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BACKGROUND AND AIMS: In one-third of patients with acute coronary syndrome (ACS), thrombosis occurs despite an intact fibrous cap (IFC) (IFC-ACS, 'plaque erosion'). Recent studies emphasize neutrophils as the immediate inflammatory response in this pathology, but their exact molecular activation patterns are still poorly understood and may represent future therapeutic targets. METHODS AND RESULTS: Thirty-two patients with IFC-ACS and matched patients with ACS with ruptured fibrous cap (RFC) (RFC-ACS) from the OPTICO-ACS study were included, and blood samples were collected from the local site of the culprit lesion and the systemic circulation. Neutrophil surface marker expression was quantified by flow cytometry. Neutrophil cytotoxicity towards endothelial cells was examined in an ex vivo co-culture assay. Secretion of active matrix metalloproteinase 9 (MMP9) by neutrophils was evaluated using zymography in supernatants and in plasma samples. Optical coherence tomography (OCT)-embedded thrombi were used for immunofluorescence analysis. Toll-like receptor 2 (TLR2) expression was higher on neutrophils from IFC-ACS than RFC-ACS patients. TLR2 stimulation increased the release of active MMP9 from local IFC-ACS-derived neutrophils, which also aggravated endothelial cell death independently of TLR2. Thrombi of IFC-ACS patients exhibited more hyaluronidase 2 with concomitant increase in local plasma levels of the TLR2 ligand: hyaluronic acid. CONCLUSION: The current study provides first in-human evidence for distinct TLR2-mediated neutrophil activation in IFC-ACS, presumably triggered by elevated soluble hyaluronic acid. Together with disturbed flow conditions, neutrophil-released MMP9 might be promoting endothelial cell loss-triggered thrombosis and therefore providing a potential future target for a phenotype-specific secondary therapeutic approach in IFC-ACS.
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Síndrome Coronario Agudo , Placa Aterosclerótica , Trombosis , Humanos , Síndrome Coronario Agudo/complicaciones , Ácido Hialurónico , Receptor Toll-Like 2 , Neutrófilos , Metaloproteinasa 9 de la Matriz , Células Endoteliales/metabolismo , Placa Aterosclerótica/patología , Fibrosis , Trombosis/complicaciones , Tomografía de Coherencia Óptica/métodos , Angiografía CoronariaRESUMEN
BACKGROUND: Immune checkpoint inhibitor (ICI) therapy is often accompanied by immune-related pathology, with an increasing occurrence of high-risk ICI-related myocarditis. Understanding the mechanisms involved in this side effect could enable the development of management strategies. In mouse models, immune checkpoints, such as PD-1 (programmed cell death protein 1), control the threshold of self-antigen responses directed against cardiac TnI (troponin I). We aimed to identify how the immunoproteasome, the main proteolytic machinery in immune cells harboring 3 distinct protease activities in the LMP2 (low-molecular-weight protein 2), LMP7 (low-molecular-weight protein 7), and MECL1 (multicatalytic endopeptidase complex subunit 1) subunit, affects TnI-directed autoimmune pathology of the heart. METHODS: TnI-directed autoimmune myocarditis (TnI-AM), a CD4+ T-cell-mediated disease, was induced in mice lacking all 3 immunoproteasome subunits (triple-ip-/-) or lacking either the gene encoding LMP2 and LMP7 by immunization with a cardiac TnI peptide. Alternatively, before induction of TnI-AM or after establishment of autoimmune myocarditis, mice were treated with the immunoproteasome inhibitor ONX 0914. Immune parameters defining heart-specific autoimmunity were investigated in experimental TnI-AM and in 2 cases of ICI-related myocarditis. RESULTS: All immunoproteasome-deficient strains showed mitigated autoimmune-related cardiac pathology with less inflammation, lower proinflammatory and chemotactic cytokines, less interleukin-17 production, and reduced fibrosis formation. Protection from TnI-directed autoimmune heart pathology with improved cardiac function in LMP7-/- mice involved a changed balance between effector and regulatory CD4+ T cells in the spleen, with CD4+ T cells from LMP7-/- mice showing a higher expression of inhibitory PD-1 molecules. Blocked immunoproteasome proteolysis, by treatment of TLR2 (Toll-like receptor 2)-engaged and TLR7 (Toll-like receptor 7)/TLR8 (Toll-like receptor 8)-engaged CD14+ monocytes with ONX 0914, diminished proinflammatory cytokine responses, thereby reducing the boost for the expansion of self-reactive CD4+ T cells. Correspondingly, in mice, ONX 0914 treatment reversed cardiac autoimmune pathology, preventing the induction and progression of TnI-AM when self-reactive CD4+ T cells were primed. The autoimmune signature during experimental TnI-AM, with high immunoproteasome expression, immunoglobulin G deposition, interleukin-17 production in heart tissue, and TnI-directed humoral autoimmune responses, was also present in 2 cases of ICI-related myocarditis, demonstrating the activation of heart-specific autoimmune reactions by ICI therapy. CONCLUSIONS: By reversing heart-specific autoimmune responses, immunoproteasome inhibitors applied to a mouse model demonstrate their potential to aid in the management of autoimmune myocarditis in humans, possibly including patients with ICI-related heart-specific autoimmunity.
