Four-and-a-half LIM domain protein-2 (FHL-2) inhibition reduces atherosclerosis in apolipoprotein E-deficient mice

Abstract

Background: Four-and-a-half LIM domain protein-2 (FHL-2), a member of the FHL family of proteins, is expressed in vascular endothelial and smooth muscle cells and negatively regulates endothelial cell survival and migration. However, its role in atherogenesis is unknown. Methods and results: To investigate the role of FHL-2 in atherosclerosis, we crossed FHL-2 knockout (FHL-2-/-) with apolipoprotein E-deficient (ApoE-/-) mice, and fed them a high-cholesterol, high-fat diet for 7 weeks. FHL-2-/-ApoE-/- mice displayed significantly less atherosclerotic plaque formation, as assessed by oil red O staining, in the aortic sinus (0.14±0.02 vs. 0.29±0.04 mm2) and aorta (6.9±0.9 vs 10.3±1%), compared with ApoE-/- mice. 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 in the aortic sinus of FHL-2-/-ApoE-/- mice, as determined by Sirius red and alpha-actin staining, respectively, compared with ApoE-/- mice. These results suggest that absence of FHL-2 promotes smaller and more stable plaques. However, relative monocyte/macrophage content within the atherosclerotic plaques, as determined by MOMA-2 immunostaining, and in spleens, as determined by FACS analysis, was equivalent in both animals groups. Decreased plaque formation in FHL-2-/-ApoE-/- mice was associated with significantly reduced aortic ICAM-1 mRNA levels by 40%. Moreover, FACS analysis of T cells in spleens showed a significant increase in CD4+CD25+Foxp3+ regulatory T cell numbers, in FHL-2-/-ApoE-/- compared with ApoE-/- mice (19.5±1.6 vs. 16.2±2% of CD3+CD4+ cells, respectively). Conclusions: These results suggest that FHL-2 may play an important role in atherogenesis by promoting plaque formation, involving upregulation of adhesion molecule expression and suppression of regulatory T cells.