Association of Circulating Autophagy Proteins ATG5 and Beclin 1 with Acute Myocardial Infarction in a Case-Control Study.

INTRODUCTION: Acute myocardial infarction (AMI) is a main contributor of sudden cardiac death worldwide. The discovery of new biomarkers that can improve AMI risk prediction meets a major clinical need for the identification of high-risk patients and the tailoring of medical treatment. Previously, we reported that autophagy a highly conserved catabolic mechanism for intracellular degradation of cellular components is involved in atherosclerotic plaque phenotype and cardiac pathological remodeling. The crucial role of autophagy in the normal and diseased heart has been well described, and its activation functions as a pro-survival process in response to myocardial ischemia. However, autophagy is dysregulated in ischemia/reperfusion injury, thus promoting necrotic or apoptotic cardiac cell death. Very few studies have focused on the plasma levels of autophagy markers in cardiovascular disease patients, even though they could be companion biomarkers of AMI injury. The aims of the present study were to evaluate (1) whether variations in plasma levels of two key autophagy regulators autophagy-related gene 5 (ATG5) and Beclin 1 (the mammalian yeast ortholog Atg6/Vps30) are associated with AMI and (2) their potential for predicting AMI risk. METHODS: The case-control study population included AMI patients (n = 100) and control subjects (n = 99) at high cardiovascular risk but without known coronary disease. Plasma levels of ATG5 and Beclin 1 were measured in the whole population study by enzyme-linked immunosorbent assay. RESULTS: Multivariate analyses adjusted on common cardiovascular factors and medical treatments, and receiver operating characteristic curves demonstrated that ATG5 and Beclin 1 levels were inversely associated with AMI and provided original biomarkers for AMI risk prediction. CONCLUSION: Plasma levels of autophagy regulators ATG5 and Beclin 1 represent relevant candidate biomarkers associated with AMI.

Genetic inhibition of CARD9 accelerates the development of atherosclerosis in mice through CD36 dependent-defective autophagy.

Caspase recruitment-domain containing protein 9 (CARD9) is a key signaling pathway in macrophages but its role in atherosclerosis is still poorly understood. Global deletion of Card9 in Apoe-/- mice as well as hematopoietic deletion in Ldlr-/- mice increases atherosclerosis. The acceleration of atherosclerosis is also observed in Apoe-/-Rag2-/-Card9-/- mice, ruling out a role for the adaptive immune system in the vascular phenotype of Card9 deficient mice. Card9 deficiency alters macrophage phenotype through CD36 overexpression with increased IL-1ß production, increased lipid uptake, higher cell death susceptibility and defective autophagy. Rapamycin or metformin, two autophagy inducers, abolish intracellular lipid overload, restore macrophage survival and autophagy flux in vitro and finally abolish the pro-atherogenic effects of Card9 deficiency in vivo. Transcriptomic analysis of human CARD9-deficient monocytes confirms the pathogenic signature identified in murine models. In summary, CARD9 is a key protective pathway in atherosclerosis, modulating macrophage CD36-dependent inflammatory responses, lipid uptake and autophagy.

Plasma levels of autophagy regulator Rubicon are inversely associated with acute coronary syndrome.

Background: The discovery of novel biomarkers that improve current cardiovascular risk prediction models of acute coronary syndrome (ACS) is needed for the identification of very high-risk patients and therapeutic decision-making. Autophagy is a highly conserved catabolic mechanism for intracellular degradation of cellular components through lysosomes. The autophagy process helps maintain cardiac homeostasis and dysregulated autophagy has been described in cardiovascular conditions. Rubicon (Run domain Beclin-1-interacting and cysteine-rich domain-containing protein) is a key regulator of autophagy with a potential role in cardiac stress. Objectives: The aims of the present study were to assess whether changes in circulating Rubicon levels are associated with ACS and to evaluate the added value of Rubicon to a clinical predictive risk model. Methods and results: The study population included ACS patients (n = 100) and control subjects (n = 99) at high to very high cardiovascular risk but without known coronary event. Plasma Rubicon levels were measured in the whole study population by enzyme-linked immunosorbent assay. Multivariate logistic regression analyses established that Rubicon levels were inversely associated with ACS. A receiver operating characteristic curve analysis demonstrated that the addition of Rubicon improved the predictive performance of the model with an increased area under the curve from 0.868 to 0.896 (p = 0.038). Conclusions: Plasma levels of the autophagy regulator Rubicon are associated with ACS and provide added value to classical risk markers for ACS.

