ANGPTL4 deficiency in haematopoietic cells promotes monocyte expansion and atherosclerosis progression

ARTICLE Received 31 Dec 2015 | Accepted 21 Jun 2016 | Published 27 Jul 2016 DOI: 10.1038/ncomms12313 OPEN ANGPTL4 deficiency in haematopoietic cells promotes monocyte expansion and atherosclerosis progression Binod Aryal1,2,3, Noemi Rotllan1,2,*, Elisa Araldi1,2,*, Cristina M. Ramı́rez1,2, Shun He4, Benjamin G. Chousterman4, Ashley M. Fenn4, Amarylis Wanschel3, Julio Madrigal-Matute3, Nikhil Warrier3, Jose L. Martı́n-Ventura5, Filip K. Swirski4, Yajaira Suárez1,2 & Carlos Fernández-Hernando1,2 Lipid accumulation in macrophages has profound effects on macrophage gene expression and contributes to the development of atherosclerosis. Here, we report that angiopoietin-like protein 4 (ANGPTL4) is the most highly upregulated gene in foamy macrophages and it’s absence in haematopoietic cells results in larger atherosclerotic plaques, characterized by bigger necrotic core areas and increased macrophage apoptosis. Furthermore, hyperlipidemic mice deficient in haematopoietic ANGPTL4 have higher blood leukocyte counts, which is associated with an increase in the common myeloid progenitor (CMP) population. ANGPTL4-deficient CMPs have higher lipid raft content, are more proliferative and less apoptotic compared with the wild-type (WT) CMPs. Finally, we observe that ANGPTL4 deficiency in macrophages promotes foam cell formation by enhancing CD36 expression and reducing ABCA1 localization in the cell surface. Altogether, these findings demonstrate that haematopoietic ANGPTL4 deficiency increases atherogenesis through regulating myeloid progenitor cell expansion and differentiation, foam cell formation and vascular inflammation. 1 Vascular Biology and Therapeutics Program, Yale University School of Medicine, New Haven, Connecticut 06520, USA. 2 Integrative Cell Signaling and Neurobiology of Metabolism Program, Section of Comparative Medicine and Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520, USA. 3 Departments of Medicine and Cell Biology, Leon H. Charney Division of Cardiology and Cell Biology, New York University School of Medicine, New York, New York 10016, USA. 4 Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA. 5 Vascular Research Lab, IIS-Fundación Jimenez-Dı́az, Universidad Autónoma de Madrid, Madrid 28040, Spain. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to C. F.-H. (email: ) or to Y.S. (). NATURE COMMUNICATIONS | 7:12313 | DOI: 10.1038/ncomms12313 | www.nature.com/naturecommunications 1 ARTICLE D NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12313 uring the early stages of atherosclerosis, modified lipoproteins, primarily oxidized low density lipoproteins (Ox-LDL) accumulate in the intima, and activate endothelial and smooth muscle cells, recruit circulating monocytes into the sub-endothelial layer. Here, monocytes differentiate into macrophages, scavenge Ox-LDL, accumulate neutral lipids and transform into foam cells1,2. Foam cell formation is a protective mechanism whereby the vessel wall rids itself of potentially harmful lipids. However, accumulation of large numbers of foam cells in the arterial wall leads to the generation of atherosclerotic plaques1. Furthermore, both macrophages and foam cells play a key role in mediating inflammatory response in athero-plaques. Apart from foam cells, the monocyte count in blood circulation independently predicts risk for coronary artery disease after adjustment for conventional risk factors3. Monocytosis and neutrophilia have been observed in animal models of atherosclerosis including pigs and rabbits, and seem to contribute to atherogenesis4,5. Previous studies have demonstrated that hyperlipidemia-induced leukocytosis in different mouse models including Abca1  /  or Abcg1  /  and Apoe  /  mice is associated with the expansion and proliferation of haematopoietic stem and multipotential progenitor cells (HSPCs) in the bone marrow (BM)6–8. Recent studies have shown that a family of proteins called angiopoietinlike proteins (ANGPTLs), particularly ANGPTL2 and ANGPTL5, are known to stimulate the expansion of haematopoietic stem cells ex vivo9–11. In addition, ANGPTL4 has been shown maintains in vivo repopulation capacity of CD34 þ human cord blood cells12. ANGPTL4 is a multifunctional protein that regulates many metabolic and non-metabolic processes through its distinct N-terminal and C-terminal domains13–17. Particularly, ANGPTL4 is a strong inhibitor of lipoprotein lipase (LPL), an enzyme that catalyses the hydrolysis of triglycerides (TG) from very LDL (VLDL) and chylomicrons, and regulates the uptake of circulating lipids into tissues18,19. As a result, overexpression of ANGPTL4 in mice leads to hypertriglyceridemia, whereas deficiency leads to lowering of circulating lipids20. Interestingly, human studies have shown that a common sequence variant near the ANGPTL4 gene is associated with decreased plasma TGs and increased high-density lipoprotein cholesterol (HDL-C) levels, and ANGPTL4 expression is positively associated with metabolic parameters including levels of insulin, fatty acids and leptin21. Although decreased lipid content is generally atheroprotective, E40K, a loss of function variant of ANGPTL4, was associated with increased coronary heart disease risk despite being associated with an atheroprotective lipid profile, suggesting that ANGPTL4 influences parameters beyond lipid levels22. ANGPTL4 is highly expressed in adipose tissues, placenta, liver, kidney and heart in humans23,24. It’s expression is regulated by fasting, PPARs, glucocorticoids, and hypoxia in a tissuespecific manner23–26. ANGPTL4 is also highly abundant in macrophages and it is induced by dietary fatty acids27. Angptl4  /  mice develop severe inflammation and accumulate foam cells in the mesenteric lymph nodes when fed a diet high in saturated fat27. This suggests that ANGPTL4 is a critical regulator of macrophage functions. Moreover, studies from overexpression or depletion of LPL in macrophages demonstrate that LPL promotes the binding and uptake of modified LDLs by macrophages and thus enhances foam cell formation28,29. ANGPTL4 can be expected to inhibit and reverse LPL-mediated effects in macrophages and atherosclerosis. However, there have been no studies addressing the direct role of macrophage ANGPTL4 during atherogenesis. Studies using global knockout or transgenic overexpression mouse models suggest both pro- and anti-atherogenic roles of ANGPTL4 (refs 30,31). These confounding observations could have resulted from diverse 2 roles of ANGPTL4 in regulating multiple metabolic parameters and inflammation, which could influence the progression of atherosclerosis. In the present study, we demonstrate that haematopoieticspecific ANGPTL4 plays a critical role in the progression of atherosclerosis. We show that haematopoietic ANGPTL4 deficiency in Ldlr  /  mice resul (...truncated)