Unknown,Transcriptomics,Genomics,Proteomics

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MicroRNA array analysis of platelets in premature coronary artery disease

ABSTRACT: Coronary artery disease (CAD) is the leading cause of human morbidity and mortality worldwide, underscoring the need to improve diagnostic strategies. Platelets play a major role, not only in the process of acute thrombosis during plaque rupture, but also in the formation of atherosclerosis itself. MicroRNAs are endogenous small non-coding RNAs that control gene expression and are expressed in a tissue and disease-specific manner. Therefore they have been proposed to be useful biomarkers. It remains unknown whether differences in miRNA expression levels in platelets can be found between patients with premature CAD and healthy controls. Methodology/Principal Findings In this case-control study we measured relative expression levels of platelet miRNAs using microarrays from 12 patients with premature CAD and 12 age- and sex-matched healthy controls. Six platelet microRNAs were significantly upregulated (miR340*, miR451, miR454*, miR545:9.1. miR615-5p and miR624*) and one miRNA (miR1280) was significantly downregulated in patients with CAD as compared to healthy controls. To validate these results, we measured the expression levels of these candidate miRNAs by qRT-PCR in platelets of individuals from two independent cohorts; validation cohort I consisted of 40 patients with premature CAD and 40 healthy controls and validation cohort II consisted of 27 patients with artery disease and 40 healthy relatives. MiR340* and miR624* were confirmed to be upregulated in patients with CAD as compared to healthy controls in both validation cohorts. Conclusion/Significance Two miRNAs in platelets are significantly upregulated in patients with CAD as compared to healthy controls. miRNA array analysis of isolated platelets from subjects with premature coronary artery disease compared to healthy control subjects.

ORGANISM(S): Homo sapiens

SUBMITTER: Brigitte Sondermeijer

PROVIDER: E-GEOD-28858 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Coronary artery disease (CAD) is the leading cause of human morbidity and mortality worldwide, underscoring the need to improve diagnostic strategies. Platelets play a major role, not only in the process of acute thrombosis during plaque rupture, but also in the formation of atherosclerosis itself. MicroRNAs are endogenous small non-coding RNAs that control gene expression and are expressed in a tissue and disease-specific manner. Therefore they have been proposed to be useful biomarkers. The aim of this study was to investigate whether differences in miRNA expression levels in platelets can be found (i) between patients with premature CAD and healthy controls and (ii) within healthy controls after and before aspirin and statin administration. In this case-control study we measured expression levels of platelet miRNAs using microarrays from 40 male patients with premature CAD and 40 age- and sex-matched healthy controls. Premature CAD was defined as a cardiac event before the age of 51 years. The patients were selected from the outpatient clinic of the Academic Medical Center (AMC) of Amsterdam, which is specialised in premature CAD. The control cohort was composed of 40 healthy Caucasian male volunteers, who were recruited by advertisement and who were matched with the cases for age and smoking habits. Individuals of this control cohort did not have a history of CVD, nor did they have a positive family history of CVD and they were not allowed to use any medication. Patients and controls were excluded when they suffered from diabetes. Most CAD patients use aspirin and statins as secondary prevention. The influence of these drugs on miRNA profiles is unknown. To assess the possible influence of medication on miRNA expression and to control for medication as confounding factor, we asked 27 volunteers in our control cohort to also use these drugs. We administered simvastatin 40 mg, once daily, for 6 weeks and during the last two weeks we added 100mg of acetyl salicylic acid, once daily. Blood samples including isolated platelets were collected at baseline in the absence of aspirin and statins and after six weeks of medication use. We also assessed platelet function using the Multiplate® Analyzer (Roche) in the absence of aspirin and statin use. In short, 300 µl whole blood was diluted with 300 µl 0.9% saline and stirred for 3 minutes at 37 ºC. Adenosine diphosphate (ADP) was added in a final concentration of 2.5 ?mol/L to initiate platelet aggregation. Aggregation was measured for 6 minutes and was reported in arbitrary aggregation units plotted against time. Also, the area under the aggregation curve (AUC) was measured. All samples were measured in the absence and presence of 200 ?mol/L indomethacin (20 min incubation with blood) to mimic the effect of aspirin use. We calculated the percentage reduction in AUC after incubation with indomethacin as an in vitro measure of the effect of aspirin use on whole blood platelet aggregation.

Project description:Monocytes and T-cells play an important role in the development of atherosclerotic coronary artery disease (CAD). Differences in transcriptional activity of these cells might reflect the individual's atherosclerotic burden. Transcriptome analysis of circulating mononuclear cells from carefully matched atherosclerotic and control patients will potentially provide insights into the pathophysiology of atherosclerosis and supply biomarkers for diagnostic purposes. From patients undergoing coronary angiography because of anginal symptoms, we carefully matched 18 patients with severe triple-vessel CAD to 13 control patients without signs of CAD on angiography. All patients were on statin and aspirin treatment. RNA from circulating CD4+ T-cells, CD14+ monocytes, lipopolysaccharide-stimulated monocytes, macrophages and CD34+ progenitor cells was subjected to genome-wide expression analysis. Only CD14+ monocytes demonstrated that a small number of genes involved in activation was overexpressed in control patients, which was verified by real-time polymerase-chain reaction. In this pilot study, cautious matching of patients with severe atherosclerotic CAD with control patients without angiographic signs of coronary atherosclerosis did not reveal differences in transcriptional activity in four out of five different mononuclear cell types. In resting monocytes from patients without overt CAD some inflammatory genes were overexpressed as compared to patients with severe CAD. Large inter-individual variability prevented the use of single differentially expressed genes as biomarkers. Keywords: disease-state analysis In total 153 arrays were analyzed with 6 technical replicates (147 biological samples). CD34+ stem cells, CD4+ T-cells, resting CD14+ monocytes, stimulated monocytes and macrophages were analyzed, all from patient with severe coronary atherosclerosis or controls that had no coronary atherosclerosis as determined angiographically, and which were carefully matched for age and gender.

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MicroRNA array analysis of platelets in premature coronary artery disease

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