Targeting Filamin A Reduces Macrophage Activity and Atherosclerosis

Abstract

Background: The actin-binding protein FLNA (filamin A) regulates signal transduction important for cell locomotion, but the role of macrophage-specific FLNA during atherogenesis has not been explored. Methods: We analyzed FLNA expression in human carotid atherosclerotic plaques by immunofluorescence. We also produced mice with Flna -deficient macrophages by breeding conditional Flna -knockout mice ( Flna o/fl ) with mice expressing Cre from the macrophage-specific lysosome M promoter ( LC ). Atherosclerosis in vivo was studied by transplanting bone marrow from male Flna o/fl / LC mice to atherogenic low-density lipoprotein receptor–deficient ( Ldlr –/– ) mice; and by infecting Flna o/fl and Flna o/fl / LC mice with AdPCSK9 (adenoviral vector overexpressing proprotein convertase subtilisin/kexin type 9). Furthermore, C57BL/6 mice were infected with AdPCSK9 and then treated with the calpain inhibitor calpeptin to inhibit FLNA cleavage. Results: We found that macrophage FLNA expression was higher in advanced than in intermediate human atherosclerotic plaques. Flna o/fl / LC macrophages proliferated and migrated less than controls; expressed lower levels of phosphorylated AKT and ERK1/2; exhibited reduced foam cell formation and lipid uptake; and excreted more lipids. The deficiency of Flna in macrophages markedly reduced the size of aortic atherosclerotic plaques in both Ldlr –/–BMT: Flnao/fl/LC and AdPCSK9-infected Flna o/fl / LC mice. Intima/media ratios and numbers of CD68-positive macrophages in atherosclerotic plaques were lower in Flna -deficient mice than in control mice. Moreover, we found that STAT3 interacts with a calpain-cleaved carboxyl-terminal fragment of FLNA. Inhibiting calpain-mediated FLNA cleavage with calpeptin in macrophages reduced nuclear levels of phosphorylated STAT3, interleukin 6 secretion, foam cell formation, and lipid uptake. Finally, calpeptin treatment reduced the size of atherosclerotic plaques in C57BL/6 mice infected with AdPCSK9. Conclusions: Genetic inactivation of Flna and chemical inhibition of calpain-dependent cleavage of FLNA impaired macrophage signaling and function, and reduced atherosclerosis in mice, suggesting that drugs targeting FLNA may be useful in the treatment of atherosclerosis.

Myriocin and d-PDMP ameliorate atherosclerosis in ApoE−/− mice via reducing lipid uptake and vascular inflammation

“…We sought to understand the major signaling pathways that underlie myriocin-and d-PDMP-induced atheroprotection. Accumulating evidence has highlighted the critical roles played by MAP kinase (p38, ERK and JNK) and nuclear factor-κB (NF-κB) signaling pathways in inflammatory responses [20]. Compared with the vehicle group, Figure 6J-L).…”

Lack of RAC1 in macrophages protects against atherosclerosis

“…All mouse experimental procedures were followed in accordance with institutional guidelines of University of Gothenburg, Sweden. To produce mice that are deficient for RAC1 in macrophages, female C57BL/6 mice homozygously expressing the floxed Rac1 gene (Rac1 fl/fl ) [6] were crossbred with male mice homozygously expressing Cre under the monocyte-specific lysozyme M promoter (LC) [12]. Mice were genotyped for Rac1 deficiency as previously described [16].…”

“…18S transcripts were included as internal loading controls. Purity of BMMs was shown by CD68 immunofluorescence staining as previously described [12]. BMMs were then stained by immunofluorescence using an anti-actin phalloidin antibody (Life Technologies) as well as FLNA CT (Bethyl Laboratories).…”

“…Secreted levels of IL-6, IL-10, IL-12 and TNF-α were detected by ELISA (eBioscience) from either mouse serum or cultured BMMs treated by LPS [10]. Using modified Boyden chambers, proliferation and migration of BMMs were assessed up to 6 days and 18 hours, respectively [12].…”

“…Secondly, RAC1 induces actin reorganization during cell membrane ruffling and phagocytosis [9] and inhibition of targeting actin dynamics in BMMs could be a potential target for treating atherosclerosis [10]. Finally, actin remodeling partly regulates lipid uptake in macrophages, RAC1 targets actin-binding protein filamin A (FLNA) to remodel the cytoskeleton [11] and FLNA is involved in both of these processes [12].…”