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.

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.

A role for 4-hydroxy-2-nonenal in premature placental senescence in preeclampsia and intrauterine growth restriction.

Premature placental senescence is a hallmark of pregnancy-related disorders such as intrauterine growth restriction (IUGR) and preeclampsia (PE), two major cause of maternal and neonatal morbidity and mortality. Oxidative stress and lipid peroxidation are involved in the pathogenesis of PE and IUGR, and may play a role in placental aging. In this study, we investigated whether 4-hydroxy-2-nonenal (HNE), a lipid peroxidation-derived aldehyde present in preeclamptic placentas, may contribute to premature senescence in placenta-related complications. Placentas from PE-affected women, exhibited several senescence patterns, such as an increased expression of phosphorylated (serine-139) histone γH2AX, a sensitive marker of double-stranded DNA breaks, the presence of lipofuscin granules, and an accumulation of high molecular weight cross-linked and ubiquitinated proteins. PE placentas showed an accumulation of acetylated proteins consistent with the presence of HNE-adducts on sirtuin 1 (SIRT1). Likewise, oxidative stress and senescence markers together with SIRT1 modification by HNE, were observed in murine placentas from mice treated with lipopolysaccharide during gestation and used as models of IUGR. The addition of HNE and ONE (4-oxo-2-nonenal), to cultured HTR-8/SVneo human trophoblasts activated the senescence-associated- ß-galactosidase, and generated an accumulation of acetylated proteins, consistent with a modification of SIRT1 by HNE. Altogether, these data emphasize the role of HNE and lipid peroxidation-derived aldehydes in premature placental senescence in PE and IUGR, and more generally in pathological pregnancies.

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.

nSMase2 (Type 2-Neutral Sphingomyelinase) Deficiency or Inhibition by GW4869 Reduces Inflammation and Atherosclerosis in Apoe-/- Mice.

OBJECTIVE: Atherosclerosis is a chronic multifactorial and inflammatory disease of large and medium arteries and the leading cause of cardiovascular diseases worldwide. The aim of this study was to investigate whether and how the nSMase2 (type 2-neutral sphingomyelinase), a key enzyme of sphingolipid metabolism, may contribute to the development of atherosclerotic lesions. APPROACH AND RESULTS: The role of nSMase2 in atherosclerosis was investigated in Apoe-/-;Smpd3fro/fro mice, mutant for nSMase2, and in Apoe-/-;Smpd3+/+ mice intraperitoneally injected with GW4869, a pharmacological nSMase2 inhibitor. The defect or inhibition of nSMase2 resulted in a reduction of atherosclerotic lesions and a decrease in macrophage infiltration and lipid deposition, although cholesterolemia remained unchanged. nSMase2 inhibition decreased the inflammatory response of murine endothelial cells to oxLDL (oxidized low-density lipoprotein), as assessed by the significant reduction of MCP-1 (monocyte chemoattractant protein 1), ICAM-1 (intercellular adhesion molecule-1), and VCAM-1 (vascular cell adhesion molecule-1) mRNA expressions and macrophage recruitment. Likewise, in RAW264.7 or in macrophages isolated from Apoe-/-/Smpd3fro/fro or Apoe-/-/Smpd3+/+ mice stimulated by lipopolysaccharides, nSMase2 inhibition resulted in a decrease in the expression of inflammatory molecules. Mechanistically, the anti-inflammatory response resulting from nSMase2 inhibition involves Nrf2 (nuclear factor [erythroid-derived 2]-like 2 or NF-E2-related factor-2) activation in both endothelial cells and macrophages, as assessed by the lack of protective effect of GW4869 in endothelial cells silenced for Nrf2 by small interfering RNAs, and in lipopolysaccharide-stimulated macrophages issued from Nrf2-KO mice. CONCLUSIONS: The genetic deficiency or inhibition of nSMase2 strongly decreases the development of atherosclerotic lesions in Apoe-/- mice, by reducing inflammatory responses through a mechanism involving the Nrf2 pathway. Inhibitors of nSMase2 may, therefore, constitute a novel approach to slow down atherosclerosis progression.

