Role of Endothelial Cell−Derived Angptl2 in Vascular Inflammation Leading to Endothelial Dysfunction and Atherosclerosis Progression Eiji Horio, Tsuyoshi Kadomatsu, Keishi Miyata, Yasumichi Arai, Kentaro Hosokawa, Yasufumi Doi, Toshiharu Ninomiya, Haruki Horiguchi, Motoyoshi Endo, Mitsuhisa Tabata, Hirokazu Tazume, Zhe Tian, Otowa Takahashi, Kazutoyo Terada, Motohiro Takeya, Hiroyuki Hao, Nobuyoshi Hirose, Takashi Minami, Toshio Suda, Yutaka Kiyohara, Hisao Ogawa, Koichi Kaikita and Yuichi Oike Arterioscler Thromb Vasc Biol. 2014;34:790-800; originally published online February 13, 2014; doi: 10.1161/ATVBAHA.113.303116 Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2014 American Heart Association, Inc. All rights reserved. Print ISSN: 1079-5642. Online ISSN: 1524-4636
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://atvb.ahajournals.org/content/34/4/790
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Role of Endothelial Cell–Derived Angptl2 in Vascular Inflammation Leading to Endothelial Dysfunction and Atherosclerosis Progression Eiji Horio, Tsuyoshi Kadomatsu, Keishi Miyata, Yasumichi Arai, Kentaro Hosokawa, Yasufumi Doi, Toshiharu Ninomiya, Haruki Horiguchi, Motoyoshi Endo, Mitsuhisa Tabata, Hirokazu Tazume, Zhe Tian, Otowa Takahashi, Kazutoyo Terada, Motohiro Takeya, Hiroyuki Hao, Nobuyoshi Hirose, Takashi Minami, Toshio Suda, Yutaka Kiyohara, Hisao Ogawa, Koichi Kaikita, Yuichi Oike Objective—Cardiovascular disease (CVD), the most common morbidity resulting from atherosclerosis, remains a frequent cause of death. Efforts to develop effective therapeutic strategies have focused on vascular inflammation as a critical pathology driving atherosclerosis progression. Nonetheless, molecular mechanisms underlying this activity remain unclear. Here, we ask whether angiopoietin-like protein 2 (Angptl2), a proinflammatory protein, contributes to vascular inflammation that promotes atherosclerosis progression. Approach and Results—Histological analysis revealed abundant Angptl2 expression in endothelial cells and macrophages infiltrating atheromatous plaques in patients with cardiovascular disease. Angptl2 knockout in apolipoprotein E–deficient mice (ApoE−/−/Angptl2−/−) attenuated atherosclerosis progression by decreasing the number of macrophages infiltrating atheromatous plaques, reducing vascular inflammation. Bone marrow transplantation experiments showed that Angptl2 deficiency in endothelial cells attenuated atherosclerosis development. Conversely, ApoE−/− mice crossed with transgenic mice expressing Angptl2 driven by the Tie2 promoter (ApoE−/−/Tie2-Angptl2 Tg), which drives Angptl2 expression in endothelial cells but not monocytes/macrophages, showed accelerated plaque formation and vascular inflammation because of increased numbers of infiltrated macrophages in atheromatous plaques. Tie2-Angptl2 Tg mice alone did not develop plaques but exhibited endothelium-dependent vasodilatory dysfunction, likely because of decreased production of endothelial cell–derived nitric oxide. Conversely, Angptl2−/− mice exhibited less severe endothelial dysfunction than did wild-type mice when fed a high-fat diet. In vitro, Angptl2 activated proinflammatory nuclear factor-κB signaling in endothelial cells and increased monocyte/macrophage chemotaxis. Conclusions—Endothelial cell–derived Angptl2 accelerates vascular inflammation by activating proinflammatory signaling in endothelial cells and increasing macrophage infiltration, leading to endothelial dysfunction and atherosclerosis progression. (Arterioscler Thromb Vasc Biol. 2014;34:790-800.) Key Words: atherosclerosis ◼ cardiovascular diseases ◼ endothelium-derived vasoconstrictor factors ◼ inflammation
C
ardiovascular disease (CVD), which remains a major cause of death worldwide, is a medical and social problem associated with adverse health outcomes, all of which decrease productive life years.1,2 Coronary heart disease (CHD) is the major common form of CVD, and its underlying pathology is atherosclerosis.3 For many years, atherosclerosis was considered simply passive accumulation of cholesterol in the vessel wall.3 The advent of therapy aimed at
reducing low-density lipoprotein-cholesterol levels decreased the number of CHD events.4 Nonetheless, some CHD events continue to occur even in subjects showing lower low-density lipoprotein-cholesterol levels lowered response to treatment,5,6 indicating that pathologies underlying atherosclerosis are more complex than anticipated. Recently, the recognition
See cover image
