ABSTRACT
RATIONALE: Metabolic syndrome (MetS) is a cluster of interrelated risk factors for cardiovascular diseases and atherosclerosis. Circulating levels of large extracellular vesicles (lEVs), submicrometer-sized vesicles released from plasma membrane, from MetS patients were shown to induce endothelial dysfunction, but their role in early stage of atherosclerosis and on vascular smooth muscle cells (SMC) remain to be fully elucidated. OBJECTIVE: To determine the mechanisms by which lEVs lead to the progression of atherosclerosis in the setting of MetS. METHODS AND RESULTS: Proteomic analysis revealed that the small GTPase, Rap1 was overexpressed in lEVs from MetS patients compared with those from non-MetS subjects. Rap1 was in GTP-associated active state in both types of lEVs, and Rap1-lEVs levels correlated with increased cardiovascular risks, including stenosis. MetS-lEVs, but not non-MetS-lEVs, increased Rap1-dependent endothelial cell permeability. MetS-lEVs significantly promoted migration and proliferation of human aortic SMC and increased expression of proinflammatory molecules and activation of ERK (extracellular signal-regulated kinase) 5/p38 pathways. Neutralization of Rap1 by specific antibody or pharmacological inhibition of Rap1 completely prevented the effects of lEVs from MetS patients. High-fat diet-fed ApoE-/- mice displayed an increased expression of Rap1 both in aortas and circulating lEVs. lEVs accumulated in plaque atherosclerotic lesions depending on the progression of atherosclerosis. lEVs from high-fat diet-fed ApoE-/- mice, but not those from mice fed with a standard diet, enhanced SMC proliferation. Human atherosclerotic lesions were enriched in lEVs expressing Rap1. CONCLUSIONS: These data demonstrate that Rap1 carried by MetS-lEVs participates in the enhanced SMC proliferation, migration, proinflammatory profile, and activation of ERK5/p38 pathways leading to vascular inflammation and remodeling, and atherosclerosis. These results highlight that Rap1 carried by MetS-lEVs may be a novel determinant of diagnostic value for cardiometabolic risk factors and suggest Rap1 as a promising therapeutic target against the development of atherosclerosis. Graphical Abstract: A graphical abstract is available for this article.
Subject(s)
Atherosclerosis/metabolism , Endothelial Cells/metabolism , Extracellular Vesicles/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Plaque, Atherosclerotic , rap1 GTP-Binding Proteins/metabolism , Adult , Aged , Aged, 80 and over , Animals , Atherosclerosis/blood , Atherosclerosis/pathology , Case-Control Studies , Cell Movement , Cell Proliferation , Cells, Cultured , Disease Models, Animal , Endothelial Cells/pathology , Female , Humans , Male , Mice, Inbred C57BL , Mice, Knockout, ApoE , Middle Aged , Mitogen-Activated Protein Kinase 7/metabolism , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/pathology , Permeability , Phosphorylation , Prognosis , Proteomics , Risk Assessment , Risk Factors , Signal Transduction , p38 Mitogen-Activated Protein Kinases/metabolism , rap GTP-Binding ProteinsABSTRACT
Abdominal aortic aneurysm (AAA) is a progressive vascular disease responsible for 1-4% of the deaths in elderly men. This study aimed to characterize specific microRNA (miRNA) expression in aneurysmal smooth muscle cells (SMCs) and macrophages in order to identify circulating miRNAs associated with AAA. We screened 850 miRNAs in aneurysmal SMCs, M1 and M2 macrophages, and in control SMCs isolated by micro-dissection from aortic biopsies using microarray analysis. In all, 92 miRNAs were detected and 10 miRNAs were selected for validation by qRT-PCR in isolated cells (n = 5), whole control and aneurysmal aorta biopsies (n = 13), and plasma from patients (n = 24) undergoing AAA (over 50 mm) repair matched to patients (n = 18) with peripheral arterial disease (PAD) with atherosclerosis but not AAA. Seven miRNAs were modulated similarly in all aneurysmal cells. The Let-7f was downregulated in aneurysmal cells compared to control SMCs with a significant lower expression in M1 compared to M2 macrophages (0.1 fold, p = 0.03), correlated with a significant downregulation in whole aneurysmal aorta compared to control aorta (0.2 fold, p = 0.03). Significant levels of circulating let-7f (p = 0.048) were found in AAA patients compared to PAD patients with no significant correlation with aortic diameter (R2 = 0.03). Our study underlines the utility of profiling isolated aneurysmal cells to identify other miRNAs for which the modulation of expression might be masked when the whole aorta is used. The results highlight let-7f as a new potential biomarker for AAA.
