Moderate beer intake downregulates inflammasome pathway gene expression in human macrophages

Inflammasomes are key components of the innate immunity system that trigger the inflammatory response. Inappropriate activity of the inflammasome system has been linked to onset and perpetuation of inflammation in atherosclerotic plaques and cardiovascular disease. Low-to-moderate beer consumption is inversely associated with cardiovascular event presentation, while high levels of alcohol intake are associated with increased cardiovascular risk. Although fermented beverages have been suggested to exert their beneficial effects through their anti-oxidant and anti-inflammatory properties, little is known regarding the capacity of beer to modulate innate immunity cell responses. To this aim, primed or activated THP-1 macrophages were conditioned with human serum obtained from a prospective two-arms longitudinal crossover study to investigate the effect of a moderate and regular daily intake of beer, either alcohol-free or traditional, in the regulation of TLR-mediated inflammatory responses in healthy but overweight individuals. Conditioned macrophages with serum obtained after four-week intervention with alcohol-free beer significantly reduced the transcription of pro-inflammatory interleukins such as IL-1β and TNF. The serum of traditional beer consumers did not exhibit the same capacity as the serum of alcohol-free beer consumers to reduce gene expression of pro-inflammatory interleukins; however, serum from traditional beer consumers showed a regulatory effect at the protein level by significantly decreasing the intracellular protein levels of pro-IL-1β in primed macrophages and preventing cleaved-IL-1β protein release

Alternative c3 complement system : Lipids and atherosclerosis

Altres ajuts: Fundación Jesus SerraAltres ajuts: Fundación de Investigación CardiovascularAltres ajuts: Fondo Europeo de Desarrollo Regional (FEDER)Familial hypercholesterolemia (FH) is increasingly associated with inflammation, a phenotype that persists despite treatment with lipid lowering therapies. The alternative C3 complement system (C3), as a key inflammatory mediator, seems to be involved in the atherosclerotic process; however, the relationship between C3 and lipids during plaque progression remains unknown. The aim of the study was to investigate by a systems biology approach the role of C3 in relation to lipoprotein levels during atherosclerosis (AT) progression and to gain a better understanding on the effects of C3 products on the phenotype and function of human lipid-loaded vascular smooth muscle cells (VSMCs). By mass spectrometry and differential proteomics, we found the extracellular matrix (ECM) of human aortas to be enriched in active components of the C3 complement system, with a significantly different proteomic signature in AT segments. Thus, C3 products were more abundant in AT-ECM than in macroscopically normal segments. Furthermore, circulating C3 levels were significantly elevated in FH patients with subclinical coronary AT, evidenced by computed tomographic angiography. However, no correlation was identified between circulating C3 levels and the increase in plaque burden, indicating a local regulation of the C3 in AT arteries. In cell culture studies of human VSMCs, we evidenced the expression of C3, C3aR (anaphylatoxin receptor) and the integrin α β receptor for C3b/iC3b (RT-PCR and Western blot). C3mRNA was up-regulated in lipid-loaded human VSMCs, and C3 protein significantly increased in cell culture supernatants, indicating that the C3 products in the AT-ECM have a local vessel-wall niche. Interestingly, C3a and iC3b (C3 active fragments) have functional effects on VSMCs, significantly reversing the inhibition of VSMC migration induced by aggregated LDL and stimulating cell spreading, organization of F-actin stress fibers and attachment during the adhesion of lipid-loaded human VSMCs. This study, by using a systems biology approach, identified molecular processes involving the C3 complement system in vascular remodeling and in the progression of advanced human atherosclerotic lesions

High-density lipoprotein remodelled in hypercholesterolaemic blood induce epigenetically driven down-regulation of endothelial HIF-1α expression in a preclinical animal model

