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BACKGROUND: Until now, the analysis of microvascular networks in the reperfused ischemic brain has been limited due to tissue transparency challenges. METHODS: Using light sheet microscopy, we assessed microvascular network remodeling in the striatum from 3 hours to 56 days post-ischemia in 2 mouse models of transient middle cerebral artery occlusion lasting 20 or 40 minutes, resulting in mild ischemic brain injury or brain infarction, respectively. We also examined the effect of a clinically applicable S1P (sphingosine-1-phosphate) analog, FTY720 (fingolimod), on microvascular network remodeling. RESULTS: Over 56 days, we observed progressive microvascular degeneration in the reperfused striatum, that is, the lesion core, which was followed by robust angiogenesis after mild ischemic injury induced by 20-minute middle cerebral artery occlusion. However, more severe ischemic injury elicited by 40-minute middle cerebral artery occlusion resulted in incomplete microvascular remodeling. In both cases, microvascular networks did not return to their preischemic state but displayed a chronically altered pattern characterized by higher branching point density, shorter branches, higher unconnected branch density, and lower tortuosity, indicating enhanced network connectivity. FTY720 effectively increased microvascular length density, branching point density, and volume density in both models, indicating an angiogenic effect of this drug. CONCLUSIONS: Utilizing light sheet microscopy together with automated image analysis, we characterized microvascular remodeling in the ischemic lesion core in unprecedented detail. This technology will significantly advance our understanding of microvascular restorative processes and pave the way for novel treatment developments in the stroke field.
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Isquemia Encefálica , Clorhidrato de Fingolimod , Ratones , Animales , Clorhidrato de Fingolimod/farmacología , Clorhidrato de Fingolimod/uso terapéutico , Infarto de la Arteria Cerebral Media/patología , Microscopía , Encéfalo/irrigación sanguínea , Microvasos/patología , Modelos Animales de EnfermedadRESUMEN
BACKGROUND: The intravenous delivery of adult neural precursor cells (NPC) has shown promising results in enabling cerebroprotection, brain tissue remodeling, and neurological recovery in young, healthy stroke mice. However, the translation of cell-based therapies to clinical settings has encountered challenges. It remained unclear if adult NPCs could induce brain tissue remodeling and recovery in mice with hyperlipidemia, a prevalent vascular risk factor in stroke patients. METHODS: Male mice on a normal (regular) diet or on cholesterol-rich Western diet were exposed to 30 min intraluminal middle cerebral artery occlusion (MCAO). Vehicle or 106 NPCs were intravenously administered immediately after reperfusion, at 3 day and 7 day post-MCAO. Neurological recovery was evaluated using the Clark score, Rotarod and tight rope tests over up to 56 days. Histochemistry and light sheet microscopy were used to examine ischemic injury and brain tissue remodeling. Immunological responses in peripheral blood and brain were analyzed through flow cytometry. RESULTS: NPC administration reduced infarct volume, blood-brain barrier permeability and the brain infiltration of neutrophils, monocytes, T cells and NK cells in the acute stroke phase in both normolipidemic and hyperlipidemic mice, but increased brain hemorrhage formation and neutrophil, monocyte and CD4+ and CD8+ T cell counts and activation in the blood of hyperlipidemic mice. While neurological deficits in hyperlipidemic mice were reduced by NPCs at 3 day post-MCAO, NPCs did not improve neurological deficits at later timepoints. Besides, NPCs did not influence microglia/macrophage abundance and activation (assessed by morphology analysis), astroglial scar formation, microvascular length or branching point density (evaluated using light sheet microscopy), long-term neuronal survival or brain atrophy in hyperlipidemic mice. CONCLUSIONS: Intravenously administered NPCs did not have persistent effects on post-ischemic neurological recovery and brain remodeling in hyperlipidemic mice. These findings highlight the necessity of rigorous investigations in vascular risk factor models to fully assess the long-term restorative effects of cell-based therapies. Without comprehensive studies in such models, the clinical potential of cell-based therapies cannot be definitely determined.