Subject(s)
Aortic Aneurysm, Abdominal/blood , Circulating MicroRNA/blood , MicroRNAs/blood , Transcriptome , Aged , Aortic Aneurysm, Abdominal/genetics , Aortic Aneurysm, Abdominal/pathology , Biomarkers/blood , Circulating MicroRNA/genetics , Down-Regulation , Humans , Macrophages/metabolism , Macrophages/pathology , MicroRNAs/genetics , Middle Aged , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathologyABSTRACT
Thoracic aortic aneurysm is a pathological local dilatation of the aorta, potentially leading to aortic rupture or dissection. The disease is a common complication of patients with bicuspid aortic valve, a congenital disorder present in 1-2% of the population. Using two dimensional fluorescence difference gel electrophoresis proteomics followed by mRNA expression, and alternative splicing analysis of the identified proteins, differences in dilated and nondilated aorta tissues between 44 patients with bicuspid and tricuspid valves was examined. The pattern of protein expression was successfully validated with LC-MS/MS. A multivariate analysis of protein expression data revealed diverging protein expression fingerprints in patients with tricuspid compared with the patients with bicuspid aortic valves. From 302 protein spots included in the analysis, 69 and 38 spots were differentially expressed between dilated and nondilated aorta specifically in patients with tricuspid and bicuspid aortic valve, respectively. 92 protein spots were differentially expressed between dilated and nondilated aorta in both phenotypes. Similarly, mRNA expression together with alternative splicing analysis of the identified proteins also showed diverging fingerprints in the two patient groups. Differential splicing was abundant but the expression levels of differentially spliced mRNA transcripts were low compared with the wild type transcript and there was no correlation between splicing and the number of spots. Therefore, the different spots are likely to represent post-translational modifications. The identification of differentially expressed proteins suggests that dilatation in patients with a tricuspid aortic valve involves inflammatory processes whereas aortic aneurysm in patients with BAV may be the consequence of impaired repair capacity. The results imply that aortic aneurysm formation in patients with bicuspid and tricuspid aortic valves involve different biological pathways leading to the same phenotype.