MiR-223 and MiR-186 Are Associated with Long-Term Mortality after Myocardial Infarction.

Background-The identification and stratification of patients at risk of fatal outcomes after myocardial infarction (MI) is of considerable interest to guide secondary prevention therapies. Currently, no accurate biomarkers are available to identify subjects who are at risk of suffering acute manifestations of coronary heart disease as well as to predict adverse events after MI. Non-coding circulating microRNAs (miRNAs) have been proposed as novel diagnostic and prognostic biomarkers in cardiovascular diseases. The aims of the study were to investigate the clinical value of a panel of circulating miRNAs as accurate biomarkers associated with MI and mortality risk prediction in patients with documented MI. Methods and Results-seven circulating plasma miRNAs were analyzed in 67 MI patients and 80 control subjects at a high cardiovascular risk but without known coronary diseases. Multivariate logistic regression analyses demonstrated that six miRNAs were independently associated with MI occurrence. Among them, miR-223 and miR-186 reliably predicted long-term mortality in MI patients, in particular miR-223 (HR 1.57 per one-unit increase, p = 0.02), after left ventricular ejection fraction (LVEF) adjustment. Kaplan-Meier survival analyses provided a predictive threshold value of miR-223 expression (p = 0.028) for long-term mortality. Conclusions-Circulating miR-223 and miR-186 are promising predictive biomarkers for long-term mortality after MI.

Metabolic and cardiovascular adaptations to an 8-wk lifestyle weight loss intervention in younger and older obese men.

The number of older obese adults is increasing worldwide. Whether obese adults show similar health benefits in response to lifestyle interventions at different ages is unknown. The study enrolled 25 obese men (body mass index: 31-39 kg/m2) in two arms according to age (30-40 and 60-70 yr old). Participants underwent an 8-wk intervention with moderate calorie restriction (∼20% below individual energy requirements) and supervised endurance training resulting in ∼5% weight loss. Body composition was measured using dual energy X-ray absorptiometry. Insulin sensitivity was assessed during a hypersinsulinemic-euglycemic clamp. Cardiometabolic profile was derived from blood parameters. Subcutaneous fat and vastus lateralis muscle biopsies were used for ex vivo analyses. Two-way repeated-measure ANOVA and linear mixed models were used to evaluate the response to lifestyle intervention and comparison between the two groups. Fat mass was decreased and bone mass was preserved in the two groups after intervention. Muscle mass decreased significantly in older obese men. Cardiovascular risk (Framingham risk score, plasma triglyceride, and cholesterol) and insulin sensitivity were greatly improved to a similar extent in the two age groups after intervention. Changes in adipose tissue and skeletal muscle transcriptomes were marginal. Analysis of the differential response to the lifestyle intervention showed tenuous differences between age groups. These data suggest that lifestyle intervention combining calorie restriction and exercise shows similar beneficial effects on cardiometabolic risk and insulin sensitivity in younger and older obese men. However, attention must be paid to potential loss of muscle mass in response to weight loss in older obese men.NEW & NOTEWORTHY Rise in obesity and aging worldwide are major trends of critical importance in public health. This study addresses a current challenge in obesity management. Do older obese adults respond differently to a lifestyle intervention composed of moderate calorie restriction and supervised physical activity than younger ones? The main conclusion of the study is that older and younger obese men similarly benefit from the intervention in terms of cardiometabolic risk.

Effects of proprotein convertase subtilisin kexin type 9 modulation in human pancreatic beta cells function.

