Molecular diagnosis of polycystic ovary syndrome in obese and non-obese women by targeted plasma miRNA profiling.

OBJECTIVE: Polycystic ovary syndrome (PCOS) is diagnosed based on the clinical signs, but its presentation is heterogeneous and potentially confounded by concurrent conditions, such as obesity and insulin resistance. miRNA have recently emerged as putative pathophysiological and diagnostic factors in PCOS. However, no reliable miRNA-based method for molecular diagnosis of PCOS has been reported. The aim of this study was to develop a tool for accurate diagnosis of PCOS by targeted miRNA profiling of plasma samples, defined on the basis of unbiased biomarker-finding analyses and biostatistical tools. METHODS: A case-control PCOS cohort was cross-sectionally studied, including 170 women classified into four groups: non-PCOS/lean, non-PCOS/obese, PCOS/lean, and PCOS/obese women. High-throughput miRNA analyses were performed in plasma, using NanoString technology and a 800 human miRNA panel, followed by targeted quantitative real-timePCR validation. Statistics were applied to define optimal normalization methods, identify deregulated biomarker miRNAs, and build classification algorithms, considering PCOS and obesity as major categories. RESULTS: The geometric mean of circulating hsa-miR-103a-3p, hsa-miR-125a-5p, and hsa-miR-1976, selected among 125 unchanged miRNAs, was defined as optimal reference for internal normalization (named mR3-method). Ten miRNAs were identified and validated after mR3-normalization as differentially expressed across the groups. Multinomial least absolute shrinkage and selection operator regression and decision-tree models were built to reliably discriminate PCOS vs non-PCOS, either in obese or non-obese women, using subsets of these miRNAs as performers. CONCLUSIONS: We define herein a robust method for molecular classification of PCOS based on unbiased identification of miRNA biomarkers and decision-tree protocols. This method allows not only reliable diagnosis of non-obese women with PCOS but also discrimination between PCOS and obesity. CAPSULE: We define a novel protocol, based on plasma miRNA profiling, for molecular diagnosis of PCOS. This tool not only allows proper discrimination of the condition in non-obese women but also permits distinction between PCOS and obesity, which often display overlapping clinical presentations.

Extra-Virgin Olive Oil Modifies the Changes Induced in Non-Nervous Organs and Tissues by Experimental Autoimmune Encephalomyelitis Models.

This study reveals the existence of oxidative stress (reactive oxygen species (ROS)) in non-nervous organs and tissues in multiple sclerosis (MS) by means of a model of experimental autoimmune encephalomyelitis (EAE) in rats. This model reproduces a similar situation to MS, as well as its relationship with intestinal microbiota starting from the changes in bacterial lipopolysaccharide levels (LPS) in the outer wall of the gram-negative bacteria. Finally, the administration of extra-virgin olive oil (EVOO), hydroxytirosol (HT), and oleic acid (OA) exert beneficial effects. Twenty-five Dark Agouti two-month-old male rats, weighing around 190 g, were distributed into the following groups: Control, EAE (experimental autoimmune encephalomyelitis group), EAE + EVOO, EAE + HT, and EAE + OA. The glutathione redox system with the EAE was measured in heart, kidney, liver, and small and large intestines. The LPS and the correlation with oxidative stress in the small and large intestines were also investigated. The results showed that (1) the oxidative damage in the EAE model affects non-nervous organs and tissues; (2) The LPS is related to inflammatory phenomena and oxidative stress in the intestinal tissue and in other organs; (3) The administration of EVOO, HT, and OA reduces the LPS levels at the same time as minimizing the oxidative damage; (4) EVOO, HT, and OA improve the disease's clinical score; and (5) on balance, EVOO offers a better neuroprotective effect.

The Mediterranean and CHO diets decrease VCAM-1 and E-selectin expression induced by modified low-density lipoprotein in HUVECs.