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Enfermedades Autoinmunes/inmunología , Modelos Animales de Enfermedad , Eliminación de Gen , Inhibidores de Puntos de Control Inmunológico/efectos adversos , Inmunidad/inmunología , Miocarditis/inmunología , Complejo de la Endopetidasa Proteasomal/inmunología , Anciano , Secuencia de Aminoácidos , Animales , Enfermedades Autoinmunes/inducido químicamente , Enfermedades Autoinmunes/genética , Cisteína Endopeptidasas/deficiencia , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/inmunología , Femenino , Humanos , Inmunidad/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Miocarditis/inducido químicamente , Miocarditis/genética , Complejo de la Endopetidasa Proteasomal/deficiencia , Complejo de la Endopetidasa Proteasomal/genéticaRESUMEN
CD146 is an adhesion molecule localized at endothelial cell junctions and facilitates cell-cell interactions. The circulating soluble form (sCD146) lacks both the intracellular and the transmembrane domains. In this study we show that CD146 expression was significantly decreased in the lung tissue of smokers with chronic obstructive pulmonary disease (COPD) and also in rats exposed to second-hand smoke (SHS). Concurrently, levels of sCD146 were increased in both the plasma and bronchoalveolar lavage fluid (BALF) of COPD patients as well as in BALF from rats exposed to SHS. Decreased or abolished CD146 protein expression in rat pulmonary micro- and macrovascular endothelial cells was found after treatment with cigarette smoke extract (CSE), proinflammatory cytokine interleukin 18 (IL-18) or after silencing CD146 expression with siRNA. The decrease in CD146 protein was accompanied by increased endothelial monolayer permeability and enhanced macrophage infiltration in vitro. In CD146 knockout (KO) mice, distinct perivascular oedema was seen and increased numbers of inflammatory cells, along with increased protein levels in BALF. Increased sCD146 was found in BALF and plasma from patients with COPD. The circulating plasma levels of sCD146 correlated positively with the presence of anti-endothelial cell antibodies (AECAs). sCD146 in combination with AECAs may be useful markers for early detection of COPD. Our study indicates that loss of CD146 function damages pulmonary endothelial integrity. This damage may represent part of the pathophysiological processes that are involved in the basic aetiology of COPD/emphysema.
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Antígeno CD146/metabolismo , Células Endoteliales/inmunología , Pulmón/irrigación sanguínea , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Enfisema Pulmonar/inmunología , Anciano , Animales , Autoanticuerpos/sangre , Biomarcadores/sangre , Líquido del Lavado Bronquioalveolar/inmunología , Antígeno CD146/análisis , Antígeno CD146/sangre , Antígeno CD146/genética , Permeabilidad Capilar , Estudios de Casos y Controles , Células Cultivadas , Modelos Animales de Enfermedad , Diagnóstico Precoz , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Regulación de la Expresión Génica , Humanos , Interleucina-18/metabolismo , Pulmón/inmunología , Pulmón/patología , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Enfermedad Pulmonar Obstructiva Crónica/sangre , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/patología , Edema Pulmonar/inmunología , Edema Pulmonar/patología , Enfisema Pulmonar/sangre , Enfisema Pulmonar/genética , Enfisema Pulmonar/patología , Interferencia de ARN , Ratas , Ratas Sprague-Dawley , Contaminación por Humo de Tabaco , TransfecciónRESUMEN
Chronic second-hand smoke (SHS) exposure comprises the main risk factor for nonsmokers to develop chronic obstructive pulmonary disease (COPD). However, the mechanisms behind the chronic inflammation and lung destruction remain incompletely understood. In this study, we show that chronic exposure of Sprague-Dawley rats to SHS results in a significant increase of proinflammatory cytokine IL-18 and chemokine (C-C motif) ligand 5 in the bronchoalveolar lavage fluid (BALF) and a significant decrease of vascular endothelial growth factor (VEGF) in the lung tissue. SHS exposure resulted in progressive alveolar airspace enlargement, cell death, pulmonary vessel loss, vessel muscularization, collagen deposition, and right ventricular hypertrophy. Alveolar macrophages displayed a foamy phenotype and a decreased expression of the natural inhibitor of IL-18, namely, IL-18 binding protein (IL-18BP). Moreover, IL-18 down-regulated the expression of VEGF receptor-1 and VEGFR receptor-2, and induced apoptosis in pulmonary microvascular endothelial cells in vitro. We also observed a trend toward increased concentrations of IL-18 in the BALF of patients with COPD. Our findings suggest that IL-18-mediated endothelial cell death may contribute to vascular destruction and disappearance in SHS-induced COPD. Moreover, IL-18 and IL-18BP are potential new targets for therapeutics.