Altres ajuts: This work was supported by the Plan Nacional de Salud (PNS) from the Spanish Ministry of Science and Innovation and funds FEDER 'Una Manera de Hacer Europa'; a grant from the Spanish Society of Cardiology (Beca FEC Investigacio'n Ba'sica/2016 to G.V); the Instituto de Salud Carlos III (ISCIII); and CIBERCV (to L.B). We thank the support of the Generalitat of Catalunya (Secretaria d'Universitats i Recerca del Departament d'Economia i Coneixement de la Generalitat,) and the Fundación Investigación Cardiovascular-Fundación Jesus Serra for their continuous support.High-density lipoproteins (HDLs) are circulating micelles that transport proteins, lipids, and miRNAs. HDL-transported miRNAs (HDL-miRNAs) have lately received attention but their effects on vascular cells are not fully understood. Additionally, whether cardiovascular risk factors affect HDL-miRNAs levels and miRNA transfer to recipient cells remains equally poorly known. Here, we have investigated the changes induced by hypercholesterolaemia on HDL-miRNA levels and its effect on recipient endothelial cells (ECs). Pigs were kept on a high-fat diet (HC; n = 10) or a normocholesterolaemic chow (NC; n = 10) for 10 days reaching cholesterol levels of 321.0 (229.7-378.5) mg/dL and 74.0 (62.5-80.2) mg/dL, respectively. HDL particles were isolated, purified, and quantified. HDL-miRNA profiling (n = 149 miRNAs) of HC- and NC-HDLs was performed by multipanel qPCR. Cell cultures of porcine aortic ECs were used to determine whether HDL-miRNAs were delivered to ECs. Potential target genes modulated by miRNAs were identified by bioinformatics and candidate miRNAs were validated by molecular analysis. In vivo effects in the coronary arteries of normocholesterolaemic swine administered HC- or NC-HDLs were analysed. Among the HDL-miRNAs, four were found in different amounts in HC- and NC-HDL (P < 0.05). miR-126-5p and -3p and miR-30b-5p (2.7×, 1.7×, and 1.3×, respectively) were found in higher levels and miR-103a-3p and miR-let-7g-5p (−1.6×, −1.4×, respectively) in lower levels in HC-HDL. miR-126-5p and -3p were transferred from HC-HDL to EC (2.5×; P < 0.05), but not from NC-HDL, by a SRB1-mediated mechanism. Bioinformatics revealed that HIF1α was the miR-126 target gene with the highest predictive value, which was accordingly found to be markedly reduced in HC-HDL-treated ECs and in miR126 mimic transfected ECs. In vivo validation confirmed that HIF1α was diminished in the coronary endothelial layer of NC pigs administered HC-HDL vs. those administered NC-HDL (P < 0.05). Hypercholesterolaemia induces changes in the miRNA content of HDL enhancing miR126 and its delivery to ECs with the consequent down-regulation of its target gene HIF1α

Efectos de la hipercolesterolemia sobre monocitos/macrófagos y la inmunidad innata

