Elena Burillo

Galectin-3, a Biomarker Linking Oxidative Stress and Inflammation With the Clinical Outcomes of Patients With Atherothrombosis

Background Galectin‐3 (Gal‐3) participates in different mechanisms involved in atherothrombosis, such as inflammation, proliferation, or macrophage chemotaxis. Thus, there have been committed intensive efforts to elucidate the function of Gal‐3 in cardiovascular (CV) diseases. The role of Gal‐3 as a circulating biomarker has been demonstrated in patients with heart failure, but its importance as a biomarker in atherothrombosis is still unknown. Methods and Results Because Gal‐3 is involved in monocyte‐to‐macrophage transition, we used fresh isolated monocytes and the in vitro model of macrophage differentiation of THP‐1 cells stimulated with phorbol myristate acetate (PMA). Gal‐3 release is increased by PMA in human monocytes and macrophages, a process involving exosomes and regulated by reactive oxygen species/NADPH oxidase activity. In asymptomatic subjects (n=199), Gal‐3 plasma levels are correlated with NADPH oxidase activity in peripheral blood mononuclear cells (r=0.476; P<0.001) and carotid intima‐media thickness (r=0.438; P<0.001), a surrogate marker of atherosclerosis. Accordingly, Gal‐3 plasma concentrations are increased in patients with carotid atherosclerosis (n=158), compared to control subjects (n=115; 14.3 [10.7 to 16.9] vs. 10.4 [8.6 to 12.5] ng/mL; P<0.001). Finally, on a 5‐year follow‐up study in patients with peripheral artery disease, Gal‐3 concentrations are significantly and independently associated with an increased risk for CV mortality (hazard ratio=2.24, 95% confidence interval: 1.06 to 4.73, P<0.05). Conclusions Gal‐3 extracellular levels could reflect key underlying mechanisms involved in atherosclerosis etiology, development, and plaque rupture, such as inflammation, infiltration of circulating cells and oxidative stress. Moreover, circulating Gal‐3 concentrations are associated with clinical outcomes in patients with atherothrombosis.

Grosor íntima-media carotídeo en sujetos sin factores de riesgo cardiovascular

El grosor intima-media de la arteria carotida permite la cuantificacion del engrosamiento arterial en fases preclinicas de la enfermedad, pero no se conoce sus valores en poblacion sin factores de riesgo cardiovascular. El objetivo es describir la distribucion y los factores determinantes del grosor intima-media carotideo en poblacion sana y sin factores de riesgo cardiovascular. Estudiamos el grosor intima-media carotideo de 138 sujetos (64 varones y 74 mujeres) de 20-79 anos de edad distribuidos homogeneamente segun edad y sexo. El limite superior de la normalidad de la media del grosor intima-media oscilo entre 0,59 y 0,95 mm en varones y entre 0,52 y 0,93 mm en mujeres. Los valores maximos oscilaron entre 0,81 y 1,11 mm en varones y entre 0,66 y 1,13 mm en mujeres. Los principales factores determinantes del grosor intima-media fueron edad, sexo masculino, presion arterial sistolica y colesterol de las lipoproteinas de baja densidad.

Erythrocytes, leukocytes and platelets as a source of oxidative stress in chronic vascular diseases: Detoxifying mechanisms and potential therapeutic options

Oxidative stress is involved in the chronic pathological vascular remodelling of both abdominal aortic aneurysm and occlusive atherosclerosis. Red blood cells (RBCs), leukocytes and platelets present in both, aneurysmal intraluminal thrombus and intraplaque haemorraghes, could be involved in the redox imbalance inside diseased arterial tissues. RBCs haemolysis may release the pro-oxidant haemoglobin (Hb), which transfers heme to tissue and low-density lipoproteins. Heme-iron potentiates molecular, cell and tissue toxicity mediated by leukocytes and other sources of reactive oxygen species (ROS). Polymorphonuclear neutrophils release myeloperoxidase and, along with activated platelets, produce superoxide mediated by NADPH oxidase, causing oxidative damage. In response to this pro-oxidant milieu, several antioxidant molecules of plasma or cell origin can prevent ROS production. Free Hb binds to haptoglobin (Hp) and once Hp-Hb complex is endocytosed by CD163, liberated heme is converted into less toxic compounds by heme oxygenase-1. Iron homeostasis is mainly regulated by transferrin, which transports ferric ions to other cells. Transferrin-bound iron is internalised via endocytosis mediated by transferrin receptor. Once inside the cell, iron is mainly stored by ferritin. Other non hemo-iron related antioxidant enzymes (e.g. superoxide dismutase, catalase, thioredoxin and peroxiredoxin) are also involved in redox modulation in vascular remodelling. Oxidative stress is a main determinant of chronic pathological remodelling of the arterial wall, partially linked to the presence of RBCs, leukocytes, platelets and oxidised fibrin within tissue and to the imbalance between pro-/anti-oxidant molecules. Understanding the complex mechanisms underlying redox imbalance could help to define novel potential targets to decrease atherothrombotic risk.

