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The role of circular RNAs (circRNAs) as biomarkers remains poorly characterized. Here, we investigated the performance of the circRNA hsa_circ_0001445 as a biomarker of coronary artery disease (CAD) in a real‐world clinical practice setting. Plasma hsa_circ_0001445 was measured in a study population of 200 consecutive patients with suspected stable CAD who had undergone coronary computed tomographic angiography (CTA). Multivariable logistic models were constructed combining conventional risk factors with established biomarkers and hsa_circ_0001445. Model robustness was internally validated by the bootstrap technique. Biomarker accuracy was evaluated using the C‐index. The integrated discrimination improvement (IDI) and net reclassification improvement (NRI) were also calculated. Risk groups were developed via classification tree models. The stability of plasma hsa_circ_0001445 was evaluated under different clinical conditions. hsa_circ_0001445 levels were associated with higher coronary atherosclerosis extent and severity with a 2‐fold increase across tertiles (28.4%‐50.0%). Levels of hsa_circ_0001445 were proportional to coronary atherosclerotic burden, even after comprehensive adjustment for cardiovascular risk factors, medications, and established biomarkers (fully adjusted OR = 0.432 for hsa_circ_0001445 as a continuous variable and fully adjusted OR = 0.277 for hsa_circ_0001445 as a binary variable). The classification of patients was improved with the incorporation of hsa_circ_0001445 into a base clinical model (CM) composed of conventional cardiovascular risk factors, showing an IDI of 0.047 and NRI of 0.482 for hsa_circ_0001445 as a continuous variable and an IDI of 0.056 and NRI of 0.373 for hsa_circ_0001445 as a binary variable. A trend toward higher discrimination capacity was also observed (C‐indexCM = 0.833, C‐indexCM+continuous hsa_circ_0001445 = 0.856 and C‐indexCM+binary hsa_circ_0001445 = 0.855). Detailed analysis of stability showed that hsa_circ_0001445 was present in plasma in a remarkably stable form. In vitro, hsa_circ_0001445 was downregulated in extracellular vesicles secreted by human coronary smooth muscle cells upon exposure to atherogenic conditions. In patients with suspected stable CAD referred for coronary CTA, plasma hsa_circ_0001445 improves the identification of coronary artery atherosclerosis.

Background Low-density lipoprotein receptor-related protein 1 (LRP1) plays a key role in fatty acid metabolism and glucose homeostasis. In the context of dyslipemia, LRP1 is upregulated in the heart. Our aim was to evaluate the impact of cardiomyocyte LRP1 deficiency on high fat diet (HFD)-induced cardiac and metabolic alterations, and to explore the potential mechanisms involved. Methods We used TnT-iCre transgenic mice with thoroughly tested suitability to delete genes exclusively in cardiomyocytes to generate an experimental mouse model with conditional Lrp1 deficiency in cardiomyocytes (TNT-iCre+-LRP1flox/flox). Findings Mice with Lrp1-deficient cardiomyocytes (cm-Lrp1−/−) have a normal cardiac function combined with a favorable metabolic phenotype against HFD-induced glucose intolerance and obesity. Glucose intolerance protection was linked to higher hepatic fatty acid oxidation (FAO), lower liver steatosis and increased whole-body energy expenditure. Proteomic studies of the heart revealed decreased levels of cardiac pro-atrial natriuretic peptide (pro-ANP), which was parallel to higher ANP circulating levels. cm-Lrp1−/− mice showed ANP signaling activation that was linked to increased fatty acid (FA) uptake and increased AMPK/ ACC phosphorylation in the liver. Natriuretic peptide receptor A (NPR-A) antagonist completely abolished ANP signaling and metabolic protection in cm-Lrp1−/− mice. Conclusions These results indicate that an ANP-dependent axis controlled by cardiac LRP1 levels modulates AMPK activity in the liver, energy homeostasis and whole-body metabolism., This work was funded by FIS PI14/01729 and FIS PI18/01584 (to VLl-C), FIS16/00139 (to JCE-G) grants from Instituto Salud Carlos III, co-financed by the European Fund for Regional Development (E.F.R.D.) and by the Ministry of Spain-MINECO (SAF2014-52223-C2-1-R to DS, co-financed by the European Fund for Regional Development (E.F.R.D.). Support was received from Ministerio de Economía y Competitividad to DdG-C (IJCI-2016-29393), Agència de Gestió d'Ajuts Universitaris i de Recerca Grant FI to ERL (FI-DGR 2013/00014), Fundació MARATÓ TV3 Project 201521-10 (to VLl-C) and Project 87/C/2016 to DS. CIBER Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM; CB07/08/0016 (FBV, JCE-G, LC); CB07/08/0017 (AZ), CIBER Fisiopatología de la Obesidad y la Nutrición (CIBEROBN; CB06/03/0001 (DS, LH); CB06/03/0017 (JCL, NR)) and CIBER Enfermedades Cardiovasculares (CIBERCV; CB16/1100403) (DdG-C, VLl-, CS, ERL and AB-G) are Instituto de Salud Carlos III (ISCIII) projects.

