RESUMEN
BACKGROUND: Pericytes are capillary-associated mural cells involved in the maintenance and stability of the vascular network. Although aging is one of the main risk factors for cardiovascular disease, the consequences of aging on cardiac pericytes are unknown. METHODS: In this study, we have combined single-nucleus RNA sequencing and histological analysis to determine the effects of aging on cardiac pericytes. Furthermore, we have conducted in vivo and in vitro analysis of RGS5 (regulator of G-protein signaling 5) loss of function and finally have performed pericytes-fibroblasts coculture studies to understand the effect of RGS5 deletion in pericytes on the neighboring fibroblasts. RESULTS: Aging reduced the pericyte area and capillary coverage in the murine heart. Single-nucleus RNA sequencing analysis further revealed that the expression of Rgs5 was reduced in cardiac pericytes from aged mice. In vivo and in vitro studies showed that the deletion of RGS5 impaired cardiac function, induced fibrosis, and morphological changes in pericytes characterized by a profibrotic gene expression signature and the expression of different ECM (extracellular matrix) components and growth factors, for example, TGFB2 and PDGFB. Indeed, culturing fibroblasts with the supernatant of RGS5-deficient pericytes induced their activation as evidenced by the increased expression of αSMA (alpha smooth muscle actin) in a TGFß (transforming growth factor beta)2-dependent mechanism. CONCLUSIONS: Our results have identified RGS5 as a crucial regulator of pericyte function during cardiac aging. The deletion of RGS5 causes cardiac dysfunction and induces myocardial fibrosis, one of the hallmarks of cardiac aging.
Asunto(s)
Fibroblastos , Fibrosis , Pericitos , Proteínas RGS , Pericitos/metabolismo , Pericitos/patología , Animales , Proteínas RGS/genética , Proteínas RGS/metabolismo , Proteínas RGS/deficiencia , Fibroblastos/metabolismo , Fibroblastos/patología , Ratones , Células Cultivadas , Envejecimiento/metabolismo , Envejecimiento/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/metabolismo , Miocardio/patología , Masculino , Técnicas de CocultivoRESUMEN
BACKGROUND: Circular RNAs (circRNAs) are generated by back splicing of mostly mRNAs and are gaining increasing attention as a novel class of regulatory RNAs that control various cellular functions. However, their physiological roles and functional conservation in vivo are rarely addressed, given the inherent challenges of their genetic inactivation. Here, we aimed to identify locus conserved circRNAs in mice and humans, which can be genetically deleted due to retained intronic elements not contained in the mRNA host gene to eventually address functional conservation. METHODS AND RESULTS: Combining published endothelial RNA-sequencing data sets with circRNAs of the circATLAS databank, we identified locus-conserved circRNA retaining intronic elements between mice and humans. CRISPR/Cas9 mediated genetic depletion of the top expressed circRNA cZfp292 resulted in an altered endothelial morphology and aberrant flow alignment in the aorta in vivo. Consistently, depletion of cZNF292 in endothelial cells in vitro abolished laminar flow-induced alterations in cell orientation, paxillin localization and focal adhesion organization. Mechanistically, we identified the protein SDOS (syndesmos) to specifically interact with cZNF292 in endothelial cells by RNA-affinity purification and subsequent mass spectrometry analysis. Silencing of SDOS or its protein binding partner Syndecan-4, or mutation of the SDOS-cZNF292 binding site, prevented laminar flow-induced cytoskeletal reorganization thereby recapitulating cZfp292 knockout phenotypes. CONCLUSIONS: Together, our data reveal a hitherto unknown role of cZNF292/cZfp292 in endothelial flow responses, which influences endothelial shape.
