RESUMEN
BACKGROUND: Cardiovascular risk in diabetes remains elevated despite glucose-lowering therapies. We hypothesized that hyperglycemia induces trained immunity in macrophages, promoting persistent proatherogenic characteristics. METHODS: Bone marrow-derived macrophages from control mice and mice with diabetes were grown in physiological glucose (5 mmol/L) and subjected to RNA sequencing (n=6), assay for transposase accessible chromatin sequencing (n=6), and chromatin immunoprecipitation sequencing (n=6) for determination of hyperglycemia-induced trained immunity. Bone marrow transplantation from mice with (n=9) or without (n=6) diabetes into (normoglycemic) Ldlr-/- mice was used to assess its functional significance in vivo. Evidence of hyperglycemia-induced trained immunity was sought in human peripheral blood mononuclear cells from patients with diabetes (n=8) compared with control subjects (n=16) and in human atherosclerotic plaque macrophages excised by laser capture microdissection. RESULTS: In macrophages, high extracellular glucose promoted proinflammatory gene expression and proatherogenic functional characteristics through glycolysis-dependent mechanisms. Bone marrow-derived macrophages from diabetic mice retained these characteristics, even when cultured in physiological glucose, indicating hyperglycemia-induced trained immunity. Bone marrow transplantation from diabetic mice into (normoglycemic) Ldlr-/- mice increased aortic root atherosclerosis, confirming a disease-relevant and persistent form of trained innate immunity. Integrated assay for transposase accessible chromatin, chromatin immunoprecipitation, and RNA sequencing analyses of hematopoietic stem cells and bone marrow-derived macrophages revealed a proinflammatory priming effect in diabetes. The pattern of open chromatin implicated transcription factor Runt-related transcription factor 1 (Runx1). Similarly, transcriptomes of atherosclerotic plaque macrophages and peripheral leukocytes in patients with type 2 diabetes were enriched for Runx1 targets, consistent with a potential role in human disease. Pharmacological inhibition of Runx1 in vitro inhibited the trained phenotype. CONCLUSIONS: Hyperglycemia-induced trained immunity may explain why targeting elevated glucose is ineffective in reducing macrovascular risk in diabetes and suggests new targets for disease prevention and therapy.
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Aterosclerosis/inmunología , Diabetes Mellitus Experimental/inmunología , Hiperglucemia/inmunología , Inmunidad Celular/inmunología , Leucocitos Mononucleares/inmunología , Macrófagos/inmunología , Animales , Aterosclerosis/patología , Células Cultivadas , Diabetes Mellitus Experimental/patología , Endarterectomía Carotidea , Humanos , Hiperglucemia/patología , Leucocitos Mononucleares/patología , Macrófagos/patología , Ratones , Ratones de la Cepa 129 , Ratones TransgénicosRESUMEN
[Figure: see text].
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Glucemia/metabolismo , Reprogramación Celular , Diabetes Mellitus/metabolismo , Metabolismo Energético , Animales , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Biomarcadores/sangre , Glucemia/efectos de los fármacos , Diabetes Mellitus/tratamiento farmacológico , Diabetes Mellitus/genética , Diabetes Mellitus/inmunología , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/genética , Epigénesis Genética , Hematopoyesis , Humanos , Hipoglucemiantes/uso terapéutico , Células Progenitoras Mieloides/inmunología , Células Progenitoras Mieloides/metabolismo , Transducción de SeñalRESUMEN
Objective- Plaque macrophages are intricately involved in atherogenesis and plaque destabilization. We sought to identify functional pathways in human plaque macrophages that are differentially regulated in respect of (1) plaque stability and (2) lipid content. We hypothesized that differentially regulated macrophage gene sets would relate to genome-wide association study variants associated with risk of acute complications of atherosclerosis. Approach and Results- Forty patients underwent carotid magnetic resonance imaging for lipid quantification before endarterectomy. Carotid plaque macrophages were procured by laser capture microdissection from (1) lipid core and (2) cap region, in 12 recently symptomatic and 12 asymptomatic carotid plaques. Applying gene set enrichment analysis, a number of gene sets were found to selectively upregulate in symptomatic plaque macrophages, which corresponded to 7 functional pathways: inflammation, lipid metabolism, hypoxic response, cell proliferation, apoptosis, antigen presentation, and cellular energetics. Predicted upstream regulators included IL-1ß, TNF-α, and NF-κB. In vivo lipid quantification by magnetic resonance imaging correlated most strongly with the upregulation of genes of the IFN/ STAT1 pathways. Cross-interrogation of gene set enrichment analysis and meta-analysis gene set enrichment of variant associations showed lipid metabolism pathways, driven by genes coding for APOE and ABCA1/G1 coincided with known risk-associated SNPs (single nucleotide polymorphisms) from genome-wide association studies. Conclusions- Macrophages from recently symptomatic carotid plaques show differential regulation of functional gene pathways. There were additional quantitative relationships between plaque lipid content and key gene sets. The data show a plausible mechanism by which known genome-wide association study risk variants for atherosclerotic complications could be linked to (1) a relevant cellular process, in (2) the key cell type of atherosclerosis, in (3) a human disease-relevant setting.