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Células-Madre Neurales , Accidente Cerebrovascular , Masculino , Animales , Ratones , Neuronas , Hemorragias Intracraneales , EncéfaloRESUMEN
Antidepressants have been reported to enhance stroke recovery independent of the presence of depressive symptoms. They have recently been proposed to exert their mood-stabilizing actions by inhibition of acid sphingomyelinase (ASM), which catalyzes the hydrolysis of sphingomyelin to ceramide. Their restorative action post-ischemia/reperfusion (I/R) still had to be defined. Mice subjected to middle cerebral artery occlusion or cerebral microvascular endothelial cells exposed to oxygen-glucose deprivation were treated with vehicle or with the chemically and pharmacologically distinct antidepressants amitriptyline, fluoxetine or desipramine. Brain ASM activity significantly increased post-I/R, in line with elevated ceramide levels in microvessels. ASM inhibition by amitriptyline reduced ceramide levels, and increased microvascular length and branching point density in wildtype, but not sphingomyelinase phosphodiesterase-1 ([Smpd1]-/-) (i.e., ASM-deficient) mice, as assessed by 3D light sheet microscopy. In cell culture, amitriptyline, fluoxetine, and desipramine increased endothelial tube formation, migration, VEGFR2 abundance and VEGF release. This effect was abolished by Smpd1 knockdown. Mechanistically, the promotion of angiogenesis by ASM inhibitors was mediated by small extracellular vesicles (sEVs) released from endothelial cells, which exhibited enhanced uptake in target cells. Proteomic analysis of sEVs revealed that ASM deactivation differentially regulated proteins implicated in protein export, focal adhesion, and extracellular matrix interaction. In vivo, the increased angiogenesis was accompanied by a profound brain remodeling response with increased blood-brain barrier integrity, reduced leukocyte infiltrates and increased neuronal survival. Antidepressive drugs potently boost angiogenesis in an ASM-dependent way. The release of sEVs by ASM inhibitors disclosed an elegant target, via which brain remodeling post-I/R can be amplified.
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Amitriptilina , Vesículas Extracelulares , Amitriptilina/metabolismo , Amitriptilina/farmacología , Animales , Antidepresivos/metabolismo , Antidepresivos/farmacología , Encéfalo/metabolismo , Ceramidas/metabolismo , Ceramidas/farmacología , Desipramina/metabolismo , Desipramina/farmacología , Células Endoteliales/metabolismo , Vesículas Extracelulares/metabolismo , Fluoxetina/metabolismo , Fluoxetina/farmacología , Isquemia/metabolismo , Ratones , ProteómicaRESUMEN
Obtained from the right cell-type, mesenchymal stromal cell (MSC)-derived small extracellular vesicles (sEVs) promote stroke recovery. Within this process, microvascular remodeling plays a central role. Herein, we evaluated the effects of MSC-sEVs on the proliferation, migration, and tube formation of human cerebral microvascular endothelial cells (hCMEC/D3) in vitro and on post-ischemic angiogenesis, brain remodeling and neurological recovery after middle cerebral artery occlusion (MCAO) in mice. In vitro, sEVs obtained from hypoxic (1% O2), but not 'normoxic' (21% O2) MSCs dose-dependently promoted endothelial proliferation, migration, and tube formation and increased post-ischemic endothelial survival. sEVs from hypoxic MSCs regulated a distinct set of miRNAs in hCMEC/D3 cells previously linked to angiogenesis, three being upregulated (miR-126-3p, miR-140-5p, let-7c-5p) and three downregulated (miR-186-5p, miR-370-3p, miR-409-3p). LC/MS-MS revealed 52 proteins differentially abundant in sEVs from hypoxic and 'normoxic' MSCs. 19 proteins were enriched (among them proteins involved in extracellular matrix-receptor interaction, focal adhesion, leukocyte transendothelial migration, protein digestion, and absorption), and 33 proteins reduced (among them proteins associated with metabolic pathways, extracellular matrix-receptor interaction, focal adhesion, and actin cytoskeleton) in hypoxic MSC-sEVs. Post-MCAO, sEVs from hypoxic MSCs increased microvascular length and branching point density in previously ischemic tissue assessed by 3D light sheet microscopy over up to 56 days, reduced delayed neuronal degeneration and brain atrophy, and enhanced neurological recovery. sEV-induced angiogenesis in vivo depended on the presence of polymorphonuclear neutrophils. In neutrophil-depleted mice, MSC-sEVs did not influence microvascular remodeling. sEVs from hypoxic MSCs have distinct angiogenic properties. Hypoxic preconditioning enhances the restorative effects of MSC-sEVs.
