RESUMEN
BACKGROUND: CALCRL (calcitonin receptor-like) protein is an important mediator of the endothelial fluid shear stress response, which is associated with the genetic risk of coronary artery disease. In this study, we functionally characterized the noncoding regulatory elements carrying coronary artery disease that risks single-nucleotide polymorphisms and studied their role in the regulation of CALCRL expression in endothelial cells. METHODS: To functionally characterize the coronary artery disease single-nucleotide polymorphisms harbored around the gene CALCRL, we applied an integrative approach encompassing statistical, transcriptional (RNA-seq), and epigenetic (ATAC-seq [transposase-accessible chromatin with sequencing], chromatin immunoprecipitation assay-quantitative polymerase chain reaction, and electromobility shift assay) analyses, alongside luciferase reporter assays, and targeted gene and enhancer perturbations (siRNA and clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) in human aortic endothelial cells. RESULTS: We demonstrate that the regulatory element harboring rs880890 exhibits high enhancer activity and shows significant allelic bias. The A allele was favored over the G allele, particularly under shear stress conditions, mediated through alterations in the HSF1 (heat shock factor 1) motif and binding. CRISPR deletion of rs880890 enhancer resulted in downregulation of CALCRL expression, whereas HSF1 knockdown resulted in a significant decrease in rs880890-enhancer activity and CALCRL expression. A significant decrease in HSF1 binding to the enhancer region in endothelial cells was observed under disturbed flow compared with unidirectional flow. CALCRL knockdown and variant perturbation experiments indicated the role of CALCRL in mediating eNOS (endothelial nitric oxide synthase), APLN (apelin), angiopoietin, prostaglandins, and EDN1 (endothelin-1) signaling pathways leading to a decrease in cell proliferation, tube formation, and NO production. CONCLUSIONS: Overall, our results demonstrate the existence of an endothelial-specific HSF (heat shock factor)-regulated transcriptional enhancer that mediates CALCRL expression. A better understanding of CALCRL gene regulation and the role of single-nucleotide polymorphisms in the modulation of CALCRL expression could provide important steps toward understanding the genetic regulation of shear stress signaling responses.
Asunto(s)
Proteína Similar al Receptor de Calcitonina , Enfermedad de la Arteria Coronaria , Células Endoteliales , Elementos de Facilitación Genéticos , Polimorfismo de Nucleótido Simple , Estrés Mecánico , Humanos , Células Endoteliales/metabolismo , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/patología , Proteína Similar al Receptor de Calcitonina/genética , Proteína Similar al Receptor de Calcitonina/metabolismo , Factores de Transcripción del Choque Térmico/genética , Factores de Transcripción del Choque Térmico/metabolismo , Mecanotransducción Celular , Células Cultivadas , Regulación de la Expresión Génica , Unión Proteica , Predisposición Genética a la Enfermedad , Sitios de UniónRESUMEN
Cerebral cavernous malformation (CCM) is a neurovascular disease that results in various neurological symptoms. Thrombi have been reported in surgically resected CCM patient biopsies, but the molecular signatures of these thrombi remain elusive. Here, we investigated the kinetics of thrombi formation in CCM and how thrombi affect the vasculature and contribute to cerebral hypoxia. We used RNA sequencing to investigate the transcriptome of mouse brain endothelial cells with an inducible endothelial-specific Ccm3 knock-out (Ccm3-iECKO). We found that Ccm3-deficient brain endothelial cells had a higher expression of genes related to the coagulation cascade and hypoxia when compared with wild-type brain endothelial cells. Immunofluorescent assays identified key molecular signatures of thrombi such as fibrin, von Willebrand factor, and activated platelets in Ccm3-iECKO mice and human CCM biopsies. Notably, we identified polyhedrocytes in Ccm3-iECKO mice and human CCM biopsies and report it for the first time. We also found that the parenchyma surrounding CCM lesions is hypoxic and that more thrombi correlate with higher levels of hypoxia. We created an in vitro model to study CCM pathology and found that human brain endothelial cells deficient for CCM3 expressed elevated levels of plasminogen activator inhibitor-1 and had a redistribution of von Willebrand factor. With transcriptomics, comprehensive imaging, and an in vitro CCM preclinical model, this study provides experimental evidence that genes and proteins related to the coagulation cascade affect the brain vasculature and promote neurological side effects such as hypoxia in CCMs. This study supports the concept that antithrombotic therapy may be beneficial for patients with CCM.
