B- and T-lymphocyte attenuator could be a new player in accelerated atherosclerosis associated with chronic kidney disease.

BACKGROUND: In chronic kidney disease (CKD), cardiovascular morbi-mortality is higher than in general population. Atherosclerotic cardiovascular disease is accelerated in CKD, but specific CKD-related risk factors for atherosclerosis are unknown. METHODS: CKD patients from the NEFRONA study were used. We performed mRNA array from blood of patients free from atheroma plaque at baseline, with (n=10) and without (n=10) de novo atherosclerotic plaque development 2 years later. Selected mRNA candidates were validated in a bigger sample (n=148). Validated candidates were investigated in vivo in an experimental model of CKD-accelerated atherosclerosis, and in vitro in murine macrophages. RESULTS: mRNA array analysis showed 92 up-regulated and 67 down-regulated mRNAs in samples from CKD patients with de novo plaque development. The functional analysis pointed to a paramount role of the immune response. The validation in a bigger sample confirmed that B- and T-lymphocyte co-inhibitory molecule (BTLA) down-regulation was associated with de novo plaque presence after 2 years. However, BTLA down-regulation was not found to be associated with atherosclerotic progression in patients with plaque already present at baseline. In a model of CKD-accelerated atherosclerosis, mRNA and protein expression levels of BTLA were significantly decreased in blood samples and atheroma plaques. Plaques from animals with CKD were bigger, had more infiltration of inflammatory cells, higher expression of IL6 and IL17 and less presence of collagen than plaques from control animals. Incubation of macrophages with rat uremic serum decreased BTLA expression. CONCLUSIONS: BTLA could be a potential biomarker or therapeutic target for atherosclerosis incidence in CKD patients.

Rewiring Vascular Metabolism Prevents Sudden Death due to Aortic Ruptures-Brief Report.

BACKGROUND: The goal of this study was to determine whether boosting mitochondrial respiration prevents the development of fatal aortic ruptures triggered by atherosclerosis and hypertension. METHODS: Ang-II (angiotensin-II) was infused in ApoE (Apolipoprotein E)-deficient mice fed with a western diet to induce acute aortic aneurysms and lethal ruptures. RESULTS: We found decreased mitochondrial respiration and mitochondrial proteins in vascular smooth muscle cells from murine and human aortic aneurysms. Boosting NAD levels with nicotinamide riboside reduced the development of aortic aneurysms and sudden death by aortic ruptures. CONCLUSIONS: Targetable vascular metabolism is a new clinical strategy to prevent fatal aortic ruptures and sudden death in patients with aortic aneurysms.

Extracellular Tuning of Mitochondrial Respiration Leads to Aortic Aneurysm.

BACKGROUND: Marfan syndrome (MFS) is an autosomal dominant disorder of the connective tissue caused by mutations in the FBN1 (fibrillin-1) gene encoding a large glycoprotein in the extracellular matrix called fibrillin-1. The major complication of this connective disorder is the risk to develop thoracic aortic aneurysm. To date, no effective pharmacologic therapies have been identified for the management of thoracic aortic disease and the only options capable of preventing aneurysm rupture are endovascular repair or open surgery. Here, we have studied the role of mitochondrial dysfunction in the progression of thoracic aortic aneurysm and mitochondrial boosting strategies as a potential treatment to managing aortic aneurysms. METHODS: Combining transcriptomics and metabolic analysis of aortas from an MFS mouse model (Fbn1c1039g/+) and MFS patients, we have identified mitochondrial dysfunction alongside with mtDNA depletion as a new hallmark of aortic aneurysm disease in MFS. To demonstrate the importance of mitochondrial decline in the development of aneurysms, we generated a conditional mouse model with mitochondrial dysfunction specifically in vascular smooth muscle cells (VSMC) by conditional depleting Tfam (mitochondrial transcription factor A; Myh11-CreERT2Tfamflox/flox mice). We used a mouse model of MFS to test for drugs that can revert aortic disease by enhancing Tfam levels and mitochondrial respiration. RESULTS: The main canonical pathways highlighted in the transcriptomic analysis in aortas from Fbn1c1039g/+ mice were those related to metabolic function, such as mitochondrial dysfunction. Mitochondrial complexes, whose transcription depends on Tfam and mitochondrial DNA content, were reduced in aortas from young Fbn1c1039g/+ mice. In vitro experiments in Fbn1-silenced VSMCs presented increased lactate production and decreased oxygen consumption. Similar results were found in MFS patients. VSMCs seeded in matrices produced by Fbn1-deficient VSMCs undergo mitochondrial dysfunction. Conditional Tfam-deficient VSMC mice lose their contractile capacity, showed aortic aneurysms, and died prematurely. Restoring mitochondrial metabolism with the NAD precursor nicotinamide riboside rapidly reverses aortic aneurysm in Fbn1c1039g/+ mice. CONCLUSIONS: Mitochondrial function of VSMCs is controlled by the extracellular matrix and drives the development of aortic aneurysm in Marfan syndrome. Targeting vascular metabolism is a new available therapeutic strategy for managing aortic aneurysms associated with genetic disorders.

