GENETIC AND EXPRESSION ANALYSIS OF CANDIDATE GENES IN CAROTID PLAQUE AND PERIPHERAL BLOOD OF PATIENTS WITH ATHEROSCLEROSIS

Atherosclerosis, a systemic pathological process, is the underlying cause of the majority of clinical cardiovascular events, such as stroke and myocardial infarction. Inflammation plays a central role in the development of atherosclerotic disease, from the early phases of lesion formation to plaque disruption, the main underlying cause of acute ischemic syndromes. Prostaglandin E2 (PGE2) is a prostanoid implicated in a variety of pathologies, whose concentration is elevated in inflammatory process. Cyclooxygenase (COX) and Prostaglandin E Synthase (PGES) are enzymes responsible for PGE2 synthesis. In this work we investigated if genes of COX-2/PGE2 pathway could be differentially expressed in carotid plaques and peripheral blood of symptomatic compared to asymptomatic patients. In order to evaluate if upregulated genes in peripheral blood of stroke patients may be specifically associated to atherothrombotic ischemic stroke, we analyzed expression of these genes also in cardioembolic stroke patients. Moreover, we investigated if specific polymorphisms in the promoter region of the upregulated genes could influence their expression. Expression of COX-2, TLR4, ACE, EP4, EP3, EPRAP and FACL4 genes was analyzed by Real Time PCR and their correlation was studied by Pearson coefficient and linear regression models. The results obtained by this study showed that TLR4 and COX-2 genes were upregulated and their expression was strongly correlated in peripheral blood of symptomatic patients. Both COX-2 and TLR4 genes are overexpressed only in atherothrombotic stroke. -2604G>A polimorphism is associated with the levels of TLR4 gene expression in peripheral blood. In particular, the presence of allele -2604A in homozygosis resulted in diminished gene expression. Analysis in silico evidenced that allele -2604A creates a putative binding site for GATA-2, a negative transcription regulator. Although these results would need to be confirmed by independent studies, they suggest that the peripheral mechanism of inflammatory injury after atherothrombotic stroke may be mediated by TLR4 through a COX-2-dependent pathway. Furthermore, the specificity of both genes for atherothrombotic stroke indicates that gene expression analysis in peripheral blood might be used as biomarkers for stroke subtype diagnosis. The deregulation of the gene TLR4 may be due by the presence of the polymorphism-2604G>A in atherosclerotic patients. Case/control studies in larger populations are warranted to determine if this variation can represent a risk factor for atherosclerosis.