Background: The LRP1 (CR9) domain and, in particular, the sequence Gly1127-Cys1140 (P3) plays a critical role in the binding and internalization of aggregated LDL (agLDL). We aimed to evaluate whether immunization with P3 reduces high-fat diet (HFD)-induced atherosclerosis. Methods: Female New Zealand White (NZW) rabbits were immunized with a primary injection and four reminder doses (R1-R4) of IrP (irrelevant peptide) or P3 conjugated to the carrier. IrP and P3-immunized rabbits were randomly divided into a normal diet group and a HFD-fed group. Anti-P3 antibody levels were determined by ELISA. Lipoprotein profile, circulating and tissue lipids, and vascular pro-inflammatory mediators were determined using standardized methods while atherosclerosis was determined by confocal microscopy studies and non-invasive imaging (PET/CT and Doppler ultrasonography). Studies treating human macrophages (hMΦ) and coronary vascular smooth muscle cells (hcVSMC) with rabbit serums were performed to ascertain the potential impact of anti-P3 Abs on the functionality of these crucial cells. Results: P3 immunization specifically induced the production of anti-P3 antibodies (Abs) and did not alter the lipoprotein profile. HFD strongly induced cholesteryl ester (CE) accumulation in the aorta of both the control and IrP groups, and their serum dose-dependently raised the intracellular CE of hMΦ and hcVSMC, promoting TNFR1 and phospho-NF-kB (p65) overexpression. These HFD pro-inflammatory effects were dramatically decreased in the aorta of P3-immunized rabbits and in hMΦ and hcVSMC exposed to the P3 rabbit serums. Microscopy studies revealed that P3 immunization reduced the percentage of lipids, macrophages, and SMCs in the arterial intima, as well as the atherosclerotic extent and lesion area in the aorta. PET/CT and Doppler ultrasonography studies showed that the average standardized uptake value (SUVmean) of the aorta and the arterial resistance index (ARI) of the carotids were more upregulated by HFD in the control and IrP groups than the P3 group. Conclusions: P3 immunization counteracts HFD-induced fatty streak formation in rabbits. The specific blockade of the LRP1 (CR9) domain with Anti-P3 Abs dramatically reduces HFD-induced intracellular CE loading and harmful coupling to pro-inflammatory signaling in the vasculature.

Epicardial adipose tissue (EAT) constitutes a novel parameter for cardiometabolic risk assessment and a target for therapy. Here, we evaluated for the first time the plasma microRNA (miRNA) profile as a source of biomarkers for epicardial fat volume (EFV). miRNAs were profiled in plasma samples from 180 patients whose EFV was quantified using multidetector computed tomography. In the screening study, 54 deregulated miRNAs were identified in patients with high EFV levels (highest tertile) compared with matched patients with low EFV levels (lowest tertile). After filtering, 12 miRNAs were selected for subsequent validation. In the validation study, miR-15b-3p, miR-22-3p, miR-148a-3p miR-148b-3p and miR-590-5p were directly associated with EFV, even after adjustment for confounding factors (p value < 0.05 for all models). The addition of miRNA combinations to a model based on clinical variables improved the discrimination (area under the receiver-operating-characteristic curve (AUC) from 0.721 to 0.787). miRNAs correctly reclassified a significant proportion of patients with an integrated discrimination improvement (IDI) index of 0.101 and a net reclassification improvement (NRI) index of 0.650. Decision tree models used miRNA combinations to improve their classification accuracy. These results were reproduced using two proposed clinical cutoffs for epicardial fat burden. Internal validation corroborated the robustness of the models. In conclusion, plasma miRNAs constitute novel biomarkers of epicardial fat burden., D.d.G.-C. was a recipient of a Juan de la Cierva-Incorporación grant from the Ministerio de Economía y Competitividad (IJCI-2016-29393). O.B. (Project 201521-10) and N.P. (Project 201716) were recipients of Fundació Marató TV3. S.B. and J.L.S.-Q. (2017-SGR-1149) and D.d.G.-C., L.N., À.V., D.V., R.L. and V.L.-C. (2017-SGR-946) are members of Quality Research Groups from Generalitat de Catalunya. S.B., J.L.S.-Q., N.P., V.L.-C. and D.d.G.-C. are members of the Group of Vascular Biology of the Spanish Society of Atherosclerosis (SEA). This work was funded by FIS PI14/01729 (to V.L.-C.), FIS PI16/00471 (to J.L.S.-Q.) & FIS PI18/01584 (to V.L.-C.) grants from Instituto Salud Carlos III, co-financed by the European Fund for Regional Development (EFRD) and by Project 201521-10 from Fundació MARATÓ TV3 (to V.L.-C.). CIBERCV (CB16/11/00403 to V.L.-C. and D.d.G.-C. and CB16/11/00276 to A.F.-G.) and CIBERDEM (CB07/08/0016 to J.L.S.-Q.) are projects from Instituto de Salud Carlos III.