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Células Endoteliales/patología , Interleucina-18/inmunología , Enfisema Pulmonar/patología , Contaminación por Humo de Tabaco/efectos adversos , Animales , Líquido del Lavado Bronquioalveolar/inmunología , Permeabilidad Capilar , Cardiomegalia/inducido químicamente , Cardiomegalia/patología , Muerte Celular , Línea Celular , Quimiocina CCL5/inmunología , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Humanos , Inmunohistoquímica , Exposición por Inhalación/efectos adversos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/inmunología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Interleucina-18/genética , Interleucina-18/metabolismo , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Enfermedad Pulmonar Obstructiva Crónica/patología , Enfisema Pulmonar/inmunología , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/patología , Ratas , Ratas Sprague-Dawley , Productos de Tabaco/efectos adversos , Factor A de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Pérdida de PesoRESUMEN
Acute coronary syndrome (ACS) remains a major cause of worldwide mortality. The syndrome occurs when blood flow to the heart muscle is decreased or blocked, causing muscle tissues to die or malfunction. There are three main types of ACS: Non-ST-elevation myocardial infarction, ST-elevation myocardial infarction, and unstable angina. The treatment depends on the type of ACS, and this is decided by a combination of clinical findings, such as electrocardiogram and plasma biomarkers. Circulating cell-free DNA (ccfDNA) is proposed as an additional marker for ACS since the damaged tissues can release DNA to the bloodstream. We used ccfDNA methylation profiles for differentiating between the ACS types and provided computational tools to repeat similar analysis for other diseases. We leveraged cell type specificity of DNA methylation to deconvolute the ccfDNA cell types of origin and to find methylation-based biomarkers that stratify patients. We identified hundreds of methylation markers associated with ACS types and validated them in an independent cohort. Many such markers were associated with genes involved in cardiovascular conditions and inflammation. ccfDNA methylation showed promise as a non-invasive diagnostic for acute coronary events. These methods are not limited to acute events, and may be used for chronic cardiovascular diseases as well.
RESUMEN
Ischemic cardiomyopathy, driven by loss of cardiomyocytes and inadequate proliferative response, persists to be a major global health problem. Using a functional high-throughput screening, we assessed differential proliferative potential of 2019 miRNAs after transient hypoxia by transfecting both miR-inhibitor and miR-mimic libraries in human iPSC-CM. Whereas miR-inhibitors failed to enhance EdU uptake, overexpression of 28 miRNAs substantially induced proliferative activity in hiPSC-CM, with an overrepresentation of miRNAs belonging to the primate-specific C19MC-cluster. Two of these miRNAs, miR-515-3p and miR-519e-3p, increased markers of early and late mitosis, indicative of cell division, and substantially alter signaling pathways relevant for cardiomyocyte proliferation in hiPSC-CM.
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Triacylglycerols (TG) are the major storage molecules of metabolic energy and fatty acids in several tissues. The final step in TG biosynthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. Lack of whole body DGAT1 is associated with reduced lipid-induced inflammation. Since one major component of atherosclerosis is chronic inflammation we hypothesized that DGAT1 deficiency might ameliorate atherosclerotic lesion development. We therefore crossbred Apolipoprotein E-deficient (ApoE(-/-)) mice with Dgat1(-/-) mice. ApoE(-/-) and ApoE(-/-)Dgat1(-/-) mice were fed Western-type diet (WTD) for 9weeks and thereafter examined for plaque formation. The mean atherosclerotic lesion area was substantially reduced in ApoE(-/-)Dgat1(-/-) compared with ApoE(-/-) mice in en face and aortic valve section analyses. The reduced lesion size was associated with decreased cholesterol uptake and absorption by the intestine, reduced plasma TG and cholesterol concentrations and increased cholesterol efflux from macrophages. The expression of adhesion molecules was reduced in aortas of ApoE(-/-)Dgat1(-/-) mice, which might be the reason for less migration capacities of monocytes and macrophages and the observed decreased amount of macrophages within the plaques. From our results we conclude that the lack of DGAT1 is atheroprotective, implicating an additional application of DGAT1 inhibitors with regard to maintaining cholesterol homeostasis and attenuating atherosclerosis.
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Aorta/metabolismo , Apolipoproteínas E/deficiencia , Aterosclerosis , Colesterol/sangre , Diacilglicerol O-Acetiltransferasa/deficiencia , Placa Aterosclerótica/sangre , Triglicéridos/sangre , Acilcoenzima A/sangre , Animales , Aorta/patología , Apolipoproteínas E/genética , Aterosclerosis/sangre , Aterosclerosis/enzimología , Aterosclerosis/genética , Movimiento Celular/genética , Células Cultivadas , Cruzamientos Genéticos , Diacilglicerol O-Acetiltransferasa/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunohistoquímica , Absorción Intestinal/genética , Mucosa Intestinal/metabolismo , Metabolismo de los Lípidos/genética , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Placa Aterosclerótica/patologíaRESUMEN
In severe pulmonary arterial hypertension (PAH), vascular lesions are composed of phenotypically altered vascular and inflammatory cells that form clusters or tumorlets. Because macrophages are found in increased numbers in intravascular and perivascular space in human PAH, here we address the question whether macrophages play a role in pulmonary vascular remodeling and whether accumulation of macrophages in the lung vasculature could be compromised by the immune system. We used the mouse macrophage cell line RAW 264.7 because these cells are resistant to apoptosis, have high proliferative capacity, and resemble cells in the plexiform lesions that tend to pile up instead of maintaining a monolayer. Cells were characterized by immunocytochemistry with cell surface markers (Lycopersicon Esculentum Lectin, CD117, CD133, FVIII, CD31, VEGFR-2, and S100). Activated, but not quiescent, T cells were able to suppress RAW 264.7 cell proliferative and migration activity in vitro. The carboxyfluorescein diacetate-labeled RAW 264.7 cells were injected into the naïve Sprague Dawley (SD) rat and athymic nude rat. Twelve days later, cells were found in the lung vasculature of athymic nude rats that lack functional T cells, contributing to vascular remodeling. No labeled RAW 264.7 cells were detected in the lungs of immune-competent SD rats. Our data demonstrate that T cells can inhibit in vitro migration and in vivo accumulation of macrophage-like cells.
