Circulating miR-6821-5p levels and coronary calcification in asymptomatic familial hypercholesterolemia patients.

BACKGROUND AND AIMS: Premature atherosclerotic cardiovascular disease (CVD) is a clinic characteristic of familial hypercholesterolemia (FH). Coronary calcium score (CCS) is a highly used imaging modality to evidence atherosclerotic plaque burden. microRNAs (miRNAs) are non-coding RNAs that epigenetically regulate gene expression. Here, we investigated whether CCS associates with a specific miRNA-signature in FH-patients. METHODS: Patients with genetic diagnosis of FH (N = 86) from the nationwide SAFEHEART-cohort were investigated by computed tomography angiography imaging and classified depending on the presence of coronary calcification in FH-CCS (+) and FH-CCS (-) groups by the Agatston score. Differential miRNA profiling was performed in two stages: first by Affymetrix microarray technology (high-throughput differential profiling-studies) and second by RT-PCR using TaqMan-technology (analytical RT-qPCR study) in plasma of the two patient groups. RESULTS: miR-193a-5p, miR-30e-5p and miR-6821-5p levels were significantly higher in FH-CCS (+) compared to FH-CCS (-). miR-6821-5p was the best miRNA to discriminate FH-patients CCS(+), according to receiver operating characteristic (ROC) analysis (AUC: 0.70 ± 0.06, p = 0.006). High miR-6821-5p levels were associated with older age (p = 0.03) and high LDL-burden (p = 0.014) using a ROC-derived cut-off value. However, miR-6821-5p did not correlate with age in either the CCS- or CCS + group. Genes involved in calcification processes were identified by in silico analysis. The relation of cell-calcification and expression levels of miR-6821-5p, BMP2 and SPP1 was validated experimentally in human vascular smooth muscle cell cultures. CONCLUSIONS: Up-regulated levels of miR-6821-5p are found in the plasma of asymptomatic FH-patients with coronary calcified atherosclerotic plaques, as well as in isolated human vascular smooth muscle cells expressing the pro-calcification genes BMP2 and SPP1. These findings highlight the impact of epigenetic regulation on the development of subclinical atherosclerosis.

miR-619-5p and cardiogenic shock in patients with ST-segment elevation myocardial infarction.

BACKGROUND: Cardiogenic shock (CS) is a severe myocardial dysfunction secondary to various cardiac conditions including ST-segment elevation acute myocardial infarction (STEMI) and associated with a high risk of death. Little is known on epigenetic determinants in CS. Here, we investigated plasma miRNAs in relation to CS stratification in STEMI-patients. METHODS: STEMI-patients (n = 49), with (CS, n = 25) and without CS (non-CS, n = 24) fulfilling inclusion criteria were included from HSCSP-cohort (Derivation-cohort). CS-miRNAs were analysed by Affymetrix-microarray and RT-PCR. Results were validated in a second cohort of CS-patients (CardShock: n = 35) with similar inclusion/exclusion criteria as the derivation cohort. In silico analysis were performed to identify potential miRNA target genes. RESULTS: Of the 5-miRNA signature obtained from microarray analysis, miR-619-5p showed higher levels in CS than in Non-CS patients (p = .003) and discriminating power for CS by ROC (AUC: .752, p = .003). miR-619-5p directly associated with risk scores [GRACE, p = .001; CardShock, p < .001]. Furthermore, miR-619-5p showed discrimination power for death in CS. Thus, miRNA levels were significantly higher in patients with mortality outcome both in the Derivation HSCSP-cohort (p = .02; AUC: .78 ± .095) and the Validation CardShock-cohort (p = .017; AUC: .737 ± .086) By in silico analysis, miR-619-5p target genes and TNF-alpha were involved in the regulation of inflammation. miR-619-5p and TNF-alpha levels discriminated mortality outcome in CS-patients during 30-day follow-up (Validation-Cohort: ROC: .812, p = .002; HR: 9.99, p = .003). CONCLUSIONS: Up-regulation of miR-619-5p is found in the plasma of STEMI-patients with CS and mortality outcome. These findings highlight the specificity of epigenetic regulation of inflammation on the disease severity of MI.

