RESUMEN
Collateral arteries are an uncommon vessel subtype that can provide alternate blood flow to preserve tissue following vascular occlusion. Some patients with heart disease develop collateral coronary arteries, and this correlates with increased survival. However, it is not known how these collaterals develop or how to stimulate them. We demonstrate that neonatal mouse hearts use a novel mechanism to build collateral arteries in response to injury. Arterial endothelial cells (ECs) migrated away from arteries along existing capillaries and reassembled into collateral arteries, which we termed "artery reassembly". Artery ECs expressed CXCR4, and following injury, capillary ECs induced its ligand, CXCL12. CXCL12 or CXCR4 deletion impaired collateral artery formation and neonatal heart regeneration. Artery reassembly was nearly absent in adults but was induced by exogenous CXCL12. Thus, understanding neonatal regenerative mechanisms can identify pathways that restore these processes in adults and identify potentially translatable therapeutic strategies for ischemic heart disease.
Asunto(s)
Circulación Colateral/fisiología , Corazón/crecimiento & desarrollo , Regeneración/fisiología , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Quimiocina CXCL12/metabolismo , Vasos Coronarios/crecimiento & desarrollo , Células Endoteliales/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neovascularización Fisiológica/fisiología , Receptores CXCR4/metabolismo , Transducción de SeñalRESUMEN
Neurovascular unit and barrier maturation rely on vascular basement membrane (vBM) composition. Laminins, a major vBM component, are crucial for these processes, yet the signaling pathway(s) that regulate their expression remain unknown. Here, we show that mural cells have active Wnt/ß-catenin signaling during central nervous system development in mice. Bulk RNA sequencing and validation using postnatal day 10 and 14 wild-type versus adenomatosis polyposis coli downregulated 1 (Apcdd1-/-) mouse retinas revealed that Lama2 mRNA and protein levels are increased in mutant vasculature with higher Wnt/ß-catenin signaling. Mural cells are the main source of Lama2, and Wnt/ß-catenin activation induces Lama2 expression in mural cells in vitro. Markers of mature astrocytes, including aquaporin 4 (a water channel in astrocyte endfeet) and integrin-α6 (a laminin receptor), are upregulated in Apcdd1-/- retinas with higher Lama2 vBM deposition. Thus, the Wnt/ß-catenin pathway regulates Lama2 expression in mural cells to promote neurovascular unit and barrier maturation.
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Vía de Señalización Wnt , beta Catenina , Animales , Ratones , Vía de Señalización Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
The transcriptional control of CNS myelin gene expression is poorly understood. Here we identify gene model 98, which we have named myelin gene regulatory factor (MRF), as a transcriptional regulator required for CNS myelination. Within the CNS, MRF is specifically expressed by postmitotic oligodendrocytes. MRF is a nuclear protein containing an evolutionarily conserved DNA binding domain homologous to a yeast transcription factor. Knockdown of MRF in oligodendrocytes by RNA interference prevents expression of most CNS myelin genes; conversely, overexpression of MRF within cultured oligodendrocyte progenitors or the chick spinal cord promotes expression of myelin genes. In mice lacking MRF within the oligodendrocyte lineage, premyelinating oligodendrocytes are generated but display severe deficits in myelin gene expression and fail to myelinate. These mice display severe neurological abnormalities and die because of seizures during the third postnatal week. These findings establish MRF as a critical transcriptional regulator essential for oligodendrocyte maturation and CNS myelination.
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Encéfalo/citología , Regulación de la Expresión Génica , Vaina de Mielina/metabolismo , Oligodendroglía/metabolismo , Factores de Transcripción/metabolismo , Animales , Encéfalo/metabolismo , Diferenciación Celular , Células Cultivadas , Ratones , Neuronas/citología , Neuronas/metabolismo , Oligodendroglía/citologíaRESUMEN
Cells derived from pluripotent sources in vitro must resemble those found in vivo as closely as possible at both transcriptional and functional levels in order to be a useful tool for studying diseases and developing therapeutics. Recently, differentiation of human pluripotent stem cells (hPSCs) into brain microvascular endothelial cells (ECs) with blood-brain barrier (BBB)-like properties has been reported. These cells have since been used as a robust in vitro BBB model for drug delivery and mechanistic understanding of neurological diseases. However, the precise cellular identity of these induced brain microvascular endothelial cells (iBMECs) has not been well described. Employing a comprehensive transcriptomic metaanalysis of previously published hPSC-derived cells validated by physiological assays, we demonstrate that iBMECs lack functional attributes of ECs since they are deficient in vascular lineage genes while expressing clusters of genes related to the neuroectodermal epithelial lineage (Epi-iBMEC). Overexpression of key endothelial ETS transcription factors (ETV2, ERG, and FLI1) reprograms Epi-iBMECs into authentic endothelial cells that are congruent with bona fide endothelium at both transcriptomic as well as some functional levels. This approach could eventually be used to develop a robust human BBB model in vitro that resembles the human brain EC in vivo for functional studies and drug discovery.