Received on: June 19, 2013; final version accepted on: January 20, 2014. From the Department of Molecular Genetics (E.H., T.K., K.M., H.H., M.E., M. Tabata, H.T., Z.T., O.T., K.T., Y.O.), Department of Cardiovascular Medicine (E.H., H.O., K.K.), and Department of Cell Pathology (M. Takeya), Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan; Division of Geriatric Medicine, Department of Internal Medicine (Y.A., N.H.) and Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine (K.H., T.S.), Keio University School of Medicine, Tokyo, Japan; Department of Environmental Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan (Y.D., T.N., Y.K.); Department of Surgical Pathology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan (H.H.); Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan (T.M.); and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Tokyo, Japan (Y.O.). The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.113.303116/-/DC1 Correspondence to Yuichi Oike, MD, PhD, Department of Molecular Genetics, Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan. E-mail [email protected] © 2014 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
DOI: 10.1161/ATVBAHA.113.303116
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Horio et al Role of Angptl2 in Cardiovascular Disease 791
Nonstandard Abbreviations and Acronyms Angptl ApoE BM CD CHD CVD eNOS FFA HCAEC IL LILRB Mgl1 NF-κB NO RT-PCR TNF-α VECs
angiopoietin-like protein apolipoprotein E bone marrow cluster of differentiation coronary heart disease cardiovascular disease endothelial NO synthase free fatty acid human coronary artery endothelial cell interleukin leukocyte immunoglobulin-like receptor subfamily B macrophage galactose-type C lectin-1 nuclear factor κB nitric oxide real-time polymerase chain reaction tumor necrosis factor-α vascular endothelial cells
that atherosclerosis progression, including plaque instability, is associated with chronic inflammation in the vessel wall is receiving much attention as a risk factor for major CHD events.7–9 Thus, to develop effective preventive and therapeutic approaches, it is necessary to identify molecular mechanisms underlying chronic inflammatory activity that may accelerate atherosclerosis progression. We recently found that angiopoietin-like protein 2 (Angptl2) plays important pathological roles in various noninfectious inflammatory diseases, such as obesity-related insulin resistance,10 rheumatoid arthritis,11 and chemically induced cancer.12 Here, we report that Angptl2 protein levels in aorta tissue from the apolipoprotein E (ApoE−/−) knockout mouse are positively correlated with atherosclerosis severity. Histological analysis demonstrated abundant Angptl2 expression in atheromatous plaques of patients with CHD, particularly in endothelial cells and infiltrated macrophages. Angptl2 deficiency in ApoE−/− (ApoE−/−/Angptl2−/−) mice attenuated atherosclerosis severity by decreasing vascular inflammation through decreasing the number of macrophages infiltrating atheromatous plaques. Interestingly, the severity of phenotypes seen in ApoE−/−/Angptl2−/− recipient mice transplanted with ApoE−/−/Angptl2+/+ bone marrow (BM) cells was also significantly reduced compared with ApoE−/−/Angptl2+/+ recipients transplanted with ApoE−/−/Angptl2+/+ BM cells, indicating that deficiency of non–BM cell-derived Angptl2 in ApoE−/− mice contributes to decreased atherosclerosis severity. Conversely, ApoE−/− mice crossed with transgenic mice expressing Angptl2 driven by the Tie2 promoter (ApoE−/−/Tie2-Angptl2 Tg), which show increased Angptl2 expression in endothelial cells but not in differentiated hematopoietic lineage cells including monocytes/macrophages, exhibited more severe atherosclerosis and increased vascular inflammation. In addition, Angptl2 activated proinflammatory nuclear factor (NF)-κB signaling in endothelial cells in vitro and increased monocyte/macrophage chemotaxis. Transgenic mice expressing Angptl2 driven by the Tie2 promoter (Tie2-Angptl2 Tg) mice alone did not develop
atheromatous plaques but did show endothelium-dependent vasodilatory dysfunction (hereafter designated endothelial dysfunction), likely because of decreased production of endothelial cell–derived nitric oxide (NO). Conversely, in mice, Angptl2 deficiency decreased endothelial dysfunction induced by obesity. Overall, our findings indicate that endothelial cell– derived Angptl2 accelerates vascular inflammation, leading to endothelial dysfunction and atherosclerosis development.