[spa] La enfermedad cardiovascular sigue siendo la principal causa de morbilidad y mortalidad en todo el mundo. La inmensa mayoría de los eventos cardiovasculares incluyendo los síndromes coronarios y accidente cerebrovascular son la causa por la rotura o erosión de las placas de ateroma en la pared arterial. La aterosclerosis es una enfermedad compleja dinámica caracterizada por la infiltración de lípidos en la pared arterial y una respuesta inflamación crónica por las células del sistema inmune innato, principalmente monocitos y macrófagos. Los altos niveles circulantes de lipoproteínas de baja densidad (LDL) son uno de los principales factores de riesgo cardiovasculares en sujetos con hipercolesterolemia familiar (FH), causada principalmente por mutaciones en LDLR afectando el metabolismo de los lípidos, que están en alto riesgo de eventos cardiovasculares prematuros. Actualmente está bien establecida la relevancia de la inflamación en el desarrollo de las placas ateroscleróticas, sin embargo, los efectos de la LDL sobre el fenotipo y la función de las células de la inmunidad innata, como monocitos y macrófagos, no son completamente entendidos. Además, la respuesta inflamatoria en los macrófagos de pacientes con HF (de SAFEHEART cohorte) y su asociación con la exposición de por vida a niveles altos de LDLc no se ha estudiado. En esta tesis, nosotros investigamos el efecto de los niveles aterogénicas LDL sobre los monocitos humanos y se demostró que la LDL facilita la diferenciación de los monocitos en macrófagos a través de un proceso que implica una expresión potenciada de moléculas de adhesión celular y la regulación disminuida de efectores de apoptosis que regulan la anoikis en el estadio temprano del macrófago. Esto sugiere un papel relevante de la LDL en la relación entre los lípidos, la inmunidad innata y la aterosclerosis. En pacientes FH, hemos demostrado que LRP5, receptor activo de internalización de lípidos, está incrementado en las células de la inmunidad innata que tiene una baja expresión del LDLR pero que conservan la función de internalización de LDL. La expresión reducida de CD163 en estos macrófagos FH también sugiere menos ateroprotección. También muestran que los macrófagos derivados de monocitos de pacientes con FH tratados de acuerdo a las guías tienen un fenotipo pro-inflamatorio sostenido, que se caracteriza por un aumento de la expresión de receptores de quimioquinas CCR3, CCR4, CXCR1 y una baja regulación de miR-505-3p, moléculas relacionadas con respuesta inflamatoria. El efecto es dependiente de la edad del paciente, lo que sugiere un patrón inflamatorio crónica en las células de la inmunidad innata que está relacionado con los cambios epigenéticas inducidos por la exposición de por vida a niveles altos de LDL.[eng] Cardiovascular disease remains a leading cause of morbidity and mortality worldwide. The vast majority of cardiovascular events including coronary syndromes and stroke are cause by the rupture or erosion of atherosclerotic plaques in the arterial wall. Atherosclerosis is a complex dynamic disease characterized by lipid infiltration in arterial wall and a chronic inflammation response by cells of the innate immune system, mainly monocytes and macrophages. High levels of circulating low-density lipoproteins (LDL) are one of the major cardiovascular risk factors and subjects with Familial Hypercholesterolemia (FH), mainly caused by LDLR mutations affecting lipid metabolism, are at high risk of premature cardiovascular events. Currently it is well established the relevance of inflammation in the development of the atherosclerotic plaques, however, the effects of LDL on the phenotype and function of cells of the innate immunity, as monocyte and macrophage, are not fully understood. Further, the inflammatory response in macrophages of FH patients (from SAFEHEART Cohort) and its association with lifetime exposure to high LDLc levels has not been studied. In this thesis, we investigated the effect of atherogenic levels LDL on human monocyte and demonstrated that LDL facilitate monocyte differentiation into macrophage through a process that involves enhanced expression of cell-adhesion molecules and downregulation of apoptosis- effectors regulating anoikis in the early stage macrophages. This suggests a relevant role of LDL in the link between lipids, innate immunity and atherosclerosis. In patients FH, we demonstrated that LRP5, active lipid-internalizing receptor, is upregulated in innate immunity cells that have downregulated LDLR but retain the LDL internalization function. The reduced CD163 expression in these FH-macrophages also suggests less atheroprotection. We also show that monocyte-derived macrophages from FH patients treated according to guidelines have a sustained pro-inflammatory phenotype, characterized by an increased expression of chemokine receptors CCR3, CCR4, CXCR1 and a down-regulation of miR-505-3p, molecules related with inflammatory response. The effect is dependent on the age of the patient, suggesting a chronic inflammatory pattern in innate immunity cells that is related to epigenetic changes induced by lifetime exposure to high levels of LDL

Systems Biology in Chronic Heart Failure-Identification of Potential miRNA Regulators

Heart failure (HF) is a complex disease entity with high clinical impact, poorly understood pathophysiology and scantly known miRNA-mediated epigenetic regulation. We have analysed miRNA patterns in patients with chronic HF (cHF) and a sex- and age-matched reference group and pursued an in silico system biology analysis to discern pathways involved in cHF pathophysiology. Twenty-eight miRNAs were identified in cHF that were up-regulated in the reference group, and eight of them were validated by RT-qPCR. In silico analysis of predicted targets by STRING protein-protein interaction networks revealed eight cluster networks (involving seven of the identified miRNAs) enriched in pathways related to cell cycle, Ras, chemokine, PI3K-AKT and TGF-β signaling. By ROC curve analysis, combined probabilities of these seven miRNAs (let-7a-5p, miR-107, miR-125a-5p, miR-139-5p, miR-150-5p, miR-30b-5p and miR-342-3p ; clusters 1-4 [C:1-4]), discriminated between HF with preserved ejection fraction (HFpEF) and HF with reduced ejection fraction (HFrEF), and ischaemic and non-ischaemic aetiology. A combination of miR-107, miR-139-5p and miR-150-5p, involved in clusters 5 and 7 (C:5+7), discriminated HFpEF from HFrEF. Pathway enrichment analysis of miRNAs present in C:1-4 (let-7a-5p, miR-125a-5p, miR-30b-5p and miR-342-3p) revealed pathways related to HF pathogenesis. In conclusion, we have identified a differential signature of down-regulated miRNAs in the plasma of HF patients and propose novel cellular mechanisms involved in cHF pathogenesis