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Comunicación Celular/inmunología , Pulmón/irrigación sanguínea , Macrófagos/citología , Arteria Pulmonar/citología , Linfocitos T/citología , Animales , Biomarcadores/análisis , Línea Celular , Movimiento Celular/inmunología , Proliferación Celular , Hipertensión Pulmonar Primaria Familiar , Fluoresceínas , Colorantes Fluorescentes , Hipertensión Pulmonar/inmunología , Hipertensión Pulmonar/patología , Hipertensión Pulmonar/fisiopatología , Inmunohistoquímica , Pulmón/citología , Pulmón/inmunología , Macrófagos/inmunología , Macrófagos/trasplante , Masculino , Ratones , Modelos Biológicos , Arteria Pulmonar/inmunología , Ratas , Ratas Desnudas , Ratas Sprague-Dawley , Linfocitos T/inmunologíaRESUMEN
RATIONALE: Vascular calcification is highly associated with cardiovascular morbidity and mortality, especially in patients with chronic kidney disease. The nuclear receptor farnesoid X receptor (FXR) has been implicated in the control of lipid, carbohydrate and bile acid metabolism in several cell types. Although recent studies have shown that FXR is also expressed in vascular smooth muscle cells, its physiological role in vasculature tissue remains obscure. OBJECTIVE: Here, we have examined the role of FXR in vascular calcification. METHODS AND RESULTS: The FXR gene, a bile acid nuclear receptor, was highly induced during osteogenic differentiation of bovine calcifying vascular cells (CVCs) and in the aorta of apolipoprotein (Apo)E(-/-) mice with chronic kidney disease which are common tissue culture and mouse model, respectively, for aortic calcification. FXR activation by a synthetic FXR agonist, 6alpha-ethyl chenodeoxycholic acid (INT-747) inhibited phosphate induced-mineralization and triglyceride accumulation in CVCs. FXR dominant negative expression augmented mineralization of CVCs and blocked the anticalcific effect of INT-747 whereas VP16FXR that is a constitutively active form reduced mineralization of CVCs. INT-747 treatment also increased phosphorylated c-Jun N-terminal kinase (JNK). SP600125 (specific JNK inhibitor) significantly induced mineralization of CVCs and alkaline phosphatase expression, suggesting that the anticalcific effect of INT-747 is attributable to JNK activation. We also found that INT-747 ameliorates chronic kidney disease induced-vascular calcification in 5/6 nephrectomized ApoE(-/-) mice without affecting the development of atherosclerosis. CONCLUSIONS: These observations provide direct evidence that FXR is a key signaling component in regulation of vascular osteogenic differentiation and, thus representing a promising target for the treatment of vascular calcification.
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Apolipoproteínas E/fisiología , Calcinosis/fisiopatología , Enfermedades Renales/fisiopatología , Receptores Citoplasmáticos y Nucleares/fisiología , Enfermedades Vasculares/fisiopatología , Animales , Aorta/citología , Aorta/efectos de los fármacos , Aorta/metabolismo , Apolipoproteínas E/genética , Calcinosis/prevención & control , Bovinos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/farmacología , Ácido Quenodesoxicólico/uso terapéutico , Enfermedad Crónica , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Noqueados , Osteogénesis/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Transducción de Señal/fisiología , Triglicéridos/metabolismoRESUMEN
Inflammasomes are crucial gatekeepers of the immune response, but their maladaptive activation associates with inflammatory pathologies. Besides canonical activation, monocytes can trigger non-transcriptional or rapid inflammasome activation that has not been well defined in the context of acute myocardial infarction (AMI). Rapid transcription-independent inflammasome activation induced by simultaneous TLR priming and triggering stimulus was measured by caspase-1 (CASP1) activity and interleukin release. Both classical and intermediate monocytes from healthy donors exhibited robust CASP1 activation, but only classical monocytes produced high mature interleukin-18 (IL18) release. We also recruited a limited number of coronary artery disease (CAD, n=31) and AMI (n=29) patients to evaluate their inflammasome function and expression profiles. Surprisingly, monocyte subpopulations isolated from blood collected during percutaneous coronary intervention (PCI) from AMI patients presented diminished CASP1 activity and abrogated IL18 release despite increased NLRP3 gene expression. This unexpected attenuated rapid inflammasome activation was accompanied by a significant increase of TNFAIP3 and IRAKM expression. Moreover, TNFAIP3 protein levels of circulating monocytes showed positive correlation with high sensitive troponin T (hsTnT), implying an association between TNFAIP3 upregulation and the severity of tissue injury. We suggest this monocyte attenuation to be a protective phenotype aftermath following a very early inflammatory wave in the ischemic area. Damage-associated molecular patterns (DAMPs) or other signals trigger a transitory negative feedback loop within newly recruited circulating monocytes as a mechanism to reduce post-injury tissue damage.
