RNY-derived small RNAs as a signature of coronary artery disease

International audienceBackgroundData from next generation sequencing technologies uncovered the existence of many classes of small RNAs. Recent studies reported that small RNAs are released by cells and can be detected in the blood. In this report, we aimed to discover the occurrence of novel circulating small RNAs in coronary artery disease (CAD).MethodsWe used high-throughput sequencing of small RNAs from human and mouse apoptotic primary macrophages, and analyzed the data by empirical Bayes moderated t-statistics to assess differential expression and the Benjamini and Hochberg method to control the false discovery rate. Results were then confirmed by Northern blot and RT-qPCR in foam cells and in two animal models for atherosclerosis, namely ApoE −/− and Ldlr −/− mouse lines. Quantitative RT-PCR to detect identified small RNAs, the RNY-derived small RNAs, was performed using sera of 263 patients with CAD compared to 514 matched healthy controls; the Student t-test was applied to statistically assess differences. Associations of small RNAs with clinical characteristics and biological markers were tested using Spearman’s rank correlations, while multivariate logistic regressions were performed to test the statistical association of small RNA levels with CAD.ResultsHere, we report that, in macrophages stimulated with pro-apoptotic or pro-atherogenic stimuli, the Ro-associated non-coding RNAs, called RNYs or Y-RNAs, are processed into small RNAs (~24–34 nt) referred to as small-RNYs (s-RNYs), including s-RNY1-5p processed from RNY1. A significant upregulation of s-RNY expression was found in aortic arches and blood plasma from ApoE −/− and Ldlr −/− mice and in serum from CAD patients (P <0.001). Biostatistical analysis revealed a positive association of s-RNY1-5p with hs-CRP and ApoB levels; however, no statistical interaction was found between either of these two markers and s-RNY1-5p in relation to the CAD status. Levels of s-RNY1-5p were also independent from statin and fibrate therapies.ConclusionOur results position the s-RNY1-5p as a relevant novel independent diagnostic biomarker for atherosclerosis-related diseases. Measurement of circulating s-RNY expression would be a valuable companion diagnostic to monitor foam cell apoptosis during atherosclerosis pathogenesis and to evaluate patient’s responsiveness to future therapeutic strategies aiming to attenuate apoptosis in foam cells in advanced atherosclerotic lesions

Serum levels of mitochondrial inhibitory factor 1 are independently associated with long-term prognosis in coronary artery disease: the GENES Study

Background Epidemiological and observational studies have established that high-density lipoprotein cholesterol (HDL-C) is an independent negative cardiovascular risk factor. However, simple measurement of HDL-C levels is no longer sufficient for cardiovascular risk assessment. Therefore, there is a critical need for novel non-invasive biomarkers that would display prognostic superiority over HDL-C. Cell surface ecto-F1-ATPase contributes to several athero-protective properties of HDL, including reverse cholesterol transport and vascular endothelial protection. Serum inhibitory factor 1 (IF1), an endogenous inhibitor of ecto-F1-ATPase, is an independent determinant of HDL-C associated with low risk of coronary artery disease (CAD). This work aimed to examine the predictive value of serum IF1 for long-term mortality in CAD patients. Its informative value was compared to that of HDL-C. Method Serum IF1 levels were measured in 577 male participants with stable CAD (age 45–74 years) from the GENES (Genetique et ENvironnement en Europe du Sud) study. Vital status was yearly assessed, with a median follow-up of 11 years and a 29.5 % mortality rate. Cardiovascular mortality accounted for the majority (62.4 %) of deaths. Results IF1 levels were positively correlated with HDL-C (rs = 0.40; P < 0.001) and negatively with triglycerides (rs = −0.21, P < 0.001) and CAD severity documented by the Gensini score (rs = −0.13; P < 0.01). Total and cardiovascular mortality were lower at the highest quartiles of IF1 (HR = 0.55; 95 % CI, 0.38–0.89 and 0.50 (0.28–0.89), respectively) but not according to HDL-C. Inverse associations of IF1 with mortality remained significant, after multivariate adjustments for classical cardiovascular risk factors (age, smoking, physical activity, waist circumference, HDL-C, dyslipidemia, hypertension, and diabetes) and for powerful biological and clinical variables of prognosis, including heart rate, ankle-brachial index and biomarkers of cardiac diseases. The 10-year mortality was 28.5 % in patients with low IF1 (<0.42 mg/L) and 21.4 % in those with high IF1 (≥0.42 mg/L, P < 0.02). Conclusions We investigated for the first time the relation between IF1 levels and long-term prognosis in CAD patients, and found an independent negative association. IF1 measurement might be used as a novel HDL-related biomarker to better stratify risk in populations at high risk or in the setting of pharmacotherapy

Régulation différentielle de la transcription des gènes murin et humain de l'apolipoprotéine A5 en réponse aux cytokines pro-inflammatoires par différents mécanismes impliquant respectivement NF-kB ou COUP-TFI (régulation transcriptionnelle du gène de l'APOA5 par RORa)