Implication of miR-155-5p and miR-143-3p in the Vascular Insulin Resistance and Instability of Human and Experimental Atherosclerotic Plaque.

(1) Background: Cardiovascular diseases (CVDs) are the main cause of death in developed countries, being atherosclerosis, a recurring process underlying their apparition. MicroRNAs (miRNAs) modulate the expression of their targets and have emerged as key players in CVDs; (2) Methods: 18 miRNAs were selected (Pubmed and GEO database) for their possible role in promoting atherosclerosis and were analysed by RT-qPCR in the aorta from apolipoprotein E-deficient (ApoE-/-) mice. Afterwards, the altered miRNAs in the aorta from 18 weeks-ApoE-/- mice were studied in human aortic and carotid samples; (3) Results: miR-155-5p was overexpressed and miR-143-3p was downregulated in mouse and human atherosclerotic lesions. In addition, a significant decrease in protein kinase B (AKT), target of miR-155-5p, and an increase in insulin-like growth factor type II receptor (IGF-IIR), target of miR-143-3p, were noted in aortic roots from ApoE-/- mice and in carotid plaques from patients with advanced carotid atherosclerosis (ACA). Finally, the overexpression of miR-155-5p reduced AKT levels and its phosphorylation in vascular smooth muscle cells, while miR-143-3p overexpression decreased IGF-IIR reducing apoptosis in vascular cells; (4) Conclusions: Our results suggest that miR-155-5p and miR-143-3p may be implicated in insulin resistance and plaque instability by the modulation of their targets AKT and IGF-IIR, contributing to the progression of atherosclerosis.

Red Blood Cells and Hemoglobin in Human Atherosclerosis and Related Arterial Diseases.

As the main particulate component of the circulating blood, RBCs play major roles in physiological hemodynamics and impact all arterial wall pathologies. RBCs are the main determinant of blood viscosity, defining the frictional forces exerted by the blood on the arterial wall. This function is used in phylogeny and ontogeny of the cardiovascular (CV) system, allowing the acquisition of vasomotricity adapted to local metabolic demands, and systemic arterial pressure after birth. In pathology, RBCs collide with the arterial wall, inducing both local retention of their membranous lipids and local hemolysis, releasing heme-Fe++ with a high toxicity for arterial cells: endothelial and smooth muscle cells (SMCs) cardiomyocytes, neurons, etc. Specifically, overloading of cells by Fe++ promotes cell death. This local hemolysis is an event associated with early and advanced stages of human atherosclerosis. Similarly, the permanent renewal of mural RBC clotting is the major support of oxidation in abdominal aortic aneurysm. In parallel, calcifications promote intramural hemorrhages, and hemorrhages promote an osteoblastic phenotypic shift of arterial wall cells. Different plasma or tissue systems are able, at least in part, to limit this injury by acting at the different levels of this system.

Impaired HDL (High-Density Lipoprotein)-Mediated Macrophage Cholesterol Efflux in Patients With Abdominal Aortic Aneurysm-Brief Report.

