RESUMO
During development or after brain injury, oligodendrocyte precursor cells (OPCs) differentiate into oligodendrocytes to supplement the number of oligodendrocytes. Although mechanisms of OPC differentiation have been extensively examined, the role of epigenetic regulators, such as histone deacetylases (HDACs) and DNA methyltransferase enzymes (DNMTs), in this process is still mostly unknown. Here, we report the differential roles of epigenetic regulators in OPC differentiation. We prepared primary OPC cultures from neonatal rat cortex. Our cultured OPCs expressed substantial amounts of mRNA for HDAC1, HDAC2, DNMT1, and DNMT3a. mRNA levels of HDAC1 and HDAC2 were both decreased by the time OPCs differentiated into myelin-basic-protein expressing oligodendrocytes. However, DNMT1 or DNMT3a mRNA level gradually decreased or increased during the differentiation step, respectively. We then knocked down those regulators in cultured OPCs with siRNA technique before starting OPC differentiation. While HDAC1 knockdown suppressed OPC differentiation, HDAC2 knockdown promoted OPC differentiation. DNMT1 knockdown also suppressed OPC differentiation, but unlike HDAC1/2, DNMT1-deficient cells showed cell damage during the later phase of OPC differentiation. On the other hand, when OPCs were transfected with siRNA for DNMT3a, the number of OPCs was decreased, indicating that DNMT3a may participate in OPC survival/proliferation. Taken together, these data demonstrate that each epigenetic regulator has different phase-specific roles in OPC survival and differentiation.
Assuntos
Epigênese Genética/fisiologia , Células Precursoras de Oligodendrócitos/fisiologia , Animais , Animais Recém-Nascidos , Diferenciação Celular , Córtex Cerebral/citologia , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , DNA Metiltransferase 3A , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Proteína Básica da Mielina/genética , Proteína Básica da Mielina/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , TransfecçãoRESUMO
Oligodendrocyte precursor cells (OPCs) give rise to oligodendrocytes in cerebral white matter. However, the underlying mechanisms that regulate this process remain to be fully defined, especially in adult brains. Recently, it has been suggested that signaling via A-kinase anchor protein 12 (AKAP12), a scaffolding protein that associates with intracellular molecules such as protein kinase A, may be involved in Schwann cell homeostasis and peripheral myelination. Here, we asked whether AKAP12 also regulates the mechanisms of myelination in the CNS. AKAP12 knockout mice were compared against wild-type (WT) mice in a series of neurochemical and behavioral assays. Compared with WTs, 2-months old AKAP12 knockout mice exhibited loss of myelin in white matter of the corpus callosum, along with perturbations in working memory as measured by a standard Y-maze test. Unexpectedly, very few OPCs expressed AKAP12 in the corpus callosum region. Instead, pericytes appeared to be one of the major AKAP12-expressing cells. In a cell culture model system, conditioned culture media from normal pericytes promoted in-vitro OPC maturation. However, conditioned media from AKAP12-deficient pericytes did not support the OPC function. These findings suggest that AKAP12 signaling in pericytes may be required for OPC-to-oligodendrocyte renewal to maintain the white matter homeostasis in adult brain. Stem Cells 2018;36:751-760.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/fisiologia , Células-Tronco Neurais/citologia , Oligodendroglia/metabolismo , Substância Branca/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Envelhecimento , Animais , Proteínas de Ciclo Celular/genética , Proliferação de Células/fisiologia , Células Cultivadas , Meios de Cultivo Condicionados , Camundongos Knockout , Bainha de Mielina/metabolismo , Neurogênese/fisiologia , Oligodendroglia/citologia , Substância Branca/citologiaRESUMO