BACKGROUND AND AIMS: Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) is an endogenous inhibitor of the LDL receptor (LDLR). Mendelian randomization studies suggest that PCSK9 deficiency increases diabetes risk, but the underlying mechanisms remain unknown. The aim of our study was to investigate whether PCSK9 or its inhibition may modulate beta cell function. METHODS: We assessed PCSK9 and insulin colocalization in human pancreatic sections by epifluorescent and confocal microscopy. We also investigated the expression and the function of PCSK9 in the human EndoC-ßH1 beta cell line, by ELISA and flow cytometry, respectively. PCSK9 was inhibited with Alirocumab or siRNA. LDLR expression and LDL uptake were assessed by flow cytometry. RESULTS: PCSK9 was expressed and secreted from beta cells isolated from human pancreas as well as from EndoC-ßH1 cells. PCSK9 secretion was enhanced by statin treatment. Recombinant PCSK9 decreased LDLR abundance at the surface of these cells, an effect abrogated by Alirocumab. Alirocumab as well as PCSK9 silencing increased LDLR expression at the surface of EndoC-ßH1 cells. Neither exogenous PCSK9, nor Alirocumab, nor PCSK9 silencing significantly altered glucose-stimulated insulin secretion (GSIS) from these cells. High-low density lipoproteins (LDL) concentrations decreased GSIS, but the addition of PCSK9 or its inhibition did not modulate this phenomenon. CONCLUSIONS: While PCSK9 regulates LDLR abundance in beta cells, inhibition of exogenous or endogenous PCSK9 does not appear to significantly impact insulin secretion. This is reassuring for the safety of PCSK9 inhibitors in terms of beta cell function.

Antioxidant and Cytoprotective Properties of Polyphenol-Rich Extracts from Antirhea borbonica and Doratoxylon apetalum against Atherogenic Lipids in Human Endothelial Cells.

The endothelial integrity is the cornerstone of the atherogenic process. Low-density lipoprotein (LDL) oxidation occurring within atheromatous plaques leads to deleterious vascular effects including endothelial cell cytotoxicity. The aim of this study was to evaluate the vascular antioxidant and cytoprotective effects of polyphenol-rich extracts from two medicinal plants from the Reunion Island: Antirhea borbonica (A. borbonica), Doratoxylon apetalum (D. apetalum). The polyphenol-rich extracts were obtained after dissolving each dry plant powder in an aqueous acetonic solution. Quantification of polyphenol content was achieved by the Folin-Ciocalteu assay and total phenol content was expressed as g gallic acid equivalent/100 g plant powder (GAE). Human vascular endothelial cells were incubated with increasing concentrations of polyphenols (1-50 µM GAE) before stimulation with oxidized low-density lipoproteins (oxLDLs). LDL oxidation was assessed by quantification of hydroperoxides and thiobarbituric acid reactive substances (TBARS). Intracellular oxidative stress and antioxidant activity (catalase and superoxide dismutase) were measured after stimulation with oxLDLs. Cell viability and apoptosis were quantified using different assays (MTT, Annexin V staining, cytochrome C release, caspase 3 activation and TUNEL test). A. borbonica and D. apetalum displayed high levels of polyphenols and limited LDL oxidation as well as oxLDL-induced intracellular oxidative stress in endothelial cells. Polyphenol extracts of A. borbonica and D. apetalum exerted a protective effect against oxLDL-induced cell apoptosis in a dose-dependent manner (10, 25, and 50 µM GAE) similar to that observed for curcumin, used as positive control. All together, these results showed significant antioxidant and antiapoptotic properties for two plants of the Reunion Island pharmacopeia, A. borbonica and D. apetalum, suggesting their therapeutic potential to prevent cardiovascular diseases by limiting LDL oxidation and protecting the endothelium.

Identification of a miRNA Based-Signature Associated with Acute Coronary Syndrome: Evidence from the FLORINF Study.

