Sashidar Bandaru1, Chandu Ala1, Reza Salimi1, Murali K Akula2, Matias Ekstrand3, Sravani Devarakonda1, Joakim Karlsson2,4, Jimmy Van den Eynden1,5, Göran Bergström3,6, Erik Larsson1, Max Levin3, Jan Borén3, Martin O Bergo2,7, Levent M Akyürek1,8. 1. Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine (S.B., C.A., R.S., S.D., J.V.d.E., E.L., L.M.A.), Sahlgrenska Academy, University of Gothenburg, Sweden. 2. Sahlgrenska Cancer Center, Sahlgrenska Academy (M.K.A., J.K., M.O.B.), Sahlgrenska Academy, University of Gothenburg, Sweden. 3. Department of Molecular and Clinical Medicine, Institute of Medicine (M.E., G.B., M.L., J.B.), Sahlgrenska Academy, University of Gothenburg, Sweden. 4. Department of Surgery, Institute of Clinical Sciences (J.K.), Sahlgrenska Academy, University of Gothenburg, Sweden. 5. Department of Human Structure and Repair, Anatomy and Embryology Unit, Ghent University, Belgium (J.V.d.E.). 6. Region Västra Götaland, Sahlgrenska University Hospital, Department of Clinical Physiology, Göteborg, Sweden (G.B.). 7. Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden (M.O.B.). 8. Region Västra Götaland, Sahlgrenska University Hospital, Department of Clinical Pathology and Cytology, Göteborg, Sweden (L.M.A.).
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.
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 humanatherosclerotic 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-deficientmice 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.
Authors: Mercy A Arkorful; Nicole Noren Hooten; Yongqing Zhang; Amirah N Hewitt; Lori Barrientos Sanchez; Michele K Evans; Douglas F Dluzen Journal: Genes (Basel) Date: 2020-05-20 Impact factor: 4.096
Authors: Sashidar Bandaru; Chandu Ala; Matias Ekstrand; Murali K Akula; Matteo Pedrelli; Xi Liu; Göran Bergström; Liliana Håversen; Jan Borén; Martin O Bergo; Levent M Akyürek Journal: PLoS One Date: 2020-09-17 Impact factor: 3.240