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Hemangioma Cavernoso del Sistema Nervioso Central , Humanos , Animales , Ratones , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Células Endoteliales/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Tromboinflamación , Factor de von Willebrand/metabolismo , Hipoxia/metabolismoRESUMEN
Lymph node (LN) lipomatosis is a common but rarely discussed phenomenon associated with aging that involves a gradual exchange of the LN parenchyma into adipose tissue. The mechanisms behind these changes and the effects on the LN are unknown. We show that LN lipomatosis starts in the medullary regions of the human LN and link the initiation of lipomatosis to transdifferentiation of LN fibroblasts into adipocytes. The latter is associated with a downregulation of lymphotoxin beta expression. We also show that isolated medullary and CD34+ fibroblasts, in contrast to the reticular cells of the T-cell zone, display an inherently higher sensitivity for adipogenesis. Progression of lipomatosis leads to a gradual loss of the medullary lymphatic network, but at later stages, collecting-like lymphatic vessels are found inside the adipose tissue. The stromal dysregulation includes a dramatic remodeling and dilation of the high endothelial venules associated with reduced density of naïve T-cells. Abnormal clustering of plasma cells is also observed. Thus, LN lipomatosis causes widespread stromal dysfunction with consequences for the immune contexture of the human LN. Our data warrant an increased awareness of LN lipomatosis as a factor contributing to decreased immune functions in the elderly and in disease. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Asunto(s)
Transdiferenciación Celular , Lipomatosis , Humanos , Anciano , Remodelación Vascular , Ganglios Linfáticos/patología , Lipomatosis/metabolismo , Lipomatosis/patología , EnvejecimientoRESUMEN
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
Cerebral Cavernous Malformation (CCM) is a brain vascular disease with various neurological symptoms. In this study, we describe the inflammatory profile in CCM and show for the first time the formation of neutrophil extracellular traps (NETs) in rodents and humans with CCM. Through RNA-seq analysis of cerebellum endothelial cells from wild-type mice and mice with an endothelial cell-specific ablation of the Ccm3 gene (Ccm3iECKO), we show that endothelial cells from Ccm3iECKO mice have an increased expression of inflammation-related genes. These genes encode proinflammatory cytokines and chemokines, as well as adhesion molecules, which promote recruitment of inflammatory and immune cells. Similarly, immunoassays showed elevated levels of these cytokines and chemokines in the cerebellum of the Ccm3iECKO mice. Consistently, both flow cytometry and immunofluorescence analysis showed infiltration of different subsets of leukocytes into the CCM lesions. Neutrophils, which are known to fight against infection through different strategies, including the formation of NETs, represented the leukocyte subset within the most pronounced increase in CCM. Here, we detected elevated levels of NETs in the blood and the deposition of NETs in the cerebral cavernomas of Ccm3iECKO mice. Degradation of NETs by DNase I treatment improved the vascular barrier. The deposition of NETs in the cavernomas of patients with CCM confirms the clinical relevance of NETs in CCM.
Asunto(s)
Trampas Extracelulares , Hemangioma Cavernoso del Sistema Nervioso Central , Animales , Proteínas Reguladoras de la Apoptosis/genética , Células Endoteliales/metabolismo , Trampas Extracelulares/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Humanos , Inflamación/patología , Proteínas de la Membrana/metabolismo , RatonesRESUMEN
[Figure: see text].