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Endotelio Vascular/citología , Células Madre Pluripotentes/citología , Factores de Transcripción/genética , Animales , Barrera Hematoencefálica , Encéfalo/irrigación sanguínea , Encéfalo/citología , Diferenciación Celular , Línea Celular , Reprogramación Celular/fisiología , Endotelio Vascular/fisiología , Expresión Génica , Humanos , Ratones Endogámicos , Células Madre Pluripotentes/fisiología , Proteína Proto-Oncogénica c-fli-1/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Análisis de la Célula Individual , Factores de Transcripción/metabolismo , Regulador Transcripcional ERG/genética , Regulador Transcripcional ERG/metabolismoRESUMEN
Neurovascular pathologies of the central nervous system (CNS), which are associated with barrier dysfunction, are leading causes of death and disability. The roles that neuronal and glial progenitors and mature cells play in CNS angiogenesis and neurovascular barrier maturation have been elucidated in recent years. Yet how neuronal activity influences these processes remains largely unexplored. Here, we discuss our current understanding of how neuronal and glial development affects CNS angiogenesis and barriergenesis, and outline future directions to elucidate how neuronal activity might influence these processes. An understanding of these mechanisms is crucial for developing new interventions to treat neurovascular pathologies.
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Barrera Hematoencefálica/citología , Barrera Hematoencefálica/metabolismo , Sistema Nervioso Central/citología , Sistema Nervioso Central/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Células Ependimogliales/citología , Células Ependimogliales/metabolismo , Femenino , Humanos , Masculino , Modelos Biológicos , Neovascularización Fisiológica/fisiologíaRESUMEN
Central nervous system (CNS) blood vessels contain a functional blood-brain barrier (BBB) that is necessary for neuronal survival and activity. Although Wnt/ß-catenin signaling is essential for BBB development, its downstream targets within the neurovasculature remain poorly understood. To identify targets of Wnt/ß-catenin signaling underlying BBB maturation, we performed a microarray analysis that identified Fgfbp1 as a novel Wnt/ß-catenin-regulated gene in mouse brain endothelial cells (mBECs). Fgfbp1 is expressed in the CNS endothelium and secreted into the vascular basement membrane during BBB formation. Endothelial genetic ablation of Fgfbp1 results in transient hypervascularization but delays BBB maturation in specific CNS regions, as evidenced by both upregulation of Plvap and increased tracer leakage across the neurovasculature due to reduced Wnt/ß-catenin activity. In addition, collagen IV deposition in the vascular basement membrane is reduced in mutant mice, leading to defective endothelial cell-pericyte interactions. Fgfbp1 is required cell-autonomously in mBECs to concentrate Wnt ligands near cell junctions and promote maturation of their barrier properties in vitro Thus, Fgfbp1 is a crucial extracellular matrix protein during BBB maturation that regulates cell-cell interactions and Wnt/ß-catenin activity.
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Barrera Hematoencefálica/embriología , Colágeno Tipo IV/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , Animales , Colágeno Tipo IV/genética , Células Endoteliales/citología , Células Endoteliales/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Ratones Transgénicos , Pericitos/citología , Pericitos/metabolismo , beta Catenina/genéticaRESUMEN
Postinfectious neuroinflammation has been implicated in multiple models of acute-onset obsessive-compulsive disorder including Sydenham chorea (SC), pediatric acute-onset neuropsychiatric syndrome (PANS), and pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection (PANDAS). These conditions are associated with a range of autoantibodies which are thought to be triggered by infections, most notably group A streptococci (GAS). Based on animal models using huma sera, these autoantibodies are thought to cross-react with neural antigens in the basal ganglia and modulate neuronal activity and behavior. As is true for many childhood neuroinflammatory diseases and rheumatological diseases, SC, PANS, and PANDAS lack clinically available, rigorous diagnostic biomarkers and randomized clinical trials. In this review article, we outline the accumulating evidence supporting the role neuroinflammation plays in these disorders. We describe work with animal models including patient-derived anti-neuronal autoantibodies, and we outline imaging studies that show alterations in the basal ganglia. In addition, we present research on metabolites, which are helpful in deciphering functional phenotypes, and on the implication of sleep in these disorders. Finally, we encourage future researchers to collaborate across medical specialties (e.g., pediatrics, psychiatry, rheumatology, immunology, and infectious disease) in order to further research on clinical syndromes presenting with neuropsychiatric manifestations.