Cardiovascular disease (CVD) is a leading cause of death worldwide. Approximately 60% of patients treated with low‐density lipoprotein (LDL)‐lowering drug treatments, with on‐target plasma cholesterol levels, are still suffering clinical acute ischemic events. Mechanisms, such as LDL aggregation, underlie extracellular and intracellular cholesterol accumulation in the vasculature. A peptide sequence (P3) of the low‐density lipoprotein receptor‐related protein 1 (LRP1) efficiently protects LDL from sphingomyelinase (SMase‐) and phospholipase A2 (PLA2)‐induced LDL aggregation. The aim is to design families of peptide derivatives from P3 with enhanced potency and proteolytic stability. New peptides are designed through in silico conformational sampling and ApoB‐100 molecular docking, and are tested in dual (biochemical‐cellular) screening assays. A total of 46 new peptides including linear, fragment, cyclic, and alanine scanning derivatives are generated through two consecutive optimization rounds. Structurally and functionally optimized peptides contain hotspot residues that are replaced by alanine. This strategy confers an increased capacity to form prone alpha‐helix conformations crucial for the electrostatic interaction with ApoB‐100. These new compounds are highly efficient at inhibiting LDL aggregation and human coronary vascular smooth muscle cell‐cholesteryl ester loading and should be studied in preclinical models of atherosclerosis., A.B.-A. and C.P. contributed equally to this work. The authors thank Dr. Ignasi Gich, Professor of Clinical Pharmacology and Therapeutics and Researcher of Sant Pau Biomedical Research Institute (IIB-SantPau) and CIBER Epidemiología y Salud Pública (CIBERESP), for this help in statistical analysis and graph representations. The Ministry of Science and Innovation of Spain, in the framework of the State Plan of Scientific and Technical Innovation Investigation 2013–2016, awarded funding to the project “DEVELOPMENT OF AN INNOVATIVE THERAPY FOR THE TREATMENT OF THE ATHEROSCLEROSIS THROUGH INHIBITION OF CHOLESTEROL VASCULAR ACCUMULATION” led by IPROTEOS SL with File No. RTC-2016-5078-1. Support was also received from the Fundació MARATÓ TV3 Project 201521-10 (to VLl-C), and FIS PI18/01584 (to VLl-C) from the Instituto de Salud Carlos III (ISCIII) and cofinanced with ERDFs. Support was also received from Ministerio de Economía y Competitividad to DdG-C (IJCI-2016-29393). CIBER Enfermedades Cardiovasculares (CIBERCV; CB16/1100403 (DdG-C, VLl-C) are projects run by the Instituto de Salud Carlos III (ISCIII). The authors also acknowledge the support from “Secretaria d’Universitats i Recerca del Departament d’Economia I Coneixement de la Generalitat de Catalunya” (2017SGR946 to VLl-C).

Aggregated LDL is the first ligand reported to interact with the cluster II CR9 domain of low-density lipoprotein receptor-related protein 1 (LRP1). In particular, the C-terminal half of domain CR9, comprising the region Gly1127-Cys1140 exclusively recognizes aggregated LDL and it is crucial for aggregated LDL binding. Our aim was to study the effect of the sequence Gly1127-Cys1140 (named peptide LP3 and its retro-enantio version, named peptide DP3) on the structural characteristics of sphingomyelinase- (SMase) and phospholipase 2 (PLA2)-modified LDL particles. Turbidimetry, gel filtration chromatography (GFC) and transmission electronic microscopy (TEM) analysis showed that LP3 and DP3 peptides strongly inhibited SMase- and PLA2-induced LDL aggregation. Nondenaturing polyacrylamide gradient gel electrophoresis (GGE), agarose gel electrophoresis and high-performance thin-layer chromatography (HPTLC) indicated that LP3 and DP3 prevented SMase-induced alterations in LDL particle size, electric charge and phospholipid content, respectively, but not those induced by PLA2. Western blot analysis showed that LP3 and DP3 counteracted changes in ApoB-100 conformation induced by the two enzymes. LDL proteomics (LDL trypsin digestion followed by mass spectroscopy) and computational modeling methods evidenced that peptides preserve ApoB-100 conformation due to their electrostatic interactions with a basic region of ApoB-100. These results demonstrate that LRP1-derived peptides are protective against LDL aggregation, even in conditions of extreme lipolysis, through their capacity to bind to ApoB-100 regions critical for ApoB-100 conformational preservation. These results suggests that these LRP1(CR9) derived peptides could be promising tools to prevent LDL aggregation induced by the main proteolytic enzymes acting in the arterial intima., The Ministry of Science and Innovation of Spain, in the framework of the State Plan of Scientific and Technical Innovation Investigation 2013–2016, awarded funding to the project “DEVELOPMENT OF AN INNOVATIVE THERAPY FOR THE TREATMENT OF THE ATHEROSCLEROSIS THROUGH INHIBITION OF CHOLESTEROL VASCULAR ACCUMULATION”, led by IPROTEOS SL with file number RTC-2016-5078-1. This project was also financed by the Ministry of Economy, Industry and Competitiveness (MINECO) in the framework of the Subprogram RETOS-COLABORACIÓN, 2016 call. The project is also co-financed by the European Union with the objective to promoting the technological development, innovation and quality research. Support was also received from SAF2017-89613R (to SV), co-financed by the European Regional Development Fund (ERDF), the Fundació la Marató de TV3 Project 201521-10 (to VLlC), FIS PI13/00364 and PI16/00471 (to JSQ) and FIS PI18/01584 (to VLlC) from the Instituto de Salud Carlos III (ISCIII) and co-financed with ERDF. Support was also received from Ministerio de Economía y Competitividad to DdG-C (IJCI-2016-29393). CIBER Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM; CB07/08/0016 (JSQ) and CIBER de Enfermedades Cardiovasculares (CIBERCV; CB16/1100403 (DdG-C, VLlC) are projects run by the Instituto de Salud Carlos III (ISCIII). The CRG/UPF Proteomics Unit is member of ProteoRed PRB3 consortium which is supported by grant PT17/0019 of the PE I+D+i 2013–2016 from the Instituto de Salud Carlos III (ISCIII) and ERDF. We also acknowledge support from the Spanish Ministry of Economy and Competitiveness, “Centro de Excelencia Severo Ochoa 2013–2017”, SEV-2012-0208, and “Secretaria d'Universitats i Recerca del Departament d'Economia I Coneixement de la Generalitat de Catalunya” (2017SGR595 to ES and 2017SGR946 to VLl-C). SB, JLS-Q, AR-U, DdG-C and VL-C are members of the Group of Vascular Biology of the Spanish Society of Atherosclerosis (SEA).