BACKGROUND: The discovery of novel biomarkers that improve risk prediction models of acute coronary syndrome (ACS) is needed to better identify and stratify very high-risk patients. MicroRNAs (miRNAs) are essential non-coding modulators of gene expression. Circulating miRNAs recently emerged as important regulators and fine-tuners of physiological and pathological cardiovascular processes; therefore, specific miRNAs expression profiles may represent new risk biomarkers. The aims of the present study were: i) to assess the changes in circulating miRNAs levels associated with ACS and ii) to evaluate the incremental value of adding circulating miRNAs to a clinical predictive risk model. METHODS AND RESULTS: The study population included ACS patients (n = 99) and control subjects (n = 103) at high to very high cardiovascular risk but without known coronary event. Based on a miRNA profiling in a matched derivation case (n = -6) control (n = 6) cohort, 21 miRNAs were selected for validation. Comparing ACS cases versus controls, seven miRNAs were significantly differentially expressed. Multivariate logistic regression analyses demonstrated that among the seven miRNAs tested, five were independently associated with the occurrence of ACS. A receiver operating characteristic curve analysis revealed that the addition of miR-122 + miR-150 + miR-195 + miR-16 to the clinical model provided the best performance with an increased area under the curve (AUC) from 0.882 to 0.924 (95% CI 0.885-0.933, p = 0.003). CONCLUSIONS: Our study identified a powerful signature of circulating miRNAs providing additive value to traditional risk markers for ACS.

Mitochondrial 4-HNE derived from MAO-A promotes mitoCa2+ overload in chronic postischemic cardiac remodeling.

Chronic remodeling postmyocardial infarction consists in various maladaptive changes including interstitial fibrosis, cardiomyocyte death and mitochondrial dysfunction that lead to heart failure (HF). Reactive aldehydes such as 4-hydroxynonenal (4-HNE) are critical mediators of mitochondrial dysfunction but the sources of mitochondrial 4-HNE in cardiac diseases together with its mechanisms of action remain poorly understood. Here, we evaluated whether the mitochondrial enzyme monoamine oxidase-A (MAO-A), which generates H2O2 as a by-product of catecholamine metabolism, is a source of deleterious 4-HNE in HF. We found that MAO-A activation increased mitochondrial ROS and promoted local 4-HNE production inside the mitochondria through cardiolipin peroxidation in primary cardiomyocytes. Deleterious effects of MAO-A/4-HNE on cardiac dysfunction were prevented by activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2), the main enzyme for 4-HNE metabolism. Mechanistically, MAO-A-derived 4-HNE bound to newly identified targets VDAC and MCU to promote ER-mitochondria contact sites and MCU higher-order complex formation. The resulting mitochondrial Ca2+ accumulation participated in mitochondrial respiratory dysfunction and loss of membrane potential, as shown with the protective effects of the MCU inhibitor, RU360. Most interestingly, these findings were recapitulated in a chronic model of ischemic remodeling where pharmacological or genetic inhibition of MAO-A protected the mice from 4-HNE accumulation, MCU oligomer formation and Ca2+ overload, thus mitigating ventricular dysfunction. To our knowledge, these are the first evidences linking MAO-A activation to mitoCa2+ mishandling through local 4-HNE production, contributing to energetic failure and postischemic remodeling.

The Multifunctional Sorting Protein PACS-2 Controls Mitophagosome Formation in Human Vascular Smooth Muscle Cells through Mitochondria-ER Contact Sites.

Mitochondria-associated ER membranes (MAMs) are crucial for lipid transport and synthesis, calcium exchange, and mitochondrial functions, and they also act as signaling platforms. These contact sites also play a critical role in the decision between autophagy and apoptosis with far reaching implications for cell fate. Vascular smooth muscle cell (VSMC) apoptosis accelerates atherogenesis and the progression of advanced lesions, leading to atherosclerotic plaque vulnerability and medial degeneration. Though the successful autophagy of damaged mitochondria promotes VSMC survival against pro-apoptotic atherogenic stressors, it is unknown whether MAMs are involved in VSMC mitophagy processes. Here, we investigated the role of the multifunctional MAM protein phosphofurin acidic cluster sorting protein 2 (PACS-2) in regulating VSMC survival following a challenge by atherogenic lipids. Using high-resolution confocal microscopy and proximity ligation assays, we found an increase in MAM contacts as in PACS-2-associated MAMs upon stimulation with atherogenic lipids. Correspondingly, the disruption of MAM contacts by PACS-2 knockdown impaired mitophagosome formation and mitophagy, thus potentiating VSMC apoptosis. In conclusion, our data shed new light on the significance of the MAM modulatory protein PACS-2 in vascular cell physiopathology and suggest MAMs may be a new target to modulate VSMC fate and favor atherosclerotic plaque stability.

