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Angiopoietin-like protein 8 accelerates atherosclerosis in ApoE−/ mice

  • Xiaolu Jiao
    Affiliations
    Beijing Anzhen Hospital, Capital Medical University, The Key Laboratory of Upper Airway Dysfunction-related Cardiovascular Diseases, Beijing, 10029, China

    Beijing Anzhen Hospital, Capital Medical University, The Key Laboratory of Remodeling-Related Cardiovascular Diseases, Ministry of Education, Beijing, 100029, China

    Beijing Institute of Heart Lung and Blood Vessel Disease, Beijing, 100029, China
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  • Yunyun Yang
    Affiliations
    Beijing Anzhen Hospital, Capital Medical University, The Key Laboratory of Upper Airway Dysfunction-related Cardiovascular Diseases, Beijing, 10029, China

    Beijing Anzhen Hospital, Capital Medical University, The Key Laboratory of Remodeling-Related Cardiovascular Diseases, Ministry of Education, Beijing, 100029, China

    Beijing Institute of Heart Lung and Blood Vessel Disease, Beijing, 100029, China
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  • Linyi Li
    Affiliations
    Beijing Anzhen Hospital, Capital Medical University, The Key Laboratory of Upper Airway Dysfunction-related Cardiovascular Diseases, Beijing, 10029, China

    Beijing Anzhen Hospital, Capital Medical University, The Key Laboratory of Remodeling-Related Cardiovascular Diseases, Ministry of Education, Beijing, 100029, China

    Beijing Institute of Heart Lung and Blood Vessel Disease, Beijing, 100029, China
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  • Huahui Yu
    Affiliations
    Beijing Anzhen Hospital, Capital Medical University, The Key Laboratory of Upper Airway Dysfunction-related Cardiovascular Diseases, Beijing, 10029, China

    Beijing Anzhen Hospital, Capital Medical University, The Key Laboratory of Remodeling-Related Cardiovascular Diseases, Ministry of Education, Beijing, 100029, China

    Beijing Institute of Heart Lung and Blood Vessel Disease, Beijing, 100029, China
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  • Yunxiao Yang
    Affiliations
    Beijing Anzhen Hospital, Capital Medical University, Department of Cardiology, Beijing, 10029, China
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  • Juan Li
    Affiliations
    Beijing Anzhen Hospital, Capital Medical University, The Key Laboratory of Upper Airway Dysfunction-related Cardiovascular Diseases, Beijing, 10029, China

    Beijing Anzhen Hospital, Capital Medical University, The Key Laboratory of Remodeling-Related Cardiovascular Diseases, Ministry of Education, Beijing, 100029, China

    Beijing Institute of Heart Lung and Blood Vessel Disease, Beijing, 100029, China
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  • Yunhui Du
    Affiliations
    Beijing Anzhen Hospital, Capital Medical University, The Key Laboratory of Upper Airway Dysfunction-related Cardiovascular Diseases, Beijing, 10029, China

    Beijing Anzhen Hospital, Capital Medical University, The Key Laboratory of Remodeling-Related Cardiovascular Diseases, Ministry of Education, Beijing, 100029, China

    Beijing Institute of Heart Lung and Blood Vessel Disease, Beijing, 100029, China
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  • Jing Zhang
    Affiliations
    Beijing Anzhen Hospital, Capital Medical University, The Key Laboratory of Upper Airway Dysfunction-related Cardiovascular Diseases, Beijing, 10029, China

    Beijing Anzhen Hospital, Capital Medical University, The Key Laboratory of Remodeling-Related Cardiovascular Diseases, Ministry of Education, Beijing, 100029, China

    Beijing Institute of Heart Lung and Blood Vessel Disease, Beijing, 100029, China
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  • Chaowei Hu
    Affiliations
    Beijing Anzhen Hospital, Capital Medical University, The Key Laboratory of Upper Airway Dysfunction-related Cardiovascular Diseases, Beijing, 10029, China

    Beijing Anzhen Hospital, Capital Medical University, The Key Laboratory of Remodeling-Related Cardiovascular Diseases, Ministry of Education, Beijing, 100029, China

    Beijing Institute of Heart Lung and Blood Vessel Disease, Beijing, 100029, China
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  • Yanwen Qin
    Correspondence
    Corresponding author. No. 2 Anzhen Road, Chaoyang District, Beijing, 100029, China.
    Affiliations
    Beijing Anzhen Hospital, Capital Medical University, The Key Laboratory of Upper Airway Dysfunction-related Cardiovascular Diseases, Beijing, 10029, China

    Beijing Anzhen Hospital, Capital Medical University, The Key Laboratory of Remodeling-Related Cardiovascular Diseases, Ministry of Education, Beijing, 100029, China

    Beijing Institute of Heart Lung and Blood Vessel Disease, Beijing, 100029, China
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Open AccessPublished:July 07, 2020DOI:https://doi.org/10.1016/j.atherosclerosis.2020.06.014

      Highlights

      • ANGPTL8 promotes the development of atherosclerosis.
      • ANGPTL8 overexpression promotes macrophages to become foam cell that accumulate more cholesterol.
      • ANGPTL8 increases uptake and decreases efflux of cholesterol in macrophages.
      • ANGPTL8 induces the expression of CD36 and SR-A, and inhibits the expression of SR-BI.