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Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/genética , Redes Reguladoras de Genes , Macrófagos/metabolismo , Placa Aterosclerótica , Transcriptoma , Adulto , Anciano , Anciano de 80 o más Años , Arterias Carótidas/diagnóstico por imagen , Arterias Carótidas/patología , Enfermedades de las Arterias Carótidas/diagnóstico por imagen , Enfermedades de las Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/patología , Femenino , Perfilación de la Expresión Génica/métodos , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Macrófagos/patología , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Fenotipo , Análisis de Componente Principal , Rotura EspontáneaRESUMEN
AIMS: Acute myocardial infarction rapidly increases blood neutrophils (<2 h). Release from bone marrow, in response to chemokine elevation, has been considered their source, but chemokine levels peak up to 24 h after injury, and after neutrophil elevation. This suggests that additional non-chemokine-dependent processes may be involved. Endothelial cell (EC) activation promotes the rapid (<30 min) release of extracellular vesicles (EVs), which have emerged as an important means of cell-cell signalling and are thus a potential mechanism for communicating with remote tissues. METHODS AND RESULTS: Here, we show that injury to the myocardium rapidly mobilizes neutrophils from the spleen to peripheral blood and induces their transcriptional activation prior to arrival at the injured tissue. Time course analysis of plasma-EV composition revealed a rapid and selective increase in EVs bearing VCAM-1. These EVs, which were also enriched for miRNA-126, accumulated preferentially in the spleen where they induced local inflammatory gene and chemokine protein expression, and mobilized splenic-neutrophils to peripheral blood. Using CRISPR/Cas9 genome editing, we generated VCAM-1-deficient EC-EVs and showed that its deletion removed the ability of EC-EVs to provoke the mobilization of neutrophils. Furthermore, inhibition of miRNA-126 in vivo reduced myocardial infarction size in a mouse model. CONCLUSIONS: Our findings show a novel EV-dependent mechanism for the rapid mobilization of neutrophils to peripheral blood from a splenic reserve and establish a proof of concept for functional manipulation of EV-communications through genetic alteration of parent cells.
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Vesículas Extracelulares , MicroARNs , Infarto del Miocardio , Ratones , Animales , Neutrófilos/metabolismo , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismo , Vesículas Extracelulares/metabolismo , Infarto del Miocardio/metabolismo , Células Endoteliales/metabolismo , MicroARNs/genética , MicroARNs/metabolismoRESUMEN
While obesity and associated metabolic complications are linked to inflammation of white adipose tissue (WAT), the causal factors remain unclear. We hypothesized that the local metabolic environment could be an important determinant. To this end, we compared metabolites released from WAT of 81 obese and non-obese women. This identified glutamine to be downregulated in obesity and inversely associated with a pernicious WAT phenotype. Glutamine administration in vitro and in vivo attenuated both pro-inflammatory gene and protein levels in adipocytes and WAT and macrophage infiltration in WAT. Metabolomic and bioenergetic analyses in human adipocytes suggested that glutamine attenuated glycolysis and reduced uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) levels. UDP-GlcNAc is the substrate for the post-translational modification O-linked ß-N-acetylglucosamine (O-GlcNAc) mediated by the enzyme O-GlcNAc transferase. Functional studies in human adipocytes established a mechanistic link between reduced glutamine, O-GlcNAcylation of nuclear proteins, and a pro-inflammatory transcriptional response. Altogether, glutamine metabolism is linked to WAT inflammation in obesity.