Altered mitochondrial quality control in Atg7-deficient VSMCs promotes enhanced apoptosis and is linked to unstable atherosclerotic plaque phenotype.

Vascular smooth muscle cells (VSMCs) are one of the main cellular determinants in arterial pathology. A large body of evidence indicates that death of VSMCs is associated with features of high-risk/vulnerable atherosclerotic plaques. Mitochondrial turnover is an essential aspect of the mitochondrial quality control in which dysfunctional mitochondria are selectively eliminated through autophagy and replaced through expansion of preexisting mitochondria. Even though successful autophagy promotes VSMC survival, it is unclear whether reduced autophagic flux affects mitochondrial quality control of VSMCs in atherosclerotic plaques. By using apolipoprotein E-deficient (ApoE-/-) mice carrying a VSMC-specific deletion of the essential autophagy gene Atg7, we show in the present study that impaired VSMC autophagy promotes an unstable plaque phenotype, as well as the accumulation of fragmented mitochondria with reduced bioenergetic efficiency and more oxidative stress. Furthermore, we demonstrate that disrupted autophagic flux is linked to defective mitophagy and biogenesis of mitochondria, which exacerbate VSMC apoptosis and in turn plaque vulnerability. Overall, our data indicate that mitochondrial quality control is a promising therapeutic target to stabilize atherosclerotic plaques.

Identifying the anti-inflammatory response to lipid lowering therapy: a position paper from the working group on atherosclerosis and vascular biology of the European Society of Cardiology.

Dysregulated lipid metabolism induces an inflammatory and immune response leading to atherosclerosis. Conversely, inflammation may alter lipid metabolism. Recent treatment strategies in secondary prevention of atherosclerosis support beneficial effects of both anti-inflammatory and lipid-lowering therapies beyond current targets. There is a controversy about the possibility that anti-inflammatory effects of lipid-lowering therapy may be either independent or not of a decrease in low-density lipoprotein cholesterol. In this Position Paper, we critically interpret and integrate the results obtained in both experimental and clinical studies on anti-inflammatory actions of lipid-lowering therapy and the mechanisms involved. We highlight that: (i) besides decreasing cholesterol through different mechanisms, most lipid-lowering therapies share anti-inflammatory and immunomodulatory properties, and the anti-inflammatory response to lipid-lowering may be relevant to predict the effect of treatment, (ii) using surrogates for both lipid metabolism and inflammation as biomarkers or vascular inflammation imaging in future studies may contribute to a better understanding of the relative importance of different mechanisms of action, and (iii) comparative studies of further lipid lowering, anti-inflammation and a combination of both are crucial to identify effects that are specific or shared for each treatment strategy.

Autophagy in Metabolic Age-Related Human Diseases.

Autophagy is a highly conserved homeostatic cellular mechanism that mediates the degradation of damaged organelles, protein aggregates, and invading pathogens through a lysosome-dependent pathway. Over the last few years, specific functions of autophagy have been discovered in many tissues and organs; however, abnormal upregulation or downregulation of autophagy has been depicted as an attribute of a variety of pathologic conditions. In this review, we will describe the current knowledge on the role of autophagy, from its regulation to its physiological influence, in metabolic age-related disorders. Finally, we propose to discuss the therapeutic potential of pharmacological and nutritional modulators of autophagy to treat metabolic diseases.

Future directions for therapeutic strategies in post-ischaemic vascularization: a position paper from European Society of Cardiology Working Group on Atherosclerosis and Vascular Biology.