      Abstract

      Background and aims

      Angiopoietin-like protein 8 (ANGPTL8) is a hormone involved in regulating lipid metabolism. Patients with coronary artery disease have markedly higher plasma levels of ANGPTL8 than controls; however, the role of ANGPTL8 in atherosclerosis has not been explored. Therefore, we explored the effects of ANGPTL8 on atherosclerosis development in a mouse model.

      Methods

      We induced experimental atherosclerosis in ApoE−/− mice ANGPTL8-knockdown. and ANGPTL8-overexpression ApoE−/− mice. We also explored the mechanism using ANGPTL8-overexpression macrophages.

      Results

      ANGPTL8 expression was increased in human and mouse atherosclerotic lesions. ANGPTL8 overexpression promoted the development of atherosclerosis whereas ANGPTL8 knockdown protected against atherosclerosis. Immunofluorescence co-staining results showed that ANGPTL8 was expressed in macrophages in atherosclerotic plaques. Compared with wild type cells, ANGPTL8-overexpressing macrophages, including bone marrow-derived macrophages and Raw 264.7 macrophages, showed enhanced foam cell formation and increased accumulation of cholesterol that was induced by increased uptake and decreased efflux of cholesterol. The results of this study also showed that ANGPTL8 induced the expression of CD36 and scavenger receptor (SR)-A, and inhibited the expression of SR-BI.

      Conclusions

      Our findings demonstrate an unanticipated role of ANGPTL8 in the development of atherosclerosis and regulation of foam cell formation. ANGPTL8 may be a promising new target for atherosclerosis.