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Proteínas Angiogénicas/metabolismo , Encéfalo/irrigación sanguínea , Células Endoteliales/metabolismo , Vesículas Extracelulares/trasplante , Infarto de la Arteria Cerebral Media/cirugía , Células Madre Mesenquimatosas/metabolismo , Microvasos/metabolismo , Neovascularización Fisiológica , Remodelación Vascular , Proteínas Angiogénicas/genética , Animales , Hipoxia de la Célula , Movimiento Celular , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Vesículas Extracelulares/metabolismo , Humanos , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/fisiopatología , Masculino , Ratones Endogámicos C57BL , MicroARNs/genética , MicroARNs/metabolismo , Microvasos/fisiopatología , Neuronas/metabolismo , Neuronas/patología , Recuperación de la Función , Transducción de Señal , Factores de TiempoRESUMEN
BACKGROUND: The newly emerged severe acute respiratory syndrome coronavirus (SARS-CoV-2) has caused a worldwide pandemic of human respiratory disease. Angiotensin-converting enzyme (ACE) 2 is the key receptor on lung epithelial cells to facilitate initial binding and infection of SARS-CoV-2. The binding to ACE2 is mediated via the spike glycoprotein present on the viral surface. Recent clinical data have demonstrated that patients with previous episodes of brain injuries are a high-risk group for SARS-CoV-2 infection. An explanation for this finding is currently lacking. Sterile tissue injuries including stroke induce the release of several inflammatory mediators that might modulate the expression levels of signaling proteins in distant organs. Whether systemic inflammation following brain injury can specifically modulate ACE2 expression in different vital tissues has not been investigated. METHODS: For the induction of brain stroke, mice were subjected to a surgical procedure for transient interruption of blood flow in the middle cerebral artery for 45 min and sacrificed after 1 and 3 days for analysis of brain, lung, heart, and kidney tissues. Gene expression and protein levels of ACE2, ACE, IL-6 and IL1ß were measured by quantitative PCR and Western blot, respectively. The level of soluble ACE2 in plasma and bronchial alveolar lavage (BAL) was measured using an immunoassay. Immune cell populations in lymphoid organs were analyzed by flow cytometry. Post-stroke pneumonia in mice was examined by bacterial cultures from lung homogenates and whole blood. RESULTS: Strikingly, 1 day after surgery, we observed a substantial increase in the protein levels of ACE2 in the lungs of stroke mice compared to sham-operated mice. However, the protein levels of ACE2 were found unchanged in the heart, kidney, and brain of these animals. In addition, we found increased transcriptional levels of alveolar ACE2 after stroke. The increased expression of ACE2 was significantly associated with the severity of behavioral deficits after stroke. The higher protein levels of alveolar ACE2 persisted until 3 days of stroke. Interestingly, we found reduced levels of soluble ACE2 in plasma but not in BAL in stroke-operated mice compared to sham mice. Furthermore, stroke-induced parenchymal and systemic inflammation was evident with the increased expression of IL-6 and IL-1ß. Reduced numbers of T-lymphocytes were present in the blood and spleen as an indicator of sterile tissue injury-induced immunosuppression. CONCLUSIONS: We demonstrate specific augmented alveolar ACE2 levels and inflammation in murine lungs after experimental stroke. These pre-clinical findings suggest that patients with brain injuries may have increased binding affinity to SARS-CoV-2 in their lungs which might explain why stroke is a risk factor for higher susceptibility to develop COVID-19.
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COVID-19 , Accidente Cerebrovascular , Animales , Humanos , Pulmón , Ratones , Peptidil-Dipeptidasa A/genética , SARS-CoV-2RESUMEN
Background and Purpose- Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) were shown to induce neurological recovery after focal cerebral ischemia in rodents and to reverse postischemic lymphopenia in peripheral blood. Since peripheral blood cells, especially polymorphonuclear neutrophils (PMNs), contribute to ischemic brain injury, we analyzed brain leukocyte responses to sEVs and investigated the role of PMNs in sEV-induced neuroprotection. Methods- Male C57Bl6/j mice were exposed to transient intraluminal middle cerebral artery occlusion. After reperfusion, vehicle or sEVs prepared from conditioned media of MSCs raised from bone marrow samples of 3 randomly selected healthy human donors were intravenously administered. sEVs obtained from normoxic and hypoxic MSCs were applied. PMNs were depleted in vehicle and MSC-sEV-treated mice. Neurological deficits, ischemic injury, blood-brain barrier integrity, peripheral blood leukocyte responses, and brain leukocyte infiltration were evaluated over 72 hours. Results- sEV preparations of all 3 donors collected from normoxic MSCs significantly reduced neurological deficits. Preparations of 2 of these donors significantly decreased infarct volume and neuronal injury. sEV-induced neuroprotection was consistently associated with a decreased brain infiltration of leukocytes, namely of PMNs, monocytes/macrophages, and lymphocytes. sEVs obtained from hypoxic MSCs (1% O2) had similar effects on neurological deficits and ischemic injury as MSC-sEVs obtained under regular conditions (21% O2) but also reduced serum IgG extravasation-a marker of blood-brain barrier permeability. PMN depletion mimicked the effects of MSC-sEVs on neurological recovery, ischemic injury, and brain PMN, monocyte, and lymphocyte counts. Combined MSC-sEV administration and PMN depletion did not have any effects superior to PMN depletion in any of the readouts examined. Conclusions- Leukocytes and specifically PMNs contribute to MSC-sEV-induced ischemic neuroprotection. Individual MSC-sEV preparations may differ in their neuroprotective activities. Potency assays are urgently needed to identify their therapeutic efficacy before clinical application. Visual Overview- An online visual overview is available for this article.