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Adipocitos , Tejido Adiposo Blanco , Glutamina , Inflamación/metabolismo , Obesidad/metabolismo , Acetilglucosamina , Adipocitos/metabolismo , Adipocitos/patología , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/patología , Adulto , Animales , Células Cultivadas , Estudios de Cohortes , Femenino , Glucosa/metabolismo , Glutamina/metabolismo , Glutamina/farmacología , Glicosilación/efectos de los fármacos , Humanos , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , N-Acetilglucosaminiltransferasas/metabolismoRESUMEN
BACKGROUND AND AIMS: Imaging studies have relied on the 'overall' volumetric quantification of perivascular adipose tissue. We sought to assess the relationship of circumferential distribution between perivascular adipose tissue and adjacent wall thickness of carotid and aortic arteries using dedicated magnetic resonance imaging sequences. METHODS: Vessel wall and perivascular adipose tissue were acquired using magnetic resonance imaging (1.5 T). Co-registered images were segmented separately, and measurements of both perivascular adipose tissue and vessel wall were obtained along radii of the vessel spaced at angles of 5° each. RESULTS: In total, 29 patients were recruited. Perivascular adipose tissue thickness of the aorta was 3.34 ± 0.79 mm with specific pattern of 'double peaks' distribution, while carotid perivascular adipose tissue had no identifiable pattern with thickness of 0.8 ± 0.91 mm. Although statistically significant, the correlation between perivascular adipose tissue thickness and wall thickness in carotid arteries with normal (r = 0.040, p = 0.001) or with abnormal wall thickness (r = -0.039, p = 0.015) was merely nominal. Similarly, perivascular adipose tissue of the aorta had very weak correlation with normal aortic wall thickness (r = 0.010, p = 0.008) but not with the abnormal ones (r = -0.05, p = 0.29). CONCLUSION: Dissociation between the spatial distribution of perivascular adipose tissue and arterial wall thickening in the aorta and carotid arteries does not support that perivascular adipose tissue has a causal role in promoting atherosclerotic plaque via a paracrine route. Yet, perivascular adipose tissue functional properties were not examined in this study.
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Tejido Adiposo/diagnóstico por imagen , Aorta Torácica/diagnóstico por imagen , Enfermedades de la Aorta/diagnóstico por imagen , Aterosclerosis/diagnóstico por imagen , Arterias Carótidas/diagnóstico por imagen , Enfermedades de las Arterias Carótidas/diagnóstico por imagen , Placa Aterosclerótica , Tejido Adiposo/patología , Anciano , Aorta Torácica/patología , Enfermedades de la Aorta/patología , Aterosclerosis/patología , Arterias Carótidas/patología , Enfermedades de las Arterias Carótidas/patología , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Valor Predictivo de las PruebasRESUMEN
Transcriptionally activated monocytes are recruited to the heart after acute myocardial infarction (AMI). After AMI in mice and humans, the number of extracellular vesicles (EVs) increased acutely. In humans, EV number correlated closely with the extent of myocardial injury. We hypothesized that EVs mediate splenic monocyte mobilization and program transcription following AMI. Some plasma EVs bear endothelial cell (EC) integrins, and both proinflammatory stimulation of ECs and AMI significantly increased VCAM-1-positive EV release. Injected EC-EVs localized to the spleen and interacted with, and mobilized, splenic monocytes in otherwise naive, healthy animals. Analysis of human plasma EV-associated miRNA showed 12 markedly enriched miRNAs after AMI; functional enrichment analyses identified 1,869 putative mRNA targets, which regulate relevant cellular functions (e.g., proliferation and cell movement). Furthermore, gene ontology termed positive chemotaxis as the most enriched pathway for the miRNA-mRNA targets. Among the identified EV miRNAs, EC-associated miRNA-126-3p and -5p were highly regulated after AMI. miRNA-126-3p and -5p regulate cell adhesion- and chemotaxis-associated genes, including the negative regulator of cell motility, plexin-B2. EC-EV exposure significantly downregulated plexin-B2 mRNA in monocytes and upregulated motility integrin ITGB2. These findings identify EVs as a possible novel signaling pathway by linking ischemic myocardium with monocyte mobilization and transcriptional activation following AMI.