      Keywords

      1. Introduction

      Atherosclerotic cardiovascular disease (ASCVD) is one of the leading causes of death and disability worldwide [
      • Tzoulaki I.
      • Castagne R.
      • Boulange C.L.
      • Karaman I.
      • Chekmeneva E.
      • et al.
      Serum metabolic signatures of coronary and carotid atherosclerosis and subsequent cardiovascular disease.
      ]. Atherosclerosis is the main underlying cause of cardiovascular disease, which is traditionally considered a lipid-driven disease [
      • Brandsma E.
      • Kloosterhuis N.J.
      • Koster M.
      • Dekker D.C.
      • Gijbels M.J.J.
      • et al.
      A proinflammatory gut microbiota increases systemic inflammation and accelerates atherosclerosis.
      ]. Foam cell formation and accumulation in the subendothelial space of the vascular wall is a hallmark of atherosclerotic lesions [
      • Wang D.
      • Yang Y.
      • Lei Y.
      • Tzvetkov N.T.
      • Liu X.
      • et al.
      Targeting foam cell formation in atherosclerosis: therapeutic potential of natural products.
      ]. The foam cells eventually collapse and disintegrate, releasing intracellular cholesterol, which is the most important component of atherosclerotic plaque, and promoting the progression of atherosclerosis. Although there are many theories for the pathogenesis of atherosclerosis, which include lipid deposition, inflammation, arterial smooth muscle proliferation, reverse cholesterol transportation and others, its development remains unclear and requires continued urgent studies.
      Recent studies showed that genetic and therapeutic antagonism of angiopoietin-like proteins (ANGPTLs) in humans and mice were associated with levels of lipid fractions and ASCVD [
      • Dewey F.E.
      • Gusarova V.
      • Dunbar R.L.
      • O'Dushlaine C.
      • Schurmann C.
      • et al.
      Genetic and pharmacologic inactivation of ANGPTL3 and cardiovascular disease.
      ,
      • Stitziel N.O.
      • Khera A.V.
      • Wang X.
      • Bierhals A.J.
      • Vourakis A.C.
      • et al.
      ANGPTL3 deficiency and protection against coronary artery disease.
      ]. A previous study found that chronic ANGPTL2 infusion accelerates formation of atherosclerotic plaque in ApoE–/– mice [
      • Farhat N.
      • Thorin-Trescases N.
      • Mamarbachi M.
      • Villeneuve L.
      • Yu C.
      • et al.
      Angiopoietin-like 2 promotes atherogenesis in mice.
      ]. A human monoclonal antibody against ANGPTL3 resulted in a greater decrease in atherosclerotic lesion area and necrotic content compared with a control antibody in dyslipidemic mice [
      • Dewey F.E.
      • Gusarova V.
      • Dunbar R.L.
      • O'Dushlaine C.
      • Schurmann C.
      • et al.
      Genetic and pharmacologic inactivation of ANGPTL3 and cardiovascular disease.
      ]. Genetic knockout of ANGPTL4 protects ApoE–/–mice against the development of atherosclerosis and strongly suppresses the ability of the macrophages to become foam cells [
      • Adachi H.
      • Fujiwara Y.
      • Kondo T.
      • Nishikawa T.
      • Ogawa R.
      • et al.
      Angptl 4 deficiency improves lipid metabolism, suppresses foam cell formation and protects against atherosclerosis.
      ].
      ANGPTL8, also known as betatrophin [
      • Abu-Farha M.
      • Abubaker J.
      • Al-Khairi I.
      • Cherian P.
      • Noronha F.
      • et al.
      Circulating angiopoietin-like protein 8 (betatrophin) association with HsCRP and metabolic syndrome.
      ], TD26 [
      • Dong X.Y.
      • Pang X.W.
      • Yu S.T.
      • Su Y.R.
      • Wang H.C.
      • et al.
      Identification of genes differentially expressed in human hepatocellular carcinoma by a modified suppression subtractive hybridization method.
      ], RIFL (re-feeding induced fat and liver) [
      • Ren G.
      • Kim J.Y.
      • Smas C.M.
      Identification of RIFL, a novel adipocyte-enriched insulin target gene with a role in lipid metabolism.
      ], lipasin [
      • Fu Z.
      • Yao F.
      • Abou-Samra A.B.
      • Zhang R.
      Lipasin, thermoregulated in brown fat, is a novel but atypical member of the angiopoietin-like protein family.
      ] and PRO1185 [
      • Li Y.
      • Teng C.
      Angiopoietin-like proteins 3, 4 and 8: regulating lipid metabolism and providing new hope for metabolic syndrome.
      ], is an atypical member of the ANGPTL family because it lacks the C-terminal fibrinogen-like domain, but shares a common coiled-coil domain at the N-terminus with ANGPTL3 and ANGPTL4 [
      • Zhang R.A.-S.A.
      A dual role of lipasin (betatrophin) in lipid metabolism and glucose homeostasis_ consensus and controversy.
      ]. The N-terminal coiled-coil domain is involved in lipid regulation [
      • Ono M.
      • Shimizugawa T.
      • Shimamura M.
      • Yoshida K.
      • Noji-Sakikawa C.
      • et al.
      Protein region important for regulation of lipid metabolism in angiopoietin-like 3 (ANGPTL3): ANGPTL3 is cleaved and activated in vivo.
      ] by directly inhibiting lipoprotein lipase (LPL) activity [
      • Quagliarini F.
      • Wang Y.
      • Kozlitina J.
      • Grishin N.V.
      • Hyde R.
      • et al.
      Atypical angiopoietin-like protein that regulates ANGPTL3.
      ]. ANGPTL8 may activate ANGPTL3. ANGPTL8 coimmunoprecipitated with the N-terminal domain of ANGPTL3 in mouse plasma and increased the appearance of N-terminal ANGPTL3 and plasma TG levels [
      • Quagliarini F.
      • Wang Y.
      • Kozlitina J.
      • Grishin N.V.
      • Hyde R.
      • et al.
      Atypical angiopoietin-like protein that regulates ANGPTL3.
      ]. ANGPTL8 also plays an important role in glucose metabolism [
      • Zielinska A.
      • Maciulewski R.
      • Siewko K.
      • Poplawska-Kita A.
      • Lipinska D.
      • et al.
      Levels of betatrophin decrease during pregnancy despite increased insulin resistance, beta-cell function and triglyceride levels.
      ].
      Although the metabolic functions of ANGPTL8 are well characterized, little is known about its pathophysiological roles in atherosclerosis, a chronic disease intimately associated with metabolic syndrome. It has been reported that patients with ASCVD have remarkably higher levels of ANGPTL8 than controls among patients with diabetes [
      • Huang Y.
      • Fang C.
      • Guo H.
      • Hu J.
      Increased angiopoietin-like protein 8 levels in patients with type 2 diabetes and cardiovascular disease.
      ], and our previous research suggested that the level of circulating ANGPTL8 is increased in patients with coronary artery disease compared with the control group among a non-diabetic population [
      • Jiao X.
      • He J.
      • Yang Y.
      • Yang S.
      • Li J.
      • et al.
      Associations between circulating full-length angiopoietin-like protein 8 levels and severity of coronary artery disease in Chinese non-diabetic patients: a case-control study.
      ]. To date, the role of ANGPTL8 in atherosclerosis has not been explored. In this study, we investigated the impact of ANGPTL8 on the pathogenesis of atherosclerosis in apolipoprotein ApoE–/– mice.