Subject(s)
Aortic Aneurysm, Thoracic/genetics , Gene Expression Regulation , Heart Valve Diseases/metabolism , Proteome/metabolism , Transcriptome , Tricuspid Valve/metabolism , Alternative Splicing , Aortic Aneurysm, Thoracic/congenital , Aortic Aneurysm, Thoracic/metabolism , Aortic Aneurysm, Thoracic/pathology , Aortic Valve/abnormalities , Aortic Valve/metabolism , Aortic Valve/pathology , Bicuspid Aortic Valve Disease , Biopsy , Case-Control Studies , Chromatography, Liquid , Electrophoresis, Gel, Two-Dimensional , Female , Gene Expression Profiling , Heart Valve Diseases/pathology , Humans , Male , Principal Component Analysis , Proteome/chemistry , Tandem Mass Spectrometry , Tricuspid Valve/pathologyABSTRACT
Abdominal aortic aneurysm (AAA) is an inflammatory disease associated with marked changes in the cellular composition of the aortic wall. This study aims to identify microRNA (miRNA) expression in aneurysmal inflammatory cells isolated by laser microdissection from human tissue samples. The distribution of inflammatory cells (neutrophils, B and T lymphocytes, mast cells) was evaluated in human AAA biopsies. We observed in half of the samples that adventitial tertiary lymphoid organs (ATLOs) with a thickness from 0.5 to 2 mm were located exclusively in the adventitia. Out of the 850 miRNA that were screened by microarray in isolated ATLOs (n = 2), 164 miRNAs were detected in ATLOs. The three miRNAs (miR-15a-3p, miR-30a-5p and miR-489-3p) with the highest expression levels were chosen and their expression quantified by RT-PCR in isolated ATLOs (n = 4), M1 (n = 2) and M2 macrophages (n = 2) and entire aneurysmal biopsies (n = 3). Except for the miR-30a-5p, a similar modulation was found in ATLOs and the two subtypes of macrophages. The modulated miRNAs were then evaluated in the plasma of AAA patients for their potential as AAA biomarkers. Our data emphasize the potential of miR-15a-3p and miR-30a-5p as biomarkers of AAA but also as triggers of ATLO evolution. Further investigations will be required to evaluate their targets in order to better understand AAA pathophysiology.
Subject(s)
Adventitia/metabolism , Aortic Aneurysm, Abdominal/pathology , MicroRNAs/metabolism , Adventitia/physiopathology , Aged , Aorta/metabolism , Aorta/pathology , Aortic Aneurysm, Abdominal/complications , Aortic Aneurysm, Abdominal/genetics , Biomarkers/metabolism , Coronary Disease/etiology , Female , Humans , Lymphocytes/cytology , Lymphocytes/metabolism , Male , MicroRNAs/isolation & purification , Middle Aged , Peripheral Arterial Disease/genetics , Peripheral Arterial Disease/pathology , Real-Time Polymerase Chain Reaction , Risk FactorsABSTRACT
In this study, we developed a novel computational approach based on protein-protein interaction networks to identify a list of proteins that might have remained undetected in differential proteomic profiling experiments. We tested our computational approach on two sets of human smooth muscle cell protein extracts that were affected differently by DNase I treatment. Differential proteomic analysis by saturation DIGE resulted in the identification of 41 human proteins. The application of our approach to these 41 input proteins consisted of four steps: (i) Compilation of a human protein-protein interaction network from public databases; (ii) calculation of interaction scores based on functional similarity; (iii) determination of a set of candidate proteins that are needed to efficiently and confidently connect the 41 input proteins; and (iv) ranking of the resulting 25 candidate proteins. Two of the three highest-ranked proteins, beta-arrestin 1, and beta-arrestin 2, were experimentally tested, revealing that their abundance levels in human smooth muscle cell samples were indeed affected by DNase I treatment. These proteins had not been detected during the experimental proteomic analysis. Our study suggests that our computational approach may represent a simple, universal, and cost-effective means to identify additional proteins that remain elusive for current 2D gel-based proteomic profiling techniques.