Objectives: To explore the diagnostic performance of circulating microRNAs (miRNAs) as biomarkers in patients with suspected stable coronary artery disease (CAD). Methods: Plasma samples were collected from 237 consecutive patients referred for coronary computed tomography angiography (CCTA). Presence, extension and severity of coronary stenosis were evaluated using the indexes: presence of diameter stenosis ≥ 50%, segment involvement score (SIS), segment stenosis score (SSS) and 3-vessel plaque score. A panel of 10 miRNAs previously associated with CAD was analysed using RT-qPCR. Multivariate analyses were used to analyse the associations between biomarkers and indexes. Discrimination was evaluated using the area under the ROC curve (AUC). Decision trees were generated using chi-squared Automatic Interaction Detector (CHAID) prediction models. Results: After comprehensive adjustment including cardiovascular risk factors, medication use, confounding factors and protein-based biomarkers (hs-TnT and hs-CRP), several circulating miRNAs were inversely associated with coronary atherosclerosis extension (SIS and 3-vessel plaque score) and severity (SSS). In the whole population, circulating miRNAs showed a poor discrimination value for all indexes (AUC = 0.539–0.644) and did not increase the discrimination capacity of a clinical model of coronary stenosis presence, extension and severity based on conventional cardiovascular risk factors. Conversely, the inclusion of circulating miRNAs in decision trees produces models that improve the classification of cases and controls in specific patient subgroups. Conclusions: This study identifies a group of circulating miRNAs that failed to improve the discrimination capacity of cardiovascular risk factors but that has the potential to define specific subpopulations of patients with suspected stable CAD., DdG‐C was a recipient of a Juan de la Cierva‐Incorporación grant from the Ministerio de Economía y Competitividad (IJCI‐2016‐29393). This work was funded by FIS PI14/01729 & FIS PI18/01584 grant from Instituto de Salud Carlos III, co‐financed by the European Fund for Regional Development (E.F.R.D.) and by Project 201521‐10 from Fundació MARATÓ TV3. CIBER Cardiovascular (CB16/11/00403 to DdG‐C and VL‐C) is a project from Instituto de Salud Carlos III.

LRP1-pPyk2 axis is essential for the upregulatory effect of hypoxia on MMP-9 activation and human VSMC (hVSMC) migration. Currently, there are not efficient models for the translational study of atherosclerosis. The morphological and physiological features of atherosclerosis are different between human and animal models, particularly in mouse models. Therefore, the aim of current investigation was to compare the effect of hypoxia on LRP1-Pyk2-MMP-9 axis in human and mouse vascular smooth muscle cells (mVSMC) and its consequences on VSMC migration. We demonstrated that hypoxic modulation of LRP1-pPyk2-MMP-9 axis is opposite between hVSMC and mVSMC. The modulation of LRP1/pPyk2 levels by hypoxia is positive in hVSMC but negative in mVSMC. We showed that the inverse effect of LRP1/pPyk2 axis is associated with a differential effect of hypoxia on MMP-9 expression and activation. Hypoxia-induced MMP-9 activation was concomitant with an increased hVSMC migratory capacity. Surprisingly, mVSMC migrate under hypoxic conditions despite the downregulatory effect of hypoxia on MMP-9 expression or activation. Our results highlight the crucial role of LRP1-pPyk2-MMP-9 axis in vascular cell migration. In addition, we propose that the extrapolation of results from animal models to humans is not suitable for this specific mechanism in hypoxia-related vascular conditions.

Our aim was to identify biophysical biomarkers of ventricular remodelling in tachycardia‐induced dilated cardiomyopathy (DCM). Our study includes healthy controls (N = 7) and DCM pigs (N = 10). Molecular analysis showed global myocardial metabolic abnormalities, some of them related to myocardial hibernation in failing hearts, supporting the translationality of our model to study cardiac remodelling in dilated cardiomyopathy. Histological analysis showed unorganized and agglomerated collagen accumulation in the dilated ventricles and a higher percentage of fibrosis in the right (RV) than in the left (LV) ventricle (P = .016). The Fourier Transform Infrared Spectroscopy (FTIR) 1st and 2nd indicators, which are markers of the myofiber/collagen ratio, were reduced in dilated hearts, with the 1st indicator reduced by 45% and 53% in the RV and LV, respectively, and the 2nd indicator reduced by 25% in the RV. The 3rd FTIR indicator, a marker of the carbohydrate/lipid ratio, was up‐regulated in the right and left dilated ventricles but to a greater extent in the RV (2.60‐fold vs 1.61‐fold, P = .049). Differential scanning calorimetry (DSC) showed a depression of the freezable water melting point in DCM ventricles – indicating structural changes in the tissue architecture – and lower protein stability. Our results suggest that the 1st, 2nd and 3rd FTIR indicators are useful markers of cardiac remodelling. Moreover, the 2nd and 3rd FITR indicators, which are altered to a greater extent in the right ventricle, are associated with greater fibrosis., This work was supported by FIS PI14/01729 and FIS PI10/01149 from the Instituto de Salud Carlos III, co-financed by the European Fund for Regional Development (E.F.R.D.) and Fundacio Marat o TV3 (201521 10). DdG-C was a recipient of a Sara Borrell grant from the Instituto de Salud Carlos III Project. (CD14/00109) and a European Society of Cardiology First Contact Initiative grant. CIBER Cardiovascular is an Instituto de Salud Carlos III Project.