Carotid Intima-Media Thickness in Subjects With No Cardiovascular Risk Factors

Measurement of the carotid intima-media thickness enables arterial wall thickening to be quantified during preclinical disease stages. However, little is known about how the thickness varies in individuals with no cardiovascular risk factors. The objective of this study was to report on the range of carotid intima-media thicknesses observed in a population of healthy subjects with no cardiovascular risk factors and to identify parameters that influence it. The carotid intima-media thickness was assessed in 138 subjects (64 men and 74 women) aged 20-79 years whose age and sex were homogeneously distributed. The upper limit of normal for the mean carotid intima-media thickness ranged from 0.59-0.95 mm in men and from 0.52-0.93 mm in women. The upper limit for the maximum thickness varied from 0.81-1.11 mm in men and from 0.66-1.13 mm in women. The main parameters determining the intima-media thickness were age, male sex, systolic blood pressure and low-density lipoprotein cholesterol level.

Apolipoprotein E gene mutations in subjects with mixed hyperlipidemia and a clinical diagnosis of familial combined hyperlipidemia.

OBJECTIVE Rare mutations in the APOE gene, undetectable with the usual genotyping technique, are responsible for dominant familial dysbetalipoproteinemia (FD) and therefore could be easily misclassified as familial combined hyperlipidemia (FCHL). We aimed to identify APOE mutations associated with dominant combined hyperlipoproteinemia and to establish their frequency in subjects with a clinical diagnosis of FCHL. METHODS AND RESULTS In 279 unrelated subjects with FCHL in whom a functional LDLR mutation was excluded, sequencing of the entire APOE gene detected 9 carriers of a rare mutation: 5 subjects (1.8%) with the R136S mutation (arginine at residue 136 changed to serine) and 4 subjects (1.4%) with the p.Leu149del mutation, a 3-bp inframe deletion that results in the loss of leucine at position 149. Both genetic defects were detected with similar frequency (2.5% and 1.3%, respectively) in an independent group of 160 FCHL subjects from other locations in Spain. Family studies demonstrated cosegregation of these APOE mutations with hyperlipoproteinemia. R136S carriers showed dysbetalipoproteinemia, while the lipid phenotype of p.Leu149del carriers was IIa or IIb. CONCLUSIONS Rare APOE mutations are responsible for approximately 3.5% of FCHL cases in our population. APOE R136S and p.Leu149del induce autosomal dominant FD and a phenotype indistinguishable from FCHL, respectively.

Beneficial effects of omega-3 fatty acids in the proteome of high-density lipoprotein proteome

BackgroundOmega-3 poly-unsaturated fatty acids (ω-3 PUFAs) have demonstrated to be beneficial in the prevention of cardiovascular disease, however, the mechanisms by which they perform their cardiovascular protection have not been clarified. Intriguingly, some of these protective effects have also been linked to HDL. The hypothesis of this study was that ω-3 PUFAs could modify the protein cargo of HDL particle in a triglyceride non-dependent mode. The objective of the study was to compare the proteome of HDL before and after ω-3 PUFAs supplemented diet.MethodsA comparative proteomic analysis in 6 smoker subjects HDL before and after a 5 weeks ω-3 PUFAs enriched diet has been performed.ResultsAmong the altered proteins, clusterin, paraoxonase, and apoAI were found to increase, while fibronectin, α-1-antitrypsin, complement C1r subcomponent and complement factor H decreased after diet supplementation with ω-3 PUFAs. Immunodetection assays confirmed these results. The up-regulated proteins are related to anti-oxidant, anti-inflammatory and anti-atherosclerotic properties of HDL, while the down-regulated proteins are related to regulation of complement activation and acute phase response.ConclusionsDespite the low number of subjects included in the study, our findings demonstrate that ω-3 PUFAs supplementation modifies lipoprotein containing apoAI (LpAI) proteome and suggest that these protein changes improve the functionality of the particle.

Proteomic Analysis of Intraluminal Thrombus Highlights Complement Activation in Human Abdominal Aortic Aneurysms

Objective—To identify proteins related to intraluminal thrombus biological activities that could help to find novel pathological mechanisms and therapeutic targets for human abdominal aortic aneurysm (AAA). Approach and Results—Tissue-conditioned media from patients with AAA were analyzed by a mass spectrometry–based strategy using liquid chromatography coupled to tandem mass spectrometry. Global pathway analysis by Ingenuity software highlighted the presence of several circulating proteins, among them were proteins from the complement system. Complement C3 concentration and activation were assessed in plasma from AAA patients (small AAA, AAA diameter=3–5 cm and large AAA, AAA diameter >5 cm), showing decreased C3 levels and activation in large AAA patients. No association of a combination of single-nucleotide polymorphisms in complement genes between large and small AAA patients was observed. Intense extracellular C3 inmunostaining, along with C9, was observed in AAA thrombus. Analysis of C3 in AAA tissue homogenates and tissue-conditioned media showed increased levels of C3 in AAA thrombus, as well as proteolytic fragments (C3a/C3c/C3dg), suggesting its local deposition and activation. Finally, the functional role of local complement activation in polymorphonuclear (PMN) cell activation was tested, showing that C3 blockade by anti-C3 antibody was able to decrease thrombus-induced neutrophil chemotaxis and reactive oxygen species production. Conclusions—A decrease of systemic C3 concentration and activity in the later stages of AAA associated with local complement retention, consumption, and proteolysis in the thrombus could induce PMN chemotaxis and activation, playing a detrimental role in AAA progression.