BACKGROUND AND PURPOSE: Endothelial progenitor cells (EPCs) have been extensively investigated as a therapeutic approach for repairing the vascular system in cerebrovascular diseases. Beyond vascular regeneration per se, EPCs may also release factors that affect the entire neurovascular unit. Here, we aim to study the effects of the EPC secretome on oligovascular remodeling in a mouse model of white matter injury after prolonged cerebral hypoperfusion. METHODS: The secretome of mouse EPCs was analyzed with a proteome array. In vitro, the effects of the EPC secretome and its factor angiogenin were assessed on primary oligodendrocyte precursor cells and mature human cerebral microvascular endothelial cells (hCMED/D3). In vivo, mice were subjected to permanent bilateral common carotid artery stenosis, then treated with EPC secretome at 24 hours and at 1 week, and cognitive outcome was evaluated with the Y maze test together with oligodendrocyte precursor cell proliferation/differentiation and vascular density in white matter at 4 weeks. RESULTS: Multiple growth factors, cytokines, and proteases were identified in the EPC secretome, including angiogenin. In vitro, the EPC secretome significantly enhanced endothelial and oligodendrocyte precursor cell proliferation and potentiated oligodendrocyte precursor cell maturation. Angiogenin was proved to be a key factor since pharmacological blockade of angiogenin signaling negated the positive effects of the EPC secretome. In vivo, treatment with the EPC secretome increased vascular density, myelin, and mature oligodendrocytes in white matter and rescued cognitive function in the mouse hypoperfusion model. CONCLUSIONS: Factors secreted by EPCs may ameliorate white matter damage in the brain by boosting oligovascular remodeling.
Assuntos
Indutores da Angiogênese/farmacologia , Estenose das Carótidas/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Progenitoras Endoteliais/metabolismo , Células Precursoras de Oligodendrócitos/efeitos dos fármacos , Ribonuclease Pancreático/farmacologia , Remodelação Vascular/efeitos dos fármacos , Substância Branca/efeitos dos fármacos , Animais , Isquemia Encefálica/metabolismo , Meios de Cultivo Condicionados , Citocinas/metabolismo , Modelos Animais de Doenças , Glutationa S-Transferase pi/metabolismo , Humanos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Proteína Básica da Mielina/metabolismo , Células Precursoras de Oligodendrócitos/metabolismo , Peptídeo Hidrolases/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Ribonuclease Pancreático/metabolismo , Substância Branca/irrigação sanguíneaRESUMO
Ischemic postconditioning is increasingly being investigated as a therapeutic approach for cerebral ischemia. However, the majority of studies are focused on the acute protection of neurons per se. Whether and how postconditioning affects multiple cells in the recovering neurovascular unit remains to be fully elucidated. Here, we asked whether postconditioning may modulate help-me signaling between injured neurons and reactive microglia. Rats were subjected to 100 min of focal cerebral ischemia, then randomized into a control versus postconditioning group. After 3 days of reperfusion, infarct volumes were significantly reduced in animals treated with postconditioning, along with better neurologic outcomes. Immunostaining revealed that ischemic postconditioning increased expression of vascular endothelial growth factor (VEGF) in neurons within peri-infarct regions. Correspondingly, we confirmed that VEGFR2 was expressed on Iba1-positive microglia/macrophages, and confocal microscopy showed that in postconditioned rats, these cells were polarized to a ramified morphology with higher expression of M2-like markers. Treating rats with a VEGF receptor 2 kinase inhibitor negated these effects of postconditioning on microglia/macrophage polarization. In vitro, postconditoning after oxygen-glucose deprivation up-regulated VEGF release in primary neuron cultures, and adding VEGF to microglial cultures partly shifted their M2-like markers. Altogether, our findings support the idea that after postconditioning, injured neurons may release VEGF as a 'help-me' signal that promotes microglia/macrophage polarization into potentially beneficial phenotypes.