Macrophages of genetically characterized familial hypercholesterolaemia patients show up-regulation of LDL-receptor-related proteins

Familial hypercholesterolaemia (FH) is a major risk for premature coronary heart disease due to severe long-life exposure to high LDL levels. Accumulation of LDL in the vascular wall triggers atherosclerosis with activation of the innate immunity system. Here, we have investigated (i) gene expression of LDLR and LRPs in peripheral blood cells (PBLs) and in differentiated macrophages of young FH-patients; and (ii) whether macrophage from FH patients have a differential response when exposed to high levels of atherogenic LDL. PBLs in young heterozygous genetically characterized FH patients have higher expression of LRP5 and LRP6 than age-matched healthy controls or patients with secondary hypercholesterolaemia. LRP1 levels were similar among groups. In monocyte-derived macrophages (MACs), LRP5 and LRP1 transcript levels did not differ between FHs and controls in resting conditions, but when exposed to agLDL, FH-MAC showed a highly significant up-regulation of LRP5, while LRP1 was unaffected. PBL and MAC cells from FH patients had significantly lower LDLR expression than control cells, independently of the lipid-lowering therapy. Furthermore, exposure of FH-MAC to agLDL resulted in a reduced expression of CD163, scavenger receptor with anti-inflammatory and atheroprotective properties. In summary, our results show for first time that LRPs, active lipid-internalizing receptors, are up-regulated in innate immunity cells of young FH patients that have functional LDLR mutations. Additionally, their reduced CD163 expression indicates less atheroprotection. Both mechanisms may play a synergic effect on the onset of premature atherosclerosis in FH patients

Systems Biology in Chronic Heart Failure—Identification of Potential miRNA Regulators

Heart failure (HF) is a complex disease entity with high clinical impact, poorly understood pathophysiology and scantly known miRNA-mediated epigenetic regulation. We have analysed miRNA patterns in patients with chronic HF (cHF) and a sex- and age-matched reference group and pursued an in silico system biology analysis to discern pathways involved in cHF pathophysiology. Twenty-eight miRNAs were identified in cHF that were up-regulated in the reference group, and eight of them were validated by RT-qPCR. In silico analysis of predicted targets by STRING protein-protein interaction networks revealed eight cluster networks (involving seven of the identified miRNAs) enriched in pathways related to cell cycle, Ras, chemokine, PI3K-AKT and TGF-β signaling. By ROC curve analysis, combined probabilities of these seven miRNAs (let-7a-5p, miR-107, miR-125a-5p, miR-139-5p, miR-150-5p, miR-30b-5p and miR-342-3p; clusters 1–4 [C:1–4]), discriminated between HF with preserved ejection fraction (HFpEF) and HF with reduced ejection fraction (HFrEF), and ischaemic and non-ischaemic aetiology. A combination of miR-107, miR-139-5p and miR-150-5p, involved in clusters 5 and 7 (C:5+7), discriminated HFpEF from HFrEF. Pathway enrichment analysis of miRNAs present in C:1–4 (let-7a-5p, miR-125a-5p, miR-30b-5p and miR-342-3p) revealed pathways related to HF pathogenesis. In conclusion, we have identified a differential signature of down-regulated miRNAs in the plasma of HF patients and propose novel cellular mechanisms involved in cHF pathogenesis

High-density lipoprotein remodelled in hypercholesterolaemic blood induce epigenetically driven down-regulation of endothelial HIF-1α expression in a preclinical animal model