Subject(s)
Muscle Proteins/metabolism , Protein Interaction Maps , Proteomics/methods , Cell Extracts , Cells, Cultured , Databases, Protein , Electrophoresis, Gel, Two-Dimensional , Humans , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/metabolism , Reproducibility of Results , SoftwareABSTRACT
Heart failure (HF) following myocardial infarction (MI) is characterized by progressive alterations of left ventricular (LV) structure and function, named LV remodelling. Although several risk factors such as infarct size have been identified, HF remains difficult to predict in clinical practice. Recently, using phosphoproteomic technology, we found that serine(208)-phosphorylated troponin T (P-Ser(208)-TnT) decreases in LV of HF rats. Our aim was to determine the performance of P-Ser(208)-TnT as plasma biomarker of HF compared to conventional cardiac biomarkers such as B-type natriuretic peptide (BNP), cardiac troponin I (cTnI), C-reactive protein (CRP) or tissue inhibitor of metalloproteinase I (TIMP-1) measured by x-MAP technology, as well as its capacity to reflect a pharmacological improvement of HF. We observed a significant increase of BNP, TnT and cTnI levels and a significant decrease of P-Ser(208)-TnT and TIMP-1 in the plasma of 2-month-MI rats compared with control rats with no modulation of CRP level. Circulating levels of P-Ser(208)-TnT were shown to be associated with most of the echocardiographic and haemodynamic parameters of cardiac function. We verified that the decrease of P-Ser(208)-TnT was not because of an excess of phosphatase activity in plasma of HF rats. Two-month-MI rats treated with the heart rate reducing agent ivabradine had improved LV function and increased plasma levels of P-Ser(208)-TnT. Thus, circulating phosphorylated troponin T is a highly sensitive biological indicator of cardiac dysfunction and has the potentiality of a new biomarker of HF post-MI, and of a surrogate marker for the efficacy of a successful treatment of HF.
Subject(s)
Biomarkers/blood , Myocardial Infarction/diagnosis , Serine/chemistry , Troponin T/blood , Animals , Humans , Male , Myocardial Infarction/blood , Phosphorylation , Rats , Rats, Wistar , Troponin T/chemistryABSTRACT
Heart failure, mostly associated with cardiac hypertrophy, is a major cause of illness and death. Oxidative stress causes accumulation of reactive oxygen species (ROS), leading to mitochondrial dysfunction, suggesting that mitochondria-targeted therapies could be effective in this context. The purpose of this work was to determine whether mitochondria-targeted therapies could improve cardiac hypertrophy induced by mitochondrial ROS. We used neonatal (NCMs) and adult (ACMs) rat cardiomyocytes hypertrophied by isoproterenol (Iso) to induce mitochondrial ROS. A decreased interaction between sirtuin 3 and superoxide dismutase 2 (SOD2) induced SOD2 acetylation on lysine 68 and inactivation, leading to mitochondrial oxidative stress and dysfunction and hypertrophy after 24 h of Iso treatment. To counteract these mechanisms, we evaluated the impact of the mitochondria-targeted antioxidant mitoquinone (MitoQ). MitoQ decreased mitochondrial ROS and hypertrophy in Iso-treated NCMs and ACMs but altered mitochondrial structure and function by decreasing mitochondrial respiration and mitophagy. The same decrease in mitophagy was found in human cardiomyocytes but not in fibroblasts, suggesting a cardiomyocyte-specific deleterious effect of MitoQ. Our data showed the importance of mitochondrial oxidative stress in the development of cardiomyocyte hypertrophy. We observed that targeting mitochondria by MitoQ in cardiomyocytes impaired the metabolism through defective mitophagy, leading to accumulation of deficient mitochondria.
ABSTRACT
An extensive body of literature suggested a possible role of the microtubule-associated protein Tau in chromatin functions and/or organization in neuronal, non-neuronal, and cancer cells. How Tau functions in these processes remains elusive. Here we report that Tau expression in breast cancer cell lines causes resistance to the anti-cancer effects of histone deacetylase inhibitors, by preventing histone deacetylase inhibitor-inducible gene expression and remodeling of chromatin structure. We identify Tau as a protein recognizing and binding to core histone when H3 and H4 are devoid of any post-translational modifications or acetylated H4 that increases the Tau's affinity. Consistent with chromatin structure alterations in neurons found in frontotemporal lobar degeneration, Tau mutations did not prevent histone deacetylase-inhibitor-induced higher chromatin structure remodeling by suppressing Tau binding to histones. In addition, we demonstrate that the interaction between Tau and histones prevents further histone H3 post-translational modifications induced by histone deacetylase-inhibitor treatment by maintaining a more compact chromatin structure. Altogether, these results highlight a new cellular role for Tau as a chromatin reader, which opens new therapeutic avenues to exploit Tau biology in neuronal and cancer cells.