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Infarto del Miocardio , Intervención Coronaria Percutánea , Humanos , Inflamasomas/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Monocitos , Infarto del Miocardio/patología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismoRESUMEN
We characterized several cellular and structural features of early stage Type II/III atherosclerotic plaques in an established model of atherosclerosis-the ApoE-deficient mouse-by using a multimodal, coregistered imaging system that integrates three nonlinear optical microscopy (NLOM) contrast mechanisms: coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), and two-photon excitation fluorescence (TPEF). Specifically, the infiltration of lipid-rich macrophages and the structural organization of collagen and elastin fibers were visualized by CARS, SHG, and TPEF, respectively, in thick tissue specimens without the use of exogenous labels or dyes. Label-free CARS imaging of macrophage accumulation was confirmed by histopathology using CD68 staining. A high-fat, high-cholesterol Western diet resulted in an approximate 2-fold increase in intimal plaque area, defined by CARS signals of lipid-rich macrophages. Additionally, analysis of collagen distribution within lipid-rich plaque regions revealed nearly a 4-fold decrease in the Western diet-fed mice, suggesting NLOM sensitivity to increased matrix metalloproteinase (MMP) activity and decreased smooth muscle cell (SMC) accumulation. These imaging results provide significant insight into the structure and composition of early stage Type II/III plaque during formation and allow for quantitative measurements of the impact of diet and other factors on critical plaque and arterial wall features.
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Apolipoproteínas E/deficiencia , Aterosclerosis , Metabolismo de los Lípidos , Macrófagos/metabolismo , Microscopía/métodos , Espectrometría Raman/métodos , Animales , Aterosclerosis/metabolismo , Aterosclerosis/patología , Células Cultivadas , Colágeno/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Humanos , Macrófagos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Espectrometría de Fluorescencia/métodosRESUMEN
Cholesteryl ester (CE) accumulation in macrophages represents a crucial event during foam cell formation, a hallmark of atherogenesis. Here we investigated the role of two previously described CE hydrolases, hormone-sensitive lipase (HSL) and KIAA1363, in macrophage CE hydrolysis. HSL and KIAA1363 exhibited marked differences in their abilities to hydrolyze CE, triacylglycerol (TG), diacylglycerol (DG), and 2-acetyl monoalkylglycerol ether (AcMAGE), a precursor for biosynthesis of platelet-activating factor (PAF). HSL efficiently cleaved all four substrates, whereas KIAA1363 hydrolyzed only AcMAGE. This contradicts previous studies suggesting that KIAA1363 is a neutral CE hydrolase. Macrophages of KIAA1363(-/-) and wild-type mice exhibited identical neutral CE hydrolase activity, which was almost abolished in tissues and macrophages of HSL(-/-) mice. Conversely, AcMAGE hydrolase activity was diminished in macrophages and some tissues of KIAA1363(-/-) but unchanged in HSL(-/-) mice. CE turnover was unaffected in macrophages lacking KIAA1363 and HSL, whereas cAMP-dependent cholesterol efflux was influenced by HSL but not by KIAA1363. Despite decreased CE hydrolase activities, HSL(-/-) macrophages exhibited CE accumulation similar to wild-type (WT) macrophages. We conclude that additional enzymes must exist that cooperate with HSL to regulate CE levels in macrophages. KIAA1363 affects AcMAGE hydrolase activity but is of minor importance as a direct CE hydrolase in macrophages.
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Ésteres del Colesterol/metabolismo , Macrófagos/enzimología , Serina Proteasas/genética , Esterol Esterasa/genética , Animales , Aterosclerosis/genética , Aterosclerosis/metabolismo , Células COS , Chlorocebus aethiops , Diglicéridos/metabolismo , Hidrólisis , Macrófagos/metabolismo , Ratones , Ratones Endogámicos , Microscopía Fluorescente , Serina Proteasas/metabolismo , Esterol Esterasa/metabolismo , TransfecciónRESUMEN
BACKGROUND: The presence of anti-endothelial cell antibodies and pathogenic T cells may reflect an autoimmune component in the pathogenesis of emphysema. Whether immune modulatory strategies can protect against the development of emphysema is not known. METHODS: Sprague Dawley rats were immunized with human umbilical vein endothelial cells (HUVEC) to induce autoimmune emphysema and treated with intrathymic HUVEC-injection and pristane. Measurements of alveolar airspace enlargement, cytokine levels, immuno histochemical, western blot analysis, and T cell repertoire of the lung tissue were performed. RESULTS: The immunomodulatory strategies protected lungs against cell death as demonstrated by reduced numbers of TUNEL and active caspase-3 positive cells and reduced levels of active caspase-3, when compared with lungs from HUVEC-immunized rats. Immunomodulatory strategies also suppressed anti-endothelial antibody production and preserved CNTF, IL-1alpha and VEGF levels. The immune deviation effects of the intrathymic HUVEC-injection were associated with an expansion of CD4+CD25+Foxp3+ regulatory T cells. Pristane treatment decreased the proportion of T cells expressing receptor beta-chain, Vß16.1 in the lung tissue. CONCLUSIONS: Our data demonstrate that interventions classically employed to induce central T cell tolerance (thymic inoculation of antigen) or to activate innate immune responses (pristane treatment) can prevent the development of autoimmune emphysema.