Altres ajuts: This work was supported by the Plan Nacional de Salud (PNS) from the Spanish Ministry of Science and Innovation and funds FEDER 'Una Manera de Hacer Europa'; a grant from the Spanish Society of Cardiology (Beca FEC Investigacio'n Ba'sica/2016 to G.V); the Instituto de Salud Carlos III (ISCIII); and CIBERCV (to L.B). We thank the support of the Generalitat of Catalunya (Secretaria d'Universitats i Recerca del Departament d'Economia i Coneixement de la Generalitat,) and the Fundación Investigación Cardiovascular-Fundación Jesus Serra for their continuous support.High-density lipoproteins (HDLs) are circulating micelles that transport proteins, lipids, and miRNAs. HDL-transported miRNAs (HDL-miRNAs) have lately received attention but their effects on vascular cells are not fully understood. Additionally, whether cardiovascular risk factors affect HDL-miRNAs levels and miRNA transfer to recipient cells remains equally poorly known. Here, we have investigated the changes induced by hypercholesterolaemia on HDL-miRNA levels and its effect on recipient endothelial cells (ECs). Pigs were kept on a high-fat diet (HC; n = 10) or a normocholesterolaemic chow (NC; n = 10) for 10 days reaching cholesterol levels of 321.0 (229.7-378.5) mg/dL and 74.0 (62.5-80.2) mg/dL, respectively. HDL particles were isolated, purified, and quantified. HDL-miRNA profiling (n = 149 miRNAs) of HC- and NC-HDLs was performed by multipanel qPCR. Cell cultures of porcine aortic ECs were used to determine whether HDL-miRNAs were delivered to ECs. Potential target genes modulated by miRNAs were identified by bioinformatics and candidate miRNAs were validated by molecular analysis. In vivo effects in the coronary arteries of normocholesterolaemic swine administered HC- or NC-HDLs were analysed. Among the HDL-miRNAs, four were found in different amounts in HC- and NC-HDL (P < 0.05). miR-126-5p and -3p and miR-30b-5p (2.7×, 1.7×, and 1.3×, respectively) were found in higher levels and miR-103a-3p and miR-let-7g-5p (−1.6×, −1.4×, respectively) in lower levels in HC-HDL. miR-126-5p and -3p were transferred from HC-HDL to EC (2.5×; P < 0.05), but not from NC-HDL, by a SRB1-mediated mechanism. Bioinformatics revealed that HIF1α was the miR-126 target gene with the highest predictive value, which was accordingly found to be markedly reduced in HC-HDL-treated ECs and in miR126 mimic transfected ECs. In vivo validation confirmed that HIF1α was diminished in the coronary endothelial layer of NC pigs administered HC-HDL vs. those administered NC-HDL (P < 0.05). Hypercholesterolaemia induces changes in the miRNA content of HDL enhancing miR126 and its delivery to ECs with the consequent down-regulation of its target gene HIF1α

Detrimental effect of hypercholesterolemia on high-density lipoprotein particle remodeling in pigs

[Background] Beneficial effects of high-density lipoproteins (HDL) seem altered in patients with symptomatic cardiovascular disease. We recently demonstrated in a swine model of ischemia-reperfusion (IR) that hypercholesterolemia abolishes HDL-related cardioprotection. [Objectives] This study sought to investigate, using the same animal model, whether the reported impairment of HDL cardioprotective function was associated with alterations in HDL remodeling and functionality. [Methods] Pigs were fed a normocholesterolemic (NC) or hypercholesterolemic (HL) diet for 10 days, reaching non-HDL cholesterol concentrations of 38.2 ± 3.5 mg/dl and 218.6 ± 27.6 mg/dl, respectively (p < 0.0001). HDLs were isolated, and lipidomics and differential proteomics tests were performed to determine HDL molecular changes. HDL functionality and particle size were determined. [Results] Using principal component analysis, we identified 255 molecular lipid species differentially clustered in NC-HDL and HL-HDL. Ninety lipid metabolites were differentially expressed, and 50 showed at least 1.5-fold variation (false discovery rate adjustment q value <0.05). HL-HDLs presented a core enriched in cholesteryl esters and a surface depleted of phosphatidylcholine species containing polyunsaturated and long-chain fatty acids, indicating the presence of mature HDL particles with low surface fluidity. Hypercholesterolemia induced an important change in HDL-transported proteins (576 spots in HL-HDL vs. 621 spots in NC-HDL). HL-HDLs showed a reduced content of lipocalin retinol binding protein 4 and apolipoprotein M and in the retinoic acid-transporter cellular retinoic acid binding protein 1 (p < 0.05 vs. NC-HDL). No changes were observed in apolipoprotein A-I content and profile. Functionally, HL-HDL showed lower antioxidant activity (−35%) and a reduced capacity to efflux cholesterol (−60%) compared to NC-HDL (p < 0.05). Hypercholesterolemia induced larger HDL particles. [Conclusions] We demonstrate that hypercholesterolemia induces HDL lipidomic changes, losing phosphatidylcholine-lipid species and gaining cholesteryl esters, and proteomic changes, with losses in cardioprotective proteins. These remodeling changes shifted HDL particles toward a dysfunctional state.The authors thank the Fundación Investigación Cardiovascular and the Fundación Jesús Serra for their continuous support and CERCA Programme/Generalitat de Catalunya.Peer reviewe