ABSTRACT
The authors have requested that the following changes be made to their paper [...].
ABSTRACT
Post-translational modifications of cardiac proteins could participate to left contractile dysfunction resulting in heart failure. Using a rat model of ischemic heart failure, we showed an accumulation of phosphorylated desmin leading to toxic aggregates in cardiomyocytes, but the cellular mechanisms are unknown. The same rat model was used to decipher the kinases involved in desmin phosphorylation and the proteolytic systems present in rat and human failing hearts. We used primary cultures of neonate rat cardiomyocytes for testing specific inhibitors of kinases and for characterizing the autophagic processes able to clear desmin aggregates. We found a significant increase of active PKCζ, no modulation of ubitiquitin-proteasome system, a defect in macroautophagy, and an activation of chaperone-mediated autophagy in heart failure rats. We validated in vitro that PKCζ inhibition induced a significant decrease of GSK3ß and of soluble desmin. In vitro activation of ubiquitination of proteins and of chaperone-mediated autophagy is able to decrease soluble and insoluble forms of desmin in cardiomyocytes. These data demonstrate a novel signaling pathway implicating activation of PKCζ in desmin phosphorylation associated with a defect of proteolytic systems in ischemic heart failure, leading to desmin aggrephagy. Our in vitro data demonstrated that ubiquitination of proteins and chaperone-mediated autophagy are required for eliminating desmin aggregates with the contribution of its chaperone protein, α-crystallin Β-chain. Modulation of the kinases involved under pathological conditions may help preserving desmin intermediate filaments structure and thus protect the structural integrity of contractile apparatus of cardiomyocytes by limiting desmin aggregates formation.
ABSTRACT
Abdominal aortic aneurysms (AAA) are defined by an increased aortic diameter and characterized by impairment of the extracellular matrix, macrophages infiltration and decreased density of smooth muscle cells. Our aim is to identify the key molecules involved in the pathogenesis of AAAs. This study investigated transcriptomic and proteomic profiles of macrophages from AAA patients (>50 mm aortic diameter) (n = 24) and peripheral arterial occlusion (PAO) patients without AAA detected (n = 18), who both needed a surgery. An antibody protein microarray, generated by printing antibodies onto membranes against proteins selected from the transcriptomic and proteomic analysis, was performed to validate the proteins differentially expressed specifically in macrophages and plasma from the same patients. We found a restricted number of proteins differentially expressed between AAA and PAO patients: TIMP-3, ADAMTS5, and ADAMTS8 that differ significantly in plasma of AAA patients compared to PAO patients, as found in the macrophages. In contrast to plasma MMP-9, soluble glycoprotein V (sGPV) and plasmin-antiplasmin complex levels, plasma TIMP-3 levels were not correlated to AAA size but interestingly correlated to sGPV, a platelet activation marker. Combining transcriptomic and proteomic is a valid approach to identify diseases causing proteins and potential biomarkers.
Subject(s)
Aortic Aneurysm, Abdominal/metabolism , Gene Expression Profiling/methods , Macrophages/metabolism , Protein Array Analysis/methods , Proteome/analysis , Antibodies/metabolism , Biomarkers , Blood Proteins/analysis , Blood Proteins/genetics , Blood Proteins/metabolism , Case-Control Studies , Cells, Cultured , Electrophoresis, Gel, Two-Dimensional , Humans , Male , Oligonucleotide Array Sequence Analysis , Peripheral Vascular Diseases , Proteins/analysis , Proteins/genetics , Proteins/metabolism , Proteome/genetics , Proteome/metabolism , Reproducibility of Results , Statistics, NonparametricABSTRACT
Unhealthy lifestyle choices, such as bad eating behaviors and cigarette smoking, have major detrimental impacts on health. However, the inter-relations between obesity and smoking are still not fully understood. We thus developed an experimental model of high-fat diet-fed obese C57BL/6 male mice chronically exposed to cigarette smoke. Our study evaluated for the first time the resulting effects of the combined exposure to unhealthy diet and cigarette smoke on several metabolic, pulmonary, intestinal, and cardiac parameters. We showed that the chronic exposure to cigarette smoke modified the pattern of body fat distribution in favor of the visceral depots in obese mice, impaired the respiratory function, triggered pulmonary inflammation and emphysema, and was associated with gut microbiota dysbiosis, cardiac hypertrophy and myocardial fibrosis.