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Barrera Hematoencefálica , Isquemia Encefálica , Vesículas Extracelulares , Células Madre Mesenquimatosas/metabolismo , Neuroprotección , Neutrófilos/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Isquemia Encefálica/sangre , Isquemia Encefálica/patología , Isquemia Encefálica/terapia , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patología , Vesículas Extracelulares/trasplante , Humanos , Masculino , Células Madre Mesenquimatosas/patología , Ratones , Neutrófilos/patologíaRESUMEN
By cleaving sphingomyelin into ceramide, which is an essential component of plasma membrane microdomains, acid sphingomyelinase (Asm) pivotally controls cell signaling. To define how the activation of the Asm/ceramide pathway, which occurs within seconds to minutes upon stress stimuli, influences brain ischemia/reperfusion (I/R) injury, we exposed male and female wildtype mice carrying both alleles of Asm's gene sphingomyelinase phosphodiesterase-1 (Smpd1+/+), heterozygously Asm-deficient mice (Smpd1+/-) and homozygously Asm-deficient mice (Smpd1-/-) of different age (8, 12 or 16 weeks) to 30, 60 or 90 min intraluminal middle cerebral artery occlusion (MCAO). For studying the contribution of brain-invading polymorphonuclear neutrophils (PMN) to I/R injury, PMNs were depleted by delivery of a PMN-specific Ly6G antibody. In male and female mice exposed to 30 min, but not 60 or 90 min MCAO, homozygous Smpd1-/- consistently increased I/R injury, blood-brain barrier permeability and brain leukocyte and PMN infiltration, whereas heterozygous Smpd1+/- reduced I/R injury. Increased abundance of the intercellular leukocyte adhesion molecule ICAM-1 was noted on cerebral microvessels of Smpd1-/- mice. PMN depletion by anti-Ly6G delivery prevented the exacerbation of I/R injury in Smpd1-/- compared with wildtype mice and reduced brain leukocyte infiltrates. Our results show that Asm tempers leukocyte entry into the reperfused ischemic brain, thereby attenuating I/R injury.
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Encéfalo/irrigación sanguínea , Infarto de la Arteria Cerebral Media/enzimología , Microvasos/enzimología , Infiltración Neutrófila , Neutrófilos/enzimología , Daño por Reperfusión/enzimología , Esfingomielina Fosfodiesterasa/deficiencia , Animales , Modelos Animales de Enfermedad , Femenino , Heterocigoto , Homocigoto , Infarto de la Arteria Cerebral Media/genética , Infarto de la Arteria Cerebral Media/patología , Molécula 1 de Adhesión Intercelular/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Microvasos/patología , Daño por Reperfusión/genética , Daño por Reperfusión/patología , Daño por Reperfusión/prevención & control , Esfingomielina Fosfodiesterasa/genética , Factores de TiempoRESUMEN
In this review, we summarize implications of the acid sphingomyelinase/ ceramide system in ischemic stroke. Acid sphingomyelinase catalyzes the formation of the bioactive sphingolipid ceramide which coalesces into membrane platforms and has a pivotal role in inflammation, cell signaling and death. Cerebral ischemia increases acid sphingomyelinase activity and elevates brain ceramide levels, which has been associated with the exacerbation of ischemic injury and deterioration of stroke outcome. In view of the fact that lowering acid sphingomyelinase activity and ceramide level was shown to protect against ischemic injury and ameliorate neurological deficits, the acid sphingomyelinase/ ceramide system might represent a promising target for stroke therapies.
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Isquemia Encefálica/tratamiento farmacológico , Ceramidas/antagonistas & inhibidores , Sistemas de Liberación de Medicamentos/métodos , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Accidente Cerebrovascular/tratamiento farmacológico , Animales , Antidepresivos/administración & dosificación , Antidepresivos/metabolismo , Isquemia Encefálica/metabolismo , Ceramidas/metabolismo , Humanos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Esfingomielina Fosfodiesterasa/metabolismo , Accidente Cerebrovascular/metabolismoRESUMEN
Empirical research on land sharing and land sparing has been criticized because preferences of local stakeholders, socio-economic aspects, a bundle of ecosystem services and the local context were only rarely integrated. Using storylines and scenarios is a common approach to include land use drivers and local contexts or to cope with the uncertainties of future developments. The objective of the presented research is to develop comparable participatory regional land use scenarios for the year 2030 reflecting land sharing, land sparing and more intermediate developments across five different European landscapes (Austria, Germany, Switzerland, The Netherlands and Spain). In order to ensure methodological consistency among the five case studies, a hierarchical multi-scale scenario approach was developed, which consisted of i) the selection of a common global storyline to frame a common sphere of uncertainty for all case studies, ii) the definition of three contrasting qualitative European storylines (representing developments for land sharing, land sparing and a balanced storyline), and iii) the development of three explorative case study-specific land use scenarios with regional stakeholders in workshops. Land use transition rules defined by stakeholders were used to generate three different spatially-explicit scenarios for each case study by means of high-resolution land use maps. All scenarios incorporated various aspects of land use and management to allow subsequent quantification of multiple ecosystem services and biodiversity indicators. The comparison of the final scenarios showed both common as well as diverging trends among the case studies. For instance, stakeholders identified further possibilities to intensify land management in all case studies in the land sparing scenario. In addition, in most case studies stakeholders agreed on the most preferred scenario, i.e. either land sharing or balanced, and the most likely one, i.e. balanced. However, they expressed some skepticism regarding the general plausibility of land sparing in a European context. It can be concluded that stakeholder perceptions and the local context can be integrated in land sharing and land sparing contexts subject to particular process design principles.