Modulation of vessel growth holds great promise for treatment of cardiovascular disease. Strategies to promote vascularization can potentially restore function in ischaemic tissues. On the other hand, plaque neovascularization has been shown to associate with vulnerable plaque phenotypes and adverse events. The current lack of clinical success in regulating vascularization illustrates the complexity of the vascularization process, which involves a delicate balance between pro- and anti-angiogenic regulators and effectors. This is compounded by limitations in the models used to study vascularization that do not reflect the eventual clinical target population. Nevertheless, there is a large body of evidence that validate the importance of angiogenesis as a therapeutic concept. The overall aim of this Position Paper of the ESC Working Group of Atherosclerosis and Vascular biology is to provide guidance for the next steps to be taken from pre-clinical studies on vascularization towards clinical application. To this end, the current state of knowledge in terms of therapeutic strategies for targeting vascularization in post-ischaemic disease is reviewed and discussed. A consensus statement is provided on how to optimize vascularization studies for the identification of suitable targets, the use of animal models of disease, and the analysis of novel delivery methods.

Interplay between hypercholesterolaemia and inflammation in atherosclerosis: Translating experimental targets into clinical practice.

Dyslipidaemia and inflammation are closely interconnected in their contribution to atherosclerosis. In fact, low-density lipoprotein (LDL)-lowering drugs have anti-inflammatory effects. The Canakinumab Antiinflammatory Thrombosis Outcome Study (CANTOS) has shown that interleukin (IL)-1ß blockade reduces the incidence of cardiovascular events in patients with previous myocardial infarction and C-reactive protein levels >2 mg/L. These data confirm the connection between lipids and inflammation, as lipids activate the Nod-like receptor protein 3 inflammasome that leads to IL-1ß activation. LDL-lowering drugs are the foundation of cardiovascular prevention. Now, the CANTOS trial demonstrates that combining them with IL-1ß blockade further decreases the incidence of cardiovascular events. However, both therapies are not at the same level, given the large evidence showing that LDL-lowering drugs reduce cardiovascular risk as opposed to only one randomized trial of IL-1ß blockade. In addition, IL-1ß blockade has only been studied in patients with C-reactive protein >2 mg/L, while the benefit of LDL-lowering is not restricted to these patients. Also, lipid-lowering drugs are not harmful even at very low ranges of LDL, while anti-inflammatory therapies may confer a higher risk of developing fatal infections and sepsis. In the future, more clinical trials are needed to explore whether targeting other inflammatory molecules, both related and unrelated to the IL-1ß pathway, reduces the cardiovascular risk. In this regard, the ongoing trials with methotrexate and colchicine may clarify whether the cardiovascular benefit of IL-1ß blockade extends to other anti-inflammatory mechanisms. A positive result would represent a major change in the future treatment of atherosclerosis.

Vascular smooth muscle cell death, autophagy and senescence in atherosclerosis.

In the present review, we describe the causes and consequences of loss of vascular smooth muscle cells (VSMCs) or their function in advanced atherosclerotic plaques and discuss possible mechanisms such as cell death or senescence, and induction of autophagy to promote cell survival. We also highlight the potential use of pharmacological modulators of these processes to limit plaque progression and/or improve plaque stability. VSMCs play a pivotal role in atherogenesis. Loss of VSMCs via initiation of cell death leads to fibrous cap thinning and promotes necrotic core formation and calcification. VSMC apoptosis is induced by pro-inflammatory cytokines, oxidized low density lipoprotein, high levels of nitric oxide and mechanical injury. Apoptotic VSMCs are characterized by a thickened basal lamina surrounding the cytoplasmic remnants of the VSMC. Inefficient clearance of apoptotic VSMCs results in secondary necrosis and subsequent inflammation. A critical determinant in the VSMC stress response and phenotypic switching is autophagy, which is activated by various stimuli, including reactive oxygen and lipid species, cytokines, growth factors and metabolic stress. Successful autophagy stimulates VSMC survival, whereas reduced autophagy promotes age-related changes in the vasculature. Recently, an interesting link between autophagy and VSMC senescence has been uncovered. Defective VSMC autophagy accelerates not only the development of stress-induced premature senescence but also atherogenesis, albeit without worsening plaque stability. VSMC senescence in atherosclerosis is likely a result of replicative senescence and/or stress-induced premature senescence in response to DNA damaging and/or oxidative stress-inducing stimuli. The finding that VSMC senescence can promote atherosclerosis further illustrates that normal, adequate VSMC function is crucial in protecting the vessel wall against atherosclerosis.