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Proteínas de Unión al ADN , Células Endoteliales , Endotelio Vascular , ARN Circular , Factores de Transcripción , Animales , Humanos , Ratones , Circulación Sanguínea , Proteínas de Unión al ADN/genética , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones Endogámicos C57BL , Unión Proteica , ARN Circular/genética , ARN Circular/metabolismo , Sindecano-4/metabolismo , Factores de Transcripción/genéticaRESUMEN
Endothelial cells play an important role in maintenance of the vascular system and the repair after injury. Under proinflammatory conditions, endothelial cells can acquire a mesenchymal phenotype by a process named endothelial-to-mesenchymal transition (EndMT), which affects the functional properties of endothelial cells. Here, we investigated the epigenetic control of EndMT. We show that the histone demethylase JMJD2B is induced by EndMT-promoting, proinflammatory, and hypoxic conditions. Silencing of JMJD2B reduced TGF-ß2-induced expression of mesenchymal genes, prevented the alterations in endothelial morphology and impaired endothelial barrier function. Endothelial-specific deletion of JMJD2B in vivo confirmed a reduction of EndMT after myocardial infarction. EndMT did not affect global H3K9me3 levels but induced a site-specific reduction of repressive H3K9me3 marks at promoters of mesenchymal genes, such as Calponin (CNN1), and genes involved in TGF-ß signaling, such as AKT Serine/Threonine Kinase 3 (AKT3) and Sulfatase 1 (SULF1). Silencing of JMJD2B prevented the EndMT-induced reduction of H3K9me3 marks at these promotors and further repressed these EndMT-related genes. Our study reveals that endothelial identity and function is critically controlled by the histone demethylase JMJD2B, which is induced by EndMT-promoting, proinflammatory, and hypoxic conditions, and supports the acquirement of a mesenchymal phenotype.
Asunto(s)
Células Endoteliales/enzimología , Transición Epitelial-Mesenquimal , Histona Demetilasas con Dominio de Jumonji/metabolismo , Células Madre Mesenquimatosas/citología , Células Endoteliales/citología , Histonas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Células Madre Mesenquimatosas/enzimología , Factor de Crecimiento Transformador beta2/metabolismoRESUMEN
Cellular specialization and interaction with other cell types in cardiac tissue is essential for the coordinated function of cell populations in the heart. The complex interplay between cardiomyocytes, endothelial cells and fibroblasts is necessary for adaptation but can also lead to pathophysiological remodeling. To understand this complex interplay, we developed 3D vascularized cardiac tissue mimetics (CTM) to study heterocellular cross-talk in hypertrophic, hypoxic and fibrogenic environments. This 3D platform responds to physiologic and pathologic stressors and mimics the microenvironment of diseased tissue. In combination with endothelial cell fluorescence reporters, these cardiac tissue mimetics can be used to precisely visualize and quantify cellular and functional responses upon stress stimulation. Utilizing this platform, we demonstrate that stimulation of α/ß-adrenergic receptors with phenylephrine (PE) promotes cardiomyocyte hypertrophy, metabolic maturation and vascularization of CTMs. Increased vascularization was promoted by conditioned medium of PE-stimulated cardiomyocytes and blocked by inhibiting VEGF or upon ß-adrenergic receptor antagonist treatment, demonstrating cardiomyocyte-endothelial cross-talk. Pathophysiological stressors such as severe hypoxia reduced angiogenic sprouting and increased cell death, while TGF ß2 stimulation increased collagen deposition concomitant to endothelial-to-mesenchymal transition. In sum, we have developed a cardiac 3D culture system that reflects native cardiac tissue function, metabolism and morphology - and for the first time enables the tracking and analysis of cardiac vascularization dynamics in physiology and pathology.
Asunto(s)
Biomimética , Neovascularización Fisiológica , Ingeniería de Tejidos , Animales , Células Cultivadas , Femenino , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Masculino , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Fenilefrina/farmacología , Ratas Sprague-Dawley , Estrés Fisiológico/efectos de los fármacosRESUMEN
BACKGROUND: The majority of the human genome comprises noncoding sequences, which are in part transcribed as long noncoding RNAs (lncRNAs). lncRNAs exhibit multiple functions, including the epigenetic control of gene expression. In this study, the effect of the lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) on atherosclerosis was examined. METHODS: The effect of MALAT1 on atherosclerosis was determined in apolipoprotein E-deficient (Apoe-/-) MALAT1-deficient (Malat1-/-) mice that were fed with a high-fat diet and by studying the regulation of MALAT1 in human plaques. RESULTS: Apoe-/- Malat1-/- mice that were fed a high-fat diet showed increased plaque size and infiltration of inflammatory CD45+ cells compared with Apoe-/- Malat1+/+ control mice. Bone marrow transplantation of Apoe-/- Malat1-/- bone marrow cells in Apoe-/- Malat1+/+ mice enhanced atherosclerotic lesion formation, which suggests that hematopoietic cells mediate the proatherosclerotic phenotype. Indeed, bone marrow cells isolated from Malat1-/- mice showed increased adhesion to endothelial cells and elevated levels of proinflammatory mediators. Moreover, myeloid cells of Malat1-/- mice displayed enhanced adhesion to atherosclerotic arteries in vivo. The anti-inflammatory effects of MALAT1 were attributed in part to reduction of the microRNA miR-503. MALAT1 expression was further significantly decreased in human plaques compared with normal arteries and was lower in symptomatic versus asymptomatic patients. Lower levels of MALAT1 in human plaques were associated with a worse prognosis. CONCLUSIONS: Reduced levels of MALAT1 augment atherosclerotic lesion formation in mice and are associated with human atherosclerotic disease. The proatherosclerotic effects observed in Malat1-/- mice were mainly caused by enhanced accumulation of hematopoietic cells.