Subject(s)
Environmental Exposure , Life Style , Obesity/etiology , Smoking/adverse effects , Adipose Tissue/metabolism , Animals , Biomarkers , Cardiomegaly/etiology , Cardiomegaly/metabolism , Cardiomegaly/pathology , Cytokines/metabolism , Disease Models, Animal , Disease Susceptibility , Energy Metabolism , Glucose/metabolism , Homeostasis , Humans , Insulin/metabolism , Lung/physiopathology , Male , Mice , Microbiota , Obesity/complications , Obesity/metabolism , Organ SpecificityABSTRACT
Clusterin (CLU) is induced in many organs after tissue injury or remodeling. Recently, we show that CLU levels are increased in plasma and left ventricle (LV) after MI, however, the mechanisms involved are not yet elucidated. On the other hand, it has been shown that the activity of the protein degradation systems (PDS) is affected after MI with a decrease in ubiquitin proteasome system (UPS) and an increase in macroautophagy. The aim of this study was to decipher if the increased CLU levels after MI are in part due to the alteration of PDS activity. Rat neonate cardiomyocytes (NCM) were treated with different modulators of UPS and macroautophagy in order to decipher their role in CLU expression, secretion, and degradation. We observed that inhibition of UPS activity in NCM increased CLU mRNA levels, its intracellular protein levels (p-CLU and m-CLU) and its secreted form (s-CLU). Macroautophagy was also induced after MG132 treatment but is not active. The inhibition of macroautophagy induction in MG132-treated NCM increased CLU mRNA and m-CLU levels, but not s-CLU compared to NCM only treated by MG132. We also demonstrate that CLU can be degraded in NCM through proteasome and lysosome by a macroautophagy independent pathway. In another hand, CLU silencing in NCM has no effect either on macroautophagy or apoptosis induced by MG132. However, the overexpression of CLU secreted isoform in H9c2 cells, but not in NCM decreased apoptosis after MG132 treatment. Finally, we observed that increased CLU levels in hypertrophied NCM and in failing human hearts are associated with proteasome inhibition and macroautophagy alteration. All these data suggest that increased CLU expression and secretion after MI is, in part, due to a defect of UPS and macroautophagy activities in the heart and may have a protective effect by decreasing apoptosis induced by proteasome inhibition.
Subject(s)
Clusterin/metabolism , Myocardial Infarction/metabolism , Proteolysis , Animals , Apoptosis/drug effects , Autophagy/drug effects , Biopsy , Gene Silencing/drug effects , Heart Failure/metabolism , Heart Failure/pathology , Humans , Hypertrophy , Leupeptins/pharmacology , Lysosomes/drug effects , Lysosomes/metabolism , Myocardium/metabolism , Myocardium/pathology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/pharmacology , Proteolysis/drug effects , Rats , Ubiquitinated Proteins/metabolism , Ubiquitination/drug effectsABSTRACT
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
ABSTRACT
Tauopathies, such as Alzheimer's disease, are characterized by intracellular aggregates of insoluble Tau proteins. Originally described as a microtubule binding protein, recent studies demonstrated additional physiological roles for Tau. The fact that a single protein can regulate multiple cellular functions has posed challenge in terms of understanding mechanistic cues behind the pathology. Here, we used tandem-affinity purification methodology coupled to mass spectrometry to identify novel interaction partners. We found that Tau interacts with DDX6, a DEAD box RNA helicase involved in translation repression and mRNA decay as well as in the miRNA pathway. Our results demonstrate that Tau increases the silencing activity of the miRNA let-7a, miR-21 and miR-124 through DDX6. Importantly, Tau mutations (P301S, P301L) found in the inherited tauopathies, frontotemporal dementia and parkinsonism linked to chromosome 17, disrupt Tau/DDX6 interaction and impair gene silencing by let-7a. Altogether, these data demonstrated a new unexpected role for Tau in regulating miRNA activity.