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Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/prevención & control , Autoinmunidad/inmunología , Citocinas/inmunología , Enfisema/inmunología , Enfisema/prevención & control , Inmunomodulación/inmunología , Animales , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND AND AIMS: The mechanisms of interindividual variation of lipid regulation by statins, such as the low-density lipoprotein cholesterol (LDL) lowering effects, are not fully understood yet. Here, we used a gut microbiota depleted mouse model to investigate the relation between the gut microbiota and the regulatory property of atorvastatin on blood lipids. METHODS: Mice (C57BL/6) with intact gut microbiota or antibiotic induced abiotic mice (ABS) were put on standard chow diet (SCD) or high fat diet (HFD) for six weeks. Atorvastatin (10 mg/kg body weight/day) or a control vehicle were applied per gavage for the last four weeks of dietary treatment. Blood lipids including total cholesterol, very low-density lipoprotein, low-density lipoprotein, high-density lipoprotein and sphingolipids were measured to probe microbiota-dependent effects of atorvastatin. The expression of genes involved in hepatic and intestinal cholesterol metabolism was analyzed with qRT-PCR. The alteration of the microbiota profile was examined using 16S rRNA qPCR in mice with intact gut microbiota. RESULTS: HFD feeding significantly increased total blood cholesterol and LDL levels, as compared to SCD in both mice with intact and depleted gut microbiota. The cholesterol lowering effect of atorvastatin was significantly attenuated in mice with depleted gut microbiota. Moreover, we observed a global shift in the abundance of several sphingolipids upon atorvastatin treatment which was absent in gut microbiota depleted mice. The regulatory effect of atorvastatin on the expression of distinct hepatic and intestinal cholesterol-regulating genes, including Ldlr, Srebp2 and Npc1l1 was altered upon depletion of gut microbiota. In response to HFD feeding, the relative abundance of the bacterial phyla Bacteroidetes decreased, while the abundance of Firmicutes increased. The altered ratio between Firmicutes to Bacteroidetes was partly reversed in HFD fed mice treated with atorvastatin. CONCLUSIONS: Our findings support a regulatory impact of atorvastatin on the gut microbial profile and, in turn, demonstrate a crucial role of the gut microbiome for atorvastatin-related effects on blood lipids. These results provide novel insights into potential microbiota-dependent mechanisms of lipid regulation by statins, which may account for variable response to statin treatment.
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AIMS: Inflammation is a key driver of atherosclerosis and myocardial infarction (MI), and beyond proteins and microRNAs (miRs), long noncoding RNAs (lncRNAs) have been implicated in inflammation control. To obtain further information on the possible role of lncRNAs in the context of atherosclerosis, we obtained comprehensive transcriptome maps of circulating immune cells (peripheral blood mononuclear cells, PBMCs) of early onset MI patients. One lncRNA significantly suppressed in post-MI patients was further investigated in a murine knockout model. METHODS AND RESULTS: Individual RNA-sequencing (RNA-seq) was conducted on PBMCs from 28 post-MI patients with a history of MI at age ≤50 years and stable disease ≥3 months before study participation, and from 31 healthy individuals without manifest cardiovascular disease or family history of MI as controls. RNA-seq revealed deregulated protein-coding transcripts and lncRNAs in post-MI PBMCs, among which nuclear enriched abundant transcript (NEAT1) was the most highly expressed lncRNA, and the only one significantly suppressed in patients. Multivariate statistical analysis of validation cohorts of 106 post-MI patients and 85 controls indicated that the PBMC NEAT1 levels were influenced (P = 0.001) by post-MI status independent of statin intake, left ventricular ejection fraction, low-density lipoprotein or high-density lipoprotein cholesterol, or age. We investigated NEAT1-/- mice as a model of NEAT1 deficiency to evaluate if NEAT1 depletion may directly and causally alter immune regulation. RNA-seq of NEAT1-/- splenocytes identified disturbed expression and regulation of chemokines/receptors, innate immunity genes, tumour necrosis factor (TNF) and caspases, and increased production of reactive oxygen species (ROS) under baseline conditions. NEAT1-/- spleen displayed anomalous Treg and TH cell differentiation. NEAT1-/- bone marrow-derived macrophages (BMDMs) displayed altered transcriptomes with disturbed chemokine/chemokine receptor expression, increased baseline phagocytosis (P < 0.0001), and attenuated proliferation (P = 0.0013). NEAT1-/- BMDMs responded to LPS with increased (P < 0.0001) ROS production and disturbed phagocytic activity (P = 0.0318). Monocyte-macrophage differentiation was deregulated in NEAT1-/- bone marrow and blood. NEAT1-/- mice displayed aortic wall CD68+ cell infiltration, and there was evidence of myocardial inflammation which could lead to severe and potentially life-threatening structural damage in some of these animals. CONCLUSION: The study indicates distinctive alterations of lncRNA expression in post-MI patient PBMCs. Regarding the monocyte-enriched NEAT1 suppressed in post-MI patients, the data from NEAT1-/- mice identify NEAT1 as a novel lncRNA-type immunoregulator affecting monocyte-macrophage functions and T cell differentiation. NEAT1 is part of a molecular circuit also involving several chemokines and interleukins persistently deregulated post-MI. Individual profiling of this circuit may contribute to identify high-risk patients likely to benefit from immunomodulatory therapies. It also appears reasonable to look for new therapeutic targets within this circuit.