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Aorta/metabolismo , Aortitis/metabolismo , Aterosclerosis/metabolismo , Células de la Médula Ósea/metabolismo , Hematopoyesis , Placa Aterosclerótica , ARN Largo no Codificante/metabolismo , Animales , Aorta/patología , Aortitis/genética , Aortitis/patología , Aterosclerosis/genética , Aterosclerosis/patología , Células de la Médula Ósea/patología , Trasplante de Médula Ósea , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Regulación hacia Abajo , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , MicroARNs/genética , MicroARNs/metabolismo , ARN Largo no Codificante/genética , Transducción de SeñalRESUMEN
OBJECTIVE: Endothelial cells play important roles in tissue homeostasis and vascularization, a function that is impaired by aging. Here, we aim to decipher the role of the microenvironment underlying the impairment of endothelial cell functions by aging. APPROACH AND RESULTS: RNA sequencing of isolated cardiac endothelial cells derived from young and 18-month-old mouse hearts revealed that aging affects the endothelial expression of genes encoding extracellular matrix proteins, specifically the laminin ß1 (Lamb1) and laminin ß2 (Lamb2) chains. Whereas Lamb1 was upregulated, Lamb2 was decreased in endothelial cells in old mice compared with young controls. A similar change in expression patterns was observed after induction of acute myocardial infarction. Mimicking aging and injury conditions by plating endothelial cells on laminin ß1-containing laminin 411 matrix impaired endothelial cell adhesion, migration, and tube formation and augmented endothelial-to-mesenchymal transition and endothelial detachment compared with laminin 421, which contains the laminin ß2 chain. Because laminins can signal via integrin receptors, we determined the activation of ITGB1 (integrin ß1). Laminin 421 coating induced a higher activation of ITGB1 compared with laminin 411. siRNA-mediated silencing of ITGB1 reduced laminin ß2-dependent adhesion, suggesting that laminin ß2 more efficiently activates ITGB1. CONCLUSIONS: Mimicking age-related modulation of laminin ß1 versus ß2 chain expression changes the functional properties and phenotype of endothelial cells. The dysregulation of the extracellular matrix during vascular aging may contribute to age-associated impairment of organ function and fibrosis.
Asunto(s)
Envejecimiento/metabolismo , Células Endoteliales/metabolismo , Laminina/metabolismo , Neovascularización Fisiológica , Factores de Edad , Envejecimiento/genética , Animales , Adhesión Celular , Movimiento Celular , Proliferación Celular , Separación Celular/métodos , Células Cultivadas , Microambiente Celular , Modelos Animales de Enfermedad , Células Endoteliales/patología , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Integrina beta1/metabolismo , Laminina/genética , Masculino , Ratones Endogámicos C57BL , Infarto del Miocardio/genética , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Fenotipo , Transducción de SeñalRESUMEN
Heart failure due to myocardial infarction is a major cause of mortality. The microRNA (miR) family let-7 is expressed during embryonic development and is up-regulated in differentiated cells. The aim of this study was to study the role of let-7 after acute myocardial infarction (AMI). We designed an antimiR to inhibit the highest expressed members of the let-7 family, let-7 a, b and c. Administration at day 0 and day 2 after AMI resulted in sustained knockdown of let-7 after 28days. Let-7 inhibition prevented deterioration of cardiac functions compared to control treatment which was especially due to improvements in the infarcted, apical cardiac segments. We observed higher contents of fibrosis in the border zone as well as increased numbers of cells positive for TCF21, which is also expressed in epicardial cells. Markers were augmented after let-7 inhibition and let-7 blocked EMT in epicardial cells in vitro. Lineage tracing in TCF21(iCre/+):R26R(tdT) mice showed abundant tomato positive cells in the infarct and border zone. In conclusion, let-7 inhibition resulted in functional benefits due to an increase in recruitment of epicardial cells and EMT.
