Lanatoside C Promotes Foam Cell Formation and Atherosclerosis

www.nature.com/scientificreports OPEN Lanatoside C Promotes Foam Cell Formation and Atherosclerosis Huairui Shi*, Xiaobo Mao*, Yucheng Zhong*, Yuzhou Liu, Xiaoqi Zhao, Kunwu Yu, Ruirui Zhu, Yuzhen Wei, Jianghao Zhu, Haitao Sun, Yi Mao & Qiutang Zeng received: 26 August 2015 accepted: 30 December 2015 Published: 29 January 2016 Lanatoside C’s impact on atherosclerosis is poorly understood. The present study was conducted to determine whether lanatoside C affects the development of atherosclerosis in apolipoprotein E-deficient (ApoE–/–) mice. ApoE–/– mice were administered either phosphate-buffered saline (PBS) containing 0.1% DMSO (the vehicle control group) or lanatoside C at low (1 mg/kg per day) or high (2 mg/ kg per day) doses, and fed a Western diet for 12 weeks. Lanatoside C dose-dependently aggravated the development of atherosclerosis in the ApoE–/– mice compared with the vehicle control group. In an effort to determine the mechanism by which lanatoside C increased atherosclerosis, we found that lanatoside C significantly promoted the uptake of oxidised low-density lipoprotein (oxLDL) and increased foamcell formation by upregulation of scavenger receptor class A (SR-A) and the class B scavenger receptor (CD36) in macrophages. Meanwhile, the effects of lanatoside C were abolished using small interfering RNA (siRNA) inhibition of peroxisome proliferator-activated receptors β/δ (PPARβ/δ). Overall, our data demonstrate that lanatoside C aggravates the development of atherosclerosis by inducing PPARβ/δ expression, which mediates upregulation of SR-A and CD36, and promotes oxLDL uptake and foam-cell formation. Atherosclerosis is a chronic disease of the large arteries that is an important cause of morbidity and mortality in industrialised nations1,2. Lipoprotein uptake by monocyte-derived macrophages is thought to be one of the earliest pathogenic events in the nascent plaque, and results in the development of early foam-cell formation3,4. The role of foam cells as the major culprit in atherosclerosis has been further demonstrated by the resistance to atherosclerosis in ApoE–/– mice5,6. This unrestricted uptake through scavenger receptor pathways, which is not limited by intracellular cholesterol levels, eventually leads to the formation of foam cells, the initial step in atherosclerosis7,8. Foam-cell formation is increased by several extracellular factors, especially uncontrolled uptake of oxidised low-density lipoprotein (oxLDL) that exceeds cholesterol influx, subsequently triggering the formation of foam cells9. The intracellular lipid homeostasis of macrophages is dynamically regulated by oxLDL uptake and cholesterol efflux. Macrophage scavenger receptor class A (SR-A) and CD36, a member of the type B family, are thought to play significant roles in foam-cell formation because of their ability to promote uptake-modified lipids, such as oxLDL7,10. The absence of CD36 and SR-A reduced 75%–90% of oxLDL uptake internalization by macrophages in some studies11–13. The removal of cellular cholesterol is critical for preventing foam-cell formation and the development of atherosclerotic lesions14–16. ATP-binding cassette (ABC) transporters (ABCA1 and ABCG1) and SR-BI, another type B scavenger receptor, protect against foam-cell formation when expressed in macrophages, by stimulating cholesterol efflux17–19. The nuclear receptor subfamily of the peroxisome proliferator-activated receptors (PPARs) family consists of α (NR1C1), β /δ (NR1C3) and γ (NR1C2) isoforms20–23. PPARs exert profound effects on the metabolism of lipoproteins, fatty acids and inflammatory responses24–28. PPARβ /δ is expressed in many tissues, particularly the gut, kidneys and heart. PPARβ /δ increases lipid accumulation by increasing expression of SR-A and CD3629,30. Cheng published a paper showing that cardiac glycoside digoxin increases PPARβ /δ expression in H9c2 cells31. Lanatoside C, as a US Food and Drug Administration (FDA)-approved cardiac glycoside, is used in the treatment of congestive heart failure and cardiac arrhythmia, and recent studies have found that lanatoside C also inhibits several negative-strand RNA viruses32,33. However, its impact on atherosclerosis is poorly understood. The present study was conducted to determine whether the cardiac glycoside lanatoside C affects the development of atherosclerosis in ApoE–/– mice. The Laboratory of Cardiovascular Immunology, Institute of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to Q.Z. (email ) Scientific Reports | 6:20154 | DOI: 10.1038/srep20154 1 www.nature.com/scientificreports/ Figure 1. Lanatoside C promotes the development of atherosclerosis in ApoE–/– mice. (A) Representative images of Oil Red O staining of en face preparations of the aortas in the different treatment groups (low-dose lanatoside C, 1 mg/kg per day; high-dose lanatoside C, 2 mg/kg per day) or vehicle control (PBS containing 0.1% DMSO). (B) Quantitative analysis of the atherosclerotic surface area of the entire aorta. (C) Cryosections of the aortic sinus stained with Oil Red O, with hematoxylin used as a counterstain. (D) The lesion sizes in the aortic roots were averaged to determine the lesion size of ten sections of the aortic sinus. The data are expressed as mean ±  SEM. One-way ANOVA was followed by the Holm-Sidak test. *P ≤  0.05; **P ≤  0.01; ***P ≤  0.001; C, n =  8; LanC-LD, n =  8; LanC-HD, n =  8; C (vehicle control); LanC-LD (low-dose lanatoside C); LanC-HD (high-dose lanatoside C). Results Lanatoside C aggravates atherosclerosis development in ApoE–/– mice. The ApoE−/− mouse is a well-established animal model for studying atherosclerosis . Our preliminary experiment showed that the different doses of lanatoside C caused various toxic reactions and death. ApoE–/– mice were given different doses of lanatoside C and observed for 48 h. A higher dose (3 mg/kg per day and 4 mg/kg per day) could increase the risk of toxic reaction and death (Table S1), but not 2mg/kg per day. To investigate the potential effects of lanatoside C on atherosclerosis, we evaluated the severity of atherosclerosis based on the morphological and histological changes that occurred in mice treated with approximately 20 μ g of lanatoside C (low-dose, 1 mg/kg per day) or 40 μ g of lanatoside C (high-dose, 2 mg/kg per day), compared to the vehicle control (PBS containing 0.1% DMSO). After the 12-week lanatoside C treatment, lanatoside C levels were measured 24 h following the final injection (lowdose lanatoside C, 0.9 ±  0.10 ng/ml; high-dose lanatoside C, 2.2 ±  0.15 ng/ml) (Fig. S1). Lanatoside C aggravated the development of atherosclerosis (Fig. 1A), and the extent of atherosclerosis in the mice receiving 2 mg/kg of lanatoside C was approximately 60% greater than in the vehicle control mice (Fig. (...truncated)