Assuntos
Isquemia Encefálica/patologia , Isquemia Encefálica/terapia , Polaridade Celular/fisiologia , Pós-Condicionamento Isquêmico/métodos , Microglia/patologia , Neurônios/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Infarto Encefálico/etiologia , Proteínas de Ligação ao Cálcio/metabolismo , Hipóxia Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Embrião de Mamíferos , Proteína Glial Fibrilar Ácida/metabolismo , Glucose/deficiência , Infusões Intraventriculares , Masculino , Proteínas dos Microfilamentos/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Oligodendrocyte precursor cells (OPCs) in the adult brain contribute to white matter homeostasis. After white matter damage, OPCs compensate for oligodendrocyte loss by differentiating into mature oligodendrocytes. However, the underlying mechanisms remain to be fully defined. Here, we test the hypothesis that, during endogenous recovery from white matter ischemic injury, astrocytes support the maturation of OPCs by secreting brain-derived neurotrophic factor (BDNF). For in vitro experiments, cultured primary OPCs and astrocytes were prepared from postnatal day 2 rat cortex. When OPCs were subjected to chemical hypoxic stress by exposing them to sublethal CoCl2 for 7 d, in vitro OPC differentiation into oligodendrocytes was significantly suppressed. Conditioned medium from astrocytes (astro-medium) restored the process of OPC maturation even under the stressed conditions. When astro-medium was filtered with TrkB-Fc to remove BDNF, the BDNF-deficient astro-medium no longer supported OPC maturation. For in vivo experiments, we analyzed a transgenic mouse line (GFAP(cre)/BDNF(wt/fl)) in which BDNF expression is downregulated specifically in GFAP(+) astrocytes. Both wild-type (GFAP(wt)/BDNF(wt/fl) mice) and transgenic mice were subjected to prolonged cerebral hypoperfusion by bilateral common carotid artery stenosis. As expected, compared with wild-type mice, the transgenic mice exhibited a lower number of newly generated oligodendrocytes and larger white matter damage. Together, these findings demonstrate that, during endogenous recovery from white matter damage, astrocytes may promote oligodendrogenesis by secreting BDNF. SIGNIFICANCE STATEMENT: The repair of white matter after brain injury and neurodegeneration remains a tremendous hurdle for a wide spectrum of CNS disorders. One potentially important opportunity may reside in the response of residual oligodendrocyte precursor cells (OPCs). OPCs may serve as a back-up for generating mature oligodendrocytes in damaged white matter. However, the underlying mechanisms are still mostly unknown. Here, we use a combination of cell biology and an animal model to report a new pathway in which astrocyte-derived BDNF supports oligodendrogenesis and regeneration after white matter damage. These findings provide new mechanistic insight into white matter physiology and pathophysiology, which would be broadly and clinically applicable to CNS disease.
Assuntos
Astrócitos/fisiologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Diferenciação Celular/fisiologia , Leucoencefalopatias/patologia , Animais , Antimutagênicos/farmacologia , Astrócitos/química , Astrócitos/metabolismo , Isquemia Encefálica/complicações , Fator Neurotrófico Derivado do Encéfalo/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Cromonas/farmacologia , Cobalto/farmacologia , Meios de Cultivo Condicionados/farmacologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Glutationa S-Transferase pi/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Leucoencefalopatias/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Morfolinas/farmacologia , Proteína Básica da Mielina/metabolismo , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Fosfopiruvato Hidratase/metabolismo , Células-Tronco/fisiologiaRESUMO
BACKGROUND AND PURPOSE: Pentraxin 3 (PTX3) is released on inflammatory responses in many organs. However, roles of PTX3 in brain are still mostly unknown. Here we asked whether and how PTX3 contributes to blood-brain barrier dysfunction during the acute phase of ischemic stroke. METHODS: In vivo, spontaneously hypertensive rats were subjected to focal cerebral ischemia by transient middle cerebral artery occlusion. At day 3, brains were analyzed to evaluate the cellular origin of PTX3 expression. Correlations with blood-brain barrier breakdown were assessed by IgG staining. In vitro, rat primary astrocytes and rat brain endothelial RBE.4 cells were cultured to study the role of astrocyte-derived PTX3 on vascular endothelial growth factor-mediated endothelial permeability. RESULTS: During the acute phase of stroke, reactive astrocytes in the peri-infarct area expressed PTX3. There was negative correlation between gradients of IgG leakage and PTX3-positive astrocytes. Cell culture experiments showed that astrocyte-conditioned media increased levels of tight junction proteins and reduced endothelial permeability under normal conditions. Removing PTX3 from astrocyte-conditioned media by immunoprecipitation increased endothelial permeability. PTX3 strongly bound vascular endothelial growth factor in vitro and was able to decrease vascular endothelial growth factor-induced endothelial permeability. CONCLUSIONS: Astrocytes in peri-infarct areas upregulate PTX3, which may support blood-brain barrier integrity by regulating vascular endothelial growth factor-related mechanisms. This response in astrocytes may comprise a compensatory mechanism for maintaining blood-brain barrier function after ischemic stroke.