Asunto(s)
Transición Epitelial-Mesenquimal , Regulación de la Expresión Génica , MicroARNs/genética , Infarto del Miocardio/genética , Infarto del Miocardio/fisiopatología , Disfunción Ventricular/genética , Animales , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Linaje de la Célula , Transición Epitelial-Mesenquimal/genética , Fibrosis , Masculino , Ratones , Ratones Transgénicos , MicroARNs/química , Infarto del Miocardio/patologíaRESUMEN
RATIONALE: Cell therapy is a promising option for the treatment of acute or chronic myocardial ischemia. The intracoronary infusion of cells imposes the potential risk of cell clotting, which may be prevented by the addition of anticoagulants. However, a comprehensive analysis of the effects of anticoagulants on the function of the cells is missing. OBJECTIVE: Here, we investigated the effects of heparin and the thrombin inhibitor bivalirudin on bone marrow-derived mononuclear cell (BMC) functional activity and homing capacity. METHODS AND RESULTS: Heparin, but not bivalirudin profoundly and dose-dependently inhibited basal and stromal cell-derived factor 1 (SDF-1)-induced BMC migration. Incubation of BMCs with 20 U/mL heparin for 30 minutes abrogated SDF-1-induced BMC invasion (16±8% of control; P<0.01), whereas no effects on apoptosis or colony formation were observed (80±33% and 100±44% of control, respectively). Pretreatment of BMCs with heparin significantly reduced the homing of the injected cells in a mouse ear-wound model (69±10% of control; P<0.05). In contrast, bivalirudin did not inhibit in vivo homing of BMCs. Mechanistically, heparin binds to both, the chemoattractant SDF-1 and its receptor, chemokine receptor 4 (CXCR4), blocking CXCR4 internalization as well as SDF-1/CXCR4 signaling after SDF-1 stimulation. CONCLUSIONS: Heparin blocks SDF-1/CXCR4 signaling by binding to the ligand as well as the receptor, thereby interfering with migration and homing of BMCs. In contrast, the thrombin inhibitor bivalirudin did not interfere with BMC homing or SDF-1/CXCR4 signaling. These findings suggest that bivalirudin but not heparin might be recommended as an anticoagulant for intracoronary infusion of BMCs for cell therapy after cardiac ischemia.
Asunto(s)
Células de la Médula Ósea/citología , Tratamiento Basado en Trasplante de Células y Tejidos , Quimiocina CXCL12/efectos de los fármacos , Heparina/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Infarto del Miocardio/terapia , Receptores CXCR4/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Anticoagulantes/farmacología , Antitrombinas/farmacología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Células Cultivadas , Quimiocina CXCL12/fisiología , Modelos Animales de Enfermedad , Femenino , Hirudinas/farmacología , Humanos , Técnicas In Vitro , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/fisiología , Ratones , Ratones Endogámicos , Fragmentos de Péptidos/farmacología , Receptores CXCR4/fisiología , Proteínas Recombinantes/farmacología , Transducción de Señal/fisiologíaRESUMEN
Hematopoietic mutations in epigenetic regulators like DNA methyltransferase 3 alpha (DNMT3A), play a pivotal role in driving clonal hematopoiesis of indeterminate potential (CHIP), and are associated with unfavorable outcomes in patients suffering from heart failure (HF). However, the precise interactions between CHIP-mutated cells and other cardiac cell types remain unknown. Here, we identify fibroblasts as potential partners in interactions with CHIP-mutated monocytes. We used combined transcriptomic data derived from peripheral blood mononuclear cells of HF patients, both with and without CHIP, and cardiac tissue. We demonstrate that inactivation of DNMT3A in macrophages intensifies interactions with cardiac fibroblasts and increases cardiac fibrosis. DNMT3A inactivation amplifies the release of heparin-binding epidermal growth factor-like growth factor, thereby facilitating activation of cardiac fibroblasts. These findings identify a potential pathway of DNMT3A CHIP-driver mutations to the initiation and progression of HF and may also provide a compelling basis for the development of innovative anti-fibrotic strategies.