Objective- The ability of HDL (high-density lipoprotein) to promote macrophage cholesterol efflux is considered the main HDL cardioprotective function. Abdominal aortic aneurysm (AAA) is usually characterized by cholesterol accumulation and macrophage infiltration in the aortic wall. Here, we aim to evaluate the composition of circulating HDL particles and their potential for promoting macrophage cholesterol efflux in AAA subjects. Approach and Results- First, we randomly selected AAA and control subjects from Spain. The AAA patients in the Spanish cohort showed lower plasma apoA-I levels concomitantly associated with low levels of plasma HDL cholesterol and the amount of preß-HDL particles. We determined macrophage cholesterol efflux to apoB-depleted plasma, which contains mature HDL, preß-HDL particles and HDL regulatory proteins. ApoB-depleted plasma from AAA patients displayed an impaired ability to promote macrophage cholesterol efflux. Next, we replicated the experiments with AAA and control subjects derived from Danish cohort. Danish AAA patients also showed lower apoA-I levels and a defective HDL-mediated macrophage cholesterol efflux. Conclusions- AAA patients show impaired HDL-facilitated cholesterol removal from macrophages, which could be mechanistically linked to AAA.

Potential role of insulin receptor isoforms and IGF receptors in plaque instability of human and experimental atherosclerosis.

BACKGROUND: Clinical complications associated with atherosclerotic plaques arise from luminal obstruction due to plaque growth or destabilization leading to rupture. We previously demonstrated that overexpression of insulin receptor isoform A (IRA) and insulin-like growth factor-I receptor (IGF-IR) confers a proliferative and migratory advantage to vascular smooth muscle cells (VSMCs) promoting plaque growth in early stages of atherosclerosis. However, the role of insulin receptor (IR) isoforms, IGF-IR or insulin-like growth factor-II receptor (IGF-IIR) in VSMCs apoptosis during advanced atherosclerosis remains unclear. METHODS: We evaluated IR isoforms expression in human carotid atherosclerotic plaques by consecutive immunoprecipitations of insulin receptor isoform B (IRB) and IRA. Western blot analysis was performed to measure IGF-IR, IGF-IIR, and α-smooth muscle actin (α-SMA) expression in human plaques. The expression of those proteins, as well as the presence of apoptotic cells, was analyzed by immunohistochemistry in experimental atherosclerosis using BATIRKO; ApoE-/- mice, a model showing more aggravated vascular damage than ApoE-/- mice. Finally, apoptosis of VSMCs bearing IR (IRLoxP+/+ VSMCs), or not (IR-/- VSMCs), expressing IRA (IRA VSMCs) or expressing IRB (IRB VSMCs), was assessed by Western blot against cleaved caspase 3. RESULTS: We observed a significant decrease of IRA/IRB ratio in human complicated plaques as compared to non-complicated regions. Moreover, complicated plaques showed a reduced IGF-IR expression, an increased IGF-IIR expression, and lower levels of α-SMA indicating a loss of VSMCs. In experimental atherosclerosis, we found a significant decrease of IRA with an increased IRB expression in aorta from 24-week-old BATIRKO; ApoE-/- mice. Furthermore, atherosclerotic plaques from BATIRKO; ApoE-/- mice had less VSMCs content and higher number of apoptotic cells. In vitro experiments showed that IGF-IR inhibition by picropodophyllin induced apoptosis in VSMCs. Apoptosis induced by thapsigargin was lower in IR-/- VSMCs expressing higher IGF-IR levels as compared to IRLoxP+/+ VSMCs. Finally, IRB VSMCs are more prone to thapsigargin-induced apoptosis than IRA or IRLoxP+/+ VSMCs. CONCLUSIONS: In advanced human atherosclerosis, a reduction of IRA/IRB ratio, decreased IGF-IR expression, or increased IGF-IIR may contribute to VSMCs apoptosis, promoting plaque instability and increasing the risk of plaque rupture and its clinical consequences.

Increased galectin-3 levels are associated with abdominal aortic aneurysm progression and inhibition of galectin-3 decreases elastase-induced AAA development.