Subject(s)
DEAD-box RNA Helicases/metabolism , MicroRNAs/metabolism , Proto-Oncogene Proteins/metabolism , tau Proteins/metabolism , Brain/metabolism , Cell Line, Tumor , DEAD-box RNA Helicases/chemistry , Humans , Mutation , Protein Interaction Domains and Motifs , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins c-myc/metabolism , Tauopathies/metabolism , tau Proteins/chemistry , tau Proteins/geneticsABSTRACT
BACKGROUND: Left ventricular remodeling (LVR) after myocardial infarction is associated with an increased risk of heart failure and death. In spite of a modern therapeutic approach, LVR remains relatively frequent and difficult to predict in clinical practice. Our aim was to identify new biomarkers of LVR and understand their involvement in its development. METHODS AND RESULTS: Proteomic analysis of plasma from the REVE-2 study (Remodelage Ventriculaire)-a study dedicated to the analysis of LVR which included 246 patients after a first anterior myocardial infarction-identified increased plasma levels of CLU (clusterin) in patients with high LVR. We used a rat model of myocardial infarction to analyze CLU expression in the LV and found a significant increase that was correlated with LVR parameters. We found increased CLU expression and secretion in primary cultures of rat neonate cardiomyocytes hypertrophied by isoproterenol. Silencing of CLU in hypertrophied neonate cardiomyocytes induced a significant decrease in cell size, ANP (atrial natriuretic peptide), and BNP (B-type natriuretic peptide) expression, associated with a decreased ERK (extracellular signal-regulated kinase) 1/2 activity, suggesting a prohypertrophic role of CLU. We then confirmed a significant increase of both intracellular p-CLU (precursor form of CLU) and m-CLU (mature form of CLU) in failing human hearts. Finally, the circulating levels of CLU (secreted form) were increased in patients with chronic heart failure who died from cardiovascular cause during a 3-year follow-up (n=99) compared with survivors (n=99). CONCLUSIONS: Our results show for the first time that plasma CLU levels are associated with LVR post-myocardial infarction, have in part a cardiac origin, and are a predictor of early death in heart failure patients.
Subject(s)
Clusterin/metabolism , Heart Failure/blood , Myocardial Infarction/blood , Ventricular Remodeling , Adult , Biomarkers/blood , Echocardiography/methods , Female , Heart/physiopathology , Heart Failure/complications , Humans , Male , Myocardial Infarction/physiopathology , ProteomicsABSTRACT
Although several risk factors such as infarct size have been identified, the progression of heart failure (HF) remains difficult to predict in clinical practice. Using an experimental rat model of post-myocardial infarction (MI), we previously identified 45 proteins differentially modulated during HF by proteomic analysis. This study sought to identify microRNAs (miRNAs) able to regulate these proteins and to test their relevance as biomarkers for HF. In silico bioinformatical analysis selected 13 miRNAs related to the 45 proteins previously identified. These miRNAs were analyzed in the rat and in cohorts of patients phenotyped for left ventricular remodeling (LVR). We identified that 3 miRNAs, miR-21-5p, miR-23a-3p and miR-222-3p, and their target Mn superoxide dismutase (SOD2) were significantly increased in LV and plasma of HF-rats. We found by luciferase activity a direct interaction of miR-222-3p with 3'UTR of SOD2. Transfection of human cardiomyocytes with miR-222-3p mimic or inhibitor induced respectively a decrease and an increase of SOD2 expression. Circulating levels of the 3 miRNAs and their target SOD2 were associated with high LVR post-MI in REVE-2 patients. We demonstrated for the first time the potential of microRNAs regulating SOD2 as new circulating biomarkers of HF.
