Immunization with the Gly1127-Cys1140 amino acid sequence of the LRP1 receptor reduces atherosclerosis in rabbits. Molecular, immunohistochemical and nuclear imaging studies.

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.

Low-density lipoprotein receptor-related protein 1 deficiency in cardiomyocytes reduces susceptibility to insulin resistance and obesity.

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.

Plasma circular RNA hsa_circ_0001445 and coronary artery disease: Performance as a biomarker.

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.

Emerging role of microRNAs in dilated cardiomyopathy: evidence regarding etiology.

Dilated cardiomyopathy (DCM) is a heart muscle disease characterized by ventricular dilation and systolic dysfunction in the absence of abnormal loading conditions or coronary artery disease. This cardiac disorder is a major health problem due to its high prevalence, morbidity, and mortality. DCM is a complex disease with a common phenotype but heterogeneous pathological mechanisms. Early etiological diagnosis and prognosis stratification is crucial for the clinical management of the patient. Advances in imaging technology and genetic tests have provided useful tools for clinical practice. Nevertheless, the assessment of the disease remains challenging. Novel noninvasive indicators are still needed to assist in decision-making. microRNAs (miRNAs), a group of small noncoding RNAs, have been identified as key mediators of cell biology. They are found in a stable form in body fluids and their concentration is altered in response to stress. Previous research has suggested that the miRNA signature constitutes a novel source of noninvasive biomarkers for a wide array of cardiovascular diseases. Specifically, several studies have reported the potential role of miRNAs as clinical indicators among the etiologies of DCM. However, this field has not been reviewed in detail. Here, we summarize the evidence of intracellular and circulating miRNAs in DCM and their usefulness in the development of novel diagnostic, prognostic and therapeutic approaches, with a focus on DCM etiology. Although the findings are still preliminary, due to methodological and technical limitations and the lack of robust population-based studies, miRNAs constitute a promising tool to assist in the clinical management of DCM.

Plasma microRNA Profiling Reveals Novel Biomarkers of Epicardial Adipose Tissue: A Multidetector Computed Tomography Study.

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.

Soluble low-density lipoprotein receptor-related protein 1 as a biomarker of coronary risk: Predictive capacity and association with clinical events.

BACKGROUND AND AIMS: We aimed to determine whether circulating sLRP1 levels are associated with future coronary events and improve the predictive capacity of the REGICOR (Registre Gironí del Cor) risk function. METHODS: We conducted a case-cohort study based on the follow-up of the REGICOR population-based cohort. Of the 5,404 participants aged between 35 and 74 years, without previous history of cardiovascular disease, 117 subjects with angina or fatal or non-fatal myocardial infarction were included, and 512 individuals were randomly selected as a subcohort (including 14 patients who presented coronary events). sLRP1 levels were measured in basal plasma samples by commercial ELISA. Hazard ratio (HR) was estimated with Cox models adjusted for potential confounding factors. Discrimination and reclassification were analyzed with the c-index and the net reclassification index (NRI), respectively. A Mendelian randomization approach was used to explore the causality of the association between sLRP1 and coronary artery disease (CAD). RESULTS: The group of participants who presented a CAD event showed higher levels of sLRP1 than the subcohort (2.45 [0.43; 8.31] vs. 2.07 [0.40; 6.65] µg/mL, p < 0.001). sLRP1 was significantly associated with CAD events even after adjustment for confounding factors (adjusted HR per standard deviation = 1.30, 95% CI: 1.01-1.67, p = 0.039). sLRP1 did not increase the predictive capacity or improve cardiovascular risk stratification of the REGICOR function. The LRP1 genetic variants associated with CAD risk were not related to sLRP1 concentration. CONCLUSIONS: Plasma sLRP1 is independently associated with the incidence of coronary events, but it does not improve the predictive capacity of the REGICOR risk function.

Molecular basis for the protective effects of low-density lipoprotein receptor-related protein 1 (LRP1)-derived peptides against LDL aggregation.

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.

Circulating microRNAs in suspected stable coronary artery disease: A coronary computed tomography angiography study.

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.

Circulating non-coding RNAs in biomarker-guided cardiovascular therapy: a novel tool for personalized medicine?

Current clinical guidelines emphasize the unmet need for technological innovations to guide physician decision-making and to transit from conventional care to personalized cardiovascular medicine. Biomarker-guided cardiovascular therapy represents an interesting approach to inform tailored treatment selection and monitor ongoing efficacy. However, results from previous publications cast some doubts about the clinical applicability of biomarkers to direct individualized treatment. In recent years, the non-coding human transcriptome has emerged as a new opportunity for the development of novel therapeutic strategies and biomarker discovery. Non-coding RNA (ncRNA) signatures may provide an accurate molecular fingerprint of patient phenotypes and capture levels of information that could complement traditional markers and established clinical variables. Importantly, ncRNAs have been identified in body fluids and their concentrations change with physiology and pathology, thus representing promising non-invasive biomarkers. Previous publications highlight the translational applicability of circulating ncRNAs for diagnosis and prognostic stratification within cardiology. Numerous independent studies have also evaluated the potential of the circulating non-coding transcriptome to predict and monitor response to cardiovascular treatment. However, this field has not been reviewed in detail. Here, we discuss the state-of-the-art research into circulating ncRNAs, specifically microRNAs and long non-coding RNAs, to support clinical decision-making in cardiovascular therapy. Furthermore, we summarize current methodological and conceptual limitations and propose future steps for their incorporation into personalized cardiology. Despite the lack of robust population-based studies and technical barriers, circulating ncRNAs emerge as a promising tool for biomarker-guided therapy.

