RESUMEN
Tumor associated macrophages (TAMs), which differentiate from circulating monocytes, are pervasive across human cancers and comprise heterogeneous populations. The contribution of tumor-derived signals to TAM heterogeneity is not well understood. In particular, tumors release both soluble factors and extracellular vesicles (EVs), whose respective impact on TAM precursors may be different. Here, we show that triple negative breast cancer cells (TNBCs) release EVs and soluble molecules promoting monocyte differentiation toward distinct macrophage fates. EVs specifically promoted proinflammatory macrophages bearing an interferon response signature. The combination in TNBC EVs of surface CSF-1 promoting survival and cargoes promoting cGAS/STING or other activation pathways led to differentiation of this particular macrophage subset. Notably, macrophages expressing the EV-induced signature were found among patients' TAMs. Furthermore, higher expression of this signature was associated with T cell infiltration and extended patient survival. Together, this data indicates that TNBC-released CSF-1-bearing EVs promote a tumor immune microenvironment associated with a better prognosis in TNBC patients.
Asunto(s)
Vesículas Extracelulares , Neoplasias de la Mama Triple Negativas , Vesículas Extracelulares/fisiología , Humanos , Macrófagos , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is presenting as a systemic disease associated with vascular inflammation and endothelial injury. Severe forms of SARS-CoV-2 infection induce acute respiratory distress syndrome (ARDS) and there is still an ongoing debate on whether COVID-19 ARDS and its perfusion defect differs from ARDS induced by other causes. Beside pro-inflammatory cytokines (such as interleukin-1 ß [IL-1ß] or IL-6), several main pathological phenomena have been seen because of endothelial cell (EC) dysfunction: hypercoagulation reflected by fibrin degradation products called D-dimers, micro- and macrothrombosis and pathological angiogenesis. Direct endothelial infection by SARS-CoV-2 is not likely to occur and ACE-2 expression by EC is a matter of debate. Indeed, endothelial damage reported in severely ill patients with COVID-19 could be more likely secondary to infection of neighboring cells and/or a consequence of inflammation. Endotheliopathy could give rise to hypercoagulation by alteration in the levels of different factors such as von Willebrand factor. Other than thrombotic events, pathological angiogenesis is among the recent findings. Overexpression of different proangiogenic factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF-2) or placental growth factors (PlGF) have been found in plasma or lung biopsies of COVID-19 patients. Finally, SARS-CoV-2 infection induces an emergency myelopoiesis associated to deregulated immunity and mobilization of endothelial progenitor cells, leading to features of acquired hematological malignancies or cardiovascular disease, which are discussed in this review. Altogether, this review will try to elucidate the pathophysiology of thrombotic complications, pathological angiogenesis and EC dysfunction, allowing better insight in new targets and antithrombotic protocols to better address vascular system dysfunction. Since treating SARS-CoV-2 infection and its potential long-term effects involves targeting the vascular compartment and/or mobilization of immature immune cells, we propose to define COVID-19 and its complications as a systemic vascular acquired hemopathy.
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COVID-19/metabolismo , Mielopoyesis , Neovascularización Patológica/metabolismo , Síndrome de Dificultad Respiratoria/metabolismo , SARS-CoV-2/metabolismo , Trombosis/metabolismo , COVID-19/patología , COVID-19/terapia , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Endoteliales/virología , Productos de Degradación de Fibrina-Fibrinógeno/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Humanos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Proteínas de la Membrana/metabolismo , Neovascularización Patológica/patología , Neovascularización Patológica/terapia , Neovascularización Patológica/virología , Síndrome de Dificultad Respiratoria/patología , Síndrome de Dificultad Respiratoria/terapia , Síndrome de Dificultad Respiratoria/virología , Trombosis/patología , Trombosis/terapia , Trombosis/virología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor de von Willebrand/metabolismoRESUMEN
BACKGROUND: Coronavirus disease 2019 (COVID-19) is a respiratory disease associated with endotheliitis and microthrombosis. OBJECTIVES: To correlate endothelial dysfunction to in-hospital mortality in a bi-centric cohort of COVID-19 adult patients. METHODS: Consecutive ambulatory and hospitalized patients with laboratory-confirmed COVID-19 were enrolled. A panel of endothelial biomarkers and von Willebrand factor (VWF) multimers were measured in each patient ≤ 48 h following admission. RESULTS: Study enrolled 208 COVID-19 patients of whom 23 were mild outpatients and 189 patients hospitalized after admission. Most of endothelial biomarkers tested were found increased in the 89 critical patients transferred to intensive care unit. However, only von Willebrand factor antigen (VWF:Ag) scaled according to clinical severity, with levels significantly higher in critical patients (median 507%, IQR 428-596) compared to non-critical patients (288%, 230-350, p < 0.0001) or COVID-19 outpatients (144%, 133-198, p = 0.007). Moreover, VWF high molecular weight multimers (HMWM) were significantly higher in critical patients (median ratio 1.18, IQR 0.86-1.09) compared to non-critical patients (0.96, 1.04-1.39, p < 0.001). Among all endothelial biomarkers measured, ROC curve analysis identified a VWF:Ag cut-off of 423% as the best predictor for in-hospital mortality. The accuracy of VWF:Ag was further confirmed in a Kaplan-Meier estimator analysis and a Cox proportional Hazard model adjusted on age, BMI, C-reactive protein and D-dimer levels. CONCLUSION: VWF:Ag is a relevant predictive factor for in-hospital mortality in COVID-19 patients. More than a biomarker, we hypothesize that VWF, including excess of HMWM forms, drives microthrombosis in COVID-19.