Subject(s)
Heart Failure/blood , MicroRNAs/blood , Superoxide Dismutase/blood , 3' Untranslated Regions , Animals , Biomarkers/blood , Disease Models, Animal , Heart Ventricles/enzymology , Male , Prognosis , Proteomics/methods , RNA Processing, Post-Transcriptional , Rats , Rats, Wistar , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Ventricular RemodelingABSTRACT
Remote ischemic preconditioning's (RIPC) ability to render the myocardium resistant to subsequent prolonged ischemia is now clearly established in different species, including humans. Strong evidence suggests that circulating humoral mediators play a key role in signal transduction, but their identities still need to be established. Our study sought to identify potential circulating RIPC mediators using a proteomic approach. Rats were exposed to 10-min limb ischemia followed by 5- (RIPC 5') or 10-min (RIPC 10') reperfusion prior to blood sampling. The control group only underwent blood sampling. Plasma samples were isolated for proteomic analysis using surface-enhanced laser desorption and ionization - time of flight - mass spectrometry (SELDI-TOF-MS). A total of seven proteins, including haptoglobin and transthyretin, were detected as up- or down-regulated in response to RIPC. These proteins had previously been identified as associated with organ protection, anti-inflammation, and various cellular and molecular responses to ischemia. In conclusion, this study indicates that RIPC results in significant modulations of plasma proteome.
Subject(s)
Ischemic Preconditioning , Plasma/metabolism , Proteome/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Amino Acid Sequence , Animals , Male , Molecular Sequence Data , Rats , Rats, WistarABSTRACT
The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.
Subject(s)
Electrophoresis, Gel, Two-Dimensional/methods , Macrophages/chemistry , Macrophages/metabolism , Proteins/chemistry , Proteomics/methods , Cell Differentiation/physiology , Electrophoresis, Polyacrylamide Gel , Fluorescent Dyes/metabolism , Humans , Isoelectric Focusing , Macrophages/cytology , Peptides/metabolism , Phenotype , Proteins/isolation & purificationABSTRACT
BACKGROUND: Remote ischemic preconditioning (RIPC) has emerged as an attractive strategy in clinical settings. Despite convincing evidence of the critical role played by circulating humoral mediators, their actual identities remain unknown. In this study, we aimed to identify RIPC-induced humoral mediators using a proteomic approach. METHODS: and Results Rats were exposed to 10-min limb ischemia followed by 5- (RIPC 5') or 10-min (RIPC 10') reperfusion prior to blood sampling. The control group only underwent blood sampling. Plasma samples were analyzed using surface-enhanced laser desorption and ionization - time of flight - mass spectrometry (SELDI-TOF-MS). Three protein peaks were selected for their significant increase in RIPC 10'. They were identified and confirmed as apolipoprotein A-I (ApoA-I). Additional rats were exposed to myocardial ischemia-reperfusion (I/R) and assigned to one of the following groups RIPC+myocardial infarction (MI) (10-min limb ischemia followed by 10-min reperfusion initiated 20 minutes prior to myocardial I/R), ApoA-I+MI (10 mg/kg ApoA-I injection 10 minutes before myocardial I/R), and MI (no further intervention). In comparison with untreated MI rats, RIPC reduced infarct size (52.2±3.7% in RIPC+MI vs. 64.9±2.6% in MI; p<0.05). Similarly, ApoA-I injection decreased infarct size (50.9±3.8%; p<0.05 vs. MI). CONCLUSIONS: RIPC was associated with a plasmatic increase in ApoA-I. Furthermore, ApoA-I injection before myocardial I/R recapitulated the cardioprotection offered by RIPC in rats. This data suggests that ApoA-I may be a protective blood-borne factor involved in the RIPC mechanism.