The neutral sphingomyelinase-2 is involved in angiogenic signaling triggered by oxidized LDL.

Capillaries of the external part of the normal arterial wall constitute the vasa vasorum network. In atherosclerotic lesions, neovascularization occurs in areas of intimal hyperplasia where it may promote plaque expansion, and intraplaque hemorrhage. Oxidized LDL that are present in atherosclerotic areas activate various angiogenic signaling pathways, including reactive oxygen species and the sphingosine kinase/sphingosine-1-phosphate pathway. We aimed to investigate whether oxidized LDL-induced angiogenesis requires neutral sphingomyelinase-2 activation and the neutral sphingomyelinase-2/sphingosine kinase-1 pathway. The role of neutral sphingomyelinase-2 in angiogenic signaling was investigated in Human Microvascular Endothelial Cells (HMEC-1) forming capillary tube on Matrigel and in vivo in the Matrigel plug assay in C57BL/6 mice and in the chicken chorioallantoic membrane model. Low concentration of human oxidized LDL elicits HMEC-1 capillary tube formation and neutral sphingomyelinase-2 activation, which were blocked by neutral sphingomyelinase-2 inhibitors, GW4869 and specific siRNA. This angiogenic effect was mimicked by low concentration of C6-Ceramide and was inhibited by sphingosine kinase-1 inhibitors. Upstream of neutral sphingomyelinase-2, oxidized LDL-induced activation required LOX-1, reactive oxygen species generation by NADPH oxidase and p38-MAPK activation. Inhibition of sphingosine kinase-1 blocked the angiogenic response and triggered HMEC-1 apoptosis. Low concentration of oxidized LDL was angiogenic in vivo, both in the Matrigel plug assay in mice and in the chorioallantoic membrane model, and was blocked by GW4869. In conclusion, low oxLDL concentration triggers sprouting angiogenesis that involves ROS-induced activation of the neutral sphingomyelinase-2/sphingosine kinase-1 pathway, and is effectively inhibited by GW4869.

Elastin Modification by 4-Hydroxynonenal in Hairless Mice Exposed to UV-A. Role in Photoaging and Actinic Elastosis.

Chronic exposure to ultraviolet (UV) radiation causes oxidative stress, which is involved in photoaging and actinic elastosis. UV and reactive oxygen species generate lipid peroxidation products, including the α, ß-unsaturated carbonyl compounds such as acrolein or 4-hydroxynonenal (4-HNE). These aldehydes can modify proteins of the extracellular matrix, but their role in the pathogenesis of photoaging is not clarified. The aim of this study was to investigate whether these aldehydes contribute to alter elastin metabolism and whether topical carbonyl scavengers delay UV-induced skin photoaging. Hairless mice (4-6-week old) daily exposed to UV-A (20 J cm(-2) per day, up to 600 J cm(-2)) exhibited the typical features of photoaging, associated with a significant increase in 4-HNE- and acrolein-adduct content, and elastotic material deposition. Immunofluorescence studies showed the accumulation of 4-HNE adducts on elastin in the dermis of UV-A-exposed mice. This was mimicked in vitro by incubating orcein-elastin with 4-HNE or acrolein, which altered its digestion by leukocyte-elastase, a feature possibly involved in the accumulation of elastotic material. A daily topical application of carnosine completely reversed the development of photoaging alterations and 4-HNE-adduct formation on elastin. These data emphasize the role of 4-HNE and acrolein in the mechanism of photoaging, and the preventive effect of carbonyl scavengers.

4-Hydroxynonenal impairs transforming growth factor-ß1-induced elastin synthesis via epidermal growth factor receptor activation in human and murine fibroblasts.