Asunto(s)
COVID-19/sangre , COVID-19/mortalidad , Pandemias , SARS-CoV-2 , Factor de von Willebrand/metabolismo , Adulto , Anciano , Biomarcadores/sangre , Biomarcadores/química , COVID-19/fisiopatología , Estudios Transversales , Endotelio Vascular/fisiopatología , Femenino , Mortalidad Hospitalaria , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Peso Molecular , Paris/epidemiología , Modelos de Riesgos Proporcionales , Multimerización de Proteína , Índice de Severidad de la Enfermedad , Trombosis/sangre , Trombosis/etiología , Factor de von Willebrand/químicaRESUMEN
BACKGROUND: The prevalence of allergy to cat is expanding worldwide. Allergen-specific immunotherapy (AIT) has advantages over symptomatic pharmacotherapy and promises long-lasting disease control in allergic patients. However, there is still a need to improve cat AIT regarding efficacy, safety, and adherence to the treatment. Here, we aim to boost immune tolerance to the major cat allergen Fel d 1 by increasing the anti-inflammatory activity of AIT with the established immunomodulatory adjuvant CpG, but at a higher dose than previously used in AIT. METHODS: Together with CpG, we used endotoxin-free Fel d 1 as therapeutic allergen throughout the study in a BALB/c model of allergy to Fel d 1, thus mimicking the conditions of human AIT trials. Multidimensional immune phenotyping including mass cytometry (CyTOF) was applied to analyze AIT-specific immune signatures. RESULTS: We show that AIT with high-dose CpG in combination with endotoxin-free Fel d 1 reverts all major hallmarks of allergy. High-dimensional CyTOF analysis of the immune cell signatures initiating and sustaining the AIT effect indicates the simultaneous engagement of both, the pDC-Treg and B-cell axis, with the emergence of a systemic GATA3+ FoxP3hi biTreg population. The regulatory immune signature also suggests the involvement of the anti-inflammatory TNF/TNFR2 signaling cascade in NK and B cells at an early stage and in Tregs later during AIT. CONCLUSION: Our results highlight the potential of CpG adjuvant in a novel formulation to be further exploited for inducing allergen-specific tolerance in patients with cat allergy or other allergic diseases.
Asunto(s)
Glicoproteínas/inmunología , Hipersensibilidad , Receptores Tipo II del Factor de Necrosis Tumoral , Alérgenos , Animales , Gatos , Desensibilización Inmunológica , Modelos Animales de Enfermedad , Humanos , Hipersensibilidad/terapia , Tolerancia Inmunológica , RatonesRESUMEN
BACKGROUND: Coronavirus disease-2019 (COVID-19), a respiratory disease has been associated with ischemic complications, coagulation disorders, and an endotheliitis. OBJECTIVES: To explore endothelial damage and activation-related biomarkers in COVID-19 patients with criteria of hospitalization for referral to intensive care unit (ICU) and/or respiratory worsening. METHODS: Analysis of endothelial and angiogenic soluble markers in plasma from patients at admission. RESULTS: Study enrolled 40 consecutive COVID-19 patients admitted to emergency department that fulfilled criteria for hospitalization. Half of them were admitted in conventional wards without any ICU transfer during hospitalization; whereas the 20 others were directly transferred to ICU. Patients transferred in ICU were more likely to have lymphopenia, decreased SpO2 and increased D-dimer, CRP and creatinine levels. In those patients, soluble E-selectin and angiopoietin-2 were significantly increased (p value at 0.009 and 0.003, respectively). Increase in SELE gene expression (gene coding for E-selectin protein) was confirmed in an independent cohort of 32 patients using a whole blood gene expression profile analysis. In plasma, we found a strong association between angiopoetin-2 and CRP, creatinine and D-dimers (with p value at 0.001, 0.001 and 0.003, respectively). ROC curve analysis identified an Angiopoietin-2 cut-off of 5000 pg/mL as the best predictor for ICU outcome (Se = 80.1%, Sp = 70%, PPV = 72.7%, NPV = 77%), further confirmed in multivariate analysis after adjustment for creatinine, CRP or D-dimers. CONCLUSION: Angiopoietin-2 is a relevant predictive factor for ICU direct admission in COVID-19 patients. This result showing an endothelial activation reinforces the hypothesis of a COVID-19-associated microvascular dysfunction.