Materials and Methods Materials and Methods are available in the online-only Supplement.
Results Circulating Angptl2 Concentrations and Angptl2 Expression Levels in Vascular Tissue Positively Correlate With Atherosclerosis Severity in Mice Circulating Angptl2 concentrations markedly increase with age in ApoE−/− mice, which exhibit atherosclerosis development (Figure 1A). Circulating Angptl2 concentrations in wild-type mice exhibiting little evidence of atherosclero sis in the aorta also increase with age, but to a lesser degree (Figure 1A). We observed that both the severity of atherosclerosis and the expression of Angptl2 protein levels in aortic vessels increased as ApoE−/− mice aged (Figure 1B and 1C). Increased serum levels of cholesterol, triglycerides, free fatty acids (FFAs), and homeostasis model assessment-insulin resistance were not exacerbated with age in ApoE−/− mice (Table I in the online-only Data Supplement). Overall, these observations suggest that, despite the fact that aging mice exhibit baseline increases in circulating Angptl2 levels, the presence of atherosclerosis exacerbates those increases, suggesting that cells in an atherosclerotic lesion may secrete Angptl2. These findings also suggest that circulating Angptl2 levels reflect atherosclerosis severity, not dyslipidemia.
Angptl2 Is Abundantly Expressed in Endothelial Cells and Macrophages Infiltrating Atherosclerotic Vessels of Patients With CHD Patients with CHD showed significantly increased levels of circulating Angptl2 compared with age-matched control subjects (Figure 2A; Table II in the online-only Data Supplement). Immunohistochemical analysis of serial sections of coronary arteries collected at autopsy from 10 patients with CHD indicated abundant Angptl2 expression in vascular endothelial cells (VECs) and in macrophages infiltrating plaque sites, and also in VECs at sites of nonatherosclerotic plaques (Figure 2B; Figure IA and IB in the online-only Data Supplement). To determine which cells express Angptl2, we undertook double-immunofluorescence staining of human coronary artery tissue using antibodies against Angptl2 and cluster of differentiation (CD)-31 (a marker of endothelial cells and some hematopoietic lineage/immune cells) or CD68 (a macrophage marker) by confocal microscopy. Both CD31+ cells and CD68+ cells coexpressed Angptl2 (Figure 2C and 2D), indicating that Angptl2 is expressed in both VECs and infiltrated macrophages in atheromatous plaque lesions in human tissues.