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Conservación de los Recursos Naturales , Ecosistema , Austria , Alemania , Países Bajos , España , SuizaRESUMEN
High-density lipoprotein (HDL) has previously been shown to promote angiogenesis. However, the mechanisms by which HDL enhances the formation of blood vessels remain to be defined. To address this, the effects of HDL on the proliferation, transwell migration and tube formation of human umbilical vein endothelial cells were investigated. By examining the abundance and phosphorylation (i.e., activation) of the vascular endothelial growth factor receptor VEGFR2 and modulating the activity of the sphingosine-1 phosphate receptors S1P1-3 and VEGFR2, we characterized mechanisms controlling angiogenic responses in response to HDL exposure. Here, we report that HDL dose-dependently increased endothelial proliferation, migration and tube formation. These events were in association with increased VEGFR2 abundance and rapid VEGFR2 phosphorylation at Tyr1054/Tyr1059 and Tyr1175 residues in response to HDL. Blockade of VEGFR2 activation by the VEGFR2 inhibitor SU1498 markedly abrogated the pro-angiogenic capacity of HDL. Moreover, the S1P3 inhibitor suramin prevented VEGFR2 expression and abolished endothelial migration and tube formation, while the S1P1 agonist CYM-5442 and the S1P2 inhibitor JTE-013 had no effect. Last, the role of S1P3 was further confirmed in regulation of S1P-induced endothelial proliferation, migration and tube formation via up-regulation and activation of VEGFR2. Together, these findings argue that HDL promotes angiogenesis via S1P3-dependent up-regulation and activation of VEGFR2 and also suggest that the S1P-S1P3-VEGFR2 signaling cascades as a novel target for HDL-modulating therapy implicated in vascular remodeling and functional recovery in atherosclerotic diseases such as myocardial infarction and ischemic stroke.
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Movimiento Celular , Proliferación Celular , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Lipoproteínas HDL/metabolismo , Neovascularización Fisiológica , Receptores de Lisoesfingolípidos/metabolismo , Transducción de Señal , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Células Endoteliales de la Vena Umbilical Humana/citología , HumanosRESUMEN
ATOH8 is a bHLH transcription factor playing roles in a variety of developmental processes such as neurogenesis, differentiation of pancreatic precursor cells, development of kidney and muscle, and differentiation of endothelial cells. PPP3CB belongs to the catalytic subunit of the serine/threonine phosphatase, calcineurin, which can dephosphorylate its substrate proteins to regulate their physiological activities. In our study, we demonstrated that ATOH8 interacts with PPP3CB in vitro with different approaches. We show that the conserved catalytic domain of PPP3CB interacts with both the N-terminus and the bHLH domain of ATOH8. Although the interaction domain of PPP3CB is conserved among all isoforms of calcineurin A, ATOH8 selectively interacts with PPP3CB instead of PPP3CA, probably due to the unique proline-rich region present in the N-terminus of PPP3CB, which controls the specificity of its interaction partners. Furthermore, we show that inhibition of the interaction with calcineurin inhibitor, cyclosporin A (CsA), leads to the retention of ATOH8 to the cytoplasm, suggesting that the interaction renders nuclear localization of ATOH8 which may be critical to control its activity as transcription factor.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Inhibidores de la Calcineurina/química , Calcineurina/química , Ciclosporina/química , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Células COS , Calcineurina/genética , Señalización del Calcio/genética , Dominio Catalítico/genética , Dominio Catalítico/fisiología , Línea Celular , Chlorocebus aethiops , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
The oculocerebrorenal syndrome of Lowe (OCRL), also called Lowe syndrome, is characterized by defects of the nervous system, the eye and the kidney. Lowe syndrome is a monogenetic X-linked disease caused by mutations of the inositol-5-phosphatase OCRL1. OCRL1 is a membrane-bound protein recruited to membranes via interaction with a variety of Rab proteins. The structural and kinetic basis of OCRL1 for the recognition of several Rab proteins is unknown. In this study, we report the crystal structure of the Rab-binding domain (RBD) of OCRL1 in complex with Rab8a and the kinetic binding analysis of OCRL1 with several Rab GTPases (Rab1b, Rab5a, Rab6a and Rab8a). In contrast to other effectors that bind their respective Rab predominantly via α-helical structure elements, the Rab-binding interface of OCRL1 consists mainly of the IgG-like ß-strand structure of the ASPM-SPD-2-Hydin domain as well as one α-helix. Our results give a deeper structural understanding of disease-causing mutations of OCRL1 affecting Rab binding.