Serum ferritin is a major determinant of lipid phenotype in familial combined hyperlipidemia and familial hypertriglyceridemia

Familial combined hyperlipidemia (FCH) and familial hypertriglyceridemia (FHTG) share pathogenic mechanisms and a high interaction with components of the metabolic syndrome. The metabolic syndrome associates increased serum ferritin concentration and high cardiovascular risk. The objective was to describe the frequency of iron overload and the relationship between serum ferritin and the phenotype in patients with FCH and FHTG. The study was composed of 211 consecutive unrelated patients aged at least 18 years with primary hypertriglyceridemia, 149 with FCH, and 62 with FHTG. The prevalence of the metabolic syndrome and hyperferritinemia was very high in both hypertriglyceridemic groups (51.7% and 20.1% in FCH and 62.9% and 16.1% in FHTG, respectively), without significant statistical differences between them. Serum ferritin concentration did not show any significant association with the number of metabolic syndrome criteria. Subjects in the highest tertile of ferritin concentration (ferritin >200 mug/L) presented higher concentrations of triglycerides and liver enzymes than subjects in the first tertile of ferritin concentration (ferritin <90 mug/L). The highest positive correlation coefficient for triglycerides was found with ferritin in FCH and in FHTG subjects (R = 0.317 [P < .001] when combined). Ferritin was also the covariate that showed the highest independent association with triglycerides in FCH and FHTG. In contrast, ferritin was not associated with carotid intima-media thickness. In summary, serum ferritin is commonly increased in FCH and in FHTG, it is not related with the presence of metabolic syndrome, and it is highly correlated with liver enzymes.

Omega-3 Fatty Acids and HDL. How Do They Work in the Prevention of Cardiovascular Disease?

Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) from marine origin have been strongly associated with cardiovascular protection, even at low doses ( < 1g/d). Despite the research performed in this promising area, basic aspects, such as the ideal doses and the mechanisms by which ω-3 PUFAs act, are not precisely defined. The best known biological property of ω-3 PUFAs is their hypotriglyceridemic effect, but other cardioprotective actions, such as reduction of arrhythmia susceptibility, antithrombotic, antiinflammatory and antioxidant effects, improvement of endothelial function, and delayed atherosclerosis development have received an increased interest in recent years. Some of these actions are also ascribable to high-density lipoproteins (HDL). Abundant epidemiological evidence links increasing HDL-cholesterol concentrations to cardiovascular protection. Recently, the protein cargo (proteome) of HDL particles has been attributed a key role in their functionality. In this review, we summarize the main effects of ω-3 PUFAs on HDL-cholesterol, HDL subfractions, and its main proteins, apolipoproteins (apo) AI and AII. The shared cardioprotective actions of ω-3 PUFAs and HDL are reviewed as well.

ApoA-I/HDL-C levels are inversely associated with abdominal aortic aneurysm progression

Abdominal aortic aneurysm (AAA) evolution is unpredictable, and there is no therapy except surgery for patients with an aortic size> 5 cm (large AAA). We aimed to identify new potential biomarkers that could facilitate prognosis and treatment of patients with AAA. A differential quantitative proteomic analysis of plasma proteins was performed in AAA patients at different stages of evolution [small AAA (aortic size=3-5 cm) vs large AAA] using iTRAQ labelling, high-throughput nano-LC-MS/MS and a novel multi-layered statistical model. Among the proteins identified, ApoA-I was decreased in patients with large AAA compared to those with small AAA. These results were validated by ELISA on plasma samples from small (n=90) and large AAA (n=26) patients (150± 3 vs 133± 5 mg/dl, respectively, p< 0.001). ApoA-I levels strongly correlated with HDL-Cholesterol (HDL-C) concentration (r=0.9, p< 0.001) and showed a negative correlation with aortic size (r=-0.4, p< 0.01) and thrombus volume (r=-0.3, p< 0.01), which remained significant after adjusting for traditional risk factors. In a prospective study, HDL-C independently predicted aneurysmal growth rate in multiple linear regression analysis (n=122, p=0.008) and was inversely associated with need for surgical repair (Adjusted hazard ratio: 0.18, 95 % confidence interval: 0.04-0.74, p=0.018). In a nation-wide Danish registry, we found lower mean HDL-C concentration in large AAA patients (n=6,560) compared with patients with aorto-iliac occlusive disease (n=23,496) (0.89± 2.99 vs 1.59± 5.74 mmol/l, p< 0.001). Finally, reduced mean aortic AAA diameter was observed in AngII-infused mice treated with ApoA-I mimetic peptide compared with saline-injected controls. In conclusion, ApoA-I/HDL-C systemic levels are negatively associated with AAA evolution. Therapies targeting HDL functionality could halt AAA formation.