Asunto(s)
Neoplasias del Sistema Nervioso Central/patología , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Propranolol/farmacología , Animales , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones NoqueadosAsunto(s)
Hemangioma Cavernoso del Sistema Nervioso Central , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Humanos , Animales , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Complejo Represivo Polycomb 1/antagonistas & inhibidores , Ratones , MasculinoRESUMEN
Innate immunity is fundamental to our defense against microorganisms. Physiologically, the intravascular innate immune system acts as a purging system that identifies and removes foreign substances leading to thromboinflammatory responses, tissue remodeling, and repair. It is also a key contributor to the adverse effects observed in many diseases and therapies involving biomaterials and therapeutic cells/organs. The intravascular innate immune system consists of the cascade systems of the blood (the complement, contact, coagulation, and fibrinolytic systems), the blood cells (polymorphonuclear cells, monocytes, platelets), and the endothelial cell lining of the vessels. Activation of the intravascular innate immune system in vivo leads to thromboinflammation that can be activated by several of the system's pathways and that initiates repair after tissue damage and leads to adverse reactions in several disorders and treatment modalities. In this review, we summarize the current knowledge in the field and discuss the obstacles that exist in order to study the cross-talk between the components of the intravascular innate immune system. These include the use of purified in vitro systems, animal models and various types of anticoagulants. In order to avoid some of these obstacles we have developed specialized human whole blood models that allow investigation of the cross-talk between the various cascade systems and the blood cells. We in particular stress that platelets are involved in these interactions and that the lectin pathway of the complement system is an emerging part of innate immunity that interacts with the contact/coagulation system. Understanding the resulting thromboinflammation will allow development of new therapeutic modalities.
Asunto(s)
Plaquetas/inmunología , Proteínas del Sistema Complemento/metabolismo , Células Endoteliales/fisiología , Inflamación/inmunología , Trombosis/inmunología , Animales , Coagulación Sanguínea , Homeostasis , Humanos , Inmunidad Innata , Calicreínas/metabolismo , Cininas/metabolismoRESUMEN
Correct organization of the vascular tree requires the balanced activities of several signaling pathways that regulate tubulogenesis and vascular branching, elongation, and pruning. When this balance is lost, the vessels can be malformed and fragile, and they can lose arteriovenous differentiation. In this review, we concentrate on the transforming growth factor (TGF)-ß/bone morphogenetic protein (BMP) pathway, which is one of the most important and complex signaling systems in vascular development. Inactivation of these pathways can lead to altered vascular organization in the embryo. In addition, many vascular malformations are related to deregulation of TGF-ß/BMP signaling. Here, we focus on two of the most studied vascular malformations that are induced by deregulation of TGF-ß/BMP signaling: hereditary hemorrhagic telangiectasia (HHT) and cerebral cavernous malformation (CCM). The first of these is related to loss-of-function mutation of the TGF-ß/BMP receptor complex and the second to increased signaling sensitivity to TGF-ß/BMP. In this review, we discuss the potential therapeutic targets against these vascular malformations identified so far, as well as their basis in general mechanisms of vascular development and stability.
Asunto(s)
Vasos Sanguíneos/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Neovascularización Fisiológica , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Malformaciones Vasculares/metabolismo , Animales , Vasos Sanguíneos/anomalías , Vasos Sanguíneos/fisiopatología , Proteínas Morfogenéticas Óseas/genética , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/fisiopatología , Humanos , Ratones Transgénicos , Mutación , Fenotipo , Factores de Riesgo , Telangiectasia Hemorrágica Hereditaria/genética , Telangiectasia Hemorrágica Hereditaria/metabolismo , Telangiectasia Hemorrágica Hereditaria/fisiopatología , Factor de Crecimiento Transformador beta/genética , Malformaciones Vasculares/genética , Malformaciones Vasculares/fisiopatologíaRESUMEN
The impact of complement activation and its possible relation to cytokine responses during malaria pathology was investigated in plasma samples from patients with confirmed Plasmodium falciparum malaria and in human whole-blood specimens stimulated with malaria-relevant agents ex vivo. Complement was significantly activated in the malaria cohort, compared with healthy controls, and was positively correlated with disease severity and with certain cytokines, in particular interleukin 8 (IL-8)/CXCL8. This was confirmed in ex vivo-stimulated blood specimens, in which complement inhibition significantly reduced IL-8/CXCL8 release. P. falciparum malaria is associated with systemic complement activation and complement-dependent release of inflammatory cytokines, of which IL-8/CXCL8 is particularly prominent.
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Activación de Complemento/inmunología , Citocinas/metabolismo , Malaria Falciparum/inmunología , Malaria Falciparum/metabolismo , Adulto , Hemoproteínas/inmunología , Hemina/inmunología , Humanos , Malaria Falciparum/epidemiología , Malaria Falciparum/fisiopatología , Plasmodium falciparum/inmunologíaRESUMEN
Xeno-transplantation of pancreatic islets represents a promising therapeutic alternative for the treatment of type 1 diabetes mellitus. However, potent innate immune responses induced shortly after the transplantation of donor islets to the recipient, comprising the Instant Blood Mediated Immune Reaction (IBMIR), exert detrimental actions on islet graft function. The coagulation and complement cascades together with the leukocyte and platelet populations are the major players in IBMIR. This innate immune attack affects dramatically islet integrity and leads to significant loss of function of the xenograft. In the present review, we focus on the mechanisms contributing to IBMIR components and address therapeutic intervention approaches to limit IBMIR by administering inhibitors in circulation, by coating the islet surface with inhibitors or by generating transgenic donor animals; these approaches could result in improved xenograft survival.