Asunto(s)
Angiopoyetina 2/sangre , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/terapia , Endotelio Vascular/metabolismo , Unidades de Cuidados Intensivos , Neumonía Viral/sangre , Neumonía Viral/terapia , Anciano , Betacoronavirus , Biomarcadores/sangre , COVID-19 , Cuidados Críticos/métodos , Selectina E/sangre , Femenino , Perfilación de la Expresión Génica , Hospitalización , Humanos , Masculino , Persona de Mediana Edad , Pandemias , Admisión del Paciente , Estudios Prospectivos , Respiración Artificial , SARS-CoV-2RESUMEN
RATIONALE: A rapid and massive influx of inflammatory cells occurs into ischemic area after myocardial infarction (MI), resulting in local release of cytokines and growth factors. Yet, the mechanisms regulating their production are not fully explored. The release of extracellular vesicles (EVs) in the interstitial space curbs important biological functions, including inflammation, and influences the development of cardiovascular diseases. To date, there is no evidence for in situ release of cardiac EVs after MI. OBJECTIVE: The present study tested the hypothesis that local EV generation in the infarcted heart coordinates cardiac inflammation after MI. METHODS AND RESULTS: Coronary artery ligation in mice transiently increases EV levels in the left ventricle when compared with sham animals. EVs from infarcted hearts were characterized as large vesicles (252±18 nm) expressing cardiomyocyte and endothelial markers and small EVs (118±4 nm) harboring exosomal markers, such as CD (cluster of differentiation) 63 and CD9. Cardiac large EVs generated after MI, but not small EVs or sham EVs, increased the release of IL (interleukin)-6, CCL (chemokine ligand) 2, and CCL7 from fluorescence-activated cell-sorted Ly6C+ cardiac monocytes. EVs of similar diameter were also isolated from fragments of interventricular septum obtained from patients undergoing aortic valve replacement, thus supporting the clinical relevance of our findings in mice. CONCLUSIONS: The present study demonstrates that acute MI transiently increases the generation of cardiac EVs characterized as both exosomes and microvesicles, originating mainly from cardiomyocytes and endothelial cells. EVs accumulating in the ischemic myocardium are rapidly taken up by infiltrating monocytes and regulate local inflammatory responses.
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Vesículas Extracelulares/patología , Infarto del Miocardio/patología , Miocarditis/etiología , Animales , Biomarcadores/metabolismo , Quimiocina CCL2/metabolismo , Quimiocina CCL7/metabolismo , Vasos Coronarios , Células Endoteliales/metabolismo , Exosomas , Vesículas Extracelulares/metabolismo , Interleucina-6/metabolismo , Ligadura , Masculino , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/complicaciones , Infarto del Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patologíaRESUMEN
While blocking tumor growth by targeting autophagy is well established, its role on the infiltration of natural killer (NK) cells into tumors remains unknown. Here, we investigate the impact of targeting autophagy gene Beclin1 (BECN1) on the infiltration of NK cells into melanomas. We show that, in addition to inhibiting tumor growth, targeting BECN1 increased the infiltration of functional NK cells into melanoma tumors. We provide evidence that driving NK cells to the tumor bed relied on the ability of autophagy-defective tumors to transcriptionally overexpress the chemokine gene CCL5 Such infiltration and tumor regression were abrogated by silencing CCL5 in BECN1-defective tumors. Mechanistically, we show that the up-regulated expression of CCL5 occurred through the activation of its transcription factor c-Jun by a mechanism involving the impairment of phosphatase PP2A catalytic activity and the subsequent activation of JNK. Similar to BECN1, targeting other autophagy genes, such as ATG5, p62/SQSTM1, or inhibiting autophagy pharmacologically by chloroquine, also induced the expression of CCL5 in melanoma cells. Clinically, a positive correlation between CCL5 and NK cell marker NKp46 expression was found in melanoma patients, and a high expression level of CCL5 was correlated with a significant improvement of melanoma patients' survival. We believe that this study highlights the impact of targeting autophagy on the tumor infiltration by NK cells and its benefit as a novel therapeutic approach to improve NK-based immunotherapy.