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Mutación/genética , Síndrome Oculocerebrorrenal/genética , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/genética , Proteínas de Unión al GTP rab/metabolismo , Membrana Celular/metabolismo , Cristalización , Cristalografía por Rayos X , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoprecipitación , Síndrome Oculocerebrorrenal/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas de Unión al GTP rab/genéticaRESUMEN
The intracellular nucleotide-binding oligomerization domain-2 (NOD2) receptor detects bacteria-derived muramyl dipeptide (MDP) and activates the transcription factor NF-κB. Here we describe the regulatome of NOD2 signaling using a systematic RNAi screen. Using three consecutive screens, we identified a set of 20 positive NF-κB regulators including the known pathway members RIPK2, RELA, and BIRC4 (XIAP) as well as FRMPD2 (FERM and PDZ domain-containing 2). FRMPD2 interacts with NOD2 via leucine-rich repeats and forms a complex with the membrane-associated protein ERBB2IP. We demonstrate that FRMPD2 spatially assembles the NOD2-signaling complex, hereby restricting NOD2-mediated immune responses to the basolateral compartment of polarized intestinal epithelial cells. We show that genetic truncation of the NOD2 leucine-rich repeat domain, which is associated with Crohn disease, impairs the interaction with FRMPD2, and that intestinal inflammation leads to down-regulation of FRMPD2. These results suggest a structural mechanism for how polarity of epithelial cells acts on intestinal NOD-like receptor signaling to mediate spatial specificity of bacterial recognition and control of immune responses.
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Acetilmuramil-Alanil-Isoglutamina/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Interferencia de ARN , Transducción de Señal , Acetilmuramil-Alanil-Isoglutamina/farmacología , Células CACO-2 , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Enfermedad de Crohn/metabolismo , Enfermedad de Crohn/patología , Enterocitos/efectos de los fármacos , Enterocitos/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Proteínas Mutantes/metabolismo , FN-kappa B/metabolismo , Proteína Adaptadora de Señalización NOD2/química , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , ARN Interferente Pequeño/metabolismo , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacos , Especificidad por Sustrato/efectos de los fármacos , Proteínas de Uniones Estrechas/químicaRESUMEN
OBJECTIVE: Therapeutic angiogenesis aims at the promotion of vascular growth, usually under conditions of atherosclerosis. It was unknown how hyperlipidemia, a risk factor that is closely associated with atherosclerosis of brain vessels in humans, influences vascular endothelial growth factor-induced angiogenesis and stroke recovery. APPROACH AND RESULTS: Wild-type and apolipoprotein-E (ApoE)(-/-) mice were kept on regular or cholesterol-rich diet for mimicking different severities of hyperlipidemia. Mice were treated intracerebroventricularly with recombinant human vascular endothelial growth factor for 21 days (0.02 µg/d) and subsequently subjected to 90-minute middle cerebral artery occlusion followed by 1 or 24 hours of reperfusion. Histochemical, autoradiographic, and regional bioluminescence techniques were used to evaluate effects of blood lipids on postischemic angiogenesis, histopathologic brain injury, cerebral blood flow, protein synthesis and energy state, and pericyte coverage of brain endothelial cells. Hyperlipidemia dose-dependently attenuated vascular endothelial growth factor-induced capillary formation and pericyte coverage of brain endothelial cells, abolishing the improvement of cerebral blood flow during subsequent stroke, resulting in the loss of the metabolic penumbra and increased brain infarction. The enhanced angiogenesis after vascular endothelial growth factor treatment was accompanied by increased expression of the adhesion protein N-cadherin, which mediates endothelial-pericytic interactions, in ischemic brain microvessels of wild-type mice on regular diet that was blunted in wild-type mice on Western diet and ApoE(-/-) mice on either diet. CONCLUSIONS: The compromised vessel formation and hemodynamics question the concept of therapeutic angiogenesis in ischemic stroke where hyperlipidemia is highly prevalent.