Abdominal aortic aneurysm (AAA) evolution is unpredictable and no specific treatment exists for AAA, except surgery to prevent aortic rupture. Galectin-3 has been previously associated with CVD, but its potential role in AAA has not been addressed. Galectin-3 levels were increased in the plasma of AAA patients (n=225) compared with the control group (n=100). In addition, galectin-3 concentrations were associated with the need for surgical repair, independently of potential confounding factors. Galectin-3 mRNA and protein expression were increased in human AAA samples compared with healthy aortas. Experimental AAA in mice was induced via aortic elastase perfusion. Mice were treated intravenously with the galectin-3 inhibitor modified citrus pectin (MCP, 10 mg/kg, every other day) or saline. Similar to humans, galectin-3 serum and aortic mRNA levels were also increased in elastase-induced AAA mice compared with control mice. Mice treated with MCP showed decreased aortic dilation, as well as elastin degradation, vascular smooth muscle cell (VSMC) loss, and macrophage content at day 14 postelastase perfusion compared with control mice. The underlying mechanism(s) of the protective effect of MCP was associated with a decrease in galectin-3 and cytokine (mainly CCL5) mRNA and protein expression. Interestingly, galectin-3 induced CCL5 expression by a mechanism involving STAT3 activation in VSMC. Accordingly, MCP treatment decreased STAT3 phosphorylation in elastase-induced AAA. In conclusion, increased galectin-3 levels are associated with AAA progression, while galectin-3 inhibition decreased experimental AAA development. Our data suggest the potential role of galectin-3 as a therapeutic target in AAA.

Oxidative Stress in Human Atherothrombosis: Sources, Markers and Therapeutic Targets.

Atherothrombosis remains one of the main causes of morbidity and mortality worldwide. The underlying pathology is a chronic pathological vascular remodeling of the arterial wall involving several pathways, including oxidative stress. Cellular and animal studies have provided compelling evidence of the direct role of oxidative stress in atherothrombosis, but such a relationship is not clearly established in humans and, to date, clinical trials on the possible beneficial effects of antioxidant therapy have provided equivocal results. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is one of the main sources of reactive oxygen species (ROS) in human atherothrombosis. Moreover, leukocyte-derived myeloperoxidase (MPO) and red blood cell-derived iron could be involved in the oxidative modification of lipids/lipoproteins (LDL/HDL) in the arterial wall. Interestingly, oxidized lipoproteins, and antioxidants, have been analyzed as potential markers of oxidative stress in the plasma of patients with atherothrombosis. In this review, we will revise sources of ROS, focusing on NADPH oxidase, but also on MPO and iron. We will also discuss the impact of these oxidative systems on LDL and HDL, as well as the value of these modified lipoproteins as circulating markers of oxidative stress in atherothrombosis. We will finish by reviewing some antioxidant systems and compounds as therapeutic strategies to prevent pathological vascular remodeling.

A Single In-Vial Dual Extraction Strategy for the Simultaneous Lipidomics and Proteomics Analysis of HDL and LDL Fractions.

A single in-vial dual extraction (IVDE) procedure for the subsequent analysis of lipids and proteins in the high-density lipoprotein (HDL) and low-density lipoprotein (LDL) fractions derived from the same biological sample is presented. On the basis of methyl-tert-butyl ether (MTBE) extraction, IVDE leads to the formation of three phases: a protein pellet at the bottom, an aqueous phase with polar compounds, and an ether phase with lipophilic compounds. After sample extraction, performed within a high-performance liquid chromatography vial insert, the ether phase was directly injected for lipid fingerprinting, while the protein pellet, after evaporation of the remaining sample, was used for proteomics analysis. Human HDL and LDL isolates were used to test the suitability of the IVDE methodology for lipid and protein analysis from a single sample in terms of data quality and matching composition to that of HDL and LDL. Subsequently, HDL and LDL fractions isolated from ApoE-KO and wild-type mice were used to validate the capacity of IVDE for revealing changes in lipid and protein abundance. Results indicate that IVDE can be successfully used for the subsequent analysis of lipids and proteins with the advantages of time saving, simplicity, and reduced sample amount.

Proteomics and metabolomics in biomarker discovery for cardiovascular diseases: progress and potential.