L'hypertriglycéridémie, considérée comme un facteur de risque indépendant de maladies cardiovasculaires, est souvent associée à des pathologies métaboliques et inflammatoires. L'apolipoprotéine A5 (APOA5), récemment découverte, exerce un rôle majeur dans l'homéostasie des triglycérides. De ce fait l'étude des facteurs régulant son expression est particulièrement intéressante. La première partie de nos travaux vise à déterminer le rôle de l'APOA5 dans l'inflammation en étudiant la régulation de son expression génique par les cytokines pro-inflammatoires et d'élucider le ou les mécanismes moléculaires impliqués. En utilisant des modèles cellulaires d'hépatomes humain et murin, nous avons initialement démontré que les cytokines pro-inflammatoire IL-1, TNFa et IL-6 régulent de manière opposée l'expression des gènes humain et murin de l'APOA5 in vitro selon différents mécanismes transcriptionnels faisant intervenir notamment les facteurs nucléaires NF- B p50 et COUP-TFI. Ensuite, des expériences réalisées sur des souris sauvages et transgéniques surexprimant le gène humain de l'APOA5, ont montré que ces cytokines modulent également l'expression des gènes humain et murin de l'APOA5 in vivo. Enfin, la mesure des concentrations en triglycérides, TNFa et apoAV plasmatiques, réalisée chez 32 enfants présentant le syndrome néphrotique idiopathique (11 en rechute et 21 en rémission), a permis de mettre en évidence une corrélation inverse entre la concentration en apoAV et les concentrations en triglycérides et TNFa chez ces patients. Cette observation suggère qu'une inhibition de l'expression du gène de l'APOA5 par le TNFa synthétisé en grande quantité chez ces patients pourrait contribuer à l'hypertriglycéridémie observée. Cette première étude a mis en évidence un nouveau mécanisme impliquant pour lapremière fois COUP-TFI dans la réponse inflammatoire. La deuxième partie de nos travaux a porté sur l'étude de la régulation du gène de l'APOA5 par les facteurs nucléaires ROR. RORa joue un rôle dans le métabolisme des lipide et l'inflammation. Nous avons d'abord démontré que seuls les facteurs RORa1 et RORa4 stimulent fortement l'activité transcriptionnelle du promoteur de l'APOA5 humaine in vitro, en se fixant directement sur un élément de réponse spécifique. Ensuite, l'infection de cellules humaines d'hépatomes par des adénovirus exprimant hRORa1 et hRORa4 a conduit à une augmentation de l'expression des ARNm de l'APOA5. En outre, nous avons montré que le gène murin de l'apoa5 n'est pas régulé par ROR. Cette partie de l'étude identifie un nouveau gène cible de RORa renforçant son rôle dans l'homéostasie des triglycérides.Hypertriglyceridemia, considered as an independent risk factor for coronary heart diseases, is often associated with metabolic and inflammatory disorders. The newly identified APOA5 is a crucial determinant of triglyceride levels and therefore the study of the factors regulating its gene expression is of significant importance. The first part of our studies aims at determining the rôle of APOA5 in inflammation by studying the regulation of its gene expression by pro-inflammatory cytokines and elucidate the molecular mechanisms involved. By using hepatic mouse and human cellular models, we initially demonstrated that the pro-inflammatory cytokines IL-1, TNFa and IL66 regulate in an opposite manner the expression of the mouse and human APOA5 genes in vitro, through different transcriptional mechanisms, involving in particular the nuclear factors NF- B and COUP-TFI. Then, experiments carried out in wild type mice and mice overexpressing human APOA5 showed that cytokines modulate mouse and human APOA5 gene expressions in vivo. Finally, the measurement of plasma triglyceride, TNFa and apoAV in 32 children with an idiopatic nephrotic syndrome ( 11 in relapse and 21 in remission) allowed to establish a negative correlation between apoAV plasma concentration and triglyceride and TNFa concentrations. This observation suggests that the inhibition of the APOA5 gene expression by the high level of TNFa in these patients could contribute to hypertriglyceridemia. This first study identifies a new mechanism involving for the first time COUP6TFI in the inflammatory response. The second part of our studies is focused on the gene regulation study of APOA5 by nuclear factors, ROR. RORa plays a rôle in lipid metabolism and inflammation. We initially demonstrated that only RORa1 and RORa4 strongly stimulate the transcriptional activity of the human APOA5 promoter in vitro through a direct binding on a specific response element. Furthermore, the infection of human hepatic cells with adenovirus expressing hRORa1 and hRORa4 induce an increase of APOA5 mRNA expression. Moreover, we demonstrated that the mouse apoa5 gene is not regulated by ROR. This part of the study identifies a new target gene for RORa strengthening its rôle in triglyceride homeostasis.LILLE2-BU Santé-Recherche (593502101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Is it time to reconcile HDL with cardiovascular diseases and beyond? An update on a paradigm shift

International audienceNo abstract availabl

Serum level of HDL particles are independently associated with long-term prognosis in patients with coronary artery disease: The GENES study

