Type II interleukin-1 receptor expression is reduced in monocytes/macrophages and atherosclerotic lesions.

Type II interleukin-1 receptor (IL-1R2) is a non-signaling decoy receptor that negatively regulates the activity of interleukin-1 (IL-1), a pro-inflammatory cytokine involved in atherogenesis. In this article we assessed the relevance of IL-1R2 in atherosclerosis by studying its expression in monocytes from hyperlipidemic patients, in THP-1 macrophages exposed to lipoproteins and in human atherosclerotic lesions. Our results showed that the mRNA and protein expression of IL-1R2 was reduced in monocytes from patients with familial combined hyperlipidemia (-30%, p<0.05). THP-1 macrophages incubated with increasing concentrations of acetylated low density (ac-LDL) and very low density (VLDL) lipoproteins also exhibit a decrease in IL-1R2 mRNA and protein levels. Pre-incubation with agents that block intracellular accumulation of lipids prevents the decrease in IL-1R2 mRNA caused by lipoproteins. Lipoproteins also prevented the increase in IL-1R1 and IL-1R2 caused by a 4-h stimulation with LPS and reduced protein expression of total and phosphorylated IL-1 receptor-associated kinase-1. Finally, IL-1R2 expression in human atherosclerotic vessels was markedly lower than in non-atherosclerotic arteries (-80%, p<0.0005). Overall, our results suggest that under atherogenic conditions, there is a decrease in IL-1R2 expression in monocytes/macrophages and in the vascular wall that may facilitate IL-1 signaling.

Atorvastatin decreases stearoyl-CoA desaturase gene expression in THP-1 macrophages incubated with oxidized LDL.

Statins, inhibitors of HMG-CoA reductase, reduce plasma low-density lipoprotein (LDL) cholesterol levels decreasing the incidence of coronary events. However, the observed benefit of statins appears to extend beyond their lipid-lowering effects. Previous studies by our group have demonstrated that atorvastatin in oxidized LDL incubated macrophages modifies the gene expression profile of certain enzymes involved in fatty acid metabolism, mainly stearoyl-CoA desaturase (SCD). SCD is a rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids and its expression is mediated by sterol regulatory element-binding protein-1 (SREBP-1). The aim of this study was to determine whether atorvastatin might affect the fatty acid composition in macrophages and if their SCD gene expression profile could explain this effect. Therefore, THP-1 macrophages were treated with atorvastatin and native or oxidized LDL, their fatty acid composition was determined by gas-chromatography, and the SCD and SREBP-1 gene expression profile was analysed using quantitative RT-PCR. We found that atorvastatin reduces the percentage of palmitoleic and oleic acids in THP-1 cells incubated with oxLDL, which could be explained by the inhibition of SCD and SREBP-1 gene expression. The observed results were reversed when mevalonate was added to THP-1 macrophages. This would suggest that inhibition of SCD in THP-1 macrophages incubated with oxLDL and the change in fatty acid composition is an important effect of atorvastatin.

Tendon xanthomas in familial hypercholesterolemia are associated with cardiovascular risk independently of the low-density lipoprotein receptor gene mutation.

OBJECTIVE: To investigate the significance of tendon xanthomas (TX) in heterozygous subjects with familial hypercholesterolemia (hFH). METHODS AND RESULTS: 951 men and women with genetic diagnosis of hFH were studied, of whom 278 (29.2%) presented TX. TX frequency increased with age from 6.9% in subjects 20 to 30 years to 38.3% at 51 to 60 years, with a decrease in those older than 60 years. Total and low-density lipoprotein (LDL) cholesterol were higher in TX+ than in TX- subjects (439.0+/-78.5 mg/dL and 363.1+/-76.5 mg/dL versus 400.6+/-73.4 and 323.3+/-71.0, respectively; P=0.001). High-density lipoprotein (HDL) cholesterol was lower in TX+ than in TX- subjects (50.4+/-15.0 mg/dL versus 53.1+/-14.8 mg/dL; P=0.005). Lp(a), apolipoprotein E genotype, and type of LDL receptor gene mutation showed no differences between groups. 102 TX+ reported premature cardiovascular disease (CVD) (36.7%) versus 93 TX- (13.8%) (P=0.001). The relative odds for premature CVD were higher in women (4.49 versus 2.26), and increased in hFH younger than 51 years to 3.60 (95% CI, 1.703 to 7.608) in men and to 17.1 (95% CI, 2.697 to 108.920) in women. In the multivariate analysis, age, male sex, LDL cholesterol, and hypertension showed significant positive association with TX, whereas body mass index showed negative association with TX. CONCLUSIONS: TX are associated with cardiovascular risk factors and higher CVD, indicating that their detection indicates the need for more aggressive lipid-lowering intervention.