Asunto(s)
Autofagia/fisiología , Quimiocina CCL5/metabolismo , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/patología , Melanoma/metabolismo , Melanoma/patología , Animales , Beclina-1/metabolismo , Línea Celular Tumoral , Humanos , Inmunoterapia/métodos , Ratones , Ratones Endogámicos C57BL , Receptor 1 Gatillante de la Citotoxidad Natural/metabolismoRESUMEN
OBJECTIVE: Previous studies suggested that microRNA-21 may be upregulated in the liver in non-alcoholic steatohepatitis (NASH), but its role in the development of this disease remains unknown. This study aimed to determine the role of microRNA-21 in NASH. DESIGN: We inhibited or suppressed microRNA-21 in different mouse models of NASH: (a) low-density lipoprotein receptor-deficient (Ldlr-/-) mice fed a high-fat diet and treated with antagomir-21 or antagomir control; (b) microRNA-21-deficient and wild-type mice fed a methionine-choline-deficient (MCD) diet; (c) peroxisome proliferation-activator receptor α (PPARα)-deficient mice fed an MCD diet and treated with antagomir-21 or antagomir control. We assessed features of NASH and determined liver microRNA-21 levels and cell localisation. MicroRNA-21 levels were also quantified in the liver of patients with NASH, bland steatosis or normal liver and localisation was determined. RESULTS: Inhibiting or suppressing liver microRNA-21 expression reduced liver cell injury, inflammation and fibrogenesis without affecting liver lipid accumulation in Ldlr-/- fed a high-fat diet and in wild-type mice fed an MCD diet. Liver microRNA-21 was overexpressed, primarily in biliary and inflammatory cells, in mouse models as well as in patients with NASH, but not in patients with bland steatosis. PPARα, a known microRNA-21 target, implicated in NASH, was decreased in the liver of mice with NASH and restored following microRNA-21 inhibition or suppression. The effect of antagomir-21 was lost in PPARα-deficient mice. CONCLUSIONS: MicroRNA-21 inhibition or suppression decreases liver injury, inflammation and fibrosis, by restoring PPARα expression. Antagomir-21 might be a future therapeutic strategy for NASH.
Asunto(s)
MicroARNs/metabolismo , Enfermedad del Hígado Graso no Alcohólico , Oligonucleótidos , PPAR alfa/metabolismo , Animales , Dieta Alta en Grasa , Perfilación de la Expresión Génica/métodos , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Metabolismo de los Lípidos , Lipoproteínas LDL/metabolismo , Ratones , MicroARNs/antagonistas & inhibidores , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/prevención & control , Oligonucleótidos/metabolismo , Oligonucleótidos/farmacología , PPAR alfa/antagonistas & inhibidoresRESUMEN
RATIONALE FOR STUDY: MicroRNAs (miRNAs) are small noncoding RNAs that regulate protein expression at post-transcriptional level. We hypothesized that a specific pool of endothelial miRNAs could be selectively regulated by flow conditions and inflammatory signals, and as such be involved in the development of atherosclerosis. OBJECTIVE: To identify miRNAs, called atheromiRs, which are selectively regulated by shear stress and oxidized low-density lipoproteins (oxLDL), and to determine their role in atherogenesis. METHODS AND RESULTS: Large-scale miRNA profiling in HUVECs identified miR-92a as an atheromiR candidate, whose expression is preferentially upregulated by the combination of low shear stress (SS) and atherogenic oxLDL. Ex vivo analysis of atheroprone and atheroprotected areas of mouse arteries and human atherosclerotic plaques demonstrated the preferential expression of miR-92a in atheroprone low SS regions. In Ldlr(-/-) mice, miR-92a expression was markedly enhanced by hypercholesterolemia, in particular in atheroprone areas of the aorta. Assessment of endothelial inflammation in gain- and loss-of-function experiments targeting miR-92a expression revealed that miR-92a regulated endothelial cell activation by oxLDL, more specifically under low SS conditions, which was associated with modulation of Kruppel-like factor 2 (KLF2), Kruppel-like factor 4 (KLF4), and suppressor of cytokine signaling 5. miR-92a expression was regulated by signal transducer and activator of transcription 3 in SS- and oxLDL-dependent manner. Furthermore, specific in vivo blockade of miR-92a expression in Ldlr(-/-) mice reduced endothelial inflammation and altered the development of atherosclerosis, decreasing plaque size and promoting a more stable lesion phenotype. CONCLUSIONS: Upregulation of miR-92a by oxLDL in atheroprone areas promotes endothelial activation and the development of atherosclerotic lesions. Therefore, miR-92a antagomir seems as a new atheroprotective therapeutic strategy.