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ADN Metiltransferasa 3A , Insuficiencia Cardíaca , Humanos , Hematopoyesis Clonal , ADN (Citosina-5-)-Metiltransferasas/genética , ADN Metiltransferasa 3A/genética , Fibroblastos , Fibrosis/genética , Fibrosis/patología , Insuficiencia Cardíaca/genética , Hematopoyesis/genética , Leucocitos Mononucleares , Mutación , Cardiopatías/genética , Cardiopatías/patologíaRESUMEN
Aging is a major risk factor for impaired cardiovascular health. Because the aging myocardium is characterized by microcirculatory dysfunction, and because nerves align with vessels, we assessed the impact of aging on the cardiac neurovascular interface. We report that aging reduces nerve density in the ventricle and dysregulates vascular-derived neuroregulatory genes. Aging down-regulates microRNA 145 (miR-145) and derepresses the neurorepulsive factor semaphorin-3A. miR-145 deletion, which increased Sema3a expression or endothelial Sema3a overexpression, reduced axon density, mimicking the aged-heart phenotype. Removal of senescent cells, which accumulated with chronological age in parallel to the decline in nerve density, rescued age-induced denervation, reversed Sema3a expression, preserved heart rate patterns, and reduced electrical instability. These data suggest that senescence-mediated regulation of nerve density contributes to age-associated cardiac dysfunction.
Asunto(s)
Envejecimiento , Senescencia Celular , Corazón , MicroARNs , Densidad Microvascular , Miocardio , Semaforina-3A , Corazón/inervación , Microcirculación , MicroARNs/genética , MicroARNs/metabolismo , Semaforina-3A/genética , Animales , Ratones , Envejecimiento/genética , Envejecimiento/patología , Masculino , Ratones Endogámicos C57BL , Senescencia Celular/genética , Miocardio/patología , AxonesRESUMEN
The regulation of bone vasculature by chronic diseases, such as heart failure is unknown. Here, we describe the effects of myocardial infarction and post-infarction heart failure on the bone vascular cell composition. We demonstrate an age-independent loss of type H endothelium in heart failure after myocardial infarction in both mice and humans. Using single-cell RNA sequencing, we delineate the transcriptional heterogeneity of human bone marrow endothelium, showing increased expression of inflammatory genes, including IL1B and MYC, in ischemic heart failure. Endothelial-specific overexpression of MYC was sufficient to induce type H bone endothelial cells, whereas inhibition of NLRP3-dependent IL-1ß production partially prevented the post-myocardial infarction loss of type H vasculature in mice. These results provide a rationale for using anti-inflammatory therapies to prevent or reverse the deterioration of bone vascular function in ischemic heart disease.
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Huesos/irrigación sanguínea , Células Endoteliales/patología , Insuficiencia Cardíaca/fisiopatología , Infarto del Miocardio/fisiopatología , Anciano , Animales , Huesos/fisiopatología , Estudios de Casos y Controles , Células Endoteliales/metabolismo , Femenino , Furanos/farmacología , Genes myc , Insuficiencia Cardíaca/etiología , Células Madre Hematopoyéticas/patología , Humanos , Indenos/farmacología , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/patología , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Infarto del Miocardio/complicaciones , Infarto del Miocardio/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Sulfonamidas/farmacologíaRESUMEN
Endothelial cells play a critical role in the adaptation of tissues to injury. Tissue ischemia induced by infarction leads to profound changes in endothelial cell functions and can induce transition to a mesenchymal state. Here we explore the kinetics and individual cellular responses of endothelial cells after myocardial infarction by using single cell RNA sequencing. This study demonstrates a time dependent switch in endothelial cell proliferation and inflammation associated with transient changes in metabolic gene signatures. Trajectory analysis reveals that the majority of endothelial cells 3 to 7 days after myocardial infarction acquire a transient state, characterized by mesenchymal gene expression, which returns to baseline 14 days after injury. Lineage tracing, using the Cdh5-CreERT2;mT/mG mice followed by single cell RNA sequencing, confirms the transient mesenchymal transition and reveals additional hypoxic and inflammatory signatures of endothelial cells during early and late states after injury. These data suggest that endothelial cells undergo a transient mes-enchymal activation concomitant with a metabolic adaptation within the first days after myocardial infarction but do not acquire a long-term mesenchymal fate. This mesenchymal activation may facilitate endothelial cell migration and clonal expansion to regenerate the vascular network.