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Corea , Trastorno Obsesivo Compulsivo , Infecciones Estreptocócicas , Animales , Niño , Humanos , Autoinmunidad , Corea/diagnóstico , Corea/complicaciones , Enfermedades Neuroinflamatorias , Infecciones Estreptocócicas/complicaciones , Infecciones Estreptocócicas/diagnóstico , Infecciones Estreptocócicas/tratamiento farmacológico , Trastorno Obsesivo Compulsivo/diagnóstico , Trastorno Obsesivo Compulsivo/psicología , Autoanticuerpos/uso terapéutico , InflamaciónRESUMEN
Antibodies against neuronal receptors and synaptic proteins are associated with a group of ill-defined central nervous system (CNS) autoimmune diseases termed autoimmune encephalitides (AE), which are characterized by abrupt onset of seizures and/or movement and psychiatric symptoms. Basal ganglia encephalitis (BGE), representing a subset of AE syndromes, is triggered in children by repeated group A Streptococcus (GAS) infections that lead to neuropsychiatric symptoms. We have previously shown that multiple GAS infections of mice induce migration of Th17 lymphocytes from the nose into the brain, causing blood-brain barrier (BBB) breakdown, extravasation of autoantibodies into the CNS, and loss of excitatory synapses within the olfactory bulb (OB). Whether these pathologies induce functional olfactory deficits, and the mechanistic role of Th17 lymphocytes, is unknown. Here, we demonstrate that, whereas loss of excitatory synapses in the OB is transient after multiple GAS infections, functional deficits in odor processing persist. Moreover, mice lacking Th17 lymphocytes have reduced BBB leakage, microglial activation, and antibody infiltration into the CNS, and have their olfactory function partially restored. Th17 lymphocytes are therefore critical for selective CNS entry of autoantibodies, microglial activation, and neural circuit impairment during postinfectious BGE.
Asunto(s)
Encéfalo/patología , Modelos Animales de Enfermedad , Encefalitis/etiología , Encefalomielitis Autoinmune Experimental/etiología , Enfermedad de Hashimoto/etiología , Trastornos del Olfato/etiología , Infecciones Estreptocócicas/complicaciones , Células Th17/inmunología , Animales , Autoanticuerpos/inmunología , Ganglios Basales/inmunología , Ganglios Basales/patología , Barrera Hematoencefálica , Encéfalo/inmunología , Encefalitis/metabolismo , Encefalitis/patología , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Femenino , Enfermedad de Hashimoto/metabolismo , Enfermedad de Hashimoto/patología , Ratones , Microglía/inmunología , Microglía/patología , Neuronas/inmunología , Neuronas/patología , Trastornos del Olfato/metabolismo , Trastornos del Olfato/patología , Percepción Olfatoria , Streptococcus pyogenes/fisiología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Células Th17/patologíaRESUMEN
Brain metastases are prevalent in various types of cancer and are often terminal, given the low efficacy of available therapies. Therefore, preventing them is of utmost clinical relevance, and prophylactic treatments are perhaps the most efficient strategy. Here, we show that systemic prophylactic administration of a toll-like receptor (TLR) 9 agonist, CpG-C, is effective against brain metastases. Acute and chronic systemic administration of CpG-C reduced tumor cell seeding and growth in the brain in three tumor models in mice, including metastasis of human and mouse lung cancer, and spontaneous melanoma-derived brain metastasis. Studying mechanisms underlying the therapeutic effects of CpG-C, we found that in the brain, unlike in the periphery, natural killer (NK) cells and monocytes are not involved in controlling metastasis. Next, we demonstrated that the systemically administered CpG-C is taken up by endothelial cells, astrocytes, and microglia, without affecting blood-brain barrier (BBB) integrity and tumor brain extravasation. In vitro assays pointed to microglia, but not astrocytes, as mediators of CpG- C effects through increased tumor killing and phagocytosis, mediated by direct microglia-tumor contact. In vivo, CpG-C-activated microglia displayed elevated mRNA expression levels of apoptosis-inducing and phagocytosis-related genes. Intravital imaging showed that CpG-C-activated microglia cells contact, kill, and phagocytize tumor cells in the early stages of tumor brain invasion more than nonactivated microglia. Blocking in vivo activation of microglia with minocycline, and depletion of microglia with a colony-stimulating factor 1 inhibitor, indicated that microglia mediate the antitumor effects of CpG-C. Overall, the results suggest prophylactic CpG-C treatment as a new intervention against brain metastasis, through an essential activation of microglia.