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Aterosclerosis/genética , Aterosclerosis/prevención & control , Regulación hacia Abajo/genética , Endotelio Vascular/metabolismo , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Animales , Aterosclerosis/patología , Endotelio Vascular/patología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Factor 4 Similar a Kruppel , Masculino , Ratones , Ratones Noqueados , MicroARNs/biosíntesis , Regulación hacia Arriba/genéticaRESUMEN
BACKGROUND: Endothelial colony forming cells (ECFC) represent a subpopulation of endothelial progenitor cells involved in endothelial repair. The activation of procoagulant mechanisms associated with the vascular wall's inflammatory responses to injury plays a crucial role in the induction and progression of atherosclerosis. However, little is known about ECFC proinflammatory potential. AIMS: To explore the role of the thrombin receptor PAR-1 proinflammatory effects on ECFC chemotaxis/recruitment capacity. METHODS AND RESULTS: The expression of 30 genes known to be associated with inflammation and chemotaxis was quantified in ECFC by real-time qPCR. PAR-1 activation with the SFLLRN peptide (PAR-1-ap) resulted in a significant increase in nine chemotaxis-associated genes expression, including CCL2 and CCL3 whose receptors are present on ECFC. Furthermore, COX-2 expression was found to be dramatically up-regulated consequently to PAR-1 activation. COX-2 silencing with the specific COX-2-siRNA also triggered down-regulation of the nine target genes. Conditioned media (c.m.) from control-siRNA- and COX-2-siRNA-transfected ECFC, stimulated or not with PAR-1-ap, were produced and tested on ECFC capacity to recruit leukocytes in vitro as well in the muscle of ischemic hindlimb in a preclinical model. The capacity of the c.m. from ECFC stimulated with PAR-1-ap to recruit leukocytes was abrogated when COX-2 gene expression was silenced in vitro (in terms of U937 cells migration and adhesion to endothelial cells) as well as in vivo. Finally, the postnatal vasculogenic stem cell derived from infantile hemangioma tumor (HemSC) incubated with PAR-1-ap increased leukocyte recruitment in Matrigel(®) implant. CONCLUSIONS: PAR-1 activation in ECFC increases chemotactic gene expression and leukocyte recruitment at ischemic sites through a COX-2-dependent mechanism.
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Quimiotaxis , Ciclooxigenasa 2/metabolismo , Leucocitos/citología , Receptor PAR-1/metabolismo , Células Madre/citología , Animales , Aterosclerosis/metabolismo , Medios de Cultivo Condicionados , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células Endoteliales/citología , Sangre Fetal/citología , Citometría de Flujo , Regulación de la Expresión Génica , Hemangioma/inmunología , Humanos , Inflamación , Leucocitos/metabolismo , Masculino , Ratones , Ratones Desnudos , ARN Interferente Pequeño/metabolismo , Células U937Asunto(s)
Antígenos CD/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentales/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Animales , Antígenos CD/genética , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Ratones , Proteínas de Neoplasias/genética , Neoplasias Experimentales/genética , Receptor de Muerte Celular Programada 1/genética , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
Infantile hemangioma (IH) is the most common tumor of infancy. Hemangioma stem cells (HemSC) are a mesenchymal subpopulation isolated from IH CD133+ cells. HemSC can differentiate into endothelial and pericyte/smooth muscle cells and form vascular networks when injected in immune-deficient mice. α6-Integrin subunit has been implicated in the tumorgenicity of glioblastoma stem cells and the homing properties of hematopoietic, endothelial, and mesenchymal progenitor cells. Therefore, we investigated the possible function(s) of α6-integrin in HemSC. We documented α6-integrin expression in IH tumor specimens and HemSC by RT-qPCR and flow cytometry. We examined the effect of blocking or silencing α6-integrin on the adhesive and proliferative properties of HemSC in vitro and the vasculogenic and homing properties of HemSC in vivo. Targeting α6-integrin in cultured HemSC inhibited adhesion to laminin but had no effect on proliferation. Vessel-forming ability in Matrigel implants and hepatic homing after i.v. delivery were significantly decreased in α6-integrin siRNA-transfected HemSC. In conclusion, α6-integrin is required for HemSC adherence to laminin, vessel formation in vivo, and for homing to the liver. Thus, we uncovered an important role for α6 integrin in the vasculogenic properties of HemSC. Our results suggest that α6-integrin expression on HemSC could be a new target for antihemangioma therapy.
Asunto(s)
Hemangioma/metabolismo , Hemangioma/patología , Integrina alfa6/metabolismo , Neovascularización Fisiológica , Células Madre/metabolismo , Células Madre/patología , Animales , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular , Preescolar , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Lactante , Laminina/farmacología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Neovascularización Fisiológica/efectos de los fármacosRESUMEN
Upregulation of hypoxia-inducible transcription factor-1α (HIF-1α), through prolyl-hydroxylase domain protein (PHD) inhibition, can be thought of as a master switch that coordinates the expression of a wide repertoire of genes involved in regulating vascular growth and remodeling. We aimed to unravel the effect of specific PHD2 isoform silencing in cell-based strategies designed to promote therapeutic revascularization in patients with critical limb ischemia (CLI). PHD2 mRNA levels were upregulated whereas that of HIF-1α were downregulated in blood cells from patients with CLI. We therefore assessed the putative beneficial effects of PHD2 silencing on human bone marrow-derived mesenchymal stem cells (hBM-MSC)-based therapy. PHD2 silencing enhanced hBM-MSC therapeutic effect in an experimental model of CLI in Nude mice, through an upregulation of HIF-1α and its target gene, VEGF-A. In addition, PHD2-transfected hBM-MSC displayed higher protection against apoptosis in vitro and increased rate of survival in the ischemic tissue, as assessed by Fluorescence Molecular Tomography. Cotransfection with HIF-1α or VEGF-A short interfering RNAs fully abrogated the beneficial effect of PHD2 silencing on the proangiogenic capacity of hBM-MSC. We finally investigated the effect of PHD2 inhibition on the revascularization potential of ischemic targeted tissues in the diabetic pathological context. Inhibition of PHD-2 with shRNAs increased postischemic neovascularization in diabetic mice with CLI. This increase was associated with an upregulation of proangiogenic and proarteriogenic factors and was blunted by concomitant silencing of HIF-1α. In conclusion, silencing of PHD2, by the transient upregulation of HIF-1α and its target gene VEGF-A, might improve the efficiency of hBM-MSC-based therapies.