Asunto(s)
Endotelio/patología , Transición Epitelial-Mesenquimal/genética , Infarto del Miocardio/patología , Miocardio/patología , Animales , Movimiento Celular/genética , Plasticidad de la Célula/genética , Proliferación Celular/genética , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/patología , Endotelio/citología , Genes Reporteros/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteínas Luminiscentes/genética , Masculino , Ratones , Ratones Transgénicos , Miocardio/citología , RNA-Seq , Análisis de la Célula IndividualRESUMEN
Circulating blood-derived vasculogenic cells improve neovascularization of ischemic tissue by a broad repertoire of potential therapeutic actions. Whereas initial studies documented that the cells incorporate and differentiate to cardiovascular cells, other studies suggested that short-time paracrine mechanisms mediate the beneficial effects. The question remains to what extent a physical incorporation is contributing to the beneficial effects of cell therapy. By using the inducible suicide gene thymidine kinase to deplete transplanted cells, we determined the contribution of physical incorporation in 3 animal models. After acute myocardial infarction, depletion of cells 14 days after infusion resulted in a reduction of capillary density and a substantial deterioration of heart function. Likewise, neovascularization of Matrigel plugs and ischemic limbs was significantly suppressed when infused cells were depleted 7 days after infusion. Induction of cell death in the previously transplanted cells reduced perfusion and led to vascular leakage as evidenced by Evans blue extravasation. These results indicate that physical incorporation and persistence of cells contribute to cell-mediated improvement of neovascularization and cardiac function. Long-term paracrine activities and/or cell intrinsic mechanisms may have contributed to the maintenance of functional improvement.
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Corazón/fisiopatología , Isquemia Miocárdica/terapia , Neovascularización Fisiológica/fisiología , Trasplante de Células Madre/métodos , Animales , Capilares/fisiopatología , Técnicas de Cultivo de Célula , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Vasos Coronarios/patología , Femenino , Genes Reporteros , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Humanos , Lentivirus , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/fisiología , Imagen por Resonancia Magnética , Ratones , Ratones Desnudos , Isquemia Miocárdica/fisiopatologíaRESUMEN
Aging is a major risk factor for cardiovascular disease. Although the impact of aging has been extensively studied, little is known regarding the aging processes in cells of the heart. Here we analyzed the transcriptomes of hearts of 12-week-old and 18-month-old mice by single-nucleus RNA-sequencing. Among all cell types, aged fibroblasts showed most significant differential gene expression, increased RNA dynamics, and network entropy. Aged fibroblasts exhibited significantly changed expression patterns of inflammatory, extracellular matrix organization angiogenesis, and osteogenic genes. Functional analyses indicated deterioration of paracrine signatures between fibroblasts and endothelial cells in old hearts. Aged heart-derived fibroblasts had impaired endothelial cell angiogenesis and autophagy and augmented proinflammatory response. In particular, expression of Serpine1 and Serpine2 were significantly increased and secreted by old fibroblasts to exert antiangiogenic effects on endothelial cells, an effect that could be significantly prevented by using neutralizing antibodies. Moreover, we found an enlarged subpopulation of aged fibroblasts expressing osteoblast genes in the epicardial layer associated with increased calcification. Taken together this study provides system-wide insights and identifies molecular changes of aging cardiac fibroblasts, which may contribute to declined heart function.