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-ß 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.

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-1ß 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-1ß in primed macrophages and preventing cleaved-IL-1ß protein release.

Alternative C3 Complement System: Lipids and Atherosclerosis.

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 αMß2 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 miR-133a levels in the circulation anticipates presentation of clinical events in familial hypercholesterolaemia patients.

AIMS: Presentation of acute events in patients with atherosclerosis remains unpredictable even after controlling for classical risk factors. MicroRNAs (miRNAs) measured in liquid biopsies could be good candidate biomarkers to improve risk prediction. Here, we hypothesized that miRNAs could predict atherosclerotic plaque progression and clinical event presentation in familial hypercholesterolaemia (FH) patients. METHODS AND RESULTS: Circulating miRNAs (plasma, exosomes, and microvesicles) were investigated by TaqMan Array and RT-qPCR assays. Patients with genetic diagnosis of FH and healthy relatives from the SAFEHEART cohort were included. A differential signature of 10 miRNA was obtained by comparing two extreme phenotypes consisting of FH patients suffering a cardiovascular event (CVE) within a 8-year follow-up period (FH-CVE, N = 42) and non-FH hypercholesterolaemic relatives from the same cohort, matched for age and treatment, without CVE during the same period (nFH-nCVE, N = 30). The validation studies included two independent groups of patients with FH background (discovery group, N = 89, validation group N = 196), developing a future CVE (FH-CVE) or not (FH-nCVE) within the same time period of follow-up. Of the 10 miRNAs initially selected, miR-133a was significantly higher in FH-CVE than in FH-nCVE patients. Receiver operating characteristic analysis confirmed miR-133a as the best microRNA for predicting CVE in FH patients (0.76 ± 0.054; P < 0.001). Furthermore, Kaplan-Meier and COX analysis showed that high plasma miR-133a levels associated to the higher risk of presenting a CVE within the next 8 years (hazard ratio 3.89, 95% confidence interval 1.88-8.07; P < 0.001). In silico analysis of curate biological interactions related miR-133a with target genes involved in regulation of the cell-membrane lipid-receptor LRP6 and inflammatory cytokines (CXCL8, IL6, and TNF). These predictions were experimentally proven in human macrophages and endothelial cells transfected with agomiR-133a. CONCLUSION: Elevated levels of miR-133a in the circulation anticipate those FH patients that are going to present a clinical CVE within the next 2 years (average). Mechanistically, miR-133a is directly related with lipid- and inflammatory signalling in key cells for atherosclerosis progression.

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

AIMS: 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). METHODS AND RESULTS: 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). CONCLUSION: 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.

Liquid Biopsies: Microvesicles in Cardiovascular Disease.

Significance: Circulating microvesicles (cMV) are small (0.1-1 µm) phospholipid-rich blebs released by almost all cell types, and their release increases with cell activation and injury, thus reflecting the state of the cell from which they are originated. Microvesicles (MV) are found in the bloodstream, and they affect the phenotype of recipient cells, after local or systemic circulation, by intercellular transfer of their molecular content. Recent Advances: Several studies suggest the use of cell-specific MV subpopulations as predictive biomarkers for cardiovascular diseases (CVDs) at different stages and degrees of severity. In this review, we describe the state of the art of cMV as noninvasive surrogate biomarkers of vascular injury and dysfunction correlated with poor clinical outcomes in CVD. Critical Issues: Despite the growing body of evidence supporting the importance of cMV as hallmarks of CVD and their utility as biomarkers of CVD, the specific roles of each phenotype of cMV in CVD burden and prognosis still remain to be elucidated and validated in large cohorts. In addition, the development of standardized and reproducible techniques is required to be used as biomarkers for disease progression in the clinical setting. Future Directions: A multipanel approach with specific cMV phenotypes, added to current biomarkers and scores, will undoubtedly provide unique prognostic information to stratify patients for appropriate therapy on the basis of their risk of atherothrombotic disease and will open a new research area as therapeutic targets for CVD. MV will add to the implementation of precision medicine by helping the cellular and molecular characterization of CVD patients.

miR-505-3p controls chemokine receptor up-regulation in macrophages: role in familial hypercholesterolemia.