Autophagy in health and disease: focus on the cardiovascular system.

Autophagy is a highly conserved mechanism of lysosome-mediated protein and organelle degradation that plays a crucial role in maintaining cellular homeostasis. In the last few years, specific functions for autophagy have been identified in many tissues and organs. In the cardiovascular system, autophagy appears to be essential to heart and vessel homeostasis and function; however defective or excessive autophagy activity seems to contribute to major cardiovascular disorders including heart failure (HF) or atherosclerosis. Here, we review the current knowledge on the role of cardiovascular autophagy in physiological and pathophysiological conditions.

Methods for Measuring Autophagy in Mice.

Autophagy is a dynamic intracellular process that mediates the degradation of damaged cytoplasmic components by the lysosome. This process plays important roles in maintaining normal cellular homeostasis and energy balance. Measuring autophagy activity is critical and although the determination of autophagic flux in isolated cells is well documented, there is a need to have reliable and quantitative assays to evaluate autophagy in whole organisms. Because mouse models have been precious in establishing the functional significance of autophagy under physiological or pathological conditions, we present in this chapter a compendium of the current available methods to measure autophagy in mice, and discuss their advantages and limitations.

Circulating miR-155, miR-145 and let-7c as diagnostic biomarkers of the coronary artery disease.

Coronary artery disease (CAD) is the most prevalent cause of mortality and morbidity worldwide and the number of individuals at risk is increasing. To better manage cardiovascular diseases, improved tools for risk prediction including the identification of novel accurate biomarkers are needed. MicroRNA (miRNA) are essential post-transcriptional modulators of gene expression leading to mRNA suppression or translational repression. Specific expression profiles of circulating miRNA have emerged as potential noninvasive diagnostic biomarkers of diseases. The aim of this study was to identify the potential diagnostic value of circulating miRNA with CAD. Circulating miR-145, miR-155, miR-92a and let-7c were selected and validated by quantitative PCR in 69 patients with CAD and 30 control subjects from the cross-sectional study GENES. The expression of miR-145, miR-155 and let-7c showed significantly reduced expression in patients with CAD compared to controls. Multivariate logistic regression analysis revealed that low levels of circulating let-7c, miR-145 and miR-155 were associated with CAD. Receiver operating curves analysis showed that let-7c, miR-145 or miR-155 were powerful markers for detecting CAD. Furthermore, we demonstrated that the combination of the three circulating miRNA managed to deliver a specific signature for diagnosing CAD.

High-density lipoprotein subclass profile and mortality in patients with coronary artery disease: Results from the GENES study.

BACKGROUND: High-density lipoproteins (HDLs) are highly heterogeneous particles, and the specific contribution of each subclass to the prediction of clinical outcome in coronary artery disease (CAD) remains controversial. OBJECTIVE: To examine the relationship between HDL subclass profile and mortality in patients with CAD, using a new and rapid electrophoretic quantitative method for the assessment of HDL particle size phenotype. METHODS: We investigated 403 patients with CAD admitted for cardiovascular examination in the context of evaluation and management of CAD. HDL subclass distribution was analysed using the Quantimetrix Lipoprint® HDL system. Cumulative survival of patients according to lipid variables was determined by the Kaplan-Meier method. The relationship between baseline variables and outcome criteria was assessed using Cox proportional hazards regression analysis. RESULTS: During follow-up (9.8±3.1 years) the mortality rate was 31.0%; 60.8% of deaths were related to CAD. The concentration of total HDL cholesterol was similar in deceased patients (42±13mg/dL) and alive patients (43±12mg/dL); the concentrations of small, intermediate and large HDL cholesterol subclasses were not significantly different in alive and deceased patients (P=0.17, P=0.34 and P=0.81, respectively). We did not observe any independent associations between overall or cardiovascular mortality and total HDL cholesterol or any HDL subclass. However, heart rate, left ventricular ejection fraction and severity score for coronary atherosclerosis were more associated with mortality than classical cardiovascular risk factors. CONCLUSIONS: HDL subclass profile is not associated with mortality in patients with CAD. Further investigations linking HDL subclass repartition with prediction of residual cardiovascular risk are required.