Elastin is a long-lived protein and a key component of connective tissues. The tissular elastin content decreases during chronological aging, and the mechanisms underlying its slow repair are not known. Lipid oxidation products that accumulate in aged tissues may generate protein dysfunction. We hypothesized that 4-hydroxynonenal (4-HNE), a highly reactive α,ß-aldehydic product generated from polyunsaturated fatty acid peroxidation, could contribute to inhibiting elastin repair by antagonizing the elastogenic signaling of transforming growth factor-ß1 (TGF-ß1) in skin fibroblasts. We report that a low 4-HNE concentration (2µmol/L) inhibits the upregulation of tropoelastin expression stimulated by TGF-ß1 in human and murine fibroblasts. The study of signaling pathways potentially involved in the regulation of elastin expression showed that 4-HNE did not block the phosphorylation of Smad3, an early step of TGF-ß1 signaling, but inhibited the nuclear translocation of Smad2. Concomitantly, 4-HNE modified and stimulated the phosphorylation of the epidermal growth factor receptor (EGFR) and subsequently ERK1/2 activation, leading to the phosphorylation/stabilization of the Smad transcriptional corepressor TGIF, which antagonizes TGF-ß1 signaling. Inhibitors of EGFR (AG1478) and MEK/ERK (PD98059), and EGFR-specific siRNAs, reversed the inhibitory effect of 4-HNE on TGF-ß1-induced nuclear translocation of Smad2 and tropoelastin synthesis. In vivo studies on aortas from aged C57BL/6 mice showed that EGFR is modified by 4-HNE, in correlation with an increased 4-HNE-adduct accumulation and decreased elastin content. Altogether, these data suggest that 4-HNE inhibits the elastogenic activity of TGF-ß1, by modifying and activating the EGFR/ERK/TGIF pathway, which may contribute to altering elastin repair in chronological aging and oxidative stress-associated aging processes.

A signaling cascade mediated by ceramide, src and PDGFRß coordinates the activation of the redox-sensitive neutral sphingomyelinase-2 and sphingosine kinase-1.

Stress-inducing agents, including oxidative stress, generate the sphingolipid mediators ceramide (Cer) and sphingosine-1-phosphate (S1P) that are involved in stress-induced cellular responses. The two redox-sensitive neutral sphingomyelinase-2 (nSMase2) and sphingosine kinase-1 (SK1) participate in transducing stress signaling to ceramide and S1P, respectively; however, whether these key enzymes are coordinately regulated is not known. We investigated whether a signaling link coordinates nSMase2 and SK1 activation by H2O2. In mesenchymal cells, H2O2 elicits a dose-dependent biphasic effect, mitogenic at low concentration (5µM), and anti-proliferative and toxic at high concentration (100µM). Low H2O2 concentration triggered activation of nSMase2 and SK1 through a nSMase2/Cer-dependent signaling pathway that acted upstream of activation of SK1. Further results implicated src and the trans-activation of PDGFRß, as supported by the blocking effect of specific siRNAs, pharmacological inhibitors, and genetically deficient cells for nSMase2, src and SK1. The H2O2-induced src/PDGFRß/SK1 signaling cascade was impaired in nSMase2-deficient fro/fro cells and was rescued by exogenous C2Cer that activated src/PDGFRß/SK1. Thus, the results define a nSMase2/SK1 signaling pathway implicated in the mitogenic response to low oxidative stress. On the other hand, high oxidative stress induced inhibition of SK1. The results also showed that the toxicity of high H2O2 concentration was comparable in control and nSMase2-deficient cells. Taken together the results identify a tightly coordinated nSMase2/SK1 pathway that mediates the mitogenic effects of H2O2 and may sense the degree of oxidative stress.

Antiatherogenic effect of bisvanillyl-hydralazone, a new hydralazine derivative with antioxidant, carbonyl scavenger, and antiapoptotic properties.