Asunto(s)
Diabetes Mellitus/terapia , Rechazo de Injerto/prevención & control , Trasplante de Islotes Pancreáticos/métodos , Transgenes , Animales , Animales Modificados Genéticamente , Antígenos CD/genética , Antígenos CD/inmunología , Factores de Coagulación Sanguínea/genética , Factores de Coagulación Sanguínea/inmunología , Plaquetas/efectos de los fármacos , Plaquetas/inmunología , Plaquetas/patología , Inactivadores del Complemento/farmacología , Proteínas del Sistema Complemento/genética , Proteínas del Sistema Complemento/inmunología , Sulfato de Dextran/farmacología , Diabetes Mellitus/genética , Diabetes Mellitus/inmunología , Diabetes Mellitus/patología , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Humanos , Trasplante de Islotes Pancreáticos/inmunología , Leucocitos/efectos de los fármacos , Leucocitos/inmunología , Leucocitos/patología , Péptidos Cíclicos/farmacología , Porcinos , Trasplante HeterólogoRESUMEN
Thromboinflammation is primarily triggered by the humoral innate immune system, which mainly consists of the cascade systems of the blood, i.e., the complement, contact/coagulation and fibrinolytic systems. Activation of these systems subsequently induces activation of endothelial cells, leukocytes and platelets, finally resulting in thrombotic and inflammatory reactions. Such reactions are triggered by a number of medical procedures, e.g., treatment with biomaterials or drug delivery devices as well as in transplantation with cells, cell clusters or whole vascularized organs. Here, we (1) describe basic mechanisms for thromboinflammation; (2) review thromboinflammatory reactions in therapeutic medicine; and (3) discuss emerging strategies to dampen thromboinflammation.
Asunto(s)
Anticoagulantes/uso terapéutico , Rechazo de Injerto/prevención & control , Factores Inmunológicos/uso terapéutico , Trombosis/prevención & control , Trasplante de Tejidos , Materiales Biocompatibles/efectos adversos , Coagulación Sanguínea/efectos de los fármacos , Factores de Coagulación Sanguínea/inmunología , Factores de Coagulación Sanguínea/metabolismo , Plaquetas/citología , Plaquetas/efectos de los fármacos , Plaquetas/inmunología , Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/inmunología , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Humanos , Inmunidad Humoral/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , Inflamación/inmunología , Inflamación/patología , Inflamación/prevención & control , Leucocitos/citología , Leucocitos/efectos de los fármacos , Leucocitos/inmunología , Trombosis/inmunología , Trombosis/patologíaRESUMEN
Treatment with mesenchymal stromal cells (MSCs) is currently of interest for a number of diseases including multiple sclerosis. MSCs are known to target inflamed tissues, but in a therapeutic setting their systemic administration will lead to few cells reaching the brain. We hypothesized that MSCs may target the brain upon intranasal administration and persist in central nervous system (CNS) tissue if expressing a CNS-targeting receptor. To demonstrate proof of concept, MSCs were genetically engineered to express a myelin oligodendrocyte glycoprotein-specific receptor. Engineered MSCs retained their immunosuppressive capacity, infiltrated into the brain upon intranasal cell administration, and were able to significantly reduce disease symptoms of experimental autoimmune encephalomyelitis (EAE). Mice treated with CNS-targeting MSCs were resistant to further EAE induction whereas non-targeted MSCs did not give such persistent effects. Histological analysis revealed increased brain restoration in engineered MSC-treated mice. In conclusion, MSCs can be genetically engineered to target the brain and prolong therapeutic efficacy in an EAE model.