Assuntos
Astrócitos/metabolismo , Barreira Hematoencefálica/metabolismo , Proteína C-Reativa/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Componente Amiloide P Sérico/metabolismo , Acidente Vascular Cerebral/metabolismo , Animais , Barreira Hematoencefálica/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Morte Celular , Meios de Cultivo Condicionados , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Infarto da Artéria Cerebral Média/patologia , Masculino , Ratos , Ratos Endogâmicos SHR , Acidente Vascular Cerebral/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Oligodendrocyte precursor cells (OPCs) play critical roles in maintaining the number of oligodendrocytes in white matter. Previously, we have shown that oxidative stress dampens oligodendrocyte regeneration after white matter damage, while a clinically proven radical scavenger, edaravone, supports oligodendrocyte repopulation. However, it is not known how edaravone exerts this beneficial effect against oxidative stress. Using in vivo and in vitro experiments, we have examined whether edaravone exhibits direct OPC-protective effects. For in vivo experiments, prolonged cerebral hypoperfusion was induced by bilateral common carotid artery stenosis in mice. OPC damage was observed on day 14 after the onset of cerebral hypoperfusion, and edaravone was demonstrated to decrease OPC death in cerebral white matter. In vitro experiments also confirmed that edaravone reduced oxidative-stress-induced OPC death. Because white matter damage is a major hallmark of many neurological diseases, and OPCs are instrumental in white matter repair after injury, our current study supports the idea that radical scavengers may provide a potential therapeutic approach for white matter related diseases.
Assuntos
Antipirina/análogos & derivados , Isquemia Encefálica/tratamento farmacológico , Sequestradores de Radicais Livres/farmacologia , Fármacos Neuroprotetores/farmacologia , Células Precursoras de Oligodendrócitos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Substância Branca/efeitos dos fármacos , Substância Branca/lesões , Animais , Antipirina/farmacologia , Edaravone , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Oligodendrocytes are generated from oligodendrocyte precursor cells (OPCs). Mechanisms of OPC differentiation have been extensively examined with two-dimensional cell culture systems. However, these cellular events may be more accurately represented using a three-dimensional (3D) model. In this study, we report the development of a novel 3D OPC culture system using gels composed of a mixture of collagen and hyaluronan, wherein cultured rat primary OPCs can proliferate and differentiate into oligodendrocytes. Our data show that the gel concentration and cell-seeding density are critical factors for the numbers of OPCs and oligodendrocytes in our 3D culture system. In addition, Notch signaling, which supports cell-to-cell communication, may also be important for OPC function in our system because a Notch inhibitor DAPT suppressed OPC proliferation and differentiation. Taken together, cultured rat OPCs can grow in collagen-/hyaluronan-based gels, and our novel 3D OPC culture system may offer a useful platform for examining the mechanisms of OPC function in vitro.
Assuntos
Técnicas de Cultura de Células/métodos , Células Precursoras de Oligodendrócitos/citologia , Animais , Astrócitos/citologia , Contagem de Células , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Células Cultivadas , Géis , Células Precursoras de Oligodendrócitos/metabolismo , Porosidade , Ratos Sprague-Dawley , Receptores Notch/metabolismo , Transdução de SinaisRESUMO
Oligodendrocytes are one of the major cell types in cerebral white matter. Under normal conditions, they form myelin sheaths that encircle axons to support fast nerve conduction. Under conditions of cerebral ischemia, oligodendrocytes tend to die, resulting in white-matter dysfunction. Repair of white matter involves the ability of oligodendrocyte precursors to proliferate and mature. However, replacement of lost oligodendrocytes may not be the only mechanism for white-matter recovery. Emerging data now suggest that coordinated signaling between neural, glial, and vascular cells in the entire neurovascular unit may be required. In this mini-review, we discuss how oligodendrocyte lineage cells participate in signaling and crosstalk with other cell types to underlie function and recovery in various experimental models of subcortical white-matter injury.