Downloaded from http://atvb.ahajournals.org/ at Universite Laval on October 10, 2014
792 Arterioscler Thromb Vasc Biol April 2014
Figure 1. Angiopoietin-like protein 2 (Angptl2) is expressed in atherosclerotic vascular tissue in mice. In all panels, the young group is defined as 20 weeks but 80 weeks. A, Serum Angptl2 levels as measured by ELISA in wild-type and apolipoprotein E–deficient (ApoE−/−) mice fed a normal chow diet (n=4–6, each group). B and C, Assessment of ApoE−/− mice fed normal chow. B, (Left) Representative photographs of Oil red O–stained aortas of mice at indicated ages. (Right) Quantitative comparison of disease severity based on atherosclerotic lesion area. C, (Upper) Western blotting analysis of Angptl2 protein in the aorta of each group (n=3–4, each group). (Lower) Quantitative evaluation of Angptl2 protein levels in aorta lysates from each group relative to the young group (n=3–4, each group). Data are normalized to heat shock cognate (Hsc)70 levels. Data are mean±SD. †P
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://atvb.ahajournals.org/content/34/4/790
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Arteriosclerosis, Thrombosis, and Vascular Biology can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Arteriosclerosis, Thrombosis, and Vascular Biology is online at: http://atvb.ahajournals.org//subscriptions/
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Data Supplement (unedited) at: http://atvb.ahajournals.org/content/suppl/2014/02/13/ATVBAHA.113.303116.DC1.html
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Arteriosclerosis, Thrombosis, and Vascular Biology can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Arteriosclerosis, Thrombosis, and Vascular Biology is online at: http://atvb.ahajournals.org//subscriptions/
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Role of Endothelial Cell–Derived Angptl2 in Vascular Inflammation Leading to Endothelial Dysfunction and Atherosclerosis Progression Eiji Horio, Tsuyoshi Kadomatsu, Keishi Miyata, Yasumichi Arai, Kentaro Hosokawa, Yasufumi Doi, Toshiharu Ninomiya, Haruki Horiguchi, Motoyoshi Endo, Mitsuhisa Tabata, Hirokazu Tazume, Zhe Tian, Otowa Takahashi, Kazutoyo Terada, Motohiro Takeya, Hiroyuki Hao, Nobuyoshi Hirose, Takashi Minami, Toshio Suda, Yutaka Kiyohara, Hisao Ogawa, Koichi Kaikita, Yuichi Oike Objective—Cardiovascular disease (CVD), the most common morbidity resulting from atherosclerosis, remains a frequent cause of death. Efforts to develop effective therapeutic strategies have focused on vascular inflammation as a critical pathology driving atherosclerosis progression. Nonetheless, molecular mechanisms underlying this activity remain unclear. Here, we ask whether angiopoietin-like protein 2 (Angptl2), a proinflammatory protein, contributes to vascular inflammation that promotes atherosclerosis progression. Approach and Results—Histological analysis revealed abundant Angptl2 expression in endothelial cells and macrophages infiltrating atheromatous plaques in patients with cardiovascular disease. Angptl2 knockout in apolipoprotein E–deficient mice (ApoE−/−/Angptl2−/−) attenuated atherosclerosis progression by decreasing the number of macrophages infiltrating atheromatous plaques, reducing vascular inflammation. Bone marrow transplantation experiments showed that Angptl2 deficiency in endothelial cells attenuated atherosclerosis development. Conversely, ApoE−/− mice crossed with transgenic mice expressing Angptl2 driven by the Tie2 promoter (ApoE−/−/Tie2-Angptl2 Tg), which drives Angptl2 expression in endothelial cells but not monocytes/macrophages, showed accelerated plaque formation and vascular inflammation because of increased numbers of infiltrated macrophages in atheromatous plaques. Tie2-Angptl2 Tg mice alone did not develop plaques but exhibited endothelium-dependent vasodilatory dysfunction, likely because of decreased production of endothelial cell–derived nitric oxide. Conversely, Angptl2−/− mice exhibited less severe endothelial dysfunction than did wild-type mice when fed a high-fat diet. In vitro, Angptl2 activated proinflammatory nuclear factor-κB signaling in endothelial cells and increased monocyte/macrophage chemotaxis. Conclusions—Endothelial cell–derived Angptl2 accelerates vascular inflammation by activating proinflammatory signaling in endothelial cells and increasing macrophage infiltration, leading to endothelial dysfunction and atherosclerosis progression. (Arterioscler Thromb Vasc Biol. 2014;34:790-800.) Key Words: atherosclerosis ◼ cardiovascular diseases ◼ endothelium-derived vasoconstrictor factors ◼ inflammation
C
ardiovascular disease (CVD), which remains a major cause of death worldwide, is a medical and social problem associated with adverse health outcomes, all of which decrease productive life years.1,2 Coronary heart disease (CHD) is the major common form of CVD, and its underlying pathology is atherosclerosis.3 For many years, atherosclerosis was considered simply passive accumulation of cholesterol in the vessel wall.3 The advent of therapy aimed at