BACKGROUND AND AIM: The oxidative modifications of low-density lipoprotein (LDL) are crucial for the atherosclerosis process. The aim of this study was to determine if the minimally modified LDL, obtained after the ingestion of three different diets, produce differential effects on the vascular cell adhesion molecule-1 (VCAM-1) and E-selectin expression in human umbilical endothelial cells (HUVECs). METHODS AND RESULTS: Twenty healthy young males were exposed to three dietary periods. Each period lasted four weeks. During the first period, all subjects consumed a saturated fat (SFA) enriched diet (38% fat, 20% SFA). The second and third dietary periods were administered following a randomized crossover design: a low fat high carbohydrates diet (CHO diet) and a Mediterranean diet. LDL particles, isolated during each dietary period, were oxidized by exposure to UV light and incubated for 48 h with HUVEC. Thereafter, 100 U/mL of TNF-alpha was added and incubation continued for 6 h. Cellular ELISA determined adhesion molecules expression. Lag time, propagation rate and total amounts of formed conjugated dienes were calculated in LDL incubated with 10mumol/L Cu(2+). When compared to the SFA diet, LDL isolated from the Mediterranean and CHO diets induced a lower expression of VCAM-1 and E-selectin in HUVECS (P<0.007). There were no differences between both lipid lowering diets. However, lag time of LDL from the Mediterranean diet was higher than with the CHO diet (P<0.042). This parameter was inversely correlated with E-selectin expression (r=-0.497; P<0.04). CONCLUSION: Our results suggest that both the Mediterranean and CHO diets may decrease the pro-inflammatory environment induced by modified LDL in endothelial cells.

The influence of lipoprotein lipase gene variation on postprandial lipoprotein metabolism.

Lipoprotein lipase (LPL) is one of the key enzymes in the metabolism of triacylglycerol-rich lipoproteins (TRL). We evaluated whether the association of LPL HindIII (H1/H2) and Serine447-Stop (S447X) polymorphisms may explain the interindividual variability observed during postprandial lipemia. Fifty-one healthy male volunteers (26 with the H2S447 genotype, 15 with the H1X447 genotype, and 10 with the H1S447 genotype) were subjected to a vitamin A-fat load test consisting of 1 g fat/kg body weight and 60,000 IU vitamin A. Blood was drawn every hour until the 6th hour and every 2 h and 30 min until the 11th hour. Data revealed that subjects that are homozygous for the H2 allele (H2H2) showed a higher postprandial response for small TRL, retinyl palmitate (RP), large TRL-RP, large TRL-B48, and small TRL-B48 levels. Furthermore, in the case of the S447X polymorphism, 447Ter carriers had a lower postprandial response for small TRL-RP, large TRL-B48, and small TRL-RP. Subjects with the LPL H2S447 genotype had higher plasma triacylglycerol, large TRL-triacylglycerol, large TRL-RP, small TRL-RP, and large TRL-B48 (P < 0.037) than H1X447 subjects. The modifications observed in postprandial lipoprotein metabolism in young normolipemic males with LPL polymorphism could be involved in the lower risk of coronary artery disease associated with the H1X447 genotype.

Effects of the human apolipoprotein A-I promoter G-A mutation on postprandial lipoprotein metabolism.

BACKGROUND: There is considerable interindividual variability in the postprandial lipid response to a fat-rich meal, and genetic factors have been considered to account for some of these effects. We previously showed that the G-A mutation 5' to the apolipoprotein (apo) A-I gene was significantly associated with the LDL-cholesterol response to diet. OBJECTIVE: We evaluated whether this effect is mediated by mechanisms involving postprandial lipoprotein metabolism. DESIGN: Twenty-eight G/G and 23 G/A healthy male subjects, homozygotes for the apo E3 allele, were subjected to a vitamin A fat-loading test. Blood was drawn at time 0 and every hour for 11 h. RESULTS: There was a significant postprandial decrease in plasma cholesterol, LDL cholesterol, and apo B in G/G subjects but not in G/A subjects. A greater postprandial response in large triacylglycerol-rich lipoproteins (TRLs) and a smaller postprandial response in large TRL apo A-IV was observed in G/A than in G/G subjects. Retinyl palmitate in large and small TRL concentrations was similar for both genotypes. No significant genotype effects were detected for triacylglycerol concentrations in plasma, small TRL fraction, and apo A-I and HDL-cholesterol concentrations. CONCLUSION: Our data suggest that the G-A mutation affects the LDL-cholesterol response to diet by mechanisms involving postprandial lipoprotein cholesterol metabolism.

Effect of simvastatin in familial hypercholesterolemia on the affinity of electronegative low-density lipoprotein subfractions to the low-density lipoprotein receptor.