Assuntos
Isquemia Encefálica/patologia , Demência Vascular/patologia , Modelos Animais de Doenças , Oligodendroglia/patologia , Acidente Vascular Cerebral Lacunar/patologia , Substância Branca/patologia , Animais , Isquemia Encefálica/complicações , Comunicação Celular , Proliferação de Células , Demência Vascular/etiologia , Humanos , Neurônios/patologia , Acidente Vascular Cerebral Lacunar/etiologiaRESUMO
Hypoxic-ischemic (HI) brain injury in newborns results in serious damage. Magnesium sulfate has been clinically used as a cyto-protective agent against HI brain injury in newborns in some countries, including Japan. However, it is not clear how magnesium exerts this effect and how it acts on the individual types of cells within the newborn brain. In this study, we exposed cultured rat oligodendrocyte precursor cells to magnesium sulfate during the period when they differentiate into oligodendrocytes, and showed that magnesium-exposed oligodendrocytes exhibited more resistance to HI injury. Our data may support the use of magnesium sulfate in the clinical setting.
Assuntos
Hipóxia-Isquemia Encefálica/patologia , Sulfato de Magnésio/farmacologia , Oligodendroglia/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Diferenciação Celular , Linhagem da Célula , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Hipóxia-Isquemia Encefálica/prevenção & controle , Oligodendroglia/patologia , RatosRESUMO
Aging and vascular comorbidities such as hypertension comprise critical cofactors that influence how the brain responds to stroke. Ischemic stress induces neurogenesis and oligodendrogenesis in younger brains. However, it remains unclear whether these compensatory mechanisms can be maintained even under pathologically hypertensive and aged states. To clarify the age-related remodeling capacity after stroke under hypertensive conditions, we assessed infarct volume, behavioral outcomes, and surrogate markers of neurogenesis and oligodendrogenesis in acute and subacute phases after transient focal cerebral ischemia in 3- and 12-month-old spontaneously hypertensive rats (SHRs). Hematoxylin and eosin staining showed that 3- and 12-month-old SHRs exhibited similar infarction volumes at both 3 and 14 days after focal cerebral ischemia. However, recovery of behavioral deficits (neurological score assessment and adhesive removal test) was significantly less in 12-month-old SHRs compared to 3-month-old SHRs. Concomitantly, numbers of nestin(+) neural stem/progenitor cells (NSPCs) near the infarct border area or subventricular zone in 12-month-old SHRs were lower than 3-month-old SHRs at day 3. Similarly, numbers of PDGFR-α(+) oligodendrocyte precursor cells (OPCs) in the corpus callosum were lower in 12-month-old SHRs at day 3. Lower levels of NSPC and OPC numbers were accompanied by lower expression levels of phosphorylated CREB. By day 14 postischemia, NSPC and OPC numbers in 12-month-old SHRs recovered to similar levels as in 3-month-old SHRs, but the numbers of proliferating NSPCs (Ki-67(+)nestin(+) cells) and proliferating OPCs (Ki-67(+)PDGFR-α(+) cells) remained lower in the older brains even at day 14. Taken together, these findings suggest that aging may also decrease poststroke compensatory responses for neurogenesis and oligodendrogenesis even under hypertensive conditions.