reducing low-density lipoprotein-cholesterol levels decreased the number of CHD events.4 Nonetheless, some CHD events continue to occur even in subjects showing lower low-density lipoprotein-cholesterol levels lowered response to treatment,5,6 indicating that pathologies underlying atherosclerosis are more complex than anticipated. Recently, the recognition
See cover image
Received on: June 19, 2013; final version accepted on: January 20, 2014. From the Department of Molecular Genetics (E.H., T.K., K.M., H.H., M.E., M. Tabata, H.T., Z.T., O.T., K.T., Y.O.), Department of Cardiovascular Medicine (E.H., H.O., K.K.), and Department of Cell Pathology (M. Takeya), Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan; Division of Geriatric Medicine, Department of Internal Medicine (Y.A., N.H.) and Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine (K.H., T.S.), Keio University School of Medicine, Tokyo, Japan; Department of Environmental Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan (Y.D., T.N., Y.K.); Department of Surgical Pathology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan (H.H.); Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan (T.M.); and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Tokyo, Japan (Y.O.). The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.113.303116/-/DC1 Correspondence to Yuichi Oike, MD, PhD, Department of Molecular Genetics, Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan. E-mail [email protected] © 2014 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
DOI: 10.1161/ATVBAHA.113.303116
Downloaded from http://atvb.ahajournals.org/ 790 at Universite Laval on October 10, 2014
Horio et al Role of Angptl2 in Cardiovascular Disease 791
Nonstandard Abbreviations and Acronyms Angptl ApoE BM CD CHD CVD eNOS FFA HCAEC IL LILRB Mgl1 NF-κB NO RT-PCR TNF-α VECs
angiopoietin-like protein apolipoprotein E bone marrow cluster of differentiation coronary heart disease cardiovascular disease endothelial NO synthase free fatty acid human coronary artery endothelial cell interleukin leukocyte immunoglobulin-like receptor subfamily B macrophage galactose-type C lectin-1 nuclear factor κB nitric oxide real-time polymerase chain reaction tumor necrosis factor-α vascular endothelial cells
that atherosclerosis progression, including plaque instability, is associated with chronic inflammation in the vessel wall is receiving much attention as a risk factor for major CHD events.7–9 Thus, to develop effective preventive and therapeutic approaches, it is necessary to identify molecular mechanisms underlying chronic inflammatory activity that may accelerate atherosclerosis progression. We recently found that angiopoietin-like protein 2 (Angptl2) plays important pathological roles in various noninfectious inflammatory diseases, such as obesity-related insulin resistance,10 rheumatoid arthritis,11 and chemically induced cancer.12 Here, we report that Angptl2 protein levels in aorta tissue from the apolipoprotein E (ApoE−/−) knockout mouse are positively correlated with atherosclerosis severity. Histological analysis demonstrated abundant Angptl2 expression in atheromatous plaques of patients with CHD, particularly in endothelial cells and infiltrated macrophages. Angptl2 deficiency in ApoE−/− (ApoE−/−/Angptl2−/−) mice attenuated atherosclerosis severity by decreasing vascular inflammation through decreasing the number of macrophages infiltrating atheromatous plaques. Interestingly, the severity of phenotypes seen in ApoE−/−/Angptl2−/− recipient mice transplanted with ApoE−/−/Angptl2+/+ bone marrow (BM) cells was also significantly reduced compared with ApoE−/−/Angptl2+/+ recipients transplanted with ApoE−/−/Angptl2+/+ BM cells, indicating that deficiency of non–BM cell-derived Angptl2 in ApoE−/− mice contributes to decreased atherosclerosis severity. Conversely, ApoE−/− mice crossed with transgenic mice expressing Angptl2 driven by the Tie2 promoter (ApoE−/−/Tie2-Angptl2 Tg), which show increased Angptl2 expression in endothelial cells but not in differentiated hematopoietic lineage cells including monocytes/macrophages, exhibited more severe atherosclerosis and increased vascular inflammation. In addition, Angptl2 activated proinflammatory nuclear factor (NF)-κB signaling in endothelial cells in vitro and increased monocyte/macrophage chemotaxis. Transgenic mice expressing Angptl2 driven by the Tie2 promoter (Tie2-Angptl2 Tg) mice alone did not develop
atheromatous plaques but did show endothelium-dependent vasodilatory dysfunction (hereafter designated endothelial dysfunction), likely because of decreased production of endothelial cell–derived nitric oxide (NO). Conversely, in mice, Angptl2 deficiency decreased endothelial dysfunction induced by obesity. Overall, our findings indicate that endothelial cell– derived Angptl2 accelerates vascular inflammation, leading to endothelial dysfunction and atherosclerosis development.