International audienceHDL-Cholesterol (HDL-C) is not an accurate surrogate marker to measure the cardioprotective functions of HDL in coronary artery diseases (CAD) patients. Hence, measurement of other HDL-related parameters may have prognostic superiority over HDL-C. In this work, we examined the predictive value of HDL particles profile for long-term mortality in CAD patients and to compare its informative value to that of HDL-C and apoA-I. HDL particles profiles were measured by nuclear magnetic resonance (NMR) spectroscopy in 214 male participants with stable CAD (45-74 years). Median follow up was 12.5 years with a 36.4% mortality rate. Cardiovascular mortality accounted for 64.5%. Mean concentrations of total HDL particles (HDL-P), small-sized HDL (SHDL-P) and apoA-I were lower in deceased than in surviving patients whereas no difference was observed according to HDL-C and large HDL particles. All NMR-HDL measures were correlated between themselves and with other HDL markers (HDL-C, apoA-I and LpA-I). In a multivariate model adjusted for cardiovascular risk factors and bioclinical variables, HDL-P and SHDL-P displayed the strongest inverse association with all-cause and cardiovascular mortality. Weaker associations were recorded for apoA-I. Based on our results, we conclude that HDL particle profile measured by NMR spectroscopy should be considered to better stratify risk in population at high risk or in the setting of pharmacotherapy

A reference measurement of circulating ATPase inhibitory factor 1 (IF1) in humans by LC-MS/MS: Comparison with conventional ELISA

International audienceATPase inhibitory factor 1 (IF1) is a 9.5 kDa protein that binds to mitochondrial and plasma membrane ATP synthase and selectively inhibits ATP hydrolysis. Recently, IF1 was identified in systemic circulation in humans. IF1 appeared as an independent determinant of HDL-cholesterol with lower levels in coronary heart disease (CHD) patients. Moreover, IF1 was also found to negatively associate with mortality in these patients, supporting the notion that circulating IF1 could be a promising biomarker of cardiovascular disease. However, in previous studies, IF1 was quantified by a non-standardized competitive enzyme-linked immunosorbent assay (ELISA). Herein, we have validated a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) enabling the accurate quantification of IF1 in human plasma. Plasma IF1 was trypsin-digested through an optimized procedure before LC-MS/MS analysis. The method was successfully validated over 4 independent experiments into the range of 100-1500 ng/mL. Intraand inter-assay variation coefficients had never exceeded 14.2% and accuracy ranged between 95% and 102% for the selected EAGGAFGK peptide marker. Subsequently, the results of the LC-MS/MS method were compared with those obtained using ELISA in 204 individuals from the GENES study. We found that IF1 plasma levels obtained using both techniques were strongly correlated (r = 0.89, p < 0.0001), while the Bland-Altman plot did not indicate any major statistically significant differences. To clinically validate LC-MS/MS, we confirmed the positive correlation between IF1 plasma levels and HDL-cholesterol (r = 0.38, p < 0.0001). Besides, we found lower IF1 plasma levels in CHD patients compared to controls (431 +/- 132 ng/mL and 555 +/- 173 ng/mL, respectively; p < 0.0001). Hence, it can be concluded that the presented LC-MS/MS analytical method provides a highly specific strategy for IF1 quantification in human plasma and could be proposed as a reference method

Apolipoprotein A5 is an inflammatory responsive gene down-regulated by tumor necrosis factor alpha and interleukin-1

Several epidemiological studies have established that elevated plasma triglyceride concentrations constitute an independent risk factor for cardiovascular diseases. In addition, systemic inflammation is associated with severe hypertriglyceridemia and previous studies have demonstrated that cytokines such as tumor necrosis factor alpha (TNFalpha;) and interleukin-1 (IL-1) can elevate plasma triglyceride levels. Recently, we identified a new apolipoprotein, APOA5, selectively expressed in the liver and showed that this gene is a crucial determinant of plasma triglyceride levels. In this study, we sought to determine whether inflammatory cytokines regulate APOA5 and consequently influence plasma triglyceride levels. We found initially that treatment of human hepatocytes with TNFalpha; or IL-1 reduced the expression of APOA5 mRNA. Subsequent, we demonstrated through transient transfection experiments that both TNFalpha; and IL-1 down-regulate human APOA5 at the transcriptional level. Further deletion analyses of the APOA5 promoter and binding assays revealed the presence of a promoter sequence, containing a PPARalpha; Response Element, responsive to cytokine stimulation. In vivo, treatment of hAPOA5 transgenic mice with TNFalpha; down-regulated the hAPOA5 gene expression in hepatocytes. In patients displaying systemic inflammation, plasma concentrations of triglycerides and ApoAV were inversely correlated. These findings demonstrate that APOA5 is an inflammatory responsive gene and constitutes a link between inflammation and triglyceride-associated cardiovascular risk

Transcriptional Regulation of Apolipoprotein A5 Gene Expression by theNuclear Receptor ROR alpha

Apolipoprotein A5 has recently been identified as a crucial determinant of plasma triglyceride levels. Our results showed that RORa up-regulates human APOA5 but has no effect on mouse apoa5 promoter. These data suggest an additional important physiological role for RORa in the regulation of genes involved in plasma triglyceride homeostasis in human and probably in the development of atherosclerosi