      2. Materials and methods

      Human atherosclerotic lesions were collected from patients undergoing carotid endarterectomy at Beijing Anzhen Hospital. Control aortic samples were obtained from heart transplantation donors in Beijing Anzhen Hospital; those with collagen disease or atherosclerotic disease were excluded as previously described [
      • Qin Y.
      • Wang Y.
      • Liu O.
      • Jia L.
      • Fang W.
      • et al.
      Tauroursodeoxycholic acid attenuates angiotensin II induced abdominal aortic aneurysm formation in apolipoprotein E-deficient mice by inhibiting endoplasmic reticulum stress.
      ]. All protocols were approved by the ethics committee of the Medical Faculty of Beijing Anzhen Hospital, Capital Medical University, and conducted in accordance with the principles of the Declaration of Helsinki. All participants gave written informed consent [
      • Qin Y.
      • Wang Y.
      • Liu O.
      • Jia L.
      • Fang W.
      • et al.
      Tauroursodeoxycholic acid attenuates angiotensin II induced abdominal aortic aneurysm formation in apolipoprotein E-deficient mice by inhibiting endoplasmic reticulum stress.
      ]. Human specimens were fixed in 10% formalin, embedded in paraffin, and sectioned at 5-μm thickness (n = 6 per group).
      Animal experiments were approved by the Institute of Institutional Animal Care and Use Committee of Capital Medical University. Animal protocols were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. ApoE–/– male mice (8 weeks of age) were fed a western diet for 12 weeks and divided into three groups: control (n = 20), ANGPTL8-overexpression (n = 20), and ANGPL8-knockdown (n = 20). All mice were housed in a room with a controlled temperature (23 ± 1 °C) and a 12-h light-dark cycle, and had free access to water and food. Details are described in the Data Supplement.
      Additional details of experimental procedures are included in the Data Supplement.
      Statistical analysis was performed using Prism 5.0 software (GraphPad Software, San Diego, CA, USA) and data are presented as mean ± standard error of the mean. In all cases, at least three independent experiments were performed. Statistical comparisons between two groups were performed using Student's t-test. For multiple comparison tests, one-way analysis of variance with Bonferroni's correction was employed. Values of p < 0.05 were considered statistically significant.

      3. Results

      3.1 Expression of ANGPTL8 is increased in human and mouse atherosclerotic lesions

      Several clinical studies have observed a significantly elevated serum level of ANGPTL8 in patients with atherosclerosis [
      • Huang Y.
      • Fang C.
      • Guo H.
      • Hu J.
      Increased angiopoietin-like protein 8 levels in patients with type 2 diabetes and cardiovascular disease.
      ,
      • Jiao X.
      • He J.
      • Yang Y.
      • Yang S.
      • Li J.
      • et al.
      Associations between circulating full-length angiopoietin-like protein 8 levels and severity of coronary artery disease in Chinese non-diabetic patients: a case-control study.
      ]. The levels of ANGPTL8 in liver and blood vessel tissues were consistently progressively elevated in ApoE–/– mice, with spontaneous development of hypercholesterolemia and atherosclerosis (Fig. 1A and B). Next, we examined the level of ANGPTL8 in atherosclerotic lesions of human carotids (Fig. 1C). The results showed that ANGPTL8 protein levels were markedly increased in plaques versus control vessels both in mice and humans. As demonstrated by immunofluorescent co-staining, ANGPTL8 in atherosclerotic lesions was expressed predominantly in macrophages, both in mouse and human carotid atherosclerotic lesions (Fig. 1D and E). These data suggested a potential role of ANGPTL8 in the development of atherosclerosis, as macrophages are one of the major cell types that contribute to atherogenesis.
      Fig. 1
      Fig. 1ANGPTL8 is expressed in macrophages and is highly expressed in atherosclerotic lesions.
      ANGPTL8 expression was assessed by Western blotting in the liver (A), vessel (B) and tissues of C57BL/6J and week-old ApoE−/− mice of the indicated ages (n = 6 in each group). Representative immunostaining and semiquantitative analysis of ANGPTL8 in human carotid atherosclerotic lesions versus control vessels (C) (n = 6 per group). Co-staining of MAC-2 (red) and ANGPTL8 (green) in 20-week-old ApoE–/– mice (D) and human carotid atherosclerotic lesions (E). Data are presented as means ± standard error of the mean. *p < 0.05 vs. control group.

      3.2 ANGPTL8 promotes the development of atherosclerosis

      To explore the pathophysiological roles of ANGPTL8 in atherosclerosis, we generated ANGPTL8-knockdown and ANGPTL8-overexpression mice. Genetic modifications were confirmed by quantitative real-time PCR and Western blot analysis of mouse liver and vessel tissues (Supplementary Fig. S1A-D). There were no differences in liver function (Supplementary Fig. S1E-F) among the three groups.
      Atherosclerotic lesion formation was assessed after 12 weeks of feeding with a Western diet. There were no obvious differences in food intake and body weight among the three groups. Analysis by Oil Red O staining in the aortic sinus showed a significant decrease in lipid accumulation in ANGPTL8-knockdown mice and a significant increase in lipid accumulation in ANGPTL8-overexpression mice compared with control mice (Fig. 2A). Additional histological evaluation showed that the plaque areas, in the aortic sinus of ANGPTL8-overexpression mice, were 2.2-fold greater than those in control mice, and were 2.1-fold lesser in ANGPTL8-knockdown mice than controls (Fig. 2B). These results suggested that ANGPTL8 may play an important role in the aggravation of atherosclerosis. Likewise, macrophage infiltration in the atherosclerotic lesion area of the aortic sinus in ANGPTL8-overexpression mice was significantly higher than that in controls, and was significantly lower in ANGPTL8-knockdown mice compared with controls (Fig. 2C). However, there were no differences in smooth muscle proliferation and collagen deposition in the atherosclerotic lesion area among the three groups (Fig. 2D and E).
      Fig. 2
      Fig. 2ANGPTL8 promotes atherosclerosis development in ApoE −/− mice fed a western diet.
      Aortas were dissected from 20-week-old ApoE–/–, ANGPTL8-knockdown ApoE–/– and ANGPTL8-overexpression ApoE–/– mice (n = 8 in each group). (A) En face staining of the entire aortas of 20-week-old mice with Oil red O. (B) Cross-sections of aortic sinus arteries of 20-week-old mice. (C) Macrophage infiltration and (D) smooth muscle proliferation in the aortic sinus as determined by immunostaining for MAC-2 and SMA-a respectively. (E) Collagen content was assessed using Masson's trichrome stain. Data are presented as means ± standard error of the mean. *p < 0.05.