The effect of simvastatin therapy on the biologic characteristics of the electronegative low-density lipoprotein (LDL) subfraction of patients with familial hypercholesterolemia (FH) was studied. Total LDL, isolated from FH plasma at 0, 3 and 6 months of simvastatin treatment, was subfractionated into electropositive LDL (LDL[+]) and electronegative LDL (LDL[-]) by anion exchange chromatography. LDL isolated from healthy normolipemic (NL) subjects was used as a control. The LDL(-) proportion was twofold higher in patients with FH than in NL subjects (17.6 +/- 1.6% vs 7.8 +/- 1.5%, respectively; p <0.05) and was progressively reduced by simvastatin therapy (15.7 +/- 1.6% at 3 months; 13.8 +/- 2.5% at 6 months; p <0.05). Both LDL subfractions from patients with FH had a higher relative cholesterol content and decreased apolipoprotein B and triglycerides than NL subfractions. Simvastatin progressively induced changes in lipid content of both LDL subfractions in patients with FH, and lipid composition was closer to these subfractions in NL subjects after 6 months of therapy. Binding displacement experiments in human fibroblasts demonstrated that LDL(-) from both groups of subjects had a lower affinity of binding to the LDL receptor that LDL(+). In addition, LDL(+) in patients with FH presented an intermediate binding affinity between LDL(-) and LDL(+) in NL subjects. Simvastatin-induced changes in LDL composition were accompanied by a progressive increase in affinity of LDL(+) and LDL(-) in patients with FH. After 6 months of therapy, LDL(+) in FH had an affinity similar to that of LDL(+) in NL subjects. The LDL(-)-induced release of chemokines interleukin-8 and monocyte chemotactic protein-1 from cultured endothelial cells was twofold higher compared with that of LDL(+). No difference in chemokine release between patients with FH and NL subjects or the effect of simvastatin were observed. We conclude that simvastatin therapy was able to modify LDL subfraction composition in subjects with FH and increase their affinity to the LDL receptor. This improvement could contribute to the observed reduction in LDL(-) proportion induced by simvastatin.

Effects of virgin olive oil phenolic compounds on LDL oxidation and vasorelaxation activity.

This study examined the efficacy of virgin olive oil phenolic extract and other phenolic compounds (oleuropein, caffeic acid) in preventing oxidative modifications of human low density lipoprotein oxidised by CuCl2. The vasorelaxant effect of these compounds on rat aortic ring with and without functional endothelium is also discussed. Olive oil phenolic extract, caffeic acid and oleuropein increased the lag time of conjugated diene formation in a concentration-dependent manner. Moreover, phenolic extract produced a vasorelaxant effect that persisted in denuded aorta and after inhibition of nitric oxide synthase by NG-methyl-L-arginine (L-NMMA) or methylene blue. Oleuropein did not produce a relaxant effect, whereas caffeic acid produced partial relaxation at concentration 0.5 g/L.

La dieta mediterránea mejora la resistencia a la oxidación de las lipoproteínas de baja densidad / Mediterranean diet improves low density lipoprotein susceptibility to oxidative modifications

Fundamento: Numerosos paneles de expertos, en especial de países anglosajones, recomiendan la dieta pobre en grasa para la prevención de enfermedades cardiovasculares. Sin embargo, la tasa de muerte por cardiopatía isquémica es baja en los países del área mediterránea, lo que puede ser debido al alto porcentaje de grasa monoinsaturada proporcionada por el aceite de oliva en la dieta. Por ello hemos comparado el efecto de ambas dietas sobre la susceptibilidad in vitro a la oxidación de las lipoproteínas de baja densidad (LDL), pieza clave en el inicio y desarrollo de la arteriosclerosis. Sujetos y métodos: Cuarenta y un sujetos varones sanos normolipémicos fueron sometidos a tres períodos de dieta, de 4 semanas de duración cada uno, consistentes en una dieta rica en grasa saturada (SAT: 38 por ciento grasa, 20 por ciento saturada), otra pobre en grasa (NCEP-I: 28 por ciento grasa, 10 por ciento saturada) y una dieta medi-terránea (38 por ciento grasa, 22 por ciento de grasa monoinsaturada). Al final de cada período dietético se determinaron las concentraciones plasmáticas de colesterol total, cLDL, cHDL, triglicéridos, apoproteínas A-I y B, *-tocoferol y la susceptibilidad a la oxidación de las LDL in vitro. Resultados: Ambas dietas hipolipemiantes produjeron un descenso significativo de las concentraciones plasmáticas de colesterol total, cLDL y apo B, mientras que sólo la dieta NCEP-I disminuyó el cHDL. La sustitución de una dieta rica en grasa saturada o de una dieta rica en hidratos de carbono por una dieta mediterránea aumentó la resistencia a la oxidación de las LDL al prolongarse el tiempo de latencia (p < 0,038) e inducir un descenso (p < 0,001) en la tasa de progresión de la curva de cinética de oxidación de las LDL. Conclusión: Nuestros resultados indican que el consumo de una dieta mediterránea rica en aceite de oliva, además de mejorar el índice aterogénico (colesterol total/cHDL), aumenta la resistencia a la oxidación de las LDL en comparación con la dieta pobre en grasa. Ello nos hace aconsejar el modelo de dieta mediterránea para la prevención de las enfermedades cardiovasculares. (AU)