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Neoplasias Encefálicas/complicaciones , Neoplasias Encefálicas/metabolismo , Microglía/metabolismo , Microglía/patología , Oligodesoxirribonucleótidos/uso terapéutico , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Factores Estimulantes de Colonias/antagonistas & inhibidores , Factores Estimulantes de Colonias/metabolismo , Femenino , Humanos , Neoplasias Pulmonares/complicaciones , Neoplasias Pulmonares/metabolismo , Masculino , Melanoma/complicaciones , Melanoma/metabolismo , Ratones , Minociclina/metabolismo , Fagocitosis/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Many patients with SARS-CoV-2 infection develop neurological signs and symptoms; although, to date, little evidence exists that primary infection of the brain is a significant contributing factor. We present the clinical, neuropathological and molecular findings of 41 consecutive patients with SARS-CoV-2 infections who died and underwent autopsy in our medical centre. The mean age was 74 years (38-97 years), 27 patients (66%) were male and 34 (83%) were of Hispanic/Latinx ethnicity. Twenty-four patients (59%) were admitted to the intensive care unit. Hospital-associated complications were common, including eight patients (20%) with deep vein thrombosis/pulmonary embolism, seven (17%) with acute kidney injury requiring dialysis and 10 (24%) with positive blood cultures during admission. Eight (20%) patients died within 24 h of hospital admission, while 11 (27%) died more than 4 weeks after hospital admission. Neuropathological examination of 20-30 areas from each brain revealed hypoxic/ischaemic changes in all brains, both global and focal; large and small infarcts, many of which appeared haemorrhagic; and microglial activation with microglial nodules accompanied by neuronophagia, most prominently in the brainstem. We observed sparse T lymphocyte accumulation in either perivascular regions or in the brain parenchyma. Many brains contained atherosclerosis of large arteries and arteriolosclerosis, although none showed evidence of vasculitis. Eighteen patients (44%) exhibited pathologies of neurodegenerative diseases, which was not unexpected given the age range of our patients. We examined multiple fresh frozen and fixed tissues from 28 brains for the presence of viral RNA and protein, using quantitative reverse-transcriptase PCR, RNAscope® and immunocytochemistry with primers, probes and antibodies directed against the spike and nucleocapsid regions. The PCR analysis revealed low to very low, but detectable, viral RNA levels in the majority of brains, although they were far lower than those in the nasal epithelia. RNAscope® and immunocytochemistry failed to detect viral RNA or protein in brains. Our findings indicate that the levels of detectable virus in coronavirus disease 2019 brains are very low and do not correlate with the histopathological alterations. These findings suggest that microglial activation, microglial nodules and neuronophagia, observed in the majority of brains, do not result from direct viral infection of brain parenchyma, but more likely from systemic inflammation, perhaps with synergistic contribution from hypoxia/ischaemia. Further studies are needed to define whether these pathologies, if present in patients who survive coronavirus disease 2019, might contribute to chronic neurological problems.
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Infarto Encefálico/patología , Encéfalo/patología , COVID-19/patología , Hipoxia-Isquemia Encefálica/patología , Hemorragias Intracraneales/patología , Lesión Renal Aguda/complicaciones , Lesión Renal Aguda/fisiopatología , Lesión Renal Aguda/terapia , Adulto , Anciano , Anciano de 80 o más Años , Bacteriemia/complicaciones , Encéfalo/metabolismo , Infarto Encefálico/complicaciones , COVID-19/complicaciones , COVID-19/fisiopatología , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Femenino , Humanos , Hipoxia-Isquemia Encefálica/complicaciones , Inflamación , Unidades de Cuidados Intensivos , Hemorragias Intracraneales/complicaciones , Masculino , Microglía/patología , Persona de Mediana Edad , Neuronas/patología , Fagocitosis , Fosfoproteínas/metabolismo , Embolia Pulmonar/complicaciones , Embolia Pulmonar/fisiopatología , ARN Viral/metabolismo , Diálisis Renal , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/metabolismo , Tasa de Supervivencia , Linfocitos T/patología , Trombosis de la Vena/complicaciones , Trombosis de la Vena/fisiopatologíaRESUMEN
Disruption of the blood-brain barrier (BBB) is a defining and early feature of multiple sclerosis (MS) that directly damages the central nervous system (CNS), promotes immune cell infiltration, and influences clinical outcomes. There is an urgent need for new therapies to protect and restore BBB function, either by strengthening endothelial tight junctions or suppressing endothelial vesicular transcytosis. Although wingless integrated MMTV (Wnt)/ß-catenin signaling plays an essential role in BBB formation and maintenance in healthy CNS, its role in BBB repair in neurologic diseases such as MS remains unclear. Using a Wnt/ß-catenin reporter mouse and several downstream targets, we demonstrate that the Wnt/ß-catenin pathway is up-regulated in CNS endothelial cells in both human MS and the mouse model experimental autoimmune encephalomyelitis (EAE). Increased Wnt/ß-catenin activity in CNS blood vessels during EAE progression correlates with up-regulation of neuronal Wnt3 expression, as well as breakdown of endothelial cell junctions. Genetic inhibition of the Wnt/ß-catenin pathway in CNS endothelium before disease onset exacerbates the clinical presentation of EAE, CD4+ T-cell infiltration into the CNS, and demyelination by increasing expression of vascular cell adhesion molecule-1 and the transcytosis protein Caveolin-1 and promoting endothelial transcytosis. However, Wnt signaling attenuation does not affect the progressive degradation of tight junction proteins or paracellular BBB leakage. These results suggest that reactivation of Wnt/ß-catenin signaling in CNS vessels during EAE/MS partially restores functional BBB integrity and limits immune cell infiltration into the CNS.