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Inductores de la Angiogénesis/administración & dosificación , Capilares/efectos de los fármacos , Circulación Cerebrovascular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Hiperlipidemias/complicaciones , Neovascularización Fisiológica/efectos de los fármacos , Fármacos Neuroprotectores/administración & dosificación , Pericitos/efectos de los fármacos , Accidente Cerebrovascular/tratamiento farmacológico , Factor A de Crecimiento Endotelial Vascular/administración & dosificación , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Autorradiografía , Biomarcadores/sangre , Velocidad del Flujo Sanguíneo , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/patología , Barrera Hematoencefálica/fisiopatología , Cadherinas/metabolismo , Capilares/patología , Capilares/fisiopatología , Permeabilidad Capilar/efectos de los fármacos , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/patología , Matriz Extracelular/metabolismo , Humanos , Hiperlipidemias/sangre , Hiperlipidemias/patología , Hiperlipidemias/fisiopatología , Bombas de Infusión Implantables , Flujometría por Láser-Doppler , Lípidos/sangre , Mediciones Luminiscentes , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pericitos/metabolismo , Pericitos/patología , Accidente Cerebrovascular/sangre , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/patología , Accidente Cerebrovascular/fisiopatología , Factores de TiempoRESUMEN
OBJECTIVE: Intimal hyperplasia is considered to be a healing response and is a major cause of vessel narrowing after injury, where migration of vascular progenitor cells contributes to pathological events, including transplant arteriosclerosis. APPROACH AND RESULTS: In this study, we used a rat aortic-allograft model to identify the predominant cell types associated with transplant arteriosclerosis and to identify factors important in their recruitment into the graft. Transplantation of labeled adventitial tissues allowed us to identify the adventitia as a major source of cells migrating to the intima. RNA microarrays revealed a potential role for monocyte chemoattractant protein 1 (MCP-1), stromal cell-derived factor 1, regulated on activation, normal T cell expressed and secreted, and interferon-inducible protein 10 in the induced vasculopathy. MCP-1 induced migration of adventitial fibroblast cells. CCR2, the receptor for MCP-1, was coexpressed with CD90, CD44, NG2, or sca-1 on mesenchymal stem cells. In vivo experiments using MCP-1-deficient and CCR2-deficient mice confirmed an important role of MCP-1 in the formation of intimal hyperplasia in a mouse model of vascular injury. CONCLUSIONS: The adventitia is a potentially important cellular source that contributes to intimal hyperplasia, and MCP-1 is a potent chemokine for the recruitment of adventitial vascular progenitor cells to intimal lesions.
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Quimiocina CCL2/metabolismo , Células Madre Mesenquimatosas/citología , Neointima/patología , Túnica Íntima/patología , Animales , Movimiento Celular , Quimiocina CCL2/genética , Hiperplasia/genética , Hiperplasia/patología , Células Madre Mesenquimatosas/metabolismo , Ratones , Modelos Animales , Miocitos del Músculo Liso/metabolismo , Neointima/metabolismo , Ratas , Sensibilidad y Especificidad , Transducción de Señal , Células Madre/citología , Células Madre/metabolismo , Trasplante Homólogo , Túnica Íntima/metabolismo , Lesiones del Sistema Vascular/patología , Lesiones del Sistema Vascular/fisiopatologíaRESUMEN
Evaluation of microvascular networks was impeded until recently by the need of histological tissue sectioning, which precluded 3D analyses. Using light-sheet microscopy, we investigated microvascular network characteristics in the peri-infarct cortex of mice 3-56 days after transient middle cerebral artery occlusion. In animal subgroups, the sphingosine-1-phosphate analog FTY720 (Fingolimod) was administered starting 24 hours post-ischemia. Light-sheet microscopy revealed a striking pattern of microvascular changes in the peri-infarct cortex, that is, a loss of microvessels, which was most prominent after 7 days and followed by the reappearance of microvessels over 56 days which revealed an increased branching point density and shortened branches. Using a novel AI-based image analysis algorithm we found that the length density of microvessels expressing the arterial specification marker α-smooth muscle actin markedly increased in the peri-infarct cortex already at 7 days post-ischemia. The length and branch density of small microvessels, but not of intermediate or large microvessels increased above pre-ischemic levels within 14-56 days. FTY720 increased the length and branch density of small microvessels. This study demonstrates long-term alterations of microvascular architecture post-ischemia indicative of increased collateralization most notably of small microvessels. Light-sheet microscopy will greatly advance the assessment of microvascular responses to restorative stroke therapies.
RESUMEN
Considerable efforts have been made to amplify angiogenesis under conditions of hypoxia and ischemia by vascular endothelial growth factor (VEGF) delivery, so far with limited success. Ischemic vascular diseases are often associated with hypercholesterolemia. To elucidate whether the exposure to blood lipids influences VEGF responses of microvessels, we characterized effects of low density lipoprotein (LDL) exposure on the proliferation, migration and tube formation of human umbilical vein endothelial cells. By examining the expression, phosphorylation and downstream signals of VEGF's receptor VEGFR2, we characterized mechanisms controlling angiogenic responses following LDL exposure. LDL attenuated endothelial proliferation, migration and tube formation in a dose-dependent way. Reduced abundance of VEGFR2 and VEGFR1 were noticed in LDL-exposed endothelial cells. In subcellular localization studies that we combined with pharmacological experiments, we showed that the loss of VEGFR2 resulted from its internalization and degradation, the latter of which required syntaxin-16-dependent endosome-trans-Golgi network trafficking. As a consequence, VEGFR2 phosphorylation and downstream signals -specifically Akt and ERK1/2 phosphorylation- were attenuated in response to VEGF treatment. VEGF only partly reversed the effects of LDL on angiogenesis under conditions of normoxia and hypoxia. Our results suggest that angiogenic responses to VEGF are compromised in hypercholesterolemia as a consequence of endosomal VEGFR2 degradation.