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Inmunidad Innata , Leucocitos Mononucleares/metabolismo , Infarto del Miocardio/metabolismo , ARN Largo no Codificante/metabolismo , Adulto , Edad de Inicio , Animales , Estudios de Casos y Controles , Diferenciación Celular , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Regulación hacia Abajo , Femenino , Humanos , Leucocitos Mononucleares/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones Noqueados , Persona de Mediana Edad , Infarto del Miocardio/genética , Infarto del Miocardio/inmunología , ARN Largo no Codificante/genética , ARN Largo no Codificante/inmunología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Bazo/inmunología , Bazo/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Factores de TiempoRESUMEN
Genome-wide association studies (GWAS) have proven a fundamental tool to identify common variants associated to complex traits, thus contributing to unveil the genetic components of human disease. Besides, the advent of GWAS contributed to expose unexpected findings that urged to redefine the framework of population genetics. First, loci identified by GWAS had small effect sizes and could only explain a fraction of the predicted heritability of the traits under study. Second, the majority of GWAS hits mapped within non-coding regions (such as intergenic or intronic regions) where new functional RNA species (such as lncRNAs or circRNAs) have started to emerge. Bigger cohorts, meta-analysis and technical improvements in genotyping allowed identification of an increased number of genetic variants associated to coronary artery disease (CAD) and cardiometabolic traits. The challenge remains to infer causal mechanisms by which these variants influence cardiovascular disease development. A tendency to assign potential causal variants preferentially to coding genes close to lead variants contributed to disregard the role of non-coding elements. In recent years, in parallel to an increased knowledge of the non-coding genome, new studies started to characterize disease-associated variants located within non-coding RNA regions. The upcoming of databases integrating single-nucleotide polymorphisms (SNPs) and non-coding RNAs together with novel technologies will hopefully facilitate the discovery of causal non-coding variants associated to disease. This review attempts to summarize the current knowledge of genetic variation within non-coding regions with a focus on long non-coding RNAs that have widespread impact in cardiometabolic diseases.
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Oxidized phospholipids (oxPAPC) induce endothelial dysfunction and atherosclerosis. Here we show that oxPAPC induce a gene network regulating serine-glycine metabolism with the mitochondrial methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2) as a causal regulator using integrative network modeling and Bayesian network analysis in human aortic endothelial cells. The cluster is activated in human plaque material and by atherogenic lipoproteins isolated from plasma of patients with coronary artery disease (CAD). Single nucleotide polymorphisms (SNPs) within the MTHFD2-controlled cluster associate with CAD. The MTHFD2-controlled cluster redirects metabolism to glycine synthesis to replenish purine nucleotides. Since endothelial cells secrete purines in response to oxPAPC, the MTHFD2-controlled response maintains endothelial ATP. Accordingly, MTHFD2-dependent glycine synthesis is a prerequisite for angiogenesis. Thus, we propose that endothelial cells undergo MTHFD2-mediated reprogramming toward serine-glycine and mitochondrial one-carbon metabolism to compensate for the loss of ATP in response to oxPAPC during atherosclerosis.
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Aminoácidos/metabolismo , Aminohidrolasas/metabolismo , Aterosclerosis/metabolismo , Células Endoteliales/metabolismo , Metilenotetrahidrofolato Deshidrogenasa (NADP)/metabolismo , Enzimas Multifuncionales/metabolismo , Fosfolípidos/química , Animales , Aorta/citología , Teorema de Bayes , Enfermedades Cardiovasculares/metabolismo , Regulación de la Expresión Génica , Técnicas Genéticas , Glicina/química , Células Endoteliales de la Vena Umbilical Humana , Humanos , Familia de Multigenes , Neovascularización Patológica , Nucleótidos/química , Oxígeno/química , Probabilidad , Purinas/química , ARN Interferente Pequeño/metabolismo , Pez CebraRESUMEN
BACKGROUND AND AIMS: High-density lipoprotein cholesterol (HDL-C) is inversely related to cardiovascular risk. HDL-C raising ester transfer protein (CETP) inhibitors, are novel therapeutics. We studied the effects of CETP inhibitors anacetrapib and evacetrapib on triglycerides, cholesterol and lipoproteins, cholesterol efflux, paraoxonase activity (PON-1), reactive oxygen species (ROS), and endothelial function in E3L and E3L.CETP mice. METHODS: Triglycerides and cholesterol were measured at weeks 5, 14 and 21 in E3L.CETP mice on high cholesterol diet and treated with anacetrapib (3 mg/kg/day), evacetrapib (3 mg/kg/day) or placebo. Cholesterol efflux was assessed ex-vivo in mice treated with CETP inhibitors for 3 weeks on a normal chow diet. Endothelial function was analyzed at week 21 in isolated aortic rings, and serum lipoproteins assessed by fast-performance liquid chromatography. RESULTS: Anacetrapib and evacetrapib increased HDL-C levels (5- and 3.4-fold, resp.) and reduced triglycerides (-39% vs. placebo, p = 0.0174). Total cholesterol levels were reduced only in anacetrapib-treated mice (-32%, p = 0.0386). Cholesterol efflux and PON-1 activity (+45% and +35% vs. control, p < 0.005, resp.) were increased, while aortic ROS production was reduced with evacetrapib (-49% vs. control, p = 0.020). Anacetrapib, but not evacetrapib, impaired endothelium dependent vasorelaxation (p < 0.05). In contrast, no such effects were observed in E3L mice for all parameters tested. CONCLUSIONS: Notwithstanding a marked rise in HDL-C, evacetrapib did not improve endothelial function, while anacetrapib impaired it, suggesting that CETP inhibition does not provide vascular protection. Anacetrapib exerts unfavorable endothelial effects beyond CETP inhibition, which may explain the neutral results of large clinical trials in spite of increased HDL-C.