Asunto(s)
Trasplante de Células/métodos , Miembro Posterior/irrigación sanguínea , Prolina Dioxigenasas del Factor Inducible por Hipoxia/antagonistas & inhibidores , Isquemia/terapia , Células Madre Mesenquimatosas/citología , Inhibidores de Prolil-Hidroxilasa/uso terapéutico , Anciano , Animales , Apoptosis/fisiología , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Procedimientos Endovasculares/métodos , Humanos , Isquemia/enzimología , Recuperación del Miembro/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Persona de Mediana Edad , TransfecciónRESUMEN
Published clinical trials in patients with ischemic diseases show limited benefit of adult stem cell-based therapy, likely due to their restricted plasticity and commitment toward vascular cell lineage. We aim to uncover the potent regenerative ability of MesP1/stage-specific embryonic antigen 1 (SSEA-1)-expressing cardiovascular progenitors enriched from human embryonic stem cells (hESCs). Injection of only 10(4) hESC-derived SSEA-1(+) /MesP1(+) cells, or their progeny obtained after treatment with VEGF-A or PDGF-BB, was effective enough to enhance postischemic revascularization in immunodeficient mice with critical limb ischemia (CLI). However, the rate of incorporation of hESC-derived SSEA-1(+) /MesP1(+) cells and their derivatives in ischemic tissues was modest. Alternatively, these cells possessed a unique miR-21 signature that inhibited phosphotase and tensin homolog (PTEN) thereby activating HIF-1α and the systemic release of VEGF-A. Targeting miR-21 limited cell survival and inhibited their proangiogenic capacities both in the Matrigel model and in mice with CLI. We next assessed the impact of mR-21 in adult angiogenesis-promoting cells. We observed an impaired postischemic angiogenesis in miR-21-deficient mice. Notably, miR-21 was highly expressed in circulating and infiltrated monocytes where it targeted PTEN/HIF-1α/VEGF-A signaling and cell survival. As a result, miR-21-deficient mice displayed an impaired number of infiltrated monocytes and a defective angiogenic phenotype that could be partially restored by retransplantation of bone marrow-derived cells from wild-type littermates. hESC-derived SSEA-1(+) /MesP1(+) cells progenitor cells are powerful key integrators of therapeutic angiogenesis in ischemic milieu and miR-21 is instrumental in this process as well as in the orchestration of the biological activity of adult angiogenesis-promoting cells.
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Isquemia/terapia , MicroARNs/metabolismo , Miocardio/metabolismo , Trasplante de Células Madre , Células Madre/metabolismo , Animales , Linaje de la Célula , Supervivencia Celular/fisiología , Miembro Posterior/irrigación sanguínea , Humanos , Ratones , Neovascularización Fisiológica/genética , Transducción de Señal/fisiología , Trasplante de Células Madre/métodosRESUMEN
OBJECTIVE: To examine the relation of endothelial microparticles (EMPs) with cardiometabolic risk in the community. BACKGROUND: Circulating EMPs are small membrane vesicles released after endothelial cell injury. Endothelial microparticles are reportedly increased among individuals with a high burden of cardiovascular risk factors. However, prior investigations have been limited to small, highly selected samples. METHODS: We studied 844 individuals without a history of cardiovascular disease in the Framingham Offspring cohort (mean age 66 ± 9 years, 57% women). We used standardized flow cytometry methods to identify and quantify circulating CD144+ and CD31+/CD41- EMPs. We then used multivariable regression analyses to investigate the relations of EMP phenotypes with cardiovascular and metabolic risk factors. RESULTS: In multivariable analyses, the following cardiovascular risk factors were associated with one or more of the circulating EMP populations: hypertension (P = 0.025 for CD144+,), elevated triglycerides (P = 0.002 for CD144+, P < 0.0001 for CD31+/CD41-), and metabolic syndrome (P < 0.0001 for CD144+,). Overall, each tertile increase in the Framingham risk score corresponded to a 9% increase in log-CD31+/CD41- EMPs (P = 0.022). Furthermore, the presence of hypertriglyceridaemic waist status was associated with 38% higher levels of CD144+ EMPs (P < 0.0001) and 46% higher levels of CD31+/CD41- EMPs (P < 0.0001). CONCLUSION: In a large community-based sample, circulating EMP levels were associated with the presence of cardiometabolic risk factors, particularly dyslipidaemia. These data underscore the potential influence of high-risk metabolic profiles on endothelial integrity.