[Aim] Circulating miRNAs have been proposed as biomarkers of ischemic heart disease (IHD). Nonetheless, most studies are based on small case-control approaches, with inadequate consideration of comorbidities and medications, and no comparison with established biomarkers. We have previously demonstrated the potential of miR-24-3p and miR-130a-3p as indicators of coronary atherosclerosis. Here, we determined the diagnostic value of both miRNAs in a real-world cohort of patients with suspected IHD., [Methods] Plasma samples were collected from 105 consecutive patients referred for computed tomography angiography (CCTA) for evaluation of suspected IHD. Presence and extension of coronary atherosclerosis were evaluated as previously described. Circulating miRNAs were analyzed using RT-qPCR., [Results] Circulating levels of miR-130a-3p were significantly reduced in patients with significant coronary stenosis (narrowing > 50 % of the reference luminal diameter) compared to patients without stenosis or non-significant stenosis (narrowing 1-49 % of the reference luminal diameter) (P < 0.050). No differences were observed for miR-24-3p or us-CRP. Circulating levels of miR-24-3p and miR-130a-3p were reduced in patients with higher extension of coronary atherosclerosis [segment involvement score (SIS) > 5] (P < 0.050). After adjustment for potential confounders (risk factors and medications), circulating miR-130a-3p levels were inversely associated with the extension of coronary atherosclerosis (P < 0.050 for all regression models). No association was observed with miR-24-3p or us-CRP. The addition of miR-130a-3p to a model of coronary atherosclerosis resulted in a higher area under the ROC curve., [Conclusions] Circulating miR-130a-3p is a potential biomarker of coronary atherosclerosis in patients with clinically suspected of IHD.

Dyslipemia has a direct impact on cardiac remodeling by altering extracellular matrix (ECM) components. One of the main ECM components is elastin, a proteic three-dimensional network that can be efficiently degraded by cysteine proteases or cathepsins. Dyslipemic status in insulin resistance and combined hyperlipoproteinemia diseases include raised levels of very low density lipoproteins (VLDL), triglyceride (TG)-cholesteryl ester (CE)-rich lipoproteins. Enhanced VLDL concentration promotes cardiomyocyte intracellular cholesteryl ester (CE) accumulation in a LRP1-dependent manner. The aim of this work was to analyze the effect of cardiomyocyte intracellular CE accumulation on tropoelastin (TE) characteristics and to investigate the role of LRP1 and cathepsin S (CatS) on these effects. Molecular studies showed that LRP1 deficiency impaired CE selective uptake and accumulation from TG-CE-rich lipoproteins (VLDL+IDL) and CE-rich lipoproteins (aggregated LDL, agLDL). Biochemical and confocal microscopic studies showed that LRP1-mediated intracellular CE accumulation increased CatS mature protein levels and induced an altered intracellular TE globule structure. Biophysical studies evidenced that LRP1-mediated intracellular CE accumulation caused a significant drop of Tg2 glass transition temperature of cardiomyocyte secreted TE. Moreover, CatS deficiency prevented the alterations in TE intracellular globule structure and on TE glass transition temperature. These results demonstrate that LRP1-mediated cardiomyocyte intracellular CE accumulation alters the structural and physical characteristics of secreted TE through an increase in CatS mature protein levels. Therefore, the modulation of LRP1-mediated intracellular CE accumulation in cardiomyocytes could impact pathological ventricular remodeling associated with insulin-resistance and combined hyperlipoproteinemia, pathologies characterized by enhanced concentrations of TG-CE-rich lipoproteins.

Aggregated low density lipoproteins (agLDL) contribute to massive intracellular cholesteryl ester (CE) accumulation in human vascular smooth muscle cells (VSMC). Our aim was to determine the conformational and physical structure of agLDL and elastic material produced either by control human VSMC or by agLDL-loaded human VSMC (agLDL-VSMC). At the conformational level scanned by FTIR spectroscopy, a new undefined, probably non-H-bonded, structure for tropoelastin produced by agLDL-VSMC is revealed. By differential scanning calorimetry, a decrease of water affinity and a drop of the glass transition associated with aggregated tropoelastin (from 200°C to 159°C) in the supernatant from agLDL VSMC are evidenced. This second phenomenon is due to an interaction between agLDL and tropoelastin as detected by the weak specific FTIR absorption band of agLDL in supernatant from agLDL-loaded VSMC.

Objective— Hypoxia disturbs vascular function by promoting extracellular matrix remodeling. Extracellular matrix integrity and composition are modulated by metalloproteinases (MMPs). Our aim was to investigate the role of low-density lipoprotein receptor–related protein 1 (LRP1) in regulating MMP-9/MMP-2 activation and vascular smooth muscle cells (VSMCs) migration in response to hypoxia, and to elucidate the LRP1-signaling pathways involved in this process. Approach and Results— Western blot analysis showed that hypoxia induced a sustained phosphorylation of proline-rich tyrosine kinase 2 concomitantly with LRP1 overexpression in human VSMCs (hVSMCs). Deletion of LRP1 using small-interfering RNA technology or treatment of hVSMCs with the Src family kinase inhibitor PP2 impaired hypoxia-induced phosphorylation of proline-rich tyrosine kinase 2 levels. Coimmunoprecipitation experiments showed that the higher amounts of phosphorylation of proline-rich tyrosine kinase 2/LRP1β immunoprecipitates in hypoxic hVSMCs were abolished in PP2-treated hVSMCs. Both LRP1 silencing and PP2 treatment were highly effective in the prevention of hypoxia-induced MMP-9 activation and hVSMC migration. Cellular subfractionation experiments revealed that PP2 effects may be caused by impairment of hypoxia-induced nuclear factor-κβ translocation to the nucleus. ELISA measurements showed that LRP1 silencing but not PP2 treatment increased interleukin-1β, interleukin-6, and monocyte chemoattractant protein-1 secretion by hypoxic hVSMCs. Conclusions— Our findings determine a crucial role of LRP1-mediated Pyk2 phosphorylation on hypoxia-induced MMP-9 activation and hVSMC migration and therefore in hypoxia-induced vascular remodeling. Both LRP1 silencing and PP2 treatments also influence hypoxia-induced proinflammatory effects in hVSMCs. Therefore, further studies are required to establish therapeutical strategies that efficiently modulate vascular remodeling and inflammation associated with hypoxia-vascular diseases.