Reactive oxygen species (ROS) generated within the vascular wall trigger low-density lipoprotein (LDL) oxidation, lipid peroxidation, and carbonyl stress that are involved in atherogenesis. We recently reported that the antihypertensive drug, hydralazine, exhibits carbonyl scavenger and antiatherogenic properties, but only moderate antioxidant activity, so that high concentrations are required for inhibiting LDL oxidation. We aimed to develop agents sharing both antioxidant and carbonyl scavenger properties. We have synthesized a new hydralazine derivative, the bisvanillyl-hydralazone (BVH). BVH strongly inhibited LDL oxidation induced by copper and by human endothelial cells (HMEC-1), and prevented the formation of macrophagic foam cells. BVH reduced both the extracellular generation of ROS (superoxide anion and hydrogen peroxide) induced by oxidized LDL (oxLDL), as well as intracellular oxidative stress and proteasome activation, NFkappaB activation, and oxLDL-mediated proinflammatory signaling. In parallel, BVH prevented the carbonyl stress induced by oxLDL on cellular proteins, and blocked the apoptotic cascade as assessed by the inhibition of Bid cleavage, cytochrome C release, and DEVDase activation. Lastly, BVH prevented atherogenesis and carbonyl stress in apoE(-/-) mice. In conclusion, BVH is the prototype of a new class of antioxidant and carbonyl scavenger agents designed for new therapeutical approaches in atherosclerosis.

Integrin alpha(v)beta(3), metalloproteinases, and sphingomyelinase-2 mediate urokinase mitogenic effect.

Plasminogen activators are implicated in the pathogenesis of several diseases such as inflammatory diseases and cancer. Beside their serine-protease activity, these agents trigger signaling pathways involved in cell migration, adhesion and proliferation. We previously reported a role for the sphingolipid pathway in the mitogenic effect of plasminogen activators, but the signaling mechanisms involved in neutral sphingomyelinase-2 (NSMase-2) activation (the first step of the sphingolipid pathway) are poorly known. This study was carried out to investigate how urokinase plasminogen activator (uPA) activates NSMase-2. We report that uPA, as well as its catalytically inactive N-amino fragment ATF, triggers the sequential activation of MMP-2, NSMase-2 and ERK1/2 in ECV304 cells that are required for uPA-induced ECV304 proliferation, as assessed by the inhibitory effect of Marimastat (a MMP inhibitor), MMP-2-specific siRNA, MMP-2 defect, and NSMase-specific siRNA. Moreover, upon uPA stimulation, uPAR, MT1-MMP, MMP-2 and NSMase-2 interacted with integrin alpha(v)beta(3), evidenced by co-immunoprecipitation and immunocytochemistry experiments. Moreover, the alpha(v)beta(3) blocking antibody inhibited the uPA-triggered MMPs/uPAR/integrin alpha(v)beta(3) interaction, NSMase-2 activation, Ki67 expression and DNA synthesis in ECV304. In conclusion, uPA triggers interaction between integrin alpha(v)beta(3), uPAR and MMPs that leads to NSMase-2 and ERK1/2 activation and cell proliferation. These findings highlight a new signaling mechanism for uPA, and suggest that, upon uPA stimulation, uPAR, MMPs, integrin alpha(v)beta(3) and NSMase-2 form a signaling complex that take part in mitogenic signaling in ECV304 cells.

Carbonyl scavenger and antiatherogenic effects of hydrazine derivatives.

Reactive carbonyl compounds (RCC) generated by polyunsaturated fatty acid oxidation alter progressively cellular and tissular proteins by forming adducts on free amino groups and thiol residues (carbonyl stress). Carbonyl scavengers may neutralize RCC, but their protective effect in atherosclerosis has not been extensively studied. We report the carbonyl scavenger and antiatherogenic properties of hydrazine derivatives, namely hydralazine, an antihypertensive drug, isoniazid, an antituberculosis agent, and two antidepressants, phenelzine and iproniazid. These drugs were poorly efficient in preventing the oxidation of LDL mediated by smooth muscle cells (SMCs), but inhibited the toxicity of UV-oxidized LDL (oxLDL) and of 4-hydroxynonenal (4-HNE). Hydrazine derivatives prevented the formation of foam cells resulting from LDL oxidation in human macrophagic U937 cells, and blocked the carbonyl stress in SMCs, by inhibiting the decrease in free amino group content, the increase in carbonylated proteins, and the formation of 4-HNE adducts on PDGFR. Experimental studies carried out on apoE-/- mice supplemented with drugs (30 mg/L in drinking water) showed a significant carbonyl stress inhibition correlated with a net reduction of atherosclerotic lesion development. In conclusion, these data indicate that hydrazine derivatives exhibit carbonyl scavenger and antiatherogenic properties, which opens novel therapeutical approaches for atherosclerosis and its cardiovascular complications.