INTRODUCTION: The process of discovering novel biomarkers and potential therapeutic targets may be shortened using proteomic and metabolomic approaches. AREAS COVERED: Several complementary strategies, each one presenting different advantages and limitations, may be used with these novel approaches. In vitro studies show how cells involved in cardiovascular disease react, although the phenotype of cultured cells differs to that occurring in vivo. Tissue analysis either in human specimens or animal models may show the proteins that are expressed in the pathological process, although the presence of structural proteins may be confounding. To identify circulating biomarkers, analyzing the secretome of cultured atherosclerotic tissue, analysis of blood cells and/or plasma may be more straightforward. However, in the latter approach, high-abundant proteins may mask small molecules that could be potential biomarkers. The study of sub-proteomes such as high-density lipoproteins may be useful to circumvent this limitation. Regarding metabolomics, most studies have been performed in small populations, and we need to perform studies in large populations in order to discover robust biomarkers. Expert commentary: It is necessary to involve the clinicians in these areas to improve the design of clinical studies, including larger populations, in order to obtain consistent novel biomarkers.

Paraoxonase-1 overexpression prevents experimental abdominal aortic aneurysm progression.

Abdominal aortic aneurysm (AAA) is a permanent dilation of the aorta due to excessive proteolytic, oxidative and inflammatory injury of the aortic wall. We aimed to identify novel mediators involved in AAA pathophysiology, which could lead to novel therapeutic approaches. For that purpose, plasma from four AAA patients and four controls were analysed by a label-free proteomic approach. Among identified proteins, paraoxonase-1 (PON1) was decreased in plasma of AAA patients compared with controls, which was further validated in a bigger cohort of samples by ELISA. The phenylesterase enzymatic activity of PON1 was also decreased in serum of AAA patients compared with controls. To address the potential role of PON1 as a mediator of AAA, experimental AAA was induced by aortic elastase perfusion in wild-type (WT) mice and human transgenic PON1 (HuTgPON1) mice. Similar to humans, PON1 activity was also decreased in serum of elastase-induced AAA mice compared with healthy mice. Interestingly, overexpression of PON1 was accompanied by smaller aortic dilation and higher elastin and vascular smooth muscle cell (VSMC) content in the AAA of HuTgPON1 compared with WT mice. Moreover, HuTgPON1 mice display decreased oxidative stress and apoptosis, as well as macrophage infiltration and monocyte chemoattractant protein-1 (MCP1) expression, in elastase-induced AAA. In conclusion, decreased circulating PON1 activity is associated with human and experimental AAA. PON1 overexpression in mice protects against AAA progression by reducing oxidative stress, apoptosis and inflammation, suggesting that strategies aimed at increasing PON1 activity could prevent AAA.

The Prognostic Value of High-Sensitive Troponin I in Stable Coronary Artery Disease Depends on Age and Other Clinical Variables.

OBJECTIVES: To study the prognostic value of high-sensitive troponin (hs-cTn) I in stable coronary artery disease. METHODS: In total, we studied 705 patients. Secondary outcomes were the incidence of: (1) acute ischemic events and (2) heart failure or death. The primary outcome was the composite of them. RESULTS: Patients with hs-cTnI >0 ng/ml (62.1%) were older, had a lower estimated glomerular filtration rate, more frequent a history of hypertension, atrial fibrillation, ejection fraction <40%, and therapy with angiotensin-converting enzyme inhibitors, diuretics and acenocumarol. The follow-up period was 2.2 ± 0.99 years. Fifty-three patients suffered an acute ischemic event, 33 died or suffered heart failure and 78 developed the primary outcome. By univariate Cox's regression analysis, hs-cTnI >0 was associated with a higher risk of developing the primary outcome [relative risk = 2.360 (1.359-4.099); p = 0.001] and heart failure or death [relative risk = 5.932 (1.806-19.482); p < 0.001], but not with acute ischemic events. Statistical significance was lost after controlling for age. By logistic regression analysis, age [relative risk = 1.026 (1.009-1.044); p = 0.003], ejection fraction <40% [relative risk = 4.099 (2.043-8.224); p < 0.001], use of anticoagulants [relative risk = 2.785 (1.049-7.395); p = 0.040] and therapy with angiotensin-converting enzyme inhibitors [relative risk = 1.471 (1.064-2.034); p = 0.020], and estimated glomerular filtration rate [relative risk = 0.988 (0.977-0.999); p = 0.027] were associated with hs-cTnI >0. CONCLUSIONS: In stable coronary disease, hs-cTnI is associated with the incidence of heart failure or death, but this relationship depends on other variables.