To analyze the impact of atherogenic lipoproteins on the miRNA signature of microvesicles derived from human coronary artery smooth muscle cells (CASMC) and to translate these results to familial hypercholesterolemia (FH) and coronary artery disease (CAD) patients.Conditioned media was collected after exposure of CASMC to atherogenic lipoproteins. Plasma samples were collected from two independent populations of diagnosed FH patients and matched normocholesterolemic controls (Study population 1, N=50; Study population 2, N=24) and a population of patients with suspected CAD (Study population 3, N=50). Extracellular vesicles were isolated and characterized using standard techniques. A panel of 30 miRNAs related to vascular smooth muscle cell (VSMC) (patho-)physiology was analyzed using RT-qPCR.Atherogenic lipoproteins significantly reduced levels of miR-15b-5p, -24-3p, -29b-3p, -130a-3p, -143-3p, -146a-3p, -222-3p, -663a levels (P0.050) in microvesicles (0.1μm-1μm in diameter) released by CASMC. Two of these miRNAs, miR-24-3p and miR-130a-3p, were reduced in circulating microvesicles from FH patients compared with normocholesterolemic controls in a pilot study (Study population 1) and in different validation studies (Study populations 1 and 2) (P0.050). Supporting these results, plasma levels of miR-24-3p and miR-130a-3p were also downregulated in FH patients compared to controls (P0.050). In addition, plasma levels of miR-130a-3p were inversely associated with coronary atherosclerosis in a cohort of suspected CAD patients (Study population 3) (P0.050).Exposure to atherogenic lipoproteins modifies the miRNA profile of CASMC-derived microvesicles and these alterations are reflected in patients with FH. Circulating miR-130a-3p emerges as a potential biomarker for coronary atherosclerosis.

Adverse cardiac remodeling after myocardial infarction (MI) causes impaired ventricular function and heart failure. Histopathological characterization is commonly used to detect the location, size and shape of MI sites. However, the information about chemical composition, physical structure and molecular mobility of peri- and infarct zones post-MI is rather limited. The main objective of this work was to explore the spatiotemporal biochemical and biophysical alterations of key cardiac components post-MI. The FTIR spectra of healthy and remote myocardial tissue shows amides A, I, II and III associated with proteins in freeze-died tissue as major absorptions bands. In infarcted myocardium, the spectrum of these main absorptions was deeply altered. FITR evidenced an increase of the amide A band and the distinct feature of the collagen specific absorption band at 1338 cm−1 in the infarct area at 21 days post-MI. At 21 days post-MI, it also appears an important shift of amide I from 1646 cm−1 to 1637 cm−1 that suggests the predominance of the triple helical conformation in the proteins. The new spectra bands also indicate an increase in proteoglycans, residues of carbohydrates in proteins and polysaccharides in ischemic areas. Thermal analysis indicates a deep increase of unfreezable water/freezable water in peri- and infarcted tissues. In infarcted tissue is evidenced the impairment of myofibrillar proteins thermal profile and the emergence of a new structure. In conclusion, our results indicate a profound evolution of protein secondary structures in association with collagen deposition and reorganization of water involved in the scar maturation of periand infarct zones post-MI., The research leading to these results received funding from the Instituto de Salud Carlos III (FIS PI14/01729 and FIS PI14/01682) that was cofinanced by the European Fund for Regional Development, and CIBER Cardiovascular (CB16/11/00403), as well as from the Ministerio de Ciencia e Innovación (SAF2011-30067-C02-01), the Fundació La Marató de TV3 (201521_10, 201502_30 and 080330), the Red de Terapia Celular-TerCel (RD12/0019/0029), the Sociedad Española de Cardiología and the Fundació Privada Daniel Bravo Andreu. DdG is a recipient of CD14/0109 from Sara Borell of the Instituto de Salud Carlos III.

Aims The myocardium accumulates intracellular lipids under ischaemic conditions, and myocardial fat deposition is closely associated with cardiac dysfunction. Our aims were to analyse the effect of hypoxia on low-density lipoprotein receptor-related protein 1 (LRP1) expression in neonatal rat ventricular myocytes (NRVM) and cardiac-derived HL-1 cells and the molecular mechanisms involved in this effect, to determine the role of LRP1 in the very low density lipoprotein (VLDL) uptake by hypoxic cardiomyocytes, and to study the effect of hypoxia on lipoprotein receptor expression and myocardial lipid profile in an in vivo porcine experimental model of acute myocardial infarction. Methods and results Thin-layer chromatography after lipid extraction showed that VLDL exposure leads to cholesteryl ester (CE) and triglyceride (TG) accumulation in a dose-dependent manner and that hypoxic conditions further increased VLDL-derived intracellular lipid accumulation in HL-1 cells. Knockdown of LRP1 through lentiviral-mediated interfering RNA specifically prevented hypoxia-induced VLDL-CE internalization in HL-1 cells and NRVM. Lipopolysaccharide (LPS)-induced LRP1 overexpression specifically increased VLDL-CE accumulation in NRVM. In addition, using double-radiolabelled [3H]CE-[14C]TG-VLDL, we found that LRP1 deficiency specifically prevented hypoxia-induced VLDL-[3H]CE uptake. Finally, in an in vivo porcine model of infarcted myocardium, ischaemic areas exhibited LRP1 protein up-regulation and intramyocardial CE overaccumulation. Conclusion Our results demonstrate that hypoxia increases LRP1 expression through HIF-1α and that LRP1 overexpression mediates hypoxia-induced VLDL-CE uptake and accumulation in cardiomyocytes.

