion TRPP/química , Sequência de Aminoácidos , Animais , Caderinas/química , Caderinas/metabolismo , Linhagem Celular , Membrana Celular/química , Humanos , Técnicas In Vitro , Rim/metabolismo , Camundongos , Modelos Moleculares , Complexos Multiproteicos/química , Mutagênese Sítio-Dirigida , Biblioteca de Peptídeos , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Tirosina Fosfatases Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Semelhantes a Receptores/metabolismo , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/química , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/química , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismo , Fator de Transcrição AP-1/metabolismoRESUMO
Hypoxia-inducible factor-1 alpha subunit (HIF-1α) is a transcriptional activator mediating adaptive cellular response to hypoxia. Normally degraded in most cell types and tissues, HIF-1α becomes stable and transcriptionally active under conditions of hypoxia. In contrast, we found that HIF-1α is continuously expressed in adult brain neurogenic zones, as well as in neural stem/progenitor cells (NSPCs) from the embryonic and post-natal mouse brain. Our in vitro studies suggest that HIF-1α does not undergo typical hydroxylation, ubiquitination, and degradation within NSPCs under normoxic conditions. Based on immunofluorescence and cell fractionation, HIF-1α is primarily sequestered in membranous cytoplasmic structures, identified by immuno-electron microscopy as HIF-1α-bearing vesicles (HBV), which may prevent HIF-1α from degradation within the cytoplasm. HIF-1α shRNAi-mediated knockdown reduced the resistance of NSPCs to hypoxia, and markedly altered the expression levels of Notch-1 and ß-catenin, which influence NSPC differentiation. These findings indicate a unique regulation of HIF-1α protein stability in NSPCs, which may have importance in NSPCs properties and function.
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Regulação da Expressão Gênica no Desenvolvimento , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Células-Tronco Neurais/metabolismo , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/fisiologia , Estabilidade Proteica/efeitos dos fármacos , RNA Interferente Pequeno/farmacologiaRESUMO
Vascular cells provide a neural stem/progenitor cell (NSPC) niche that regulates expansion and differentiation of NSPCs within the germinal zones of the embryonic and adult brain under both physiologic and pathologic conditions. Here, we examined the NSPC-endothelial cell (NSPC/EC) interaction under conditions of ischemia, both in vitro and after intracerebral transplantation. In culture, embryonic mouse NSPCs supported capillary morphogenesis and protected ECs from cell death induced by serum starvation or by transient oxygen and glucose deprivation (OGD). Neural stem/progenitor cells constitutively expressed hypoxia-inducible factor 1alpha (HIF-1alpha) transcription factor and vascular endothelial growth factor (VEGF), both of which were increased approximately twofold after the exposure of NSPCs to OGD. The protective effects of NSPCs on ECs under conditions of serum starvation and hypoxia were blocked by pharmacological inhibitors of VEGF signaling, SU1498 and Flt-1-Fc. After intracerebral transplantation, NSPCs continued to express HIF-1alpha and VEGF, and promoted microvascular density after focal ischemia. These studies support a role for NSPCs in stabilization of vasculature during ischemia, mediated via HIF-1alpha-VEGF signaling pathways, and suggest therapeutic application of NSPCs to promote revascularization and repair after brain injury.
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Células Endoteliais/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Isquemia/etiologia , Morfogênese , Neurônios/fisiologia , Células-Tronco/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Embrião de Mamíferos , Hipóxia , Camundongos , Neovascularização Fisiológica , Transdução de SinaisRESUMO
Polycystins are plasma membrane proteins that are expressed in kidney epithelial cells and associated with the progression of ADPKD (autosomal dominant polycystic kidney disease). A polycystin multiprotein complex, including adherens junction proteins, is thought to play an important role in cell polarity and differentiation. Sucrose gradient analyses and immunoprecipitation studies of primary human kidney epithelial cells showed the polycystins and their associated proteins E-cadherin and beta-catenin distributed in a complex with the raft marker flotillin-2, but not caveolin-1, in high-density gradient fractions. The integrity of the polycystin multiprotein complex was sensitive to cholesterol depletion, as shown by cyclodextrin treatment of immunoprecipitated complexes. The overexpressed C-terminus of polycystin-1 retained the ability to associate with flotillin-2. Flotillin-2 was found to contain CRAC (cholesterol recognition/interaction amino acid) cholesterol-binding domains and to promote plasma membrane cholesterol recruitment. Based on co-association of signalling molecules, such as Src kinases and phosphatases, we propose that the polycystin multiprotein complex is embedded in a cholesterol-containing signalling microdomain specified by flotillin-2, which is distinct from classical light-buoyant-density, detergent-resistant domains.
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Colesterol/metabolismo , Células Epiteliais/metabolismo , Rim/citologia , Proteínas de Membrana/metabolismo , Proteínas/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Animais , Caderinas/metabolismo , Caveolina 1/metabolismo , Linhagem Celular , Células Cultivadas , Humanos , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/química , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Estrutura Terciária de Proteína , Proteínas/química , Canais de Cátion TRPP , beta Catenina/metabolismoRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is typified by the accumulation of fluid-filled cysts and abnormalities in renal epithelial cell function. The disease is principally caused by mutations in the gene encoding polycystin-1, a large basolateral plasma membrane protein expressed in kidney epithelial cells. Our studies reveal that, in normal kidney cells, polycystin-1 forms a complex with the adherens junction protein E-cadherin and its associated catenins, suggesting a role in cell adhesion or polarity. In primary cells from ADPKD patients, the polycystin-1/polycystin-2/E-cadherin/beta-catenin complex was disrupted and both polycystin-1 and E-cadherin were depleted from the plasma membrane as a result of the increased phosphorylation of polycystin-1. The loss of E-cadherin was compensated by the transcriptional upregulation of the normally mesenchymal N-cadherin. Increased cell surface N-cadherin in the disease cells in turn stabilized the continued plasma membrane localization of beta-catenin in the absence of E-cadherin. The results suggest that enhanced phosphorylation of polycystin-1 in ADPKD cells precipitates changes in its localization and its ability to form protein complexes that are critical for the stabilization of adherens junctions and the maintenance of a fully differentiated polarized renal epithelium.
Assuntos
Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Rim/metabolismo , Doenças Renais Policísticas/metabolismo , Proteínas/metabolismo , Junções Aderentes/metabolismo , Caderinas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Perfilação da Expressão Gênica , Humanos , Fosforilação , Canais de Cátion TRPP , Transativadores/metabolismo , beta CateninaRESUMO
Establishment and maintenance of a polarized epithelium relies on the integration of signaling cascades, acquisition of specialized trafficking circuits and establishment of a unique cytoarchitecture. Defects in any of these processes can adversely affect cell polarity and cause defects in specific organs and systemic disease. Mutations that disrupt the proper transport of individual plasma membrane proteins, or inactivate components of the epithelial-specific trafficking machinery, have severe functional consequences. Links between renal diseases and defects in trafficking, differentiation or signaling, highlight the delicate balance between these parameters which, when altered, precipitates a loss of renal function.