The presence of activating IgG Fc receptors in macrophages aggravates the development of experimental abdominal aortic aneurysm. / La presencia de receptores Fc de IgG activadores en macrófagos agrava el desarrollo de aneurisma aórtico abdominal experimental.

INTRODUCTION: Abdominal aortic aneurysm (AAA) is a multifactorial, degenerative disease characterized by progressive aortic dilation and chronic activation of inflammation, proteolytic activity, and oxidative stress in the aortic wall. The immune response triggered by antibodies against antigens present in the vascular wall participates in the formation and progression of AAA through mechanisms not completely understood. This work analyses the function of specific IgG receptors (FcγR), especially those expressed by monocytes/macrophages, in the development of experimental AAA. METHODS: In the elastase-induced AAA model, the abdominal aortas from wildtype and FcγR deficient mice with/without macrophage adoptive transfer were analysed by histology and quantitative PCR. In vitro, mouse macrophages were transfected with RNA interference of FcγRIV/CD16.2 or treated with Syk kinase inhibitor before stimulation with IgG immune complexes. RESULTS: Macrophage adoptive transfer in FcγR deficient mice increased the susceptibility to AAA development. Mice receiving macrophages with functional FcγR exhibited higher aortic diameter increase, higher content of macrophages and B lymphocytes, and upregulated expression of chemokine CCL2, cytokines (TNF-α and IL-17), metalloproteinase MMP2, prooxidant enzyme NADPH oxidase-2, and the isoforms FcγRIII/CD16 and FcγRIV/CD16.2. In vitro, both FcγRIV/CD16.2 gene silencing and Syk inhibition reduced cytokines and reactive oxygen species production induced by immune complexes in macrophages. CONCLUSIONS: Activation of macrophage FcγR contributes to AAA development by inducing mediators of inflammation, proteolysis, and oxidative stress. Modulation of FcγR or effector molecules may represent a potential target for AAA treatment.

Role of LCN2 in a murine model of hindlimb ischemia and in peripheral artery disease patients, and its potential regulation by miR-138-5P.

BACKGROUND AND AIMS: Peripheral arterial disease (PAD) is a leading cause of morbimortality worldwide. Lipocalin-2 (LCN2) has been associated with higher risk of amputation or mortality in PAD and might be involved in muscle regeneration. Our aim is to unravel the role of LCN2 in skeletal muscle repair and PAD. METHODS AND RESULTS: WT and Lcn2-/- mice underwent hindlimb ischemia. Blood and crural muscles were analyzed at the inflammatory and regenerative phases. At day 2, Lcn2-/- male mice, but not females, showed increased blood and soleus muscle neutrophils, and elevated circulating pro-inflammatory monocytes (p < 0.05), while locally, total infiltrating macrophages were reduced (p < 0.05). Moreover, Lcn2-/- soleus displayed an elevation of Cxcl1 (p < 0.001), and Cxcr2 (p < 0.01 in males), and a decrease in Ccl5 (p < 0.05). At day 15, Lcn2 deficiency delayed muscle recovery, with higher density of regenerating myocytes (p < 0.04) and arterioles (αSMA+, p < 0.025). Reverse target prediction analysis identified miR-138-5p as a potential regulator of LCN2, showing an inverse correlation with Lcn2 mRNA in skeletal muscles (rho = -0.58, p < 0.01). In vitro, miR-138-5p mimic reduced Lcn2 expression and luciferase activity in murine macrophages (p < 0.05). Finally, in human serum miR-138-5p was inversely correlated with LCN2 (p ≤ 0.001 adjusted, n = 318), and associated with PAD (Odds ratio 0.634, p = 0.02, adjusted, PAD n = 264, control n = 54). CONCLUSIONS: This study suggests a possible dual role of LCN2 in acute and chronic conditions, with a probable role in restraining inflammation early after skeletal muscle ischemia, while being associated with vascular damage in PAD, and identifies miR-138-5p as one potential post-transcriptional regulator of LCN2.

CCL20 is increased in hypercholesterolemic subjects and is upregulated by LDL in vascular smooth muscle cells: role of NF-κB.