Assuntos
Envelhecimento/metabolismo , Infarto Encefálico/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese , Oligodendroglia/metabolismo , Envelhecimento/patologia , Animais , Infarto Encefálico/patologia , Masculino , Células-Tronco Neurais/patologia , Oligodendroglia/patologia , Ratos , Ratos Endogâmicos SHRRESUMO
Oligodendrocytes, which are the main cell type in cerebral white matter, are generated from their precursor cells (oligodendrocyte precursor cells: OPCs). However, the differentiation from OPCs to oligodendrocytes is disturbed under stressed conditions. Therefore, drugs that can improve oligodendrocyte regeneration may be effective for white matter-related diseases. Here we show that a vasoactive peptide adrenomedullin (AM) promotes the in vitro differentiation of OPCs under pathological conditions. Primary OPCs were prepared from neonatal rat brains, and differentiated into myelin-basic-protein expressing oligodendrocytes over time. This in vitro OPC differentiation was inhibited by prolonged chemical hypoxic stress induced by non-lethal CoCl(2) treatment. However, AM promoted the OPC differentiation under the hypoxic stress conditions, and the AM receptor antagonist AM(22-52) canceled the AM-induced OPC differentiation. In addition, AM treatment increased the phosphorylation level of Akt in OPC cultures, and correspondingly, the PI3K/Akt inhibitor LY294002 blocked the AM-induced OPC differentiation. Taken together, AM treatment rescued OPC maturation under pathological conditions via an AM-receptor-PI3K/Akt pathway. Oligodendrocytes play critical roles in white matter by forming myelin sheath. Therefore, AM signaling may be a promising therapeutic target to boost oligodendrocyte regeneration in CNS disorders.
Assuntos
Adrenomedulina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proteína Básica da Mielina/metabolismo , Células-Tronco Neurais/citologia , Oligodendroglia/citologia , Fragmentos de Peptídeos/farmacologia , Animais , Hipóxia Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Receptores de Adrenomedulina/metabolismoRESUMO
Pericytes are embedded within basal lamina and play multiple roles in the perivascular niche in brain. Recently, oligodendrocyte precursor cells (OPCs) have also been reported to associate with cerebral endothelium. Is it possible that within this gliovascular locus, there may also exist potential spatial and functional interactions between pericytes and OPCs? Here, we demonstrated that in the perivascular region of cerebral white matter, pericytes and OPCs may attach and support each other. Immunostaining showed that pericytes and OPCs are localized in close contact with each other in mouse white matter at postnatal days 0, 60 and 240. Electron microscopic analysis confirmed that pericytes attached to OPCs via basal lamina in the perivascular region. The close proximity between these two cell types was also observed in postmortem human brains. Functional interaction between pericytes and OPCs was assessed by in vitro media transfer experiments. When OPC cultures were treated with pericyte-conditioned media, OPC number increased. Similarly, pericyte number increased when pericytes were maintained in OPC-conditioned media. Taken together, our data suggest a potential anatomical and functional interaction between pericytes and OPCs in cerebral white matter.
Assuntos
Córtex Cerebral/citologia , Oligodendroglia/fisiologia , Pericitos/fisiologia , Células-Tronco/fisiologia , Substância Branca/citologia , Idoso , Animais , Comunicação Celular , Proliferação de Células , Células Cultivadas , Corpo Caloso/citologia , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Oligodendroglia/citologia , Pericitos/citologia , Ratos Sprague-Dawley , Especificidade da Espécie , Células-Tronco/citologiaRESUMO
Adult mammalian brain can be plastic after injury and disease. Therefore, boosting endogenous repair mechanisms would be a useful therapeutic approach for neurological disorders. Isoxazole-9 (Isx-9) has been reported to enhance neurogenesis from neural stem/progenitor cells (NSPCs). However, the effects of Isx-9 on other types of progenitor/precursor cells remain mostly unknown. In this study, we investigated the effects of Isx-9 on the three major populations of progenitor/precursor cells in brain: NSPCs, oligodendrocyte precursor cells (OPCs), and endothelial progenitor cells (EPCs). Cultured primary NSPCs, OPCs, or EPCs were treated with various concentrations of Isx-9 (6.25, 12.5, 25, 50 µM), and their cell numbers were counted in a blinded manner. Isx-9 slightly increased the number of NSPCs and effectively induced neuronal differentiation of NSPCs. However, Isx-9 significantly decreased OPC number in a concentration-dependent manner, suggesting cytotoxicity. Isx-9 did not affect EPC cell number. But in a matrigel assay of angiogenesis, Isx-9 significantly inhibited tube formation in outgrowth endothelial cells derived from EPCs. This potential anti-tube-formation effect of Isx-9 was confirmed in a brain endothelial cell line. Taken together, our data suggest that mechanisms and targets for promoting stem/progenitor cells in the central nervous system may significantly differ between cell types.