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Sistema Nervioso Central/citología , Encefalomielitis Autoinmune Experimental/terapia , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Administración Intranasal , Animales , Sistema Nervioso Central/inmunología , Encefalomielitis Autoinmune Experimental/patología , Ingeniería Genética , Humanos , Inflamación/patología , Inflamación/prevención & control , Inflamación/terapia , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Glicoproteína Mielina-Oligodendrócito/genética , Glicoproteína Mielina-Oligodendrócito/metabolismoRESUMEN
BACKGROUND: Cerebral cavernous malformation (CCM) is a disease associated with an elevated risk of focal neurological deficits, seizures, and hemorrhagic stroke. The disease has an inflammatory profile and improved knowledge of CCM pathology mechanisms and exploration of candidate biomarkers will enable new non-invasive treatments. METHODS: We analyzed protein signatures in human CCM tissue samples by using a highly specific and sensitive multiplexing technique, proximity extension assay. FINDINGS: Data analysis revealed CCM specific proteins involved in endothelial dysfunction/inflammation/activation, leukocyte infiltration/chemotaxis, hemostasis, extracellular matrix dysfunction, astrocyte and microglial cell activation. Biomarker expression profiles matched bleeding status, especially with higher levels of inflammatory markers and activated astrocytes in ruptured than non-ruptured samples, some of these biomarkers are secreted into blood or urine. Furthermore, analysis was also done in a spatially resolving manner by separating the lesion area from the surrounding brain tissue. Our spatial studies revealed that although appearing histologically normal, the CCM border areas were pathological when compared to control brain tissues. Moreover, the functional relevance of CD93, ICAM-1 and MMP9, markers related to endothelial cell activation and extracellular matrix was validated by a murine pre-clinical CCM model. INTERPRETATION: Here we present a novel strategy for proteomics analysis on human CCMs, offering a possibility for high-throughput protein screening acquiring data on the local environment in the brain. Our data presented here describe CCM relevant brain proteins and specifically those which are secreted can serve the need of circulating CCM biomarkers to predict cavernoma's risk of bleeding.
Asunto(s)
Biomarcadores , Hemangioma Cavernoso del Sistema Nervioso Central , Molécula 1 de Adhesión Intercelular , Proteómica , Humanos , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Proteómica/métodos , Biomarcadores/metabolismo , Biomarcadores/análisis , Animales , Ratones , Molécula 1 de Adhesión Intercelular/metabolismo , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Femenino , Adulto , Persona de Mediana Edad , Encéfalo/metabolismo , Encéfalo/patología , Proteínas de la Membrana , Proteínas Proto-Oncogénicas , Proteínas Reguladoras de la ApoptosisRESUMEN
BACKGROUND: Cerebral Cavernous Malformation (CCM) is a rare cerebrovascular disease, characterized by the presence of multiple vascular malformations that may result in intracerebral hemorrhages (ICHs), seizure(s), or focal neurological deficits (FND). Familial CCM (fCCM) is due to loss of function mutations in one of the three independent genes KRIT1 (CCM1), Malcavernin (CCM2), or Programmed Cell death 10 (PDCD10/CCM3). The aim of this study was to identify plasma protein biomarkers of fCCM to assess the severity of the disease and predict its progression. METHODS: Here, we have investigated plasma samples derived from n = 71 symptomatic fCCM patients (40 female/31 male) and n = 17 healthy donors (HD) (9 female/8 male) of the Phase 1/2 Treat_CCM trial, using multiplexed protein profiling approaches. FINDINGS: Biomarkers as sCD14 (p = 0.00409), LBP (p = 0.02911), CXCL4 (p = 0.038), ICAM-1 (p = 0.02013), ANG2 (p = 0.026), CCL5 (p = 0.00403), THBS1 (p = 0.0043), CRP (p = 0.0092), and HDL (p = 0.027), were significantly different in fCCM compared to HDs. Of note, sENG (p = 0.011), THBS1 (p = 0.011) and CXCL4 (p = 0.011), were correlated to CCM genotype. sROBO4 (p = 0.014), TM (p = 0.026) and CRP (p = 0.040) were able to predict incident adverse clinical events, such as ICH, FND or seizure. GDF-15, FLT3L, CXCL9, FGF-21 and CDCP1, were identified as predictors of the formation of new MRI-detectable lesions over 2-year follow-up. Furthermore, the functional relevance of ang2, thbs1, robo4 and cdcp1 markers was validated by zebrafish pre-clinical model of fCCM. INTERPRETATION: Overall, our study identifies a set of biochemical parameters to predict CCM progression, suggesting biological interpretations and potential therapeutic approaches to CCM disease. FUNDING: Italian Medicines Agency, Associazione Italiana per la Ricerca sul Cancro (AIRC), ERC, Leducq Transatlantic Network of Excellence, Swedish Research Council.