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Enfermedades Cardiovasculares/patología , Micropartículas Derivadas de Células/patología , Endotelio Vascular/patología , Síndrome Metabólico/patología , Anciano , Antígenos CD/metabolismo , Células Endoteliales/patología , Femenino , Humanos , Estudios Longitudinales , Masculino , Factores de RiesgoRESUMEN
Migration of circulating leukocytes from the vasculature into the surrounding tissue is an important component of the inflammatory response. Among the cell surface molecules identified as contributing to leukocyte extravasation is VCAM-1, expressed on activated vascular endothelium, which participates in all stages of leukocyte-endothelial interaction by binding to leukocyte surface expressed integrin VLA-4. However, not all VLA-4-mediated events can be linked to VCAM-1. A novel interaction between VLA-4 and endothelial Lutheran (Lu) blood group antigens and basal cell adhesion molecule (BCAM) proteins has been recently shown, suggesting that Lu/BCAM may have a role in leukocyte recruitments in inflamed tissues. Here, we assessed the participation of Lu/BCAM in the immunopathogenesis of crescentic glomerulonephritis. High expression of Lu/BCAM in glomeruli of mice with rapidly progressive glomerulonephritis suggests a potential role for the local expression of Lu/BCAM in nephritogenic recruitment of leukocytes. Genetic deficiency of Lu/BCAM attenuated glomerular accumulation of T cells and macrophages, crescent formation, and proteinuria, correlating with reduced fibrin and platelet deposition in glomeruli. Furthermore, we found a pro-adhesive interaction between human monocyte α4ß1 integrin and Lu/BCAM proteins. Thus, Lu/BCAM may have a critical role in facilitating the accumulation of monocytes and macrophages, thereby exacerbating renal injury.
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Enfermedad por Anticuerpos Antimembrana Basal Glomerular/metabolismo , Adhesión Celular , Riñón/metabolismo , Glicoproteínas de Membrana/metabolismo , Monocitos/metabolismo , Animales , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/genética , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/inmunología , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/patología , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/prevención & control , Autoanticuerpos , Moléculas de Adhesión Celular , Quimiotaxis de Leucocito , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Integrina alfa4beta1/metabolismo , Riñón/inmunología , Riñón/ultraestructura , Sistema del Grupo Sanguíneo Lutheran , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Monocitos/inmunología , Unión Proteica , Insuficiencia Renal/genética , Insuficiencia Renal/inmunología , Insuficiencia Renal/metabolismo , Insuficiencia Renal/prevención & control , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/metabolismo , Factores de TiempoRESUMEN
OBJECTIVES: We studied whether plasma levels of angiogenic factors VEGF and placental growth factor (PlGF) in coronary artery disease patients or undergoing cardiac surgery are modified, and whether those factors modulate endothelial progenitor's angiogenic potential. METHODS AND RESULTS: A total of 143 patients' plasmas from two different studies were analyzed (30 coronary artery disease patients, 30 patients with stable angina, coupled with 30 age and sex-matched controls; 53 patients underwent cardiac surgery). Among factors screened, only PlGF was found significantly increased in these pathological populations. PlGF-1 and PlGF-2 were then tested on human endothelial-colony-forming cells (ECFCs). We found that PlGF-1 and PlGF-2 induce VEGFR1 phosphorylation and potentiate ECFCs tubulogenesis in vitro. ECFCs VEGFR1 was further inhibited using a specific small interfering RNA (siRNA) and the chemical compound 4321. We then observed that the VEGFR1-siRNA and the compound 4321 decrease ECFCs tubulogenesis potential in vitro. Finally, we tested the compound 4321 in the preclinical Matrigel(®)-plug model with C57Bl/6J mice as well as in the murine hindlimb ischemia model. We found that 4321 inhibited the plug vascularization, attested by the hemoglobin content and the VE-Cadherin expression level and that 4321 inhibited the post-ischemic revascularization. CONCLUSION: PlGF plasma levels were found increased in cardiovascular patients. Disrupting PlGF/VEGFR1 pathway could modulate ECFC-induced tubulogenesis, the cell type responsible for newly formed vessels in vivo.