      3.3 ANGPTL8 increases the plasma levels of TG instead of LDL-C

      Because ANGPTL8 is an important metabolic regulator, we next investigated the levels of plasma lipids under ANGPTL8-knockdown and ANGPTL8-overexpression in ApoE–/– mice. The results showed that the levels of plasma TG in ANGPTL8-overexpression mice were significantly higher compared with control mice, whereas the levels of plasma TG in ANGPTL8-knockdown mice were lower in ANGPTL8-overexpression mice than controls (Fig. 3A). The levels of plasma apolipoprotein (Apo) A1 were lower in ANGPTL8-overexpression mice and higher in ANGPTL8-knockdown mice compared with controls (Fig. 3E). There were no differences in the levels of plasma TC, LDL-C, HDL-C, ApoB and glucose (Fig. 3B–D, F-G) among the three groups.
      Fig. 3
      Fig. 3ANGPTL8 increases the plasma levels of TG instead of LDL-C.
      Eight-week-old ApoE–/–, ANGPTL8-knockdown ApoE–/– and ANGPTL8-overexpression ApoE–/– mice were fed a western diet and euthanized at 20 weeks. Plasma samples were collected for measurement of triglycerides (TG; A), total cholesterol (TC; B), low-density lipoprotein cholesterol (LDL-C; C), high-density lipoprotein cholesterol (HDL-C; D), apolipoprotein A-1 (ApoA-1; E), apolipoprotein B (AopB; F) and glucose (GLU; G). Data are presented as means ± standard error of the mean. *p < 0.05.

      3.4 ANGPTL8 promotes macrophage foam cell formation

      The experiments described above showed that ANGPTL8 was expressed in macrophages and that there were more macrophages in the lesions of ANGPTL8-overexpression mice and less macrophages in the lesions of ANGPTL8-knockdown mice compared with controls. Macrophages play an important role in inflammation, and inflammation enhances atherogenesis. We examined the expression of several inflammatory factors. Quantitative real-time PCR analysis demonstrated that expression of IL-1b, MCP-1 and TNF-α was equivalent among the three groups (Supplementary Fig. S2 A-C). Likewise, there were no differences in the circulating levels of these proinflammatory chemokines among the three groups (Supplementary Fig. S2 D-F).
      Macrophage-derived foam cell formation is a crucial step in the pathogenesis of atherosclerosis. We assessed the possible involvement of ANGPTL8 in foam cell formation and found a time-dependent increase in ANGPTL8 protein levels in Ox-LDL-treated bone marrow-derived macrophages (BMDMs) (Fig. 4A). To further explore the role of ANGPTL8 in foam cell formation, we transfected BMDMs with an ANGPTL8-overexpression lentiviral vector. Cells overexpressing ANGPTL8 were confirmed by Western blot and quantitative real-time PCR analysis (Supplementary Fig. S3). ANGPTL8-overexpression and control BMDMs were incubated with Ox-LDL to induce foam cell formation. Cells overexpressing ANGPTL8 showed enhanced foam cell formation (Fig. 4B) and accumulated more cholesterol (Fig. 4C) compared with control cells. We also transfected Raw 264.7 macrophages with ANGPTL8-overexpression lentiviral vector (Supplementary Fig. S4) to further verify the role of ANGPTL8 in foam cell formation. The results also found that ANGPTL8 protein levels were increased in Ox-LDL-treated Raw 264.7 macrophages (Supplementary Fig. S5A). ANGPTL8-overexpression Raw 264.7 cells also showed enhanced foam cell formation and accumulated more cholesterol (Supplementary Fig. S5B-C). These results indicated that ANGPTL8 promotes foam cell formation.
      Fig. 4
      Fig. 4ANGPTL8 promotes foam cell formation in primary bone marrow-derived macrophages (BMDMs).
      (A) ANGPTL8 expression was assessed by Western blotting in BMDMs incubated with oxidized low-density lipoprotein (Ox-LDL; 50 μg/mL) for the indicated time. (B) Increased foam cell formation and (C) increased accumulation of cholesterol in ANGPTL8-overexpressing BMDMs after treatment with Ox-LDL (50 μg/mL). (D) Total uptake of Dil-Ox-LDL was quantified in wild type and ANGPTL8-overexpressing BMDMs. (E) Fluorescence intensity in cells incubated with Dil-Ox-LDL (20 μg/mL) for 24 h was assessed using a microplate reader. (F) Cholesterol efflux to high-density lipoprotein (HDL) in BMDMs was detected for each group. Data are presented as means ± standard error of the mean of three biologically independent experiments. *p < 0.05.