E-cadherin/beta-catenin/T-cell factor pathway is involved in smooth muscle cell proliferation elicited by oxidized low-density lipoprotein.

The E-cadherin/beta-catenin/T-cell factor (Tcf) signaling pathway plays a crucial role in embryogenesis and carcinogenesis and has recently emerged in atherosclerosis. The aim of this work was to investigate whether this signaling pathway is involved in smooth muscle cell proliferation induced by oxidized low-density lipoprotein (LDL). In human aortic smooth muscle cells, mitogenic concentration of mildly oxidized LDL induced the activation of beta-catenin, as assessed by the dissociation of the beta-catenin/cadherin complex, and the concomitant rise of active beta-catenin in the cytosol. The oxidized LDL-induced rise of active beta-catenin required metalloproteinase activation, as well as epidermal growth factor receptor and Src signaling, as assessed by the use of pharmacological inhibitors and cells overexpressing a SrcK-inactive form. The concomitant phosphatidylinositol 3-kinase/Akt activation and glycogen synthase kinase 3-beta phosphorylation induced the inhibition of the proteasomal degradation of beta-catenin. Then active beta-catenin associated with Tcf4 and translocated into the nucleus. This enhanced the expression of the cell cycle activator cyclin D1. This crucial role of beta-catenin in the mitogenic effect of oxidized LDL was confirmed by silencing beta-catenin by specific small interfering RNA that blocked DNA synthesis. Immunohistochemistry staining of stable and disrupted plaques from carotid endarterectomy sections showed a correlation between active beta-catenin and Ki67, a proliferation marker, and a more intense staining in the smooth muscle cell layer surrounding the lipid core of disrupted plaques. In conclusion, the beta-catenin pathway is required for the mitogenic effect of oxidized LDL on human aortic smooth muscle cells. This study highlights the putative important role of the E-cadherin/beta-catenin/Tcf signaling pathway in atherosclerosis.

Desensitization of platelet-derived growth factor receptor-beta by oxidized lipids in vascular cells and atherosclerotic lesions: prevention by aldehyde scavengers.

The platelet-derived growth factor receptor-beta (PDGFRbeta) signaling pathway regulates smooth muscle cell (SMC) migration and proliferation and plays a role in the vascular wall response to injury. Oxidized low-density lipoprotein (oxLDL) in atherosclerotic lesions can activate the PDGFRbeta pathway, but the long-term effects of oxLDL on PDGFRbeta function are not well understood. We found that oxLDL induced a dual effect on PDGFRbeta signaling. Initial activation of the PDGFR was followed by desensitization of the receptor. PDGFRbeta desensitization was not attributable to PDGFRbeta degradation or changes in localization to the caveolae but instead resulted from decreased PDGF binding and inhibition of PDGFRbeta tyrosine kinase activity. This inhibition was associated with formation of (4HNE)- and acrolein-PDGFRbeta adducts and was mimicked by preincubation of cells with 4HNE. These PDGFRbeta adducts were also detected in aortae of apolipoprotein-deficient mice and hypercholesterolemic rabbits and in human carotid plaques. The aldehyde scavengers DNPH and Hydralazine prevented both oxLDL- and 4HNE-induced structural modification and PDGFRbeta signaling dysfunction in cells and in vivo. OxLDL inhibition of PDGF signaling may contribute to defective SMC proliferation and decrease the stability of a vulnerable plaque.