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Diferenciación Celular , Células Endoteliales/metabolismo , Células Madre/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Procedimientos Quirúrgicos Cardíacos , Diferenciación Celular/efectos de los fármacos , Ensayos de Migración Celular , Proliferación Celular/efectos de los fármacos , Colágeno/metabolismo , Ensayo de Unidades Formadoras de Colonias , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/patología , Combinación de Medicamentos , Células Endoteliales/efectos de los fármacos , Miembro Posterior/irrigación sanguínea , Miembro Posterior/patología , Humanos , Isquemia/patología , Laminina/metabolismo , Proteínas de la Membrana/sangre , Ratones Endogámicos C57BL , Neovascularización Fisiológica/efectos de los fármacos , Fosforilación/efectos de los fármacos , Proteoglicanos/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/farmacología , Células Madre/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/sangreRESUMEN
COVID-19 and infectious diseases have been included in strategic development goals (SDG) of United Nations (UN). The SARS-CoV-2 pandemic has unveiled complex pathophysiological mechanisms underpinning COVID-19, notably inducing a systemic acquired vascular hemopathy characterized by endothelial dysfunction and intussusceptive angiogenesis, a rapid vascular remodeling process identified as a hallmark in severe COVID-19 cases affecting pulmonary and cardiac tissues. Stem cell migration have been proposed as significant regulators of this neoangiogenic process. In a monocentric cross-sectional study, through spectral flow cytometry analysis of peripheral blood mononuclear cells, we identified a distinct stem cell subpopulation mobilized in critical COVID-19. Indeed, by an unsupervised analysis generating a UMAP representation we highlighted eleven different clusters in critical and non-critical COVID-19 patients. Only one cluster was significantly associated to critical COVID-19 compared to non-critical patients. This cluster expressed the markers: CD45dim, CD34+, CD117+, CD147+, and CD143+, and were negative for CD133. Higher level of expression of hemangioblast markers CD143 were found in critical COVID-19 patients. This population, indicative of hemangioblast-like cells, suggests a key role in COVID-19-related neoangiogenesis, potentially driving the severe vascular complications observed. Our findings underscore the need for further investigation into the contributions of adult stem cells in COVID-19 pathology, offering new insights into therapeutic targets and interventions.
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COVID-19 , SARS-CoV-2 , Humanos , COVID-19/patología , COVID-19/metabolismo , Masculino , Femenino , Persona de Mediana Edad , Estudios Transversales , Hemangioblastos/metabolismo , Anciano , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Biomarcadores/metabolismo , Adulto , Leucocitos Mononucleares/metabolismo , Células Madre/metabolismo , Angiogénesis , BasiginaRESUMEN
DNA methylation (DNAme) is a key epigenetic mark that regulates critical biological processes maintaining overall genome stability. Given its pleiotropic function, studies of DNAme dynamics are crucial, but currently available tools to interfere with DNAme have limitations and major cytotoxic side effects. Here, we present cell models that allow inducible and reversible DNAme modulation through DNMT1 depletion. By dynamically assessing whole genome and locus-specific effects of induced passive demethylation through cell divisions, we reveal a cooperative activity between DNMT1 and DNMT3B, but not of DNMT3A, to maintain and control DNAme. We show that gradual loss of DNAme is accompanied by progressive and reversible changes in heterochromatin, compartmentalization, and peripheral localization. DNA methylation loss coincides with a gradual reduction of cell fitness due to G1 arrest, with minor levels of mitotic failure. Altogether, this system allows DNMTs and DNA methylation studies with fine temporal resolution, which may help to reveal the etiologic link between DNAme dysfunction and human disease.
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ADN (Citosina-5-)-Metiltransferasa 1 , Metilación de ADN , ADN Metiltransferasa 3A , Epigenómica , Humanos , División Celular , Heterocromatina/genética , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN Metiltransferasa 3A/genética , Línea CelularRESUMEN
Mucosal-associated invariant T (MAIT) cells harbor evolutionarily conserved TCRs, suggesting important functions. As human and mouse MAIT functional programs appear distinct, the evolutionarily conserved MAIT functional features remain unidentified. Using species-specific tetramers coupled to single-cell RNA sequencing, we characterized MAIT cell development in six species spanning 110 million years of evolution. Cross-species analyses revealed conserved transcriptional events underlying MAIT cell maturation, marked by ZBTB16 induction in all species. MAIT cells in human, sheep, cattle, and opossum acquired a shared type-1/17 transcriptional program, reflecting ancestral features. This program was also acquired by human iNKT cells, indicating common differentiation for innate-like T cells. Distinct type-1 and type-17 MAIT subsets developed in rodents, including pet mice and genetically diverse mouse strains. However, MAIT cells further matured in mouse intestines to acquire a remarkably conserved program characterized by concomitant expression of type-1, type-17, cytotoxicity, and tissue-repair genes. Altogether, the study provides a unifying view of the transcriptional features of innate-like T cells across evolution.