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Envejecimiento/fisiología , Fibroblastos , Corazón/fisiología , Miocardio/citología , Transcriptoma , Animales , Fibroblastos/química , Fibroblastos/metabolismo , Fibroblastos/fisiología , Masculino , Ratones , Serpinas/genética , Serpinas/metabolismo , Transcriptoma/genética , Transcriptoma/fisiología , Calcificación Vascular/genética , Calcificación Vascular/metabolismoRESUMEN
Aims: To determine the bioequivalence of several T1 mapping sequences in myocardial characterization of diffuse myocardial fibrosis. Methods and results: We performed an intra-individual sequence comparison of three types of T1 mapping sequences [MOdified Look-Locker Inversion recovery (MOLLI), Shortened MOdified Look-Locker Inversion recovery ((sh)MOLLI), and SAturation recovery single-SHot Acquisition (SASHA)]. We employed two model diseases of diffuse interstitial fibrosis [patients with non-ischaemic dilated cardiomyopathy (NIDCM), n = 32] and aortic stenosis [(AS), n = 25)]. Twenty-six healthy individuals served as controls. Relationship with collagen volume fraction (CVF) was assessed using endomyocardial biopsies (EMB) intraoperatively in 12 AS patients. T2 mapping (GraSE) was also performed. Myocardial native T1 with MOLLI and shMOLLI showed, firstly, an excellent discriminatory accuracy between health and disease [area under the curves (P-value): 0.94 (0.88-0.99); 0.87 (0.79-0.94); 0.61 (0.49-0.72)], secondly, relationship between histological CVF [native T1 MOLLI vs. shMOLLI vs. SASHA: r = 0.582 (P = 0.027), r = 0.524 (P = 0.046), r = 0.443 (P = 0.150)], and thirdly, with native T2 [r = 0.628(P < 0.001), r = 0.459 (P = 0.003), r = 0.211 (P = 0.083)]. The respective relationships for extracellular volume fraction with CVF [r = 0.489 (P = 0.044), r = 0.417 (0.071), r = 0.353 (P = 0.287)] were significant for MOLLI, but not other sequences. In AS patients, native T2 was significantly higher compared to controls, and associated with levels of C-reactive protein and troponin. Conclusion: T1 mapping sequences differ in their bioequivalence for discrimination between health and disease as well as associations with diffuse myocardial fibrosis.
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Mapeo del Potencial de Superficie Corporal/métodos , Cardiomiopatías/diagnóstico por imagen , Insuficiencia Cardíaca/diagnóstico por imagen , Interpretación de Imagen Asistida por Computador/métodos , Imagen por Resonancia Cinemagnética/métodos , Adulto , Anciano , Cardiomiopatías/patología , Estudios de Cohortes , Medios de Contraste , Femenino , Insuficiencia Cardíaca/patología , Humanos , Masculino , Persona de Mediana Edad , Fantasmas de Imagen , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Estudios Retrospectivos , Sensibilidad y EspecificidadRESUMEN
Excessive leukocyte infiltration causes severe tissue damage in a variety of inflammatory diseases. The initial step in leukocyte extravasation is mediated by selectins and oligosaccharides on their glycoconjugate ligands. Human milk is a rich source of lactose-derived oligosaccharides that are partly absorbed in the intestine and excreted with the urine. As these components contain binding determinants for the selectins we investigated whether human milk oligosaccharides are able to affect leukocyte rolling and adhesion to endothelial cells under dynamic conditions. Therefore, monocytes, lymphocytes, or neutrophils isolated from human peripheral blood were passed over TNF-alpha-activated HUVEC under shear stress. The influence of different oligosaccharide fractions was determined by video-microscopy and compared with the effects of various individual oligosaccharides. Within a physiological range (12.5 - 125 microg/ml) the acidic fraction significantly inhibited leukocyte rolling and adhesion (up to 24.0% and 52.8%, respectively) in a concentration-dependent manner. These effects were even more pronounced than those achieved by soluble sialyl-Lewis x, a physiological binding determinant for selectins. Several active components within the oligosaccharide fraction of human milk were identified, e.g. 3'-sialyl-lactose and 3'-sialyl-3-fucosyl-lactose. These results indicate that specific oligosaccharides in human milk may serve as anti-inflammatory components and might therefore contribute to the lower incidence of inflammatory diseases in human milk-fed infants.