Genetic variants in CCNB1 associated with differential gene transcription and risk of coronary in-stent restenosis.

BACKGROUND: The development of diagnostic tools to assess restenosis risk after stent deployment may enable the intervention to be tailored to the individual patient, for example, by targeting the use of drug-eluting stent to high-risk patients, with the goal of improving safety and reducing costs. The CCNB1 gene (encoding cyclin B1) positively regulates cell proliferation, a key component of in-stent restenosis. Therefore, we hypothesized that single-nucleotide polymorphisms in CCNB1 may serve as useful tools in risk stratification for in-stent restenosis. METHODS AND RESULTS: We identified 3 single-nucleotide polymorphisms in CCNB1 associated with increased restenosis risk in a cohort of 284 patients undergoing coronary angioplasty and stent placement (rs350099: TT versus CC+TC; odds ratio [OR], 1.82; 95% confidence interval [CI], 1.09-3.03; P=0.023; rs350104: CC versus CT+TT; OR, 1.82; 95% CI, 1.02-3.26; P=0.040; and rs164390: GG versus GT+TT; OR, 2.27; 95% CI, 1.33-3.85; P=0.002). These findings were replicated in another cohort study of 715 patients (rs350099: TT versus CC+TC; OR, 1.88; 95% CI, 0.92-3.81; P=0.080; rs350104: CC versus CT+TT; OR, 2.23; 95% CI, 1.18-4.25; P=0.016; and rs164390: GG versus GT+TT; OR, 1.87; 95% CI, 1.03-3.47; P=0.040). Moreover, the haplotype containing all 3 risk alleles is associated with higher CCNB1 mRNA expression in circulating lymphocytes and increased in-stent restenosis risk (OR, 1.43; 95% CI, 1.00-1.823; P=0.039). The risk variants of rs350099, rs350104, and rs164390 are associated with increased reporter gene expression through binding of transcription factors nuclear factor-Y, activator protein 1, and specificity protein 1, respectively. CONCLUSIONS: Allele-dependent transcriptional regulation of CCNB1 associated with rs350099, rs350104, and rs164390 affects the risk of in-stent restenosis. These findings reveal these common genetic variations as attractive diagnostic tools in risk stratification for restenosis.

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.

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.

Overexpression of the CXCL3 gene in response to oxidized low-density lipoprotein is associated with the presence of tendon xanthomas in familial hypercholesterolemia.

To examine if overexpression of certain chemokines and proinflammatory cytokines in response to oxidized low-density lipoprotein could be involved in the onset and development of tendon xanthomas (TX), we quantified IL-1beta, TNF-alpha, and IL-8 and compared gene expression of PPAR-gamma, NF-kappaBIA, IL-8, IL-1beta, CXCL3, tryptase, and TNF-alpha in macrophages of familial hypercholesterolemia subjects with and without TX stimulated with oxidized low-density lipoprotein at 1, 3, 6, and 18 h of incubation. We propose that chemokines belonging to the CXC family could play an important role in the etiology of TX, with CXCL3 being a possible biological marker of onset and development of TX.

Individual variation of scavenger receptor expression in human macrophages with oxidized low-density lipoprotein is associated with a differential inflammatory response.

Atherosclerosis is an inflammatory disease in which oxidized low-density lipoprotein (oxLDL) plays important roles. Scavenger receptors (SR) CD36, SR-A, and LOX-1 uptake over 90% of the oxLDL leading to foam cell formation and secretion of inflammatory cytokines. To investigate whether the interindividual differences in macrophage SR gene expression could determine the inflammatory variability in response to oxLDL, we quantified the gene and protein expression of SR and inflammatory molecules from macrophages isolated from 18 volunteer subjects and incubated with oxLDL for 1, 3, 6, and 18 h. The individual gene expression profile of the studied SR at 1 h of incubation was highly variable, showing a wide fold-change range: CD36: -3.57-4.22, SR-A: -5.0-4.43, and LOX-1: -1.56-75.32. We identified subjects as high and low responders depending on whether their SR gene expression was above or below the median, showing a different inflammation response pattern. CD36 and LOX-1 gene expression correlated positively with IL-1beta; SR-A correlated negatively with IL-8 and positively with PPARgamma and NF-kappaBIotaA. These results were confirmed in the same subjects 3 mo after the first sampling. Furthermore, a negative correlation existed between CD36 and SR-A at protein level after 18 h of oxLDL incubation (R = -0.926, p = 0.024). These data would suggest that the type of SR could determine the macrophage activation: more proinflammatory when associated to CD36 and LOX-1 than when associated with SR-A.

