Comparative platelet releasate proteome profiling of acute coronary syndrome versus stable coronary artery disease
ABSTRACT: Upon activation, platelets release a host of soluble and vesicular signals, collectively termed the ‘platelet releasate’ (PR). The contents of this PR play a significant role in haemostasis, inflammation, and pathologic sequelae. Despite this, proteomic studies investigating the PR in coronary artery disease have not been performed. We undertook a comparative label-free quantitative (LFQ) proteomic profiling of the 1U/ml thrombin-induced PR from 13 acute coronary syndrome (ACS-STEMI) versus 14 stable angina pectoris patients using a tandem mass spectrometry approach. We identified differentially released platet proteins including tetranectin (CLEC3B), protein disulfide-isomerase-A3 (PDIA3), coagulation factor V (F5) and fibronectin (FN1). Strikingly, all 9 differential proteins were associated with the GO cellular component term ‘extracellular vesicle’ and reduced levels of EVs were detected in plasma of ACS-STEMI patients. Network analysis revealed 3 PR proteins either reduced (F5; FN1) or absent (CLEC3B) in ACS-STEMI patients, which are strongly connected to both the clotting cascade and major druggable targets on platelets. This moderated signature highlights the possible basis of platelet dysfunction in ACS-STEMI and may prove useful for non-invasive risk assessment of the progression of coronary artery disease.
Project description:Inflammatory mechanisms and immune cells are involved in acute coronary syndromes (ACS) and may lead to acute plaque rupture. However, the local expression of the different genes potentially involved is largely unknown. We therefore performed an Affymetrix analysis of genes expressed in white blood cells obtained from an occluding coronary thrombus or peripheral blood of patients with ST-elevation myocardial infarction. Thrombi of ACS patients were harvested from the site of coronary occlusion. Leukocytes were isolated by Ficoll centrifugation. Peripheral blood leukocytes (PBL) were treated in a similar fashion and mRNA was extracted from both cells.
Project description:Whole-genome gene expression analysis has been successfully utilized to diagnose, prognosticate, and identify potential therapeutic targets for cardiovascular disease. However, the utility of this approach to identify outcome-related genes and dysregulated pathways following first-time myocardial infarction (AMI) remains unknown and may offer a novel strategy to detect affected expressome networks that predict long-term outcome. Whole-genome microarray and targeted cytokine expression profiling on blood samples from normal cardiac function controls and first-time AMI patients within 48-hours post-MI revealed expected differential gene expression profiles enriched for inflammation and immune-response pathways in AMI patients. To determine molecular signatures at the time of AMI that could prognosticate long-term outcomes, transcriptional profiles from sub-groups of AMI patients with (n=5) or without (n=22) any recurrent events over an 18-month follow-up were compared. This analysis identified 559 differentially expressed genes. Bioinformatic analysis of this differential gene set for associated pathways revealed 1) increasing disease severity in AMI patients is associated with a decreased expression of the developmental epithelial-to-mesenchymal transition, and 2) modulation of cholesterol transport genes that include ABCA1, CETP, APOA1, and LDLR is associated with clinical outcome. In conclusion, differentially regulated genes and modulated pathways were identified that predicted recurrent cardiovascular outcomes in first-time AMI patients. This cell-based approach for risk stratification in AMI warrants a larger study to determine the role of metabolic remodeling and regenerative processes required for optimal outcomes. A validated transcriptome assay could represent a novel, non-invasive platform to anticipate modifiable pathways and therapeutic targets to optimize long-term outcome for AMI patients. The overall experimental design includes blood samples from 21 control and 31 myocardial infaction patient groups. Among the 31patients, 5 patients have recurrent events. Microarray were peformed on the blood samples and comparisons of control vs patient and patients with recurrent events vs patients without recurrent events were performed to identify differential genes related to disease or patients groups with recurrent events for the following bioinformatic analysis.
Project description:We have employed whole genome microarray expression profiling as a discovery platform to identify genes involved in plaque rupture. Human coronary artery endothelial cells (ECs) were stimulated in vitro for 12 hours with plasma obtained from the coronary sinus (CS) and the aorta (Ao) of patients with ACS (n=8), or patients with stable angina (SA, n=4). For each patient, gene expression profile was evaluated by microarray technology in ECs exposed to plasma obtained from CS and compared to that of ECs exposed to plasma sampled from Ao. In patient with ACS we found 684 genes up-regulated and 283 down-regulated was observed as compared to patients with SA. Functional and network analyses of statistically significant gene showed that the up regulated genes were associated to pathways IL17 Signaling. To validate the microarray data the RNAs were used for Real Time PCR experiment. Human coronary artery endothelial cells (ECs) were stimulated in vitro for 12 hours with plasma obtained from the coronary sinus (CS) and the aorta (Ao) of patients with ACS (n=8), or patients with stable angina (SA, n=4). For each patient, gene expression profile was evaluated by microarray technology in ECs exposed to plasma obtained from CS and compared to that of ECs exposed to plasma sampled from Ao.