OBJECTIVE: Our aim was to analyze the regulation of CC Chemokine ligand 20 (CCL20) by LDL in human vascular smooth muscle cells (VSMC). METHODS AND RESULTS: In asymptomatic subjects, circulating CCL20 levels were higher in patients with hypercholesterolemia (18.5±3.2 versus 9.1±1.3 pg/mL; P<0.01). LDL induced the expression of CCL20 in VSMC in a dose- and time-dependent manner. Increased levels of CCL20 secreted by LDL-treated VSMC significantly induced human lymphocyte migration, an effect reduced by CCL20 silencing. The upregulation of CCL20 by LDL was dependent on the activation of kinase signaling pathways and NF-κB. By site-directed mutagenesis, electrophoretic mobility shift assay, and chromatin immunoprecipitation, we identified a NF-κB site (-80/-71) in CCL20 promoter critical for LDL responsiveness. Lysophosphatidic acid mimicked the upregulation of CCL20 induced by LDL, and minimal oxidation of LDL increased the ability of LDL to induce CCL20 through a mechanism that involves lysophosphatidic acid receptors. CCL20 was overexpressed in atherosclerotic lesions from coronary artery patients, colocalizing with VSMC. CCL20 was detected in conditioned media from healthy human aorta and its levels were significantly higher in secretomes from carotid endarterectomy specimens. CONCLUSION: This study identifies CCL20 in atherosclerotic lesions and recognizes this chemokine as a mediator highly sensitive to the inflammatory response elicited by LDL.

Proteomic analysis of polymorphonuclear neutrophils identifies catalase as a novel biomarker of abdominal aortic aneurysm: potential implication of oxidative stress in abdominal aortic aneurysm progression.

OBJECTIVE: Polymorphonuclear neutrophils (PMNs) play a main role in abdominal aortic aneurysm (AAA) progression. We have analyzed circulating PMNs isolated from AAA patients and controls by a proteomic approach to identify proteins potentially involved in AAA pathogenesis. METHODS AND RESULTS: PMNs from 8 AAA patients (4 large AAA >5 cm and 4 small AAA 3-5 cm) and 4 controls were analyzed by 2D differential in-gel electrophoresis. Among differentially expressed spots, several proteins involved in redox balance were identified by mass spectrometry (eg, cyclophilin, thioredoxin reductase, catalase). Diminished catalase expression and activity were observed in PMNs from AAA patients compared with controls. In contrast, PMNs from AAA patients displayed higher H(2)O(2) and myeloperoxidase levels than PMNs from controls. Moreover, a significant decrease in catalase mRNA levels was observed in PMNs after phorbol 12-myristate 13-acetate incubation. Catalase plasma levels were also decreased in large (n=47) and small (n=56) AAA patients compared with controls (n=34). We observed catalase expression in AAA thrombus and thrombus-conditioned medium, associated with PMN infiltration. Furthermore, increased H(2)O(2) levels were observed in AAA thrombus-conditioned medium compared with the media layer. CONCLUSIONS: Diminished catalase levels in circulating PMNs and plasma are observed in AAA patients, supporting an important role of oxidative stress in AAA evolution.

Metabolomic study of plasma of patients with abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is an important health problem, both because of AAA rupture and death and because of increased cardiovascular mortality. Identification of new biomarkers of AAA may suggest novel pathological mechanisms and targets for new medical treatments to slow AAA progression. Metabolic changes in AAA patients were mainly related to carbohydrate and lipid metabolism and many of these changes can be associated with a situation of insulin resistance (which can be related to metabolic syndrome) together with altered amino acid metabolism. For the first time, metabolites that can be associated with differential metabolism by the gut microflora of AAA patients have also been found. Moreover, aminomalonic acid in plasma has been shown to be the metabolite with the biggest difference between patients suffering from large aneurysm (>5 cm) and controls.

Role of Extracellular Vesicles as Potential Diagnostic and/or Therapeutic Biomarkers in Chronic Cardiovascular Diseases.