      3.5 ANGPTL8 promotes the uptake of modified lipoproteins and inhibits cholesterol efflux

      To investigate whether increased uptake of modified forms of LDL could account for the enhanced foam cell formation in ANGPTL8-overexpression macrophages, we performed uptake assays with Dil-labeled Ox-LDL. Immunofluorescence revealed greatly increased uptake in ANGPTL8-overexpression cells compared with control cells both in BMDMs (Fig. 4D and E) and Raw 264.7 (Supplementary Fig. S5D).
      Next, we examined whether ANGPTL8 is involved in cholesterol efflux using NBD-labeled cholesterol to analyze the efflux. The results showed that ANGPTL8 had negative effects on HDL-mediated cholesterol efflux capacity in BMDMs (Fig. 4F) and Raw 264.7 (Supplementary Fig. S5F).

      3.6 ANGPTL8 alters the expression of scavenger receptor

      To gain insights into potential mechanisms by which ANGPTL8 promoted foam cell formation, we assessed the expression of receptors involved in Ox-LDL uptake and transporters involved in cholesterol efflux. The expression levels of scavenger receptors CD36 and SR-A, which are the principal receptors responsible for the uptake of modified lipoproteins, were significantly increased in macrophages overexpressing ANGPTL8. Conversely, the levels of SR-BI, which is the transporter responsible for cholesterol efflux, were decreased significantly. However, the levels of ATP-binding cassette transporter A1 (ABCA1), the transporter responsible for cholesterol efflux, were unchanged (Fig. 5A and B). Importantly, the protein levels in blood vessels and the liver in mice showed a similar expression tendency (Fig. 6A and B). These results indicated that ANGPTL8 regulates the expression of scavenger receptors, promotes the uptake of modified lipoproteins, and inhibits cholesterol efflux.
      Fig. 5
      Fig. 5ANGPTL8 regulates the expression of scavenger receptors in macrophages.
      Expression of CD36, scavenger receptor (SR)-A, SR-BI and ATP-binding cassette reporter A1(ABCA1) was assessed by Western blotting in BMDMs (A) and RAW 267.4 cells (B) incubated with oxidized low-density lipoprotein (Ox-LDL; 50 μg/mL) for 24 h.
      Fig. 6
      Fig. 6ANGPTL8 regulates the expression of scavenger receptors in tissues of ApoE–/– mice.
      Expression of CD36, SR-A, SR-BI and ABCA1 was assessed by Western blotting of blood vessels (A) and liver tissues (B) of ApoE–/–, ANGPTL8-knockdown ApoE–/–, and ANGPTL8-overexpression ApoE–/– mice. Data are presented as means ± standard error of the mean of three biologically independent experiments. *p < 0.05.