Asunto(s)
Hemangioma Cavernoso del Sistema Nervioso Central , Animales , Humanos , Masculino , Femenino , Hemangioma Cavernoso del Sistema Nervioso Central/etiología , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Asociadas a Microtúbulos/genética , Pez Cebra/metabolismo , Biomarcadores , Convulsiones , Antígenos de Neoplasias , Moléculas de Adhesión CelularRESUMEN
Multipotent mesenchymal stromal cells (MSCs) are tested in numerous clinical trials. Questions have been raised concerning fate and function of these therapeutic cells after systemic infusion. We therefore asked whether culture-expanded human MSCs elicit an innate immune attack, termed instant blood-mediated inflammatory reaction (IBMIR), which has previously been shown to compromise the survival and function of systemically infused islet cells and hepatocytes. We found that MSCs expressed hemostatic regulators similar to those produced by endothelial cells but displayed higher amounts of prothrombotic tissue/stromal factors on their surface, which triggered the IBMIR after blood exposure, as characterized by formation of blood activation markers. This process was dependent on the cell dose, the choice of MSC donor, and particularly the cell-passage number. Short-term expanded MSCs triggered only weak blood responses in vitro, whereas extended culture and coculture with activated lymphocytes increased their prothrombotic properties. After systemic infusion to patients, we found increased formation of blood activation markers, but no formation of hyperfibrinolysis marker D-dimer or acute-phase reactants with the currently applied dose of 1.0-3.0 × 10(6) cells per kilogram. Culture-expanded MSCs trigger the IBMIR in vitro and in vivo. Induction of IBMIR is dose-dependent and increases after prolonged ex vivo expansion. Currently applied doses of low-passage clinical-grade MSCs elicit only minor systemic effects, but higher cell doses and particularly higher passage cells should be handled with care. This deleterious reaction can compromise the survival, engraftment, and function of these therapeutic cells.
Asunto(s)
Células Madre Mesenquimatosas/citología , Tratamiento Basado en Trasplante de Células y Tejidos , Células Endoteliales/citología , Humanos , Células Madre Mesenquimatosas/inmunología , Trombosis/sangreRESUMEN
BACKGROUND: Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). In the murine experimental autoimmune encephalomyelitis (EAE) model of MS, T regulatory (Treg) cell therapy has proved to be beneficial, but generation of stable CNS-targeting Tregs needs further development. Here, we propose gene engineering to achieve CNS-targeting Tregs from naïve CD4 cells and demonstrate their efficacy in the EAE model. METHODS: CD4+ T cells were modified utilizing a lentiviral vector system to express a chimeric antigen receptor (CAR) targeting myelin oligodendrocyte glycoprotein (MOG) in trans with the murine FoxP3 gene that drives Treg differentiation. The cells were evaluated in vitro for suppressive capacity and in C57BL/6 mice to treat EAE. Cells were administered by intranasal (i.n.) cell delivery. RESULTS: The engineered Tregs demonstrated suppressive capacity in vitro and could efficiently access various regions in the brain via i.n cell delivery. Clinical score 3 EAE mice were treated and the engineered Tregs suppressed ongoing encephalomyelitis as demonstrated by reduced disease symptoms as well as decreased IL-12 and IFNgamma mRNAs in brain tissue. Immunohistochemical markers for myelination (MBP) and reactive astrogliosis (GFAP) confirmed recovery in mice treated with engineered Tregs compared to controls. Symptom-free mice were rechallenged with a second EAE-inducing inoculum but remained healthy, demonstrating the sustained effect of engineered Tregs. CONCLUSION: CNS-targeting Tregs delivered i.n. localized to the CNS and efficiently suppressed ongoing inflammation leading to diminished disease symptoms.
