ANA BELEN ARROYO RODRIGUEZ, ALBERTO DEL MONTE, NURIA GARCÍA BARBERÁ, MARÍA CALEPRICO, SALAM SALLOUM ASFAR, RAÚL TERUEL MONTOYA, VICENTE VICENTE GARCÍA, VANESSA ROLDAN SCHILLING, ROCÍO GONZÁLEZ-CONEJERO HILLA, VICENTE ANDRÉS, CONSTANTINO MARTÍNEZ GÓMEZ,

• [GI/IMIB/C001/2011] HEMATOLOGÍA Y ONCOLOGÍA MÉDICA CLÍNICO-EXPERIMENTAL

Atherosclerosis is a chronic inflammatory disorder of the vessel wall in which monocytes and macrophages are recruited in atherosclerotic plaques where they further activate, among other the IRAK1/TRAF6/NF-kB pathway. Mir146a is a negative regulator of this pathway, and its overexpression decreases inflammation and arteriosclerosis. Recently, we have shown that the functional allelic variant rs2431697 (TT) of MIR146A gene is associated with lower levels of miR-146a in monocytes and predicts adverse cardiovascular events in anticoagulated patients with atrial fibrillation to promote an increased inflammatory state. The objective of this study was to evaluate the role of miR-146a in the hematopoietic compartment in the development of atherosclerosis in a mouse model.

Thirty mice LDLr -/- irradiated with lethal doses of ? rays were transplanted with bone marrow (BM) of wild-type mice (WT) or miR146a-/- (N = 3 per genotype) (0.5e106 cells/mouse). The effectiveness and efficiency of transplantation was evaluated by flow cytometry after 1 month. After two months of fat-rich diet and cholesterol, the animals were sacrificed. Plasma was obtained for chemistry, hematology and NETosis studies by quantifying dsDNA (Sytox Green) and elastase activity (ELISA). F4/80 (macrophage marker), miR146a-5p, Irak1, and Traf6 mRNA expression were measured by qRT-PCR in thoracic artery. Plaque size was quantified by Oil-Red staining in aortic arch and by immunohistochemistry in heart valves.

After the diet, hematological count and lipid profile were not different in groups with or without miR-146a. We observed significantly higher levels of dsDNA in miR-146a -/- vs. WT mice (348 vs. 177 ng / ml; p = 0.03) as well as elastase levels (229 vs. 113 ng / ml; p = 0.002). There were no significant differences in atherosclerotic lesion size between groups. Aortic macrophage infiltration was higher in mice deficient for miR-146a (p=0.031). Irak1 and Traf6 expression were significantly higher in mice with miR-146a hematopoietic deficiency (p = 0.01 and p=0.039, respectively).

miR-146a deficiency exclusively in the hematopoietic compartment is not sufficient to aggravate atherosclerosis formation in our murine model. However, deficiency of miR-146a appears to modulate both its targets in hematopoietic cells and NETosis processes. Additional studies are needed to clarify the role of miR-146a in NETosis and to assess whether systemic or conditional (e.g. endothelium) deficiency of this miRNA contribute to the formation of atherosclerotic lesions and increase the risk of cardiovascular disease in patients with atrial fibrillation.