      4. Discussion

      Despite intensive research on the metabolic functions of ANGPTL8, its role in the cardiovascular system has barely been explored. This study provides novel evidence that ANGPTL8 overexpression causes a marked exacerbation of atherosclerosis while ANGPTL8 knockdown protects against atherosclerosis, suggesting that ANGPTL8 plays an important role in vascular diseases.
      ANGPTL8 is an atypical member of the ANGPTLs and recent studies showed that both genetic and therapeutic antagonism of ANGPTLs in humans and mice was associated with lipid metabolism and atherosclerotic cardiovascular disease [
      • Adachi H.
      • Fujiwara Y.
      • Kondo T.
      • Nishikawa T.
      • Ogawa R.
      • et al.
      Angptl 4 deficiency improves lipid metabolism, suppresses foam cell formation and protects against atherosclerosis.
      ]. Low-frequency missense variants in the ANGPTL4 (E40K) gene protect against the risk of coronary artery disease [
      • Myocardial Infarction G.
      • Investigators C.A.E.C.
      • Stitziel N.O.
      • Stirrups K.E.
      • Masca N.G.
      • et al.
      Coding variation in ANGPTL4, LPL, and SVEP1 and the risk of coronary disease.
      ] and ANGPTL3 loss-of-function mutations lower the risk of coronary artery disease in humans [
      • Stitziel N.O.
      • Khera A.V.
      • Wang X.
      • Bierhals A.J.
      • Vourakis A.C.
      • et al.
      ANGPTL3 deficiency and protection against coronary artery disease.
      ]. Experimental evidence has demonstrated that anti-ANGPTL3 therapies have an important anti-atherosclerotic effect [
      • Dewey F.E.
      • Gusarova V.
      • Dunbar R.L.
      • O'Dushlaine C.
      • Schurmann C.
      • et al.
      Genetic and pharmacologic inactivation of ANGPTL3 and cardiovascular disease.
      ]; indeed, the role of ANGPTL3 inhibitors in cardiovascular disease may be to regulate lipid metabolism [
      • Geladari E.
      • Tsamadia P.
      • Vallianou N.G.
      ANGPTL3 inhibitors- their role in cardiovascular disease through regulation of lipid metabolism.
      ]. ANGPTL4 deficiency improved lipid metabolism and protected against atherosclerosis [
      • Adachi H.
      • Fujiwara Y.
      • Kondo T.
      • Nishikawa T.
      • Ogawa R.
      • et al.
      Angptl 4 deficiency improves lipid metabolism, suppresses foam cell formation and protects against atherosclerosis.
      ]. ANGPTL8 plays an important role in lipid metabolism. Although many clinical studies have shown that ANGPTL8 is associated with the levels of circulating TG, TC, LDL-C and HDL-C, in ANGPTL8 knockout mice, only plasma TG levels are significantly reduced, while cholesterol levels are unaltered [
      • Wang Y.
      • Quagliarini F.
      • Gusarova V.
      • Gromada J.
      • Valenzuela D.M.
      • et al.
      Mice lacking ANGPTL8 (Betatrophin) manifest disrupted triglyceride metabolism without impaired glucose homeostasis.
      ]. Previous studies have shown that serum TG levels in ANGPTL8 null mice were one-third of those in wide type mice. Another study showed that adenoviral ANGPTL8 overexpression in mice increased serum TG levels [
      • Zhang R.
      Lipasin, a novel nutritionally-regulated liver-enriched factor that regulates serum triglyceride levels.
      ]. In this study, we found that the levels of circulating TG were higher in ANGPTL8-overexpression mice and lower in ANGPTL8-knockdown mice compared with the controls. This suggests that ANGPTL8 promotes atherosclerosis partly by increasing plasma TG.
      ANGPTLs could affect the development of atherosclerosis through other ways besides affecting blood lipids. Analysis of the protein structure of ANGPTL3 has led to the hypothesis that, beyond its role in lipid metabolism, this molecule may have a proinflammatory and proangiogenic effect, as well as a negative effect on cholesterol efflux [
      • Lupo M.G.
      • Ferri N.
      Angiopoietin-like 3 (ANGPTL3) and atherosclerosis: lipid and non-lipid related effects.
      ]. Adachi et al. reported that ANGPTL4 deficiency suppressed foam cell formation and protected against atherosclerosis [
      • Adachi H.
      • Fujiwara Y.
      • Kondo T.
      • Nishikawa T.
      • Ogawa R.
      • et al.
      Angptl 4 deficiency improves lipid metabolism, suppresses foam cell formation and protects against atherosclerosis.
      ]. In contrast, Georgiadi et al. reported that recombinant ANGPTL4 significantly decreased uptake of Ox-LDL by macrophages, suppressing foam cell formation to reduce atherosclerosis development [
      • Georgiadi A.
      • Wang Y.
      • Stienstra R.
      • Tjeerdema N.
      • Janssen A.
      • et al.
      Overexpression of angiopoietin-like protein 4 protects against atherosclerosis development.
      ]. Further research is needed to clarify the role of ANGPTL4 in foam cell formation. ANGPTL8 is thought to have similar functions to ANGPTL3 and ANGPTL4 because their N-terminal domains share 20% sequence identity [
      • Li Y.
      • Teng C.
      Angiopoietin-like proteins 3, 4 and 8: regulating lipid metabolism and providing new hope for metabolic syndrome.
      ]. Current evidence supports a notion that ANGPTL8 may activate ANGPTL3 [
      • Quagliarini F.
      • Wang Y.
      • Kozlitina J.
      • Grishin N.V.
      • Hyde R.
      • et al.
      Atypical angiopoietin-like protein that regulates ANGPTL3.
      ] and ANGPTL8 also requires ANGPTL3 for its effects on LPL [
      • Haller J.F.
      • Mintah I.J.
      • Shihanian L.M.
      • Stevis P.
      • Buckler D.
      • et al.
      ANGPTL8 requires ANGPTL3 to inhibit lipoprotein lipase and plasma triglyceride clearance.
      ]. ANGPTL8 also can form complexes with either ANGPTL3 or ANGPTL4 when the proteins are refolded together from their denatured states [
      • Kovrov O.
      • Kristensen K.K.
      • Larsson E.
      • Ploug M.
      • Olivecrona G.
      On the mechanism of angiopoietin-like protein 8 for control of lipoprotein lipase activity.
      ]. A previous study reported that ANGPTL8 has a negative effect on HDL-mediated cholesterol efflux capacity in THP-1 cell models [
      • Luo M.
      • Zhang Z.
      • Peng Y.
      • Wang S.
      • Peng D.
      The negative effect of ANGPTL8 on HDL-mediated cholesterol efflux capacity.