Familial hypercholesterolemia (FH) conveys a high risk of premature atherosclerosis as a result of lifelong exposure to high LDL cholesterol levels that are not fully reduced by standard-of-care lipid-lowering treatment. Inflammatory mediators have played a role in the progression of atherosclerotic lesions. Here, we investigated whether innate immunity cells in patients with FH have a specific proinflammatory phenotype that is distinct from that of cells in normal participants. To this end, miR-505-3p-a microRNA related to chronic inflammation-and its target genes were investigated in monocyte-derived macrophages (MACs) of patients with FH (FH-MACs) and non-FH controls (co-MACs). On the basis of the profiler PCR array analysis of agomiR-505-3p-transfected MACs, we identified the chemokine receptors, CCR3, CCR4, and CXCR1, as genes that are regulated by miR-505-3p via the transcription factor, RUNX1. miR-505-3p was significantly down-regulated, whereas CCR3, CCR4, CXCR, and RUNX1 were increased in FH-MAC compared with co-MAC, with the increase being more evident in the proinflammatory M1-like FH-MAC. Chemokine receptor levels were unrelated to LDL plasma levels at entry, but correlated with age in patients with FH, not in controls. In summary, we demonstrate for first time to our knowledge that MACs from FH-MACs have an inflammatory phenotype that is characterized by the up-regulation of CCR3, CCR4, and CXCR1 under the control of miR-505-3p. These results suggest a chronic inflammatory condition in FH innate immunity cells that is not reverted by standard lipid-lowering treatment.-Escate, R., Mata, P., Cepeda, J. M., Padró, T., Badimon, L. miR-505-3p controls chemokine receptor up-regulation in macrophages: role in familial hypercholesterolemia.

miR-505-3p controls chemokine receptor up-regulation in macrophages: role in familial hypercholesterolemia.

Familial hypercholesterolemia (FH) conveys a high risk of premature atherosclerosis as a result of lifelong exposure to high LDL cholesterol levels that are not fully reduced by standard-of-care lipid-lowering treatment. Inflammatory mediators have played a role in the progression of atherosclerotic lesions. Here, we investigated whether innate immunity cells in patients with FH have a specific proinflammatory phenotype that is distinct from that of cells in normal participants. To this end, miR-505-3p-a microRNA related to chronic inflammation-and its target genes were investigated in monocyte-derived macrophages (MACs) of patients with FH (FH-MACs) and non-FH controls (co-MACs). On the basis of the profiler PCR array analysis of agomiR-505-3p-transfected MACs, we identified the chemokine receptors, CCR3, CCR4, and CXCR1, as genes that are regulated by miR-505-3p via the transcription factor, RUNX1. miR-505-3p was significantly down-regulated, whereas CCR3, CCR4, CXCR, and RUNX1 were increased in FH-MAC compared with co-MAC, with the increase being more evident in the proinflammatory M1-like FH-MAC. Chemokine receptor levels were unrelated to LDL plasma levels at entry, but correlated with age in patients with FH, not in controls. In summary, we demonstrate for first time to our knowledge that MACs from FH-MACs have an inflammatory phenotype that is characterized by the up-regulation of CCR3, CCR4, and CXCR1 under the control of miR-505-3p. These results suggest a chronic inflammatory condition in FH innate immunity cells that is not reverted by standard lipid-lowering treatment.-Escate, R., Mata, P., Cepeda, J.M., Padró, T., Badimon, L. miR-505-3p controls chemokine receptor up-regulation in macrophages: role in familial hypercholesterolemia. FASEB J. 32, 601-612 (2018). www.fasebj.org.

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.

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.