Materials and Methods Materials and Methods are available in the online-only Supplement.
Results Circulating Angptl2 Concentrations and Angptl2 Expression Levels in Vascular Tissue Positively Correlate With Atherosclerosis Severity in Mice Circulating Angptl2 concentrations markedly increase with age in ApoE−/− mice, which exhibit atherosclerosis development (Figure 1A). Circulating Angptl2 concentrations in wild-type mice exhibiting little evidence of atherosclero sis in the aorta also increase with age, but to a lesser degree (Figure 1A). We observed that both the severity of atherosclerosis and the expression of Angptl2 protein levels in aortic vessels increased as ApoE−/− mice aged (Figure 1B and 1C). Increased serum levels of cholesterol, triglycerides, free fatty acids (FFAs), and homeostasis model assessment-insulin resistance were not exacerbated with age in ApoE−/− mice (Table I in the online-only Data Supplement). Overall, these observations suggest that, despite the fact that aging mice exhibit baseline increases in circulating Angptl2 levels, the presence of atherosclerosis exacerbates those increases, suggesting that cells in an atherosclerotic lesion may secrete Angptl2. These findings also suggest that circulating Angptl2 levels reflect atherosclerosis severity, not dyslipidemia.
Angptl2 Is Abundantly Expressed in Endothelial Cells and Macrophages Infiltrating Atherosclerotic Vessels of Patients With CHD Patients with CHD showed significantly increased levels of circulating Angptl2 compared with age-matched control subjects (Figure 2A; Table II in the online-only Data Supplement). Immunohistochemical analysis of serial sections of coronary arteries collected at autopsy from 10 patients with CHD indicated abundant Angptl2 expression in vascular endothelial cells (VECs) and in macrophages infiltrating plaque sites, and also in VECs at sites of nonatherosclerotic plaques (Figure 2B; Figure IA and IB in the online-only Data Supplement). To determine which cells express Angptl2, we undertook double-immunofluorescence staining of human coronary artery tissue using antibodies against Angptl2 and cluster of differentiation (CD)-31 (a marker of endothelial cells and some hematopoietic lineage/immune cells) or CD68 (a macrophage marker) by confocal microscopy. Both CD31+ cells and CD68+ cells coexpressed Angptl2 (Figure 2C and 2D), indicating that Angptl2 is expressed in both VECs and infiltrated macrophages in atheromatous plaque lesions in human tissues.
Downloaded from http://atvb.ahajournals.org/ at Universite Laval on October 10, 2014
792 Arterioscler Thromb Vasc Biol April 2014
Figure 1. Angiopoietin-like protein 2 (Angptl2) is expressed in atherosclerotic vascular tissue in mice. In all panels, the young group is defined as 20 weeks but 80 weeks. A, Serum Angptl2 levels as measured by ELISA in wild-type and apolipoprotein E–deficient (ApoE−/−) mice fed a normal chow diet (n=4–6, each group). B and C, Assessment of ApoE−/− mice fed normal chow. B, (Left) Representative photographs of Oil red O–stained aortas of mice at indicated ages. (Right) Quantitative comparison of disease severity based on atherosclerotic lesion area. C, (Upper) Western blotting analysis of Angptl2 protein in the aorta of each group (n=3–4, each group). (Lower) Quantitative evaluation of Angptl2 protein levels in aorta lysates from each group relative to the young group (n=3–4, each group). Data are normalized to heat shock cognate (Hsc)70 levels. Data are mean±SD. †P