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Linfocitos T CD4-Positivos/metabolismo , Células Endoteliales/metabolismo , Esclerosis Múltiple/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Caveolina 1/metabolismo , Sistema Nervioso Central/metabolismo , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Humanos , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Transgénicos , Esclerosis Múltiple/genética , Transcitosis , beta Catenina/genéticaRESUMEN
The adult quiescent blood-brain barrier (BBB), a structure organised by endothelial cells through interactions with pericytes, astrocytes, neurons and microglia in the neurovascular unit, is highly regulated but fragile at the same time. In the past decade, there has been considerable progress in understanding not only the molecular pathways involved in BBB development, but also BBB breakdown in neurological diseases. Specifically, the Wnt/ß-catenin, retinoic acid and sonic hedgehog pathways moved into the focus of BBB research. Moreover, angiopoietin/Tie2 signalling that is linked to angiogenic processes has gained attention in the BBB field. Blood vessels play an essential role in initiation and progression of many diseases, including inflammation outside the central nervous system (CNS). Therefore, the potential influence of CNS blood vessels in neurological diseases associated with BBB alterations or neuroinflammation has become a major focus of current research to understand their contribution to pathogenesis. Moreover, the BBB remains a major obstacle to pharmaceutical intervention in the CNS. The complications may either be expressed by inadequate therapeutic delivery like in brain tumours, or by poor delivery of the drug across the BBB and ineffective bioavailability. In this review, we initially describe the cellular and molecular components that contribute to the steady state of the healthy BBB. We then discuss BBB alterations in ischaemic stroke, primary and metastatic brain tumour, chronic inflammation and Alzheimer's disease. Throughout the review, we highlight common mechanisms of BBB abnormalities among these diseases, in particular the contribution of neuroinflammation to BBB dysfunction and disease progression, and emphasise unique aspects of BBB alteration in certain diseases such as brain tumours. Moreover, this review highlights novel strategies to monitor BBB function by non-invasive imaging techniques focussing on ischaemic stroke, as well as novel ways to modulate BBB permeability and function to promote treatment of brain tumours, inflammation and Alzheimer's disease. In conclusion, a deep understanding of signals that maintain the healthy BBB and promote fluctuations in BBB permeability in disease states will be key to elucidate disease mechanisms and to identify potential targets for diagnostics and therapeutic modulation of the BBB.
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Barrera Hematoencefálica/citología , Barrera Hematoencefálica/patología , Animales , Barrera Hematoencefálica/diagnóstico por imagen , Barrera Hematoencefálica/metabolismo , HumanosRESUMEN
BACKGROUND: Pregnancy is a sex-specific risk factor for causing hemorrhagic stroke (HS) in young adults. Unique physiological characteristics during pregnancy may alter the relative risk for HS in pregnant/postpartum (PP) women compared to HS in other young women. We compared patient characteristics and HS subtypes between young non-pregnant and PP women. METHODS: We reviewed the medical records of all women 18-45 years old admitted to our center with HS from October 15, 2008 through March 31, 2015, and compared patient characteristics and stroke mechanisms using logistic regression. RESULTS: Of the 130 young women with HS during the study period, 111 were non-PP women, and 19 PP women. PP women had lower proportions of vascular risk factors such as hypertension, prior stroke, and smoking, and a higher proportion of migraine (36.8 vs. 14.4%, p = 0.01). After adjusting for hypertension, smoking, migraine, prior stroke and prior myocardial infarction, PP women had lower odds of having an underlying vascular lesion (OR 0.14, 95% CI 0.04-0.44, p = 0.0009) and a higher proportion of the reversible cerebral vasoconstriction syndrome (RCVS) as cause of their HS. CONCLUSIONS: Women with pregnancy-associated HS had fewer cerebrovascular risk factors, lower odds of having -underlying vascular lesions, and higher proportion of -migraine and RCVS compared with similar-aged non--pregnant women. Pregnancy-associated HS appears to represent a unique pathophysiological process, requiring targeted study.