Sobreexpresión génica de citocinas proinflamatorias en macrófagos de sujetos con hipercolesterolemia familiar y xantomas tendinosos / Proinflammatory cytokine gene overexpression in macrophages of patients with familial hypercholesterolemia and tendon xanthomas

Introducción y objetivo. Entre el 30 y el 50% de los sujetos con diagnóstico genético de hipercolesterolemia familiar (HF) heterocigota presentan xantomas tendinosos (XT), pero el mecanismo por el cual unos sujetos HF desarrollan XT y otros no se desconoce. Previamente, nuestro grupo de investigación ha demostrado que los macrófagos de sujetos HF con y sin XT desarrollan una respuesta inflamatoria diferente frente a lipoproteínas de baja densidad oxidadas (LDLox). Por ello, el objetivo de este trabajo fue analizar la expresión génica de diversas moléculas inflamatorias que podrían estar involucradas en la aparición y desarrollo de XT. Material y métodos. Se seleccionó a 10 pacientes con diagnóstico genético de HF, en los que se midió el diámetro anteroposterior del tendón de Aquiles mediante ecografía de alta resolución. Se aislaron sus monocitos a partir de 40 ml de sangre periférica. Una vez diferenciados a macrófagos, se suplementaron con 50 µg/ml de LDLox durante 1, 3, 6 y 18 h. Mediante RT-PCR en tiempo real, se analizó la expresión de los genes PPAR*, IL-8, IL-1ß, CXCL3, triptasa, NF-*BIA y TNF-*. Resultados y conclusión. Los sujetos HF con XT (HF XT+) mostraron una tendencia a sobreexpresar el gen IL-8 tras 18 h de incubación con LDLox, mientras que el grupo de sujetos HF sin XT (HF XT­) tendió a sobreexpresar el gen TNF-* tras 1 h de incubación con LDLox. El gen CXCL3 se sobreexpresó significativamente en todos los tiempos de incubación en el grupo HF XT+. Además, se halló una correlación positiva entre la expresión de CXCL3 y el tamaño del tendón de Aquiles, que fue máxima a 3 h del tratamiento con LDLox (R = 0,782; p = 0,008). Estos resultados sugieren que CXCL3 podría desempeñar un papel importante en la etiología de los xantomas, y se puede considerar como un posible marcador predictor de estos depósitos lipídicos (AU)

Introduction and objective. Approximately 30%-50% of patients with genetic diagnosis of heterozygous familial hypercholesterolemia (FH) present tendon xanthomas (TX), but the mechanism by which some subjects develop TX and others do not is unknown. Previously, we have shown that macrophages of FH subjects with and without TX develop a different inflammatory response to oxidized LDL (oxLDL). Therefore, the objective of this work was to analyze the gene expression of several inflammatory molecules that could be involved in the onset and development of TX. Material and methods. Ten FH patients were selected, and the antero-posterior Achilles tendon diameter was measured with high resolution sonography. Their monocytes were isolated from 40 ml of peripheral blood. When they were differentiated to macrophages, were supplemented with 50 µg/ml of oxLDL for 1, 3, 6 and 18 hours. The gene expression of PPAR*, IL-8, IL-1ß, CXCL3, tryptase, NF-*BIA and TNF-* was analyzed with real time RT-PCR. Results and conclusion. The FH subjects with TX (FH TX+) showed a tendency to over-express IL-8 gene after 18 h of incubation with oxLDL, while FH subjects without TX (FH TX­) tended to over-express TNF-* gene after 1 h of incubation. CXCL3 gene was significantly over-expressed at all incubation times with oxLDL in FH TX+ group. Furthermore, a positive correlation was found between CXCL3 gene expression and Achilles tendon size, being maximum at 3h of treatment with oxLDL (R = 0.782; p = 0.008). These results would suggest that CXCL3 could play an important role in the ethiology of xanthomas and could be considered as a possible predictor marker of these lipid deposits (AU)