Assuntos
Diferenciação Celular , Células Progenitoras Endoteliais/efeitos dos fármacos , Isoxazóis/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Animais , Biomarcadores , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/metabolismo , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese , Neurônios/citologia , Neurônios/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , RatosRESUMO
Matrix metalloproteinase-9 (MMP-9) plays key roles in the brain pathophysiology, especially in blood-brain barrier (BBB) breakdown. Therefore, inhibiting MMP-9 activity may be a promising therapy for protecting brains in cerebrovascular diseases. Here we show that in a mouse prolonged cerebral hypoperfusion model, a clinically proven radical scavenger edaravone suppressed MMP-9 and reduced BBB damage in cerebral white matter. Prolonged cerebral hypoperfusion was induced by bilateral common carotid artery stenosis in male adult C57BL/6J mice (10 weeks old). After 7 days of cerebral hypoperfusion, white matter region (e.g. corpus callosum) exhibited significant BBB leakage, assessed by IgG staining. Correspondingly, immunostaining and western blotting showed that MMP-9 was upregulated in the white matter. Edaravone treatment (3mg/kg, i.p. at days 0 and 3) inhibited both BBB leakage and MMP-9 increase. Under the early phase of cerebral hypoperfusion conditions, oligodendrocyte precursor cells (OPCs) mainly contribute to the MMP-9 increase, but our immunostaining data showed that very little OPCs expressed MMP-9 in the edaravone-treated animals at day 7. Therefore, in vitro studies with primary rat OPCs were conducted to examine whether edaravone would directly suppressed MMP-9 expressions in OPCs. OPC cultures were exposed to sub-lethal CoCl2 for 7 days to induce prolonged chemical hypoxic stress. Prolonged chemical hypoxic stress increased MMP-9 expression in OPCs, and radical scavenging with edaravone (10µM for 7 days) ameliorated the increase. Taken together, our proof-of-concept study demonstrates that radical scavengers may provide a potential therapeutic approach for white matter injury by suppressing BBB damage.
Assuntos
Antipirina/análogos & derivados , Barreira Hematoencefálica/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Hipóxia Encefálica/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Animais , Antipirina/farmacologia , Barreira Hematoencefálica/metabolismo , Artéria Carótida Primitiva , Estenose das Carótidas/complicações , Estenose das Carótidas/metabolismo , Estenose das Carótidas/fisiopatologia , Morte Celular , Sobrevivência Celular , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/metabolismo , Circulação Cerebrovascular/efeitos dos fármacos , Edaravone , Hipóxia Encefálica/etiologia , Hipóxia Encefálica/patologia , Hipóxia Encefálica/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Regulação para Cima , Substância Branca/irrigação sanguínea , Substância Branca/efeitos dos fármacos , Substância Branca/metabolismoRESUMO
Pericytes are vascular mural cells embedded within the basal lamina of blood micro-vessels. Within the neurovascular unit, pericytes play important roles in regulating neurovascular homeostasis by secreting soluble factors, such as matrix metalloproteinases (MMPs). However, little is known about the regulatory signaling pathways in brain pericytes. Here we show that transforming growth factor-ß1 (TGF-ß1) induces MMP-9 upregulation in pericytes via p38 mitogen-activated protein (MAP) kinase signaling. Cultured human brain vascular pericytes were used in this study. When the brain pericytes were treated with purified human TGF-ß1 (0.1-10ng/mL for 24h), the levels of MMP-2 and MMP-9 in culture media were significantly increased in a concentration dependent manner as measured by gelatin zymography. WST assay confirmed that TGF-ß1 did not affect cell survival of the brain pericytes. A TGF-ß-receptor inhibitor SB431542 (0.5-5µM) decreased the TGF-ß1-induced upregulation of MMP-2 and MMP-9. To assess the underlying intracellular mechanisms, we focused on p38 MAP kinase signaling, which is one of the major downstream kinases for TGF-ß1. A well-validated p38 MAP kinase inhibitor SB203580 (0.5-5µM) cancelled the effect of TGF-ß1 in upregulation of MMP-9 but not MMP-2. Western blotting confirmed that TGF-ß1 treatment increased the level of p38 MAP kinase phosphorylation in pericytes, and again, the TGF-ß-receptor inhibitor SB431542 (0.5-5µM) blocked the TGF-ß1-induced phosphorylation of p38 MAP kinase. Both TGF-ß1 and MMP-9 are major neurovascular mediators, and therefore, our current finding may suggest a novel mechanism for how pericytes regulate neurovascular homeostasis.