The maintenance of sarcoplasmic reticulum Ca2+ ATPase (SERCA2) activity is crucial for cardiac function and SERCA2 is dramatically reduced in the heart exposed to hypoxic/ischemic conditions. Previous work from our group showed that hypoxia upregulates the phosphorylated form of the Ca2+-dependent nonreceptor protein tyrosine kinase (PTK) proline-rich tyrosine kinase 2 (pPyk2) protein levels in a low-density lipoprotein receptor-related protein (LRP1)-dependent manner. Pyk2 in turn may modulate SERCA2 in cardiomyocytes although this remains controversial. We therefore aimed to investigate the role of LRP1 on hypoxia-induced SERCA2 depletion in cardiomyocytes and to establish LRP1 signalling mechanisms involved. Western blot analysis showed that hypoxia reduced SERCA2 concomitantly with a sustained increase in LRP1 and pPyk2 protein levels in HL-1 cardiomyocytes. By impairing hypoxia-induced Pyk2 phosphorylation and HIF-1 alpha accumulation, LRP1 deficiency prevented SERCA2 depletion and reduction of the sarcoplasmic reticulum calcium content in cardiomyocytes. Moreover, the inhibition of Pyk2 phosphorylation (with the Src-family inhibitor PP2) or the specific silencing of Pyk2 (with siRNA-anti Pyk2) preserved low HIF-1 alpha and high SERCA2 levels in HL-1 cardiomyocytes exposed to hypoxia. We determined that the LRP1/Pyk2 axis represses SERCA2 mRNA expression via HIF-1 alpha since HIF-1 alpha overexpression abolished the protective effect of LRP1 deficiency on SERCA2 depletion. Our findings show a crucial role of LRP1/Pyk2/HIF-1 alpha in hypoxia-induced cardiomyocyte SERCA2 downregulation, a pathophysiological process closely associated with heart failure. (C) 2015 Elsevier Ltd. All rights reserved.

Dyslipemia has a direct impact on cardiac remodeling by altering extracellular matrix (ECM) components. One of the main ECM components is elastin, a proteic three-dimensional network that can be efficiently degraded by cysteine proteases or cathepsins. Dyslipemic status in insulin resistance and combined hyperlipoproteinemia diseases include raised levels of very low density lipoproteins (VLDL), triglyceride (TG)-cholesteryl ester (CE)-rich lipoproteins. Enhanced VLDL concentration promotes cardiomyocyte intracellular cholesteryl ester (CE) accumulation in a LRP1-dependent manner. The aim of this work was to analyze the effect of cardiomyocyte intracellular CE accumulation on tropoelastin (TE) characteristics and to investigate the role of LRP1 and cathepsin S (CatS) on these effects. Molecular studies showed that LRP1 deficiency impaired CE selective uptake and accumulation from TG-CE-rich lipoproteins (VLDL+IDL) and CE-rich lipoproteins (aggregated LDL, agLDL). Biochemical and confocal microscopic studies showed that LRP1-mediated intracellular CE accumulation increased CatS mature protein levels and induced an altered intracellular TE globule structure. Biophysical studies evidenced that LRP1-mediated intracellular CE accumulation caused a significant drop of Tg2 glass transition temperature of cardiomyocyte secreted TE. Moreover, CatS deficiency prevented the alterations in TE intracellular globule structure and on TE glass transition temperature. These results demonstrate that LRP1-mediated cardiomyocyte intracellular CE accumulation alters the structural and physical characteristics of secreted TE through an increase in CatS mature protein levels. Therefore, the modulation of LRP1-mediated intracellular CE accumulation in cardiomyocytes could impact pathological ventricular remodeling associated with insulin-resistance and combined hyperlipoproteinemia, pathologies characterized by enhanced concentrations of TG-CE-rich lipoproteins.

Lipoprotein receptor expression plays a crucial role in the pathophysiology of adipose tissue in in vivo models of diabetes. However, there are no studies in diabetic patients. The aims of this study were to analyze (a) low-density lipoprotein receptor-related protein 1 (LRP1) and very low-density lipoprotein receptor (VLDLR) expression in epicardial and subcutaneous fat from type 2 diabetes mellitus compared with nondiabetic patients and (b) the possible correlation between the expression of these receptors and plasmatic parameters. Adipose tissue biopsy samples were obtained from diabetic (n = 54) and nondiabetic patients (n = 22) undergoing cardiac surgery before the initiation of cardiopulmonary bypass. Adipose LRP1 and VLDLR expression was analyzed at mRNA level by real-time PCR and at protein level by Western blot analysis. Adipose samples were also subjected to lipid extraction, and fat cholesterol ester, triglyceride, and free cholesterol contents were analyzed by thin-layer chromatography. LRP1 expression was higher in epicardial fat from diabetic compared with nondiabetic patients (mRNA 17.63 +/- A 11.37 versus 7.01 +/- A 4.86; P = 0.02; protein 11.23 +/- A 7.23 versus 6.75 +/- A 5.02, P = 0.04). VLDLR expression was also higher in epicardial fat from diabetic patients but only at mRNA level (231.25 +/- A 207.57 versus 56.64 +/- A 45.64, P = 0.02). No differences were found in the expression of LRP1 or VLDLR in the subcutaneous fat from diabetic compared with nondiabetic patients. Epicardial LRP1 and VLDLR mRNA overexpression positively correlated with plasma triglyceride levels (R (2) = 0.50, P = 0.01 and R (2) = 0.44, P = 0.03, respectively) and epicardial LRP1 also correlated with plasma glucose levels (R (2) = 0.33, P = 0.03). These results suggest that epicardial overexpression of certain lipoprotein receptors such as LRP1 and VLDLR expression may play a key role in the alterations of lipid metabolism associated with type 2 diabetes mellitus.