Cardiovascular diseases (CVDs) are the first cause of death worldwide. In recent years, there has been great interest in the analysis of extracellular vesicles (EVs), including exosomes and microparticles, as potential mediators of biological communication between circulating cells/plasma and cells of the vasculature. Besides their activity as biological effectors, EVs have been also investigated as circulating/systemic biomarkers in different acute and chronic CVDs. In this review, the role of EVs as potential diagnostic and prognostic biomarkers in chronic cardiovascular diseases, including atherosclerosis (mainly, peripheral arterial disease, PAD), aortic stenosis (AS) and aortic aneurysms (AAs), will be described. Mechanistically, we will analyze the implication of EVs in pathological processes associated to cardiovascular remodeling, with special emphasis in their role in vascular and valvular calcification. Specifically, we will focus on the participation of EVs in calcium accumulation in the pathological vascular wall and aortic valves, involving the phenotypic change of vascular smooth muscle cells (SMCs) or valvular interstitial cells (IC) to osteoblast-like cells. The knowledge of the implication of EVs in the pathogenic mechanisms of cardiovascular remodeling is still to be completely deciphered but there are promising results supporting their potential translational application to the diagnosis and therapy of different CVDs.

NT-proBNP Levels Influence the Prognostic Value of Mineral Metabolism Biomarkers in Coronary Artery Disease.

Background. Mineral metabolism (MM) system and N-terminal pro-brain natriuretic peptide (NT-ProBNP) have been shown to add prognostic value in patients with stable coronary artery disease (SCAD). However, the influence of NT-ProBNP on the prognostic role of MM in patients with SCAD has not been shown yet. The objective of this study is to assess the influence of NT-ProBNP on the prognostic role of MM markers in patients with SCAD. Methods: We analyzed the prognostic value of MM markers (parathormone (PTH), klotho, phosphate, calcidiol (25-hydroxyvitamin D3), and fibroblast growth factor-23) in 964 patients with SCAD and NT-ProBNP > 125 pg/mL vs. patient with NT-ProBNP ≤ 125 pg/mL included in five hospitals in Spain. The main outcome was the combination of death, heart failure, and ischemic events (any acute coronary syndrome, ischemic stroke, or transient ischemic attack). Results: A total of 622 patients had NT-proBNP > 125 pg/mL and 342 patients had NT-ProBNP ≤ 125 pg/mL. The median follow-up was 5.1 years. In the group of NT-proBNP > 125 pg/mL, the patients were older, and there were more females and smokers than in the group of patients with normal NT-proBNP. Additionally, the proportion of patients with hypertension, atrial fibrillation, ejection fraction < 40%, cerebrovascular attack, or prior coronary artery bypass graft was higher in the high NT-proBNP group. In the high NT-proBNP patients, the predictors of poor prognosis were PTH (HR = 1.06 (1.01−1.10), p < 0.001) and NT-proBNP (HR = 1.02 (1.01−1.03), p = 0.011), along with age (HR = 1.039 (1.02−1.06), p < 0.001), prior coronary artery bypass graft (HR = 1.624 (1.02−2.59), p = 0.041), treatment with statins (HR = 0.32 (0.19−0.53), p < 0.001), insulin (HR = 2.49 (1.59−4.09), p < 0.001), angiotensin receptor blockers (HR = 1.73 (1.16−2.56), p = 0.007), nitrates (HR = 1.65 (1.10−2.45), p = 0.014), and proton pump inhibitors (HR = 2.75 (1.74−4.36), p < 0.001). In the NT-proBNP ≤ 125 pg/mL subgroup, poor prognosis predictors were plasma levels of non-high-density lipoprotein (non-HDL) cholesterol (HR = 1.01 (1.00−1.02), p = 0.014) and calcidiol (HR = 0.96 (0.92−0.99), p = 0.045), as well as treatment with verapamil (HR = 11.28 (2.54−50.00), p = 0.001), and dihydropyridines (HR = 3.16 (1.63−6.13), p = 0.001). Conclusion: In patients with SCAD and NT-ProBNP > 125 pg/mL, PTH and NT-ProBNP, which are markers related to ventricular damage, are predictors of poor outcome. In the subgroup of patients with NT-ProBNP ≤ 125 pgm/L, calcidiol and non-HDL cholesterol, which are more related to vascular damage, are the independent predictors of poor outcome. Then, in patients with SCAD, baseline NT-ProBNP may influence the type of biomarker that is effective in risk prediction.