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Hemorragias Intracraneales/epidemiología , Complicaciones Cardiovasculares del Embarazo/epidemiología , Accidente Cerebrovascular/epidemiología , Adulto , Factores de Edad , Comorbilidad , Femenino , Humanos , Hemorragias Intracraneales/diagnóstico , Persona de Mediana Edad , Ciudad de Nueva York/epidemiología , Embarazo , Complicaciones Cardiovasculares del Embarazo/diagnóstico , Medición de Riesgo , Factores de Riesgo , Factores Sexuales , Accidente Cerebrovascular/diagnóstico , Adulto JovenRESUMEN
BACKGROUND: The mechanism of leukocyte transendothelial migration (TEM) across the highly restrictive blood-brain barrier (BBB) remains enigmatic, with paracellular TEM thought to require leukocytes to somehow navigate the obstructive endothelial tight junctions (TJs). Transient interactions between TJ proteins on the respective leukocyte and endothelial surfaces have been proposed as one mechanism for TEM. Given the expanding role of extracellular vesicles (EVs) in intercellular communication, we investigated whether EVs derived from brain microvascular endothelial cells (BMEC) of the BBB may play a role in transferring a major TJ protein, claudin-5 (CLN-5), to leukocytes as a possible basis for such a mechanism during neuroinflammation. METHODS: High-resolution 3D confocal imaging was used to highlight CLN-5 immunoreactivity in the central nervous system (CNS) and on leukocytes of mice with the neuroinflammatory condition experimental autoimmune encephalomyelitis (EAE). Both Western blotting of circulating leukocytes from wild-type mice and fluorescence imaging of leukocyte-associated eGFP-CLN-5 in the blood and CNS of endothelial-targeted, Tie-2-eGFP-CLN-5 transgenic mice were used to confirm the presence of CLN-5 protein on these cells. EVs were isolated from TNF-α-stimulated BMEC cultures and blood plasma of Tie-2-eGFP-CLN-5 mice with EAE and evaluated for CLN-5 protein by Western blotting and fluorescence-activated cell sorting (FACS), respectively. Confocal imaging and FACS were used to detect binding of endothelial-derived EVs from these two sources to leukocytes in vitro. Serial electron microscopy (serial EM) and 3D contour-based surface reconstruction were employed to view EV-like structures at the leukocyte:BBB interface in situ in inflamed CNS microvessels. RESULTS: A subpopulation of leukocytes immunoreactive for CLN-5 on their surface was seen to infiltrate the CNS of mice with EAE and reside in close apposition to inflamed vessels. Confocal imaging of immunostained samples and Western blotting established the presence of CLN-5+ leukocytes in blood as well, implying these cells are present prior to TEM. Moreover, imaging of inflamed CNS vessels and the associated perivascular cell infiltrates from Tie-2-eGFP-CLN-5 mice with EAE revealed leukocytes bearing the eGFP label, further supporting the hypothesis CLN-5 is transferred from endothelial cells to circulating leukocytes in vivo. Western blotting of BMEC-derived EVs, corresponding in size to both exosomes and microvesicles, and FACS analysis of plasma-derived EVs from Tie-2-eGFP-CLN-5 mice with EAE validated expression of CLN-5 by EVs of endothelial origin. Confocal imaging and FACS further revealed both PKH-67-labeled EVs from cultured BMECs and eGFP-CLN-5+ EVs from plasma of Tie-2-eGFP-CLN-5 mice with EAE can bind to leukocytes. Lastly, serial EM and 3D contour-based surface reconstruction revealed a close association of EV-like structures between the marginating leukocytes and BMECs in situ during EAE. CONCLUSIONS: During neuroinflammation, CLN-5+ leukocytes appear in the CNS, and both CLN-5+ leukocytes and CLN-5+ EVs are detected in the blood. As endothelial cells transfer CLN-5+ to leukocytes in vivo, and EVs released from BMEC bind to leukocytes in vitro, EVs may serve as the vehicles to transfer CLN-5 protein at sites of leukocyte:endothelial contact along the BBB. This action may be a prelude to facilitate TEM through the formation of temporary TJ protein bridges between these two cell types.
