Ay also express ARIA in atherosclerotic plaque. We also confirmed the
Ay also express ARIA in atherosclerotic plaque. We also confirmed the ARIA expression in CD68-positive macrophages by immunofluorescent double staining (Fig. 1C). Furthermore, we discovered that ARIA expression in the aorta of ApoE-deficient mice significantly improved during a high-cholesterol diet program (HCD) feeding as compared with that throughout a typical chow feeding (Fig. 1D). These final results suggest that ARIAVOLUME 290 Number six FEBRUARY six,3786 JOURNAL OF BIOLOGICAL CHEMISTRYARIA Modifies AtherosclerosisFIGURE 1. ARIA regulates PI3KAkt signaling in macrophages. A, quantitative evaluation of ARIA mRNA expression. ARIA was expressed in mouse PMs at a level comparable with mouse aortic endothelial cells (AECs). RAW, NIH3T3, and C2C12 are cell lines for mouse macrophages, fibroblasts, and myoblasts, respectively. Highest expression was detected in mouse endothelial cell line, C166 (n three each). B, immunohistochemistry for ARIA and CD68 in human atherosclerotic plaque. ARIA staining was detected in endothelial cells as indicated by arrowheads. CD68-positive macrophages seem to become optimistic for ARIA staining (arrows). Bar: one hundred m. C, immunofluorescent staining for ARIA (green) and CD68 (red) in human atherosclerotic plaque. The majority of the CD68-positive macrophages are also positive for ARIA. Bar: one hundred m. D, expression of ARIA inside the aortas of ApoE-deficient mice fed ERĪ± Purity & Documentation either HCD or regular chow (NC) for the indicated duration (n four every single). E, immunoblotting for Akt and ARIA-FLAG. Akt BRD4 Accession activity was significantly lowered in RAW macrophages overexpressing ARIA (ARIA-OE). , p 0.05 (n eight each and every). F, immunoblotting for Akt and ARIA-FLAG. Akt activity was substantially decreased in PMs overexpressing ARIA (ARIA-OE). , p 0.01 (n 9 every single). G, immunoblotting for Akt. PMs isolated from ARIA-deficient mice (ARIA ) showed significantly enhanced Akt activity as compared with that in WT macrophages. p-Akt, phospho-Akt; t-Akt, total Akt. , p 0.01 (n 6 every). Error bars in a and D indicate mean S.E.has a possible part inside the improvement of atherosclerosis by modulating macrophage functions. We previously reported that ARIA regulates PI3KAkt signaling in endothelial cells and cardiomyocytes in a cell-autonomous fashion (20, 21). Consequently, we examined irrespective of whether ARIA regulates PI3KAkt signaling in macrophages too. Overexpression of ARIA substantially lowered phosphorylation of Akt in RAW264.7 macrophages (Fig. 1E). Overexpression of ARIA in PMs also decreased Akt phosphorylation (Fig. 1F), whereas genetic loss of ARIA substantially enhanced Akt phosphorylation in PMs (Fig. 1G). These benefits strongly recommend that ARIA also regulates PI3KAkt signaling in macrophages inside a cell-autonomous manner. ARIA Modulates Macrophage Foam Cell Formation–Recently, the critical role of Akt3 in the regulation of macrophage foam cell formation has been reported. Akt3 accelerates the degradation of ACAT-1 that catalyzes the esterification of absolutely free cholesterols for storage into cytoplasmic lipid droplets. Accordingly,FEBRUARY six, 2015 VOLUME 290 NUMBERloss of Akt3 enhanced macrophage foam cell formation by increasing ACAT-1 expression. Mainly because ARIA regulates PI3K Akt signaling in macrophages, we explored whether ARIA modulates macrophage foam cell formation. PMs isolated from WT and ARIA mice exhibited a equivalent uptake of acetylated LDL (Fig. 2A). Nevertheless, PMs isolated from ARIA mice showed a substantial reduction in foam cell formation as compared with PMs from WT mice (Fig. 2B). Inhibition of PI3K ab.