Assuntos
Encéfalo/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Pericitos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Benzamidas/farmacologia , Encéfalo/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Fármacos do Sistema Nervoso Central/farmacologia , Dioxóis/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Imidazóis/farmacologia , Sistema de Sinalização das MAP Quinases , Pericitos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Recombinantes/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Trophic coupling between cerebral endothelium and their neighboring cells is required for the development and maintenance of blood-brain barrier (BBB) function. Here we report that oligodendrocyte precursor cells (OPCs) secrete soluble factor TGF-ß1 to support BBB integrity. Firstly, we prepared conditioned media from OPC cultures and added them to cerebral endothelial cultures. Our pharmacological experiments showed that OPC-conditioned media increased expressions of tight-junction proteins and decreased in vitro BBB permeability by activating TGB-ß-receptor-MEK/ERK signaling pathway. Secondly, our immuno-electron microscopic observation revealed that in neonatal mouse brains, OPCs attach to cerebral endothelial cells via basal lamina. And finally, we developed a novel transgenic mouse line that TGF-ß1 is knocked down specifically in OPCs. Neonates of these OPC-specific TGF-ß1 deficient mice (OPC-specific TGF-ß1 partial KO mice: PdgfraCre/Tgfb1flox/wt mice or OPC-specific TGF-ß1 total KO mice: PdgfraCre/Tgfb1flox/flox mice) exhibited cerebral hemorrhage and loss of BBB function. Taken together, our current study demonstrates that OPCs increase BBB tightness by upregulating tight junction proteins via TGF-ß signaling. Although astrocytes and pericytes are well-known regulators of BBB maturation and maintenance, these findings indicate that OPCs also play a pivotal role in promoting BBB integrity.
Assuntos
Barreira Hematoencefálica , Células-Tronco Neurais/citologia , Oligodendroglia/citologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Ratos , Ratos Sprague-DawleyRESUMO
White matter dysfunction is an important part of many CNS disorders including multiple sclerosis (MS) and vascular dementia. Within injured areas, myelin loss and oligodendrocyte death may trigger endogenous attempts at regeneration. However, during disease progression, remyelination failure may eventually occur due to impaired survival/proliferation, migration/recruitment, and differentiation of oligodendrocyte precursor cells (OPCs). The ventricular-subventricular zone (V-SVZ) and the subgranular zone (SGZ) are the main sources of neural stem/progenitor cells (NSPCs), which can give rise to neurons as well as OPCs. Under normal conditions in the adult brain, the V-SVZ progenitors generate a large number of neurons with a small number of oligodendrocyte lineage cells. However, after demyelination, the fate of V-SVZ-derived progenitor cells shifts from neurons to OPCs, and these newly generated OPCs migrate to the demyelinating lesions to ease white matter damage. In this mini-review, we will summarize the recent studies on extrinsic (e.g., vasculature, extracellular matrix (ECM), cerebrospinal fluid (CSF)) and intrinsic (e.g., transcription factors, epigenetic modifiers) factors, which mediate oligodendrocyte generation from the V-SVZ progenitor cells. A deeper understanding of the mechanisms that regulate the fate of V-SVZ progenitor cells may lead to new therapeutic approaches for ameliorating white matter dysfunction and damage in CNS disorders.