      ]. ApoA1, the main protein component of HDL-C (65%–70%), promotes cellular cholesterol efflux and cholesterol transportation from peripheral tissues to the liver [
      • Chen B.D.
      • Chen X.C.
      • Yang Y.N.
      • Gao X.M.
      • Ma X.
      • et al.
      Apolipoprotein A1 is associated with SYNTAX score in patients with a non-ST segment elevation myocardial infarction.
      ]. The results of our study showed the levels of ApoA1 were higher in ANGPTL8-knockdown mice and lower in ANGPTL8-overexpression mice. The findings of our study suggested that ANGPTL8 may play a role in cellular cholesterol efflux. Our data also revealed an unexpected enhancement effect of ANGPTL8 overexpression on foam cell formation that was related to improved uptake and impaired efflux of cholesterol. It is therefore suggested that ANGPTL8 may play a role in the formation of foam cells either directly or indirectly (by interfering with ANGPTL3 or ANGPTL4).
      The scavenger receptors in macrophages play critical roles in foam cell formation by affecting cholesterol uptake and efflux of [
      • Patten D.A.
      • Shetty S.
      More than just a removal service: scavenger receptors in leukocyte trafficking.
      ]. Our present study clearly demonstrated that ANGPTL8 increased the expression of SR-A and CD36, which are the primary receptors responsible for cholesterol uptake, and decreased the expression of SR-BI, which is one of the key factors in cholesterol efflux. To the best of our knowledge, many signal transduction pathways are involved in the expression of scavenger receptor. The activation of the Akt pathway could inhibit the activity of the SR-BI promoter, decreasing the expression of SR-BI [
      • Y X.
      • M K.
      • I H.
      • C W.M.
      • L J.
      • et al.
      Regulation of scavenger receptor class BI gene expression by angiotensin II in vascular endothelial cells.
      ]. Activation of the ERK/MAPK pathway could profoundly upregulate the expression of CD36 and suppress the expression of ABCG1 and SRBI in macrophages, thereby facilitating the cholesterol influx capacity of macrophages [
      • S S.
      • D D.
      • H S.
      • J N.
      High-density lipoprotein from subjects with coronary artery disease promotes macrophage foam cell formation: role of scavenger receptor CD36 and ERK/MAPK signaling.
      ]. Blocking ERK phosphorylation could alleviate ox-LDL-dependent up-regulation of CD36 expression and inhibit the formation of foam cell and inflammation [
      • Zq L.
      • Xy H.
      • Cy H.
      • Zs Z.
      • Y C.
      • et al.
      Geniposide protects against ox-LDL-induced foam cell formation through inhibition of MAPKs and NF-kB signaling pathways.
      ]. It is reported that ANGPTL8 could activate the Akt and ERK signal transduction pathway. Over-expression of ANGPTL8 enhanced the insulin-stimulated activation of the Akt-GSK3β or Akt-FoxO1 pathway, no matter whether the cells were insulin resistant or not [
      • Guo X.R.
      • Wang X.L.
      • Chen Y.
      • Yuan Y.H.
      • Chen Y.M.
      • et al.
      ANGPTL8/betatrophin alleviates insulin resistance via the Akt-GSK3 beta or Akt-FoxO1 pathway in HepG2 cells.
      ]. ANGPTL8 activated the ERK signal transduction pathway in hepatocytes, adipocytes, and pancreatic β-cells [
      • Z Y.
      • L S.
      • D W.
      • X C.
      • W H.
      • et al.
      Angiopoietin-like protein 8 (betatrophin) is a stress-response protein that down-regulates expression of adipocyte triglyceride lipase.
      ]. We inferred that ANGPTL8 increased the expression of SR-A and CD36, decreased the expression of SR-BI and promoted the formation of foam cells, maybe by activating the Atk and ERK transduction pathway. This should be confirmed in future studies.
      Our rodent data indicated that the levels of ANGPTL8 in hepatic and vessel tissues were markedly increased in mice with development of atherosclerosis, and the expression of ANGPTL8 in hepatic tissues was higher than in vessel tissues. It is suggested that the liver may be the major site for the production ANGPTL8 in response to atherosclerosis.
      There are some limitations to this study. First, the role of ANGPTL8 in the promotion of atherosclerosis was only characterized using total ANGPTL8 overexpression and knockdown mice, instead of mice with conditional ANGPTL8 knockout/overexpression in macrophages, which does not allow to observe a direct effect on atherosclerosis of ANGPTL8 in macrophages. Second, we only used male mice because of the high success rate and good reproducibility in this model. However, certain cardiovascular characteristics, such as blood pressure, serum lipid profile, endothelial function, and abundance of thrombotic plaques differ between females and males. Third, the relationship between scavenger receptors and ANGPTL8 could have been further examined in this mouse model. We plan to explore the specific roles of the scavenger receptor by exposing SR-A, SR-BI and CD36 knockout mice.

      4.1 Conclusions

      In summary, our study identifies a prominent role for ANGPTL8 in the development of atherosclerotic lesions and provides a mechanism explaining the link between ANGPTL8 and atherosclerosis, which is partly attributable to the acceleration of foam cell formation induced by increasing the uptake and decreasing the efflux of cholesterol. These findings provide an impetus for further analysis of the role of ANGPTL8 in other metabolic diseases, and point to ANGPTL8 as a target for therapeutic interventions in cardiovascular disease.

      Financial support

      This study was supported by the National Natural Science Foundation of China (Grant Nos. 81670331 , 81970224 , 81870335 ) and the Beijing Natural Science Foundation (Grant No. 7192030 ).

      Authors contribution statement

      XLJ performed the experiments. YYY and LYJ prepared the human samples. HUY,YXY and JL performed animal model. YHD, JZ and CWH performed data analysis.YWQ designed the study and prepared the manuscript. All authors read and approvedthe final manuscript.

      Declaration of competing interest

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

      Acknowledgments

      We thank Michelle Kahmeyer-Gabbe, PhD, from Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for the English editing of this manuscript.

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