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Hipercolesterolemia/fisiopatología , Hipoxia/fisiopatología , Lipoproteínas LDL/farmacología , Neovascularización Fisiológica/fisiología , Factor A de Crecimiento Endotelial Vascular/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Análisis de Varianza , Western Blotting , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Proliferación Celular/efectos de los fármacos , Cartilla de ADN/genética , Relación Dosis-Respuesta a Droga , Endosomas/metabolismo , Técnica del Anticuerpo Fluorescente , Vectores Genéticos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inmunoprecipitación , Lentivirus , Neovascularización Fisiológica/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Red trans-Golgi/metabolismoRESUMEN
Light-sheet fluorescence microscopy (LSFM) can produce high-resolution tomograms of tissue vasculature with high accuracy. However, data processing and analysis is laborious due to the size of the datasets. Here, we introduce VesselExpress, an automated software that reliably analyzes six characteristic vascular network parameters including vessel diameter in LSFM data on average computing hardware. VesselExpress is â¼100 times faster than other existing vessel analysis tools, requires no user interaction, and integrates batch processing and parallelization. Employing an innovative dual Frangi filter approach, we show that obesity induces a large-scale modulation of brain vasculature in mice and that seven other major organs differ strongly in their 3D vascular makeup. Hence, VesselExpress transforms LSFM from an observational to an analytical working tool.
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Imagenología Tridimensional , Programas Informáticos , Animales , Ratones , Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos , Encéfalo/diagnóstico por imagenRESUMEN
BACKGROUND AND OBJECTIVES: Experimental studies indicate shared molecular pathomechanisms in cerebral hypoxia-ischemia and autoimmune neuroinflammation. This has led to clinical studies investigating the effects of immunomodulatory therapies approved in multiple sclerosis on inflammatory damage in stroke. So far, mutual and combined interactions of autoimmune, CNS antigen-specific inflammatory reactions and cerebral ischemia have not been investigated so far. METHODS: Active MOG35-55 experimental autoimmune encephalomyelitis (EAE) was induced in male C57Bl/6J mice. During different phases of EAE, transient middle cerebral artery occlusion (tMCAO, 60 minutes) was induced. Brain tissue was analyzed for infarct size and immune cell infiltration. Multiplex gene expression analysis was performed for 186 genes associated with neuroinflammation and hypoxic-ischemic damage. RESULTS: Mice with severe EAE disease showed a substantial reduction in infarct size after tMCAO. Histopathologic analysis showed less infiltration of CD45+ hematopoietic cells in the infarct core of severely diseased acute EAE mice; this was accompanied by an accumulation of Arginase1-positive/Iba1-positive cells. Gene expression analysis indicated an involvement of myeloid cell-driven anti-inflammatory mechanisms in the attenuation of ischemic injury in severely diseased mice exposed to tMCAO in the acute EAE phase. DISCUSSION: CNS autoantigen-specific autoimmunity has a protective influence on primary tissue damage after experimental stroke, indicating a very early involvement of CNS antigen-specific, myeloid cell-associated anti-inflammatory immune mechanisms that mitigate ischemic injury in the acute EAE phase.
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Encefalomielitis Autoinmune Experimental , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Animales , Infarto , Masculino , Ratones , Ratones Endogámicos C57BL , Células Mieloides/metabolismo , Células Mieloides/patología , Enfermedades NeuroinflamatoriasRESUMEN
Following ischemic stroke, polymorphonuclear neutrophils (PMNs) are rapidly recruited to the ischemic brain tissue and exacerbate stroke injury by release of reactive oxygen species (ROS), proteases and proinflammatory cytokines. PMNs may aggravate post-ischemic microvascular injury by obstruction of brain capillaries, contributing to reperfusion deficits in the stroke recovery phase. Thus, experimental studies which specifically depleted PMNs by delivery of anti-Ly6G antibodies or inhibited PMN brain entry, e.g., by CXC chemokine receptor 2 (CXCR2) or very late antigen-4 (VLA-4) blockade in the acute stroke phase consistently reduced neurological deficits and infarct volume. Although elevated PMN responses in peripheral blood are similarly predictive for large infarcts and poor stroke outcome in human stroke patients, randomized controlled clinical studies targeting PMN brain infiltration did not improve stroke outcome or even worsened outcome due to serious complications. More recent studies showed that PMNs have decisive roles in post-ischemic angiogenesis and brain remodeling, most likely by promoting extracellular matrix degradation, thereby amplifying recovery processes in the ischemic brain. In this minireview, recent findings regarding the roles of PMNs in ischemic brain injury and post-ischemic brain remodeling are summarized.