Asunto(s)
Endotelio Vascular/citología , Linfocitos/citología , Leche Humana/química , Monocitos/citología , Neutrófilos/citología , Oligosacáridos/química , Adhesión Celular , Células Cultivadas , Cromatografía , Relación Dosis-Respuesta a Droga , Endotelio Vascular/metabolismo , Endotoxinas/metabolismo , Humanos , Inflamación , Espectrometría de Masas , Oligosacáridos/farmacología , Unión Proteica , Antígeno Sialil Lewis X , Factor de Necrosis Tumoral alfa/metabolismo , Venas Umbilicales/citologíaRESUMEN
OBJECTIVE: To assess the effect of long-term pharmacological inhibition of miR-21 in a model of metabolic syndrome and obesity. METHODS: Aged db/db mice were treated with locked nucleic acid-modified anti-miRs directed against miR-21 (LNA-21), control LNAs or PBS for 18 weeks. Cardiac function was assessed by echocardiography and the effect on body weight and white adipose tissue (WAT) was evaluated. RESULTS: MiR-21 expression was efficiently inhibited in the heart and WAT with no apparent liver toxicity or deterioration of kidney function. MiR-21 inhibition had no effect on cardiac hypertrophy as well as systolic and diastolic cardiac functions. However, levels of cardiac collagen 1 were modestly reduced in LNA-21 treated mice. MiR-21 inhibition reduced body weight, as well as adipocyte size and serum triglycerides were significantly decreased. The miR-21 targets TGFß-receptor 2 (TGFBR2) and phosphatase and tensin homolog (PTEN) were derepressed in WAT of LNA-21 treated mice and Sprouty1 and 2 were increased after miR-21 inhibition. CONCLUSIONS: Long-term treatment with LNA-21 is safe and efficiently suppresses miR-21 expression. Cardiac function was not affected. LNA-21 treatment led to a significant weight loss and reduces adipocyte size as well as derepression of the targets TGFRB2, PTEN, and Sprouty1 and 2.
Asunto(s)
MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Obesidad/genética , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/metabolismo , Animales , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Cardiomiopatías Diabéticas/genética , Cardiomiopatías Diabéticas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Síndrome Metabólico/complicaciones , Síndrome Metabólico/genética , Síndrome Metabólico/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Obesidad/complicaciones , Obesidad/metabolismo , Oligonucleótidos/farmacologíaRESUMEN
Severe endothelial abnormalities are a prominent feature in sepsis with cytokines such as tumor necrosis factor (TNF)alpha being implicated in the pathogenesis. As mimic to inflammation, human umbilical vascular endothelial cells (HUVEC) were incubated with TNFalpha for 22 h, in the absence or presence of the omega-6 fatty acid (FA), arachidonic acid (AA), or the alternative omega-3 FA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). TNFalpha caused marked alterations in the PUFA profile and long chain PUFA content of total phospholipids (PL) decreased. In contrast, there was a compensatory increase in mead acid [MA, 20:3(omega-9)], the hallmark acid of the essential fatty acid deficiency (EFAD) syndrome. Corresponding changes were noted in phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, but not in the sphingomyelin fraction. Supplementation with AA, EPA, or DHA markedly increased the respective FA contents in the PL pools, suppressed the increase in MA, and resulted in a shift either toward further predominance of omega-6 or predominance of omega-3 FA. We conclude that short-term TNFalpha incubation of HUVEC causes an EFAD state hitherto only described for long-term malnutrition, and that endothelial cells are susceptible to differential influence by omega-3 versus omega-6 FA supplementation under these conditions.
Asunto(s)
Endotelio Vascular/metabolismo , Ácidos Grasos Esenciales/metabolismo , Ácidos Grasos Omega-3/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Células Cultivadas , Ácidos Grasos/metabolismo , Humanos , Técnicas In Vitro , Cinética , Imitación Molecular , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilinositoles/metabolismo , Fosfatidilserinas/metabolismo , Fosfolípidos/metabolismo , Esfingomielinas/metabolismoRESUMEN
Monocyte-endothelium interaction is a fundamental process in many acute and chronic inflammatory diseases. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are fish oil-derived alternative (omega-3) precursor fatty acids implicated in the suppression of inflammatory events. We investigated their influence on rolling and adhesion of monocytes to human umbilical vein endothelial cells (HUVEC) under laminar flow conditions in vitro. Exposure of HUVEC to tumor necrosis factor (TNF-alpha) strongly increased 1) surface expression of intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), and E-selectin, 2) platelet-activating factor (PAF) synthesis as assessed by thrombin challenge, and 3) rate of rolling and adhesion of monocytes. Preincubation of HUVEC with EPA or DHA markedly suppressed PAF synthesis, monocyte rolling, and adherence, whereas expression of endothelial adhesion molecules was unchanged. Also, PAF receptor antagonists markedly suppressed the adhesion rate of monocytes, and EPA or DHA revealed no additional inhibitory capacity. In contrast, arachidonic acid partially reversed the effect of the antagonist. We conclude that omega-3 fatty acids suppress rolling and adherence of monocytes on activated endothelial cells in vitro by affecting endothelial PAF generation.