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Anticolesterolemiantes/farmacología , Benzodiazepinas/farmacología , Proteínas de Transferencia de Ésteres de Colesterol/antagonistas & inhibidores , HDL-Colesterol/sangre , Dislipidemias/tratamiento farmacológico , Endotelio Vascular/efectos de los fármacos , Oxazolidinonas/farmacología , Vasodilatación/efectos de los fármacos , Animales , Anticolesterolemiantes/toxicidad , Apolipoproteína E3/genética , Arildialquilfosfatasa/sangre , Benzodiazepinas/toxicidad , Biomarcadores/sangre , Proteínas de Transferencia de Ésteres de Colesterol/genética , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Dislipidemias/sangre , Dislipidemias/genética , Dislipidemias/fisiopatología , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Femenino , Predisposición Genética a la Enfermedad , Humanos , Ratones Transgénicos , Oxazolidinonas/toxicidad , Fenotipo , Especies Reactivas de Oxígeno/metabolismo , Triglicéridos/sangre , Regulación hacia ArribaRESUMEN
Hypertension represents a major cardiovascular risk factor. The pathophysiology of increased blood pressure (BP) is not yet completely understood. Transcriptome profiling offers possibilities to uncover genetics effects on BP. Based on 2 populations including 2549 individuals, a meta-analyses of monocytic transcriptome-wide profiles were performed to identify transcripts associated with BP. Replication was performed in 2 independent studies of whole-blood transcriptome data including 1990 individuals. For identified candidate genes, a direct link between long-term changes in BP and gene expression over time and by treatment with BP-lowering therapy was assessed. The predictive value of protein levels encoded by candidate genes for subsequent cardiovascular disease was investigated. Eight transcripts (CRIP1, MYADM, TIPARP, TSC22D3, CEBPA, F12, LMNA, and TPPP3) were identified jointly accounting for up to 13% (95% confidence interval, 8.7-16.2) of BP variability. Changes in CRIP1, MYADM, TIPARP, LMNA, TSC22D3, CEBPA, and TPPP3 expression associated with BP changes-among these, CRIP1 gene expression was additionally correlated to measures of cardiac hypertrophy. Assessment of circulating CRIP1 (cystein-rich protein 1) levels as biomarkers showed a strong association with increased risk for incident stroke (hazard ratio, 1.06; 95% confidence interval, 1.03-1.09; P=5.0×10-5). Our comprehensive analysis of global gene expression highlights 8 novel transcripts significantly associated with BP, providing a link between gene expression and BP. Translational approaches further established evidence for the potential use of CRIP1 as emerging disease-related biomarker.
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Proteínas Portadoras/genética , Hipertensión , Proteínas con Dominio LIM/genética , Accidente Cerebrovascular , Adulto , Presión Sanguínea/genética , Determinación de la Presión Sanguínea/métodos , Determinación de la Presión Sanguínea/estadística & datos numéricos , Proteínas Potenciadoras de Unión a CCAAT/genética , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Humanos , Hipertensión/diagnóstico , Hipertensión/genética , Masculino , Proteínas Proteolipídicas Asociadas a Mielina y Linfocito/genética , Proteínas de Transporte de Nucleósidos , Poli(ADP-Ribosa) Polimerasas/genética , Polimorfismo de Nucleótido Simple , Factores de Riesgo , Accidente Cerebrovascular/epidemiología , Accidente Cerebrovascular/etiología , Accidente Cerebrovascular/prevención & control , Factores de Transcripción/genéticaRESUMEN
Numerous studies have examined the role of microRNAs (miRNAs) in cell homeostasis and cardiovascular disease and have markedly improved our understanding of RNA biology in general and the potential role of miRNAs in atherosclerosis. In atherosclerosis, several miRNAs, such as miR-33a,b, miR-92a, miR-126 and others, have been identified that are relevant mediators of pathological processes, including regulation of cholesterol and lipid biosynthesis, lipoprotein metabolism and cholesterol efflux, but also immune responses, endothelial cell biology and vascular function. Further understanding of the specific roles of miRNAs in the distinct cell types involved in atherosclerosis initiation, progression and resolution may reveal new intervention strategies for the prevention and treatment of atherosclerotic cardiovascular disease.