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Sistema Nervioso Central/patología , Encefalomielitis Autoinmune Experimental/patología , Células Endoteliales/patología , Endotelio Vascular/metabolismo , Vesículas Extracelulares/metabolismo , Glicoproteínas de Membrana/metabolismo , Animales , Células Cultivadas , Sistema Nervioso Central/diagnóstico por imagen , Citocinas/metabolismo , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/sangre , Encefalomielitis Autoinmune Experimental/inmunología , Células Endoteliales/ultraestructura , Endotelio Vascular/ultraestructura , Vesículas Extracelulares/ultraestructura , Femenino , Leucocitos/metabolismo , Proteínas de Membrana de los Lisosomas , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Glicoproteína Mielina-Oligodendrócito/inmunología , Glicoproteína Mielina-Oligodendrócito/toxicidad , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/toxicidadRESUMEN
Hereditary hypotrichosis simplex is a rare autosomal dominant form of hair loss characterized by hair follicle miniaturization. Using genetic linkage analysis, we mapped a new locus for the disease to chromosome 18p11.22, and identified a mutation (Leu9Arg) in the adenomatosis polyposis down-regulated 1 (APCDD1) gene in three families. We show that APCDD1 is a membrane-bound glycoprotein that is abundantly expressed in human hair follicles, and can interact in vitro with WNT3A and LRP5-two essential components of Wnt signalling. Functional studies show that APCDD1 inhibits Wnt signalling in a cell-autonomous manner and functions upstream of beta-catenin. Moreover, APCDD1 represses activation of Wnt reporters and target genes, and inhibits the biological effects of Wnt signalling during both the generation of neurons from progenitors in the developing chick nervous system, and axis specification in Xenopus laevis embryos. The mutation Leu9Arg is located in the signal peptide of APCDD1, and perturbs its translational processing from the endoplasmic reticulum to the plasma membrane. APCDD1(L9R) probably functions in a dominant-negative manner to inhibit the stability and membrane localization of the wild-type protein. These findings describe a novel inhibitor of the Wnt signalling pathway with an essential role in human hair growth. As APCDD1 is expressed in a broad repertoire of cell types, our findings indicate that APCDD1 may regulate a diversity of biological processes controlled by Wnt signalling.
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Hipotricosis/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mutación Puntual/genética , Proteínas Wnt/antagonistas & inhibidores , Animales , Diferenciación Celular , Línea Celular , Proliferación Celular , Embrión de Pollo , Mapeo Cromosómico , Cromosomas Humanos Par 18/genética , Genes Dominantes/genética , Genes Reporteros/genética , Cabello/crecimiento & desarrollo , Cabello/metabolismo , Folículo Piloso/crecimiento & desarrollo , Folículo Piloso/metabolismo , Folículo Piloso/patología , Humanos , Hipotricosis/metabolismo , Hipotricosis/patología , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/deficiencia , Proteínas de la Membrana , Ratones , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Neuronas/citología , Neuronas/metabolismo , Cuero Cabelludo , Transducción de Señal , Piel , Médula Espinal/citología , Células Madre/citología , Células Madre/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteínas de Xenopus/deficiencia , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Xenopus laevis/genética , Xenopus laevis/metabolismo , beta Catenina/metabolismoRESUMEN
Interactions among neuronal, glial and vascular components are crucial for retinal angiogenesis and blood-retinal barrier (BRB) maturation. Although synaptic dysfunction precedes vascular abnormalities in many retinal pathologies, how neuronal activity, specifically glutamatergic activity, regulates retinal angiogenesis and BRB maturation remains unclear. Using in vivo genetic studies in mice, single-cell RNA-sequencing and functional validation, we show that deep plexus angiogenesis and paracellular BRB maturation are delayed in Vglut1 -/- retinas where neurons fail to release glutamate. In contrast, deep plexus angiogenesis and paracellular BRB maturation are accelerated in Gnat1 -/- retinas where constitutively depolarized rods release excessive glutamate. Norrin expression and endothelial Norrin/ß-catenin signaling are downregulated in Vglut1 -/- retinas, and upregulated in Gnat1 -/- retinas. Pharmacological activation of endothelial Norrin/ß-catenin signaling in Vglut1 -/- retinas rescued defects in deep plexus angiogenesis and paracellular BRB maturation. Our findings demonstrate that glutamatergic neuronal activity regulates retinal angiogenesis and BRB maturation by modulating endothelial Norrin/ß-catenin signaling.