Interplay between epicardial adipose tissue, metabolic and cardiovascular diseases / Interacción entre tejido adiposo epicárdico, enfermedades metabólicas y cardiovasculares

Cardiovascular disease is the primary cause of death in obese and diabetic patients. In these groups of patients, the alterations of epicardial adipose tissue (EAT) contribute to both vascular and myocardial dysfunction. Therefore, it is of clinical interest to determine the mechanisms by which EAT influences cardiovascular disease. Two key factors contribute to the tight intercommunication among EAT, coronary arteries and myocardium. One is the close anatomical proximity between these tissues. The other is the capacity of EAT to secrete cytokines and other molecules with paracrine and vasocrine effects on the cardiovascular system. Epidemiological studies have demonstrated that EAT thickness is associated with not only metabolic syndrome but also atherosclerosis and heart failure. The evaluation of EAT using imaging modalities, although effective, presents several disadvantages including radiation exposure, limited availability and elevated costs. Therefore, there is a clinical interest in EAT as a source of new biomarkers of cardiovascular and endocrine alterations. In this review, we revise the mechanisms involved in the protective and pathological role of EAT and present the molecules released by EAT with greater potential to become biomarkers of cardiometabolic alterations

Las enfermedades cardiovasculares son la primera causa de muerte en pacientes obesos y diabéticos. Las alteraciones del tejido adiposo epicárdico (TAE) contribuyen a la disfunción vascular y del miocardio en estos pacientes. Es por tanto de interés clínico determinar los mecanismos por los cuales el TAE influye en la enfermedad cardiovascular. Aquí resumimos los mecanismos que subyacen a la asociación entre TAE, síndrome metabólico y enfermedades cardiovasculares. Dos factores contribuyen a la estrecha intercomunicación entre el TAE, las arterias coronarias y el miocardio. Uno es la estrecha proximidad anatómica entre estos tejidos. El otro es la capacidad del TAE para secretar citocinas con efectos paracrinos y vasocrinos en el sistema cardiovascular. Estudios epidemiológicos han demostrado que el grosor del TAE está asociado no solo con el síndrome metabólico sino también con la aterosclerosis y la insuficiencia cardíaca. La evaluación del TAE utilizando técnicas de imagen, aunque eficaz presenta desventajas tales como la exposición a la radiación, la disponibilidad limitada y los costes elevados. Por lo tanto, existe un interés clínico en el TAE como fuente de nuevos biomarcadores de alteraciones cardiovasculares y endocrinas. En este artículo, revisamos los mecanismos implicados en el papel protector y patológico del TAE y presentamos las moléculas liberadas por el TAE con mayor potencial para convertirse en biomarcadores de alteraciones cardiometabólicas

Circular RNAs in Blood.

Recent advances in RNA sequencing and bioinformatic analysis have allowed the development of a new research field: circular RNAs (circRNAs). These members of the non-coding transcriptome are generated by backsplicing, which results in a covalently closed, single-stranded RNA molecule. To date, thousands of circRNAs have been identified in different human cell types. CircRNAs are evolutionarily conserved, highly stable, cell-/developmental stage-specific and have longer half-lives compared with linear RNAs. Interestingly, different studies have demonstrated that circRNAs are abundantly expressed in the bloodstream. In this chapter, we review the current knowledge of circRNA biology in blood cells and the cell-free compartment, including extracellular vesicles. The potential clinical application of blood circRNAs in the biomarker and therapy fields is also discussed. Finally, perspectives for future studies are proposed.

Interplay between epicardial adipose tissue, metabolic and cardiovascular diseases.

Cardiovascular disease is the primary cause of death in obese and diabetic patients. In these groups of patients, the alterations of epicardial adipose tissue (EAT) contribute to both vascular and myocardial dysfunction. Therefore, it is of clinical interest to determine the mechanisms by which EAT influences cardiovascular disease. Two key factors contribute to the tight intercommunication among EAT, coronary arteries and myocardium. One is the close anatomical proximity between these tissues. The other is the capacity of EAT to secrete cytokines and other molecules with paracrine and vasocrine effects on the cardiovascular system. Epidemiological studies have demonstrated that EAT thickness is associated with not only metabolic syndrome but also atherosclerosis and heart failure. The evaluation of EAT using imaging modalities, although effective, presents several disadvantages including radiation exposure, limited availability and elevated costs. Therefore, there is a clinical interest in EAT as a source of new biomarkers of cardiovascular and endocrine alterations. In this review, we revise the mechanisms involved in the protective and pathological role of EAT and present the molecules released by EAT with greater potential to become biomarkers of cardiometabolic alterations.

Structural basis for genome wide recognition of 5-bp GC motifs by SMAD transcription factors.

Smad transcription factors activated by TGF-ß or by BMP receptors form trimeric complexes with Smad4 to target specific genes for cell fate regulation. The CAGAC motif has been considered as the main binding element for Smad2/3/4, whereas Smad1/5/8 have been thought to preferentially bind GC-rich elements. However, chromatin immunoprecipitation analysis in embryonic stem cells showed extensive binding of Smad2/3/4 to GC-rich cis-regulatory elements. Here, we present the structural basis for specific binding of Smad3 and Smad4 to GC-rich motifs in the goosecoid promoter, a nodal-regulated differentiation gene. The structures revealed a 5-bp consensus sequence GGC(GC)|(CG) as the binding site for both TGF-ß and BMP-activated Smads and for Smad4. These 5GC motifs are highly represented as clusters in Smad-bound regions genome-wide. Our results provide a basis for understanding the functional adaptability of Smads in different cellular contexts, and their dependence on lineage-determining transcription factors to target specific genes in TGF-ß and BMP pathways.