ABSENCE OF FOUR-AND-A-HALF LIM DOMAIN PROTEIN 2 DECREASES ATHEROSCLEROSIS IN APOE-/- MICE: ROLE OF MONOCYTIC IMMUNE CELLS

Abstract

Four-and-a-half LIM domain protein-2 (FHL2) is expressed in endothelial and vascular smooth muscle cells. It negatively regulates endothelial cell survival and migration, but its role in atherogenesis is unknown. To investigate the role of FHL2 in atherosclerosis, FHL2-deficient (FHL2-/-) mice were crossed with ApoE-deficient (ApoE -/-) mice, to generate ApoE/FHL2-/- mice. After 7 weeks of high fat diet, ApoE/FHL2-/- mice had significantly smaller (P<0.05) atherosclerotic plaques than ApoE-/- mice in the aortic sinus (0.14±0.02 vs. 0.29±0.04 mm2), the brachiocephalic artery (0.03±0.008 vs. 0.07±0.01 mm2) and the aorta (6.9±0.9 vs 10.3±1%), assessed by oil red O staining. This was associated with enhanced collagen (16±2 vs 8.6±3 %) and smooth muscle cell (4.5±0.8 vs 1.8±0.5%) contents within the plaques of ApoE/FHL-2-/- mice. Moreover, macrophage content within plaques was reduced 2-fold in ApoE/FHL2-/- vs ApoE-/- mice (P<0.05). This could be explained, in part, by the 40% reduction in aortic ICAM-1 mRNA and 55% reduction in VCAM-1 protein expression in the plaque. Furthermore, aortic gene expression of Cx3cl1 and Ccl5 was increased in ApoE/FHL2-/- vs ApoE-/- mice, suggesting that different monocyte populations might be recruited to the plaques. Peritoneal thioglycollate injection elicited equivalent numbers of monocytes and macrophages in both groups, but a significantly lower number of pro-inflammatory Ly6C high monocytes were recruited in ApoE/FHL2-/- vs ApoE-/- mice (8±3 vs 18±4%). Furthermore, mRNA levels of Cx3cr1 were 2-fold higher in monocytes from ApoE/FHL2-/- mice compared with ApoE-/- mice. Finally, we also investigated the potential importance of myeloid cell FHL2 deficiency in atherosclerosis. ApoE-/- or ApoE/FHL2-/- mice were lethally irradiated and transplanted with ApoE-/- or ApoE/FHL2-/- bone marrow. After recovery and 7 weeks of high fat diet, both chimeric groups developed smaller plaques than ApoE-/- mice transplanted with ApoE-/- bone marrow. These results suggest that FHL2 in both myeloid and vascular cells may play an important role in atherosclerosis by promoting pro-inflammatory chemokine production, adhesion molecule expression, and pro-inflammatory monocyte recruitment.