RESUMEN
Interactions among neuronal, glial, and vascular components are crucial for retinal angiogenesis and blood-retinal barrier (BRB) maturation. Although synaptic dysfunction precedes vascular abnormalities in many retinal pathologies, how neuronal activity, specifically glutamatergic activity, regulates retinal angiogenesis and BRB maturation remains unclear. Using in vivo genetic studies in mice, single-cell RNA sequencing (scRNA-seq), and functional validation, we show that deep plexus angiogenesis and paracellular BRB maturation are delayed in Vglut1-/- retinas where neurons fail to release glutamate. By contrast, deep plexus angiogenesis and paracellular BRB maturation are accelerated in Gnat1-/- retinas, where constitutively depolarized rods release excessive glutamate. Norrin expression and endothelial Norrin/ß-catenin signaling are downregulated in Vglut1-/- retinas and upregulated in Gnat1-/- retinas. Pharmacological activation of endothelial Norrin/ß-catenin signaling in Vglut1-/- retinas rescues defects in deep plexus angiogenesis and paracellular BRB maturation. Our findings demonstrate that glutamatergic neuronal activity regulates retinal angiogenesis and BRB maturation by modulating endothelial Norrin/ß-catenin signaling.
Asunto(s)
Barrera Hematorretinal , Proteínas del Ojo , Ácido Glutámico , Proteínas del Tejido Nervioso , Transducción de Señal , beta Catenina , Animales , Barrera Hematorretinal/metabolismo , beta Catenina/metabolismo , Ratones , Ácido Glutámico/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Ojo/metabolismo , Proteínas del Ojo/genética , Transducción de Señal/fisiología , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Neuronas/metabolismo , Ratones Noqueados , Neovascularización Retiniana/metabolismo , Retina/metabolismo , Ratones Endogámicos C57BL , AngiogénesisRESUMEN
The blood-brain barrier (BBB), formed by specialized brain microvascular endothelial cells (BMECs), regulates brain function in health and disease. In vitro modeling of the human BBB is limited by the lack of robust protocols to generate BMECs from human iPSCs (hiPSCs). Here, we report generation of reprogrammed BMECs (rBMECs) through combining hiPSC differentiation into BBB-primed endothelial cells (bpECs) and reprogramming with two BBB transcription factors, FOXF2 and ZIC3. rBMECs express a subset of the BBB gene repertoire including tight junctions and transporters, exhibit higher paracellular barrier properties, lower caveolar-mediated transcytosis, and equivalent p-glycoprotein activity compared to primary HBMECs, and can be activated by oligomeric Aß42. We then generated an hiPSC-derived 3D neurovascular system that incorporates rBMECs, pericytes, and astrocytes using the MIMETAS platform. This novel 3D system closely resembles the in vivo BBB at structural and functional levels and can be used to study pathogenic mechanisms of neurological diseases.
RESUMEN
Stroke is a devastating cause of global morbidity and mortality. Ischemic brain injury triggers a profound local and systemic immune response that participates in stroke pathophysiology. In turn, this immune response has emerged as a potential therapeutic target. In order to maximize its therapeutic potential, it is critical to understand how the immune response to ischemic brain injury is affected by age - the strongest non-modifiable risk factor for stroke. The development of multi-omics and single-cell technologies has provided a more comprehensive characterization of transcriptional and cellular changes that occur during aging. In this review, we summarize recent advances in our understanding of how age-related immune alterations shape differential stroke outcomes in older versus younger organisms, highlighting studies in both experimental mouse models and patient cohorts. Wherever possible, we emphasize outstanding questions that present important avenues for future investigation with therapeutic value for the aging population.
Asunto(s)
Lesiones Encefálicas , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Ratones , Animales , Accidente Cerebrovascular/terapia , Envejecimiento , InmunidadRESUMEN
The stability of tight junctions (TJs) between endothelial cells (ECs) is essential to maintain blood-brain barrier (BBB) function in the healthy brain. Following ischemic stroke, TJ strand dismantlement due to protein degradation leads to BBB dysfunction, yet the mechanisms driving this process are poorly understood. Here, we show that endothelial-specific ablation of Rab7a, a small GTPase that regulates endolysosomal protein degradation, reduces stroke-induced TJ strand disassembly resulting in decreased paracellular BBB permeability and improved neuronal outcomes. Two pro-inflammatory cytokines, TNFα and IL1ß, but not glucose and oxygen deprivation, induce Rab7a activation via Ccz1 in brain ECs in vitro, leading to increased TJ protein degradation and impaired paracellular barrier function. Silencing Rab7a in brain ECs in vitro reduces cytokine-driven endothelial barrier dysfunction by suppressing degradation of a key BBB TJ protein, Claudin-5. Thus, Rab7a activation by inflammatory cytokines promotes degradation of select TJ proteins leading to BBB dysfunction after ischemic stroke.