Asunto(s)
Ingeniería Celular/métodos , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/terapia , Factores de Transcripción Forkhead/administración & dosificación , Terapia Genética/métodos , Receptores de Antígenos de Linfocitos T/administración & dosificación , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Administración Intranasal , Animales , Línea Celular , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/metabolismo , Encefalomielitis Autoinmune Experimental/genética , Femenino , Factores de Transcripción Forkhead/genética , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Vectores Genéticos/inmunología , Lentivirus/genética , Lentivirus/inmunología , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T Reguladores/trasplanteRESUMEN
BACKGROUND: Prolonged cold ischemia is frequently associated with a greater risk of delayed graft function and enhanced graft failure. We hypothesized that media, combining a high oxygen-dissolving capacity with specific qualities of organ preservation solutions, would be more efficient in reducing immediate ischemia-reperfusion injury from organs stored long term compared with standard preservation media. METHODS: Kidneys retrieved from brain-dead pigs were flushed using either cold histidine-tryptophan-ketoglutarate (HTK) or oxygen-precharged emulsion composed of 75% HTK and 25% perfluorohexyloctane. After 18 h of cold ischemia the kidneys were transplanted into allogeneic recipients and assessed for adenosine triphosphate content, morphology, and expression of genes related to hypoxia, environmental stress, inflammation, and apoptosis. RESULTS: Compared with HTK-flushed kidneys, organs preserved using oxygen-precharged HTK-perfluorohexyloctane emulsion had increased elevated adenosine triphosphate content and a significantly lower gene expression of hypoxia inducible factor-1α, vascular endothelial growth factor, interleukin-1α, tumor necrosis factor-α, interferon-α, JNK-1, p38, cytochrome-c, Bax, caspase-8, and caspase-3 at all time points assessed. In contrast, the mRNA expression of Bcl-2 was significantly increased. CONCLUSIONS: The present study has demonstrated that in brain-dead pigs the perfusion of kidneys with oxygen-precharged HTK-perfluorohexyloctane emulsion results in significantly reduced inflammation, hypoxic injury, and apoptosis and cellular integrity and energy content are well maintained. Histologic examination revealed less tubular, vascular, and glomerular changes in the emulsion-perfused tissue compared with the HTK-perfused counterparts. The concept of perfusing organs with oxygen-precharged emulsion based on organ preservation media represents an efficient alternative for improved organ preservation.
Asunto(s)
Trasplante de Riñón , Riñón/irrigación sanguínea , Oxígeno/farmacología , Daño por Reperfusión/prevención & control , Adenosina Trifosfato/metabolismo , Animales , Muerte Encefálica , Citocinas/genética , Emulsiones , Femenino , Glucosa/uso terapéutico , Masculino , Manitol/uso terapéutico , Cloruro de Potasio/uso terapéutico , Procaína/uso terapéutico , ARN Mensajero/análisis , PorcinosRESUMEN
BACKGROUND: Tumor vessels in glioma are molecularly and functionally abnormal, contributing to treatment resistance. Proteins differentially expressed in glioma vessels can change vessel phenotype and be targeted for therapy. ELTD1 (Adgrl4) is an orphan member of the adhesion G-protein-coupled receptor family upregulated in glioma vessels and has been suggested as a potential therapeutic target. However, the role of ELTD1 in regulating vessel function in glioblastoma is poorly understood. METHODS: ELTD1 expression in human gliomas and its association with patient survival was determined using tissue microarrays and public databases. The role of ELTD1 in regulating tumor vessel phenotype was analyzed using orthotopic glioma models and ELTD1-/- mice. Endothelial cells isolated from murine gliomas were transcriptionally profiled to determine differentially expressed genes and pathways. The consequence of ELTD1 deletion on glioma immunity was determined by treating tumor-bearing mice with PD-1-blocking antibodies. RESULTS: ELTD1 levels were upregulated in human glioma vessels, increased with tumor malignancy, and were associated with poor patient survival. Progression of orthotopic gliomas was not affected by ELTD1 deletion, however, tumor vascular function was improved in ELTD1-/- mice. Bioinformatic analysis of differentially expressed genes indicated increased inflammatory response and decreased proliferation in tumor endothelium in ELTD1-/- mice. Consistent with an enhanced inflammatory response, ELTD1 deletion improved T-cell infiltration in GL261-bearing mice after PD-1 checkpoint blockade. CONCLUSION: Our data demonstrate that ELTD1 participates in inducing vascular dysfunction in glioma, and suggest that targeting of ELTD1 may normalize the vessels and improve the response to immunotherapy.