Project description:We aim to determine blood transcriptome-based molecular signature of acute coronary syndrome (ACS), and to identify novel serum biomarkers for early stage ST-segment-elevation myocardial infarction (STEMI) We obtained peripheral blood from the patients with ACS who visited emergency department within 4 hours after the onset of chest pain: a set of blood samples of patients with STEMI (n=7) before and 7 days after the primary percutaneous coronary intervention (n=7) and normal control (n=10)
Project description:We aim to determine blood transcriptome-based molecular signature of acute coronary syndrome (ACS), and to identify novel serum biomarkers for early stage ST-segment-elevation myocardial infarction (STEMI) We obtained peripheral blood from the patients with ACS who visited emergency department within 4 hours after the onset of chest pain: ST-elevation myocardial infarction (STEMI, n=7), Non-ST-elevation MI (NSTEMI, n=10) and unstable angina (UA, n=9), and normal control (n=7)
Project description:Angiotensin-(1-7) (Ang-(1-7)) is an endogenous heptapeptide from the renin-angiotensin system. The cardioprotective role of Ang-(1-7) has been described due to its anti-inflammatory and anti-fibrotic activities. In this context, we investigated the impact of the oral formulation of Ang-(1-7) vehiculized in hydroxypropyl β-cyclodextrin (HPβCD) on cardiac proteome remodeling after experimental myocardial infarction. For this, Wistar male rats were submitted to short- (7 days) or long-term (60 days) oral treatment with HPβCD/Ang-(1-7) after induction of experimental myocardial infarction (MI)
Project description:Bone marrow and peripheral blood were obtained from patients after myocardial infarction. Erythrocytes were removed by NH4CL lysis to finally obtain nucleated bone marrow cells and peripheral blood leukocytes. After RNA isolation, RNA from three patients was pooled and the RNA expression profile of both organism parts were compared.
Project description:Acquired drug resistance represents a major challenge in chemo-therapy treatment for various types of cancers. We have found that the retinoid X receptor–selective agonist bexarotene (LGD1069, Targretin) was efficacious in treating chemo-resistant cancer cells. The goal of this microarray study was to understand the mechanism of bexarotene’s role in overcoming acquired drug resistance using human breast cancer cells MDA-MB-231 as a model system and paclitaxel as model compound. After MDA-MB-231 cells were repeatedly treated with paclitaxel for 8 cycles with each cycle including a 3-day treatment with 30 nM paclitaxel and followed by a 7-day exposure to control medium, MDA cells resistant to paclitaxel were developed and their growth was no longer inhibited by paclitaxel treatment. Those MDA cells with acquired drug resistance, when treated with paclitaxel and bexarotene in combination, could regain their sensitivity and their growth were again inhibited. Therefore, RNA samples from parental MDA-MB-231 cells, paclitaxel-resistant MDA cells treated with vehicle, paclitaxel alone or in combination with bexarotene, were used for perform global gene expression profiling with Affymetrix HG-U133A gene chips. Keywords: Drug Treatment MDA-MB-231 cells were exposed to regimens on a 10-day cycle: a 3-day treatment with 30 nM paclitaxel and followed by a 7-day exposure to control medium. Paclitaxel resistant MDA-MB-231 cells (MDA-PR) were established within 8 cycles of such treatment (80 days). These MDA-PR cells were then treated with vehicle control, paclitaxel along, or the combination of 30 nM paclitaxel ( 3 days on and 7 days off) and 1 µM Targretin (10 days on) in a new 10-day cycle for 3 months. Thus, there are four treatment groups, parent MDA cells, MDA-PR, MDA-PR treated with paclitaxel, MDA-PR treated with paclitaxel and bexarotene, and each group had four biological replicates.
Project description:Chinese medicine is a complex system guided by traditional Chinese medicine (TCM) theories, which has proven to be especially effective in treating chronic and complex diseases. However, the underlying modes of action (MOA) are not always systematically investigated. Herein, a systematic study was designed to elucidate the multi-compound, multi-target and multi-pathway MOA of a Chinese medicine ,QSYQ, on myocardial infarction. Male Sprague Dawley rat model of myocardial infarction were administered QSYQ intragastrically for 7 days while the control group was not treated. The differentially expressed genes (DEGs) were identified from myocardial infarction rat model treated with QSYQ, followed by constructing a cardiovascular disease (CVD)-related multilevel compound-target-pathway network connecting main compounds to those DEGs supported by literature evidences and the pathways that are functionally enriched. Three conditions were compared with three replicates each: (1) sham, i.e. without left anterior descending coronary artery (LAD) ligation; (2) model, with LAD ligation; (3) QSYQ, with LAD ligation and treated with QSYQ intragastrically for 7 days, the dosage was 105.6 mg/kg once a day. Rats were sacrificed after 7 days of i.g. administration under 10% chloral hydrate anesthesia (300mg/kg). Three tissue samples on the border between infarct and non-infarct area were dissected from left ventricles of each group. The tissue samples were stored at -80℃ refrigerator. Total RNA was extracted using TRIZol Reagent (Invitrogen) and purified using RNeasy Mini kit (QIAGEN), following manufacturers’ protocols. RNA quality was evaluated using an Agilent 2100 Bioanalyzer and electrophoresis in 2% (w/v) agarose gels. Only RNA with RNA integrity numbers (RINs) greater than 7.0 and 28SrRNA/18S rRNA ratio greater than 0.7 was used for microarray analyses. Whole genome microarray analysis was performed using Affymetrix rat Genome 230 2.0 chips.
Project description:Inflammatory mechanisms and immune cells are involved in acute coronary syndromes (ACS) and may lead to acute plaque rupture. However, the local expression of the different genes potentially involved is largely unknown. We therefore performed an Affymetrix analysis of genes expressed in white blood cells obtained from an occluding coronary thrombus or peripheral blood of patients with ST-elevation myocardial infarction. Overall design: Thrombi of ACS patients were harvested from the site of coronary occlusion. Leukocytes were isolated by Ficoll centrifugation. Peripheral blood leukocytes (PBL) were treated in a similar fashion and mRNA was extracted from both cells.