Sterol regulatory element-binding proteins (SREBPs) negatively modulate the expression of the CD91/low-density lipoprotein receptor-related protein (LRP1), a carrier and signaling receptor that mediates the endocytosis of more than 40 structurally and functionally distinct ligands. The aim of this work was to analyze whether lipopolysaccharide (LPS) can regulate LRP1 expression through SREBPs in human monocyte-derived macrophages (HMDM). LPS led to LRP1 mRNA and protein inhibition in a dose-and time-dependent manner. Concomitantly, a strong upregulation of SREBP-1 mRNA and SREBP-1 nuclear protein levels was observed in LPS-treated HMDM. The specific silencing of SREBP-1 efficiently prevented LRP1 reduction caused by LPS. SREBP-1 mRNA and nuclear protein levels remained high in HMDM treated with LPS unexposed or exposed to LDL. Native (nLDL) or aggregated LDL (agLDL) per se downregulated SREBP-2 expression levels and increased LRP1 expression. However, lipoproteins did not significantly alter the effect of LPS on SREBP-1 and LRP1 expression. Collectively, these data support that lipoproteins and LPS exert their modulatory effect on LRP1 expression through different SREBP isoforms, SREBP-2 and SREBP-1, respectively. These results highlight a crucial role of SREBP1 as a mediator of the downregulatory effects of LPS on LRP1 expression in human macrophages, independently of the absence or presence of modified lipoproteins. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Aggregated low-density lipoprotein (agLDL), one of the main LDL modifications in the arterial intima, contributes to massive intracellular cholesteryl ester (CE) accumulation in human vascular smooth muscle cells (VSMC), which are major producers of elastin in the vascular wall. Our aim was to analyze the levels, physical structure, and molecular mobility of tropoelastin produced by agLDL-loaded human VSMC (agLDL-VSMC) versus that produced by control VSMC. Western blot analysis demonstrated that agLDL reduced VSMC-tropoelastin protein levels by increasing its degradation rate. Moreover, our results demonstrated increased levels of precursor and mature forms of cathepsin S in agLDL-VSMC. Fourier transform infrared analysis revealed modifications in the secondary structures of tropoelastin produced by lipid-loaded VSMCs. Thermal and dielectric analyses showed that agLDL-VSMC tropoelastin has decreased glass transition temperatures and distinct chain dynamics that, in addition to a loss of thermal stability, lead to strong changes in its mechanical properties. In conclusion, agLDL lipid loading of human vascular cells leads to an increase in cathepsin S production concomitantly with a decrease in cellular tropoelastin protein levels and dramatic changes in secreted tropoelastin physical structure. Therefore, VSMC-lipid loading likely determines alterations in the mechanical properties of the vascular wall and plays a crucial role in elastin loss during atherosclerosis.

[Cardiovascular Research, Volume 94, Issue 3, 1 June 2012, Pages 469–479; doi:10.1093/cvr/cvs136.] The published version …

Background: Idiopathic dilated cardiomyopathy (IDCM) is characterized by adverse ventricular remodeling attributed to altered activity of extracellular matrix metalloproteinase (MMP). MMP overactivation is linked to changes in extracellular signal-regulated kinases (ERK), reportedly modulated by the low-density lipoprotein receptor-related protein 1 (LRP1) receptor. The aim of this work was to compare the levels, membrane distribution and interactions of LRP1, ERK1,2 and MMP2/9 in control and IDCM myocardium. Methods: Left ventricle samples from IDCM patients and control subjects were collected to analyze gene and protein expression by Real-time PCR and Western blot, respectively. Fractions enriched in cholesterol, Flotillin-1 and Caveolin-3 (rafts) were isolated from the remaining membrane (non-rafts) by sucrose gradient ultracentrifugation. We assessed the formation of LRP1-ERK1,2 complexes and MMP activity by immunoprecipitation and zymography, respectively. Results: In control myocardium, LRP1 was exclusively found in non-rafts while activation of ERK1,2 was preferentially detected in rafts. LRP1/p-ERK1,2 complexes were almost undetectable in rafts and non-rafts. In contrast, in IDCM myocardium, LRP1 moved to rafts and ERK1,2 activation was found in raft and non-raft fractions. Moreover, LRP1/p-ERK1,2 complexes were also found in both membrane fractions, although the amount was higher in non-rafts where MMP9 overactivation was exclusively detected. Conclusions: The presented findings demonstrate a differential membrane compartmentalisation of ERK signaling in IDCM myocardium. The movement of LRP1 to rafts and the concomitant increase in non-raft-related ERK1,2/MMP9 activation may have crucial clinical implications in the progression of disease. (C) 2014 Elsevier Ireland Ltd. All rights reserved.