P642Identification of LOX-1 and Legumain as novel biomarkers in acute coronary syndromes using differential gene expression comparing coronary thrombi with peripheral blood

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Abstract

Introduction

Leukocytes play a key role in acute coronary syndromes (ACS). We hypothesized that gene expression profiling of leukocytes from the site of coronary occlusion in direct comparison with peripheral leukocytes from patients with ACS might help identify novel biomarkers involved in plaque rupture.

Methods

Thrombi (Thr) were aspirated during primary percutaneous coronary intervention (PCI), snap-frozen, and stored at -80°C. Peripheral blood mononuclear cells (PBMC) from the same patients were extracted and treated accordingly. Total RNA was isolated from thrombi and PBL. cDNA samples were hybridized to Affimetrix GeneChip Human Genome U133 Plus 2.0 arrays. An Affymetrix GeneChip Scanner was used to measure the fluorescent intensity emitted by the labelled target. Differentially expressed genes were confirmed by RT-PCR on the same materials used for the gene array. Confirmation on the protein level on individual cells was performed by flow cytometry on fresh thrombi and blood subjected to mechanical and lysis protocols. In addition, ELISAs were performed on sera from matched patients with ST-elevation MI (n=19), stable CAD (n=18) and healthy controls (n=13), respectively.

Results

Of a total of 14 patients recruited, after quality controls gene expression profiles from 4 patients were used. Among 54'675 probe sets 200 genes were differentially upregulated in Thr compared to PBMC as defined by a more than 2-fold increase in expression, statistical significance (p ≤ 0.01) and false discovery rate (FDR ≤ 0.05). Among those, LOX-1 showed a 105-fold (p=9.84E-06) and Legumain a 50-fold upregulation (p=0.000424). Flow cytometric analysis (n=5) confirmed a marked upregulation of LOX-1 predominantly on monocytes/macrophages and similarly for Legumain on granulocytes. Soluble LOX-1 was not different between patients with STEMI, stable CAD and healthy controls.

Conclusion

This pilot study identified two novel cell-bound biomarkers in patients with ACS – Legumain and LOX-1 - using an unbiased gene expression approach. Current studies address their role in diagnostic differentiation in patients with ACS and in the pathophysiology of plaque rupture.

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