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From the Department of Cardiovascular Medicine (M.I., K.E., Q.Z., K.H., K.O., Y.I., A.T., K.S.) and the Department of Medical Biophysics and Radiation Biology (S.K., T.T.), Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; and the Gladstone Institute of Cardiovascular Disease (I.F.C.), San Francisco, Calif.
ABSTRACT
Angiotensin II (Ang II) is implicated in atherogenesis by activating inflammatory responses in arterial wall cells. Ang II accelerates the atherosclerotic process in hyperlipidemic apoE–/– mice by recruiting and activating monocytes. Monocyte chemoattractant protein-1 (MCP-1) controls monocyte-mediated inflammation through its receptor, CCR2. The roles of leukocyte-derived CCR2 in the Ang II-induced acceleration of the atherosclerotic process, however, are not known. We hypothesized that deficiency of leukocyte-derived CCR2 suppresses Ang II-induced atherosclerosis.
Methods and Results— A bone marrow transplantation technique (BMT) was used to develop apoE–/– mice with and without deficiency of CCR2 in leukocytes (BMT-apoE–/–CCR2+/+ and BMT-apoE–/–CCR2–/– mice). Compared with BMT-apoE–/–CCR2+/+ mice, Ang II-induced increases in atherosclerosis plaque size and abdominal aortic aneurysm formation were suppressed in BMT-apoE–/–CCR2–/– mice. This suppression was associated with a marked decrease in monocyte-mediated inflammation and inflammatory cytokine expression.
Conclusion— Leukocyte-derived CCR2 is critical in Ang II-induced atherosclerosis and abdominal aneurysm formation. The present data suggest that vascular inflammation mediated by CCR2 in leukocytes is a reasonable target of therapy for treatment of atherosclerosis.
We determined the role of leukocyte-derived CCR2 (the receptor for monocyte chemoattractant protein-1) in the angiotensin II (Ang II)-induced acceleration of atherosclerotic process. We show that Ang II-induced atherosclerotic process was blunted in hypercholesterolemic apoE–/– mice with deficiency of leukocyte-derived CCR2. These data suggest that leukocyte-derived CCR2 is critical in Ang II-induced atherosclerosis.
Key Words: atherosclerosis ? angiotensin II ? inflammation ? leukocytes
Introduction
Chronic inflammatory processes have an important role in atherosclerotic plaque progression, destabilization, and subsequent rupture/thrombosis, resulting in acute coronary syndrome.1,2 Therefore, identification of the critical inflammatory pathway involved in atherosclerotic plaque progression and destabilization might aid in the development of novel therapeutic strategies to reduce atherothrombotic events.
The renin-angiotensin system is now recognized as an important therapeutic target of atherosclerotic vascular disease.3,4 Angiotensin II (Ang II) induces the production of reactive oxidative species and stimulates the expression of adhesion molecules (vascular cell adhesion molecule-1) and chemokines (monocyte chemoattractant protein-1 ).3–5 Infusion of Ang II into hypercholesterolemic mice dramatically accelerates the atherosclerotic process, leading to the development of extensive atherosclerotic plaque formation and abdominal aortic aneurysm (AAA).6,7 The Ang II-mediated acceleration of atherogenesis is characterized by the recruitment and activation of monocytes/macrophages and the degradation of elastin and collagen layers, suggesting that Ang II changes the lesion composition into a more destabilized phenotype. MCP-1 is a C-C chemokine that controls monocyte recruitment to the site of inflammation through its receptor, C-C chemokine receptor (CCR) 2.8–10 We recently demonstrated that blockade of the MCP-1 pathway by transfection of mutant MCP-1 gene limits Ang II-induced progression and destabilization of atherosclerotic lesions in hyperlipidemic apoE–/– mice.11 We and others also demonstrated that blockade or abrogation of MCP-1 or CCR2 attenuates hyperlipidemia-induced atherosclerosis in mice,12–15 and that CCR2-deficient (CCR2–/–) mice display reduced neointimal formation after arterial injury.16 Overall, these studies provide ample evidence for a decisive role of MCP-1/CCR2 in atherosclerosis formation, progression, and destabilization.
The role of MCP-1 and/or CCR2 in atherogenesis might be more complex. Ang II is thought to accelerate atherogenesis by stimulating MCP-1 expression and function in multiple cell types in atherosclerotic lesions such as endothelial cells, smooth muscle cells, and leukocytes. Because CCR2 is present in these cell types, activation of the MCP-1–CCR2 pathway mediates recruitment and activation of monocytes,17 endothelial migration and angiogenesis,18,19 and migration/proliferation of vascular smooth muscle cells.20 It is impossible, however, to dissect the relative pathobiologic role of leukocytes versus nonleukocyte cells in the arterial wall using the systemic absence or blockade of MCP-1/CCR2. The aim of this study was to address the role of CCR2 on leukocytes in Ang II-mediated acceleration of the atherosclerotic process. We used bone marrow cell transplantation (BMT) techniques to create a murine model with a leukocyte-derived CCR2 deficiency and demonstrated the essential role of leukocyte-derived CCR2 in Ang II-induced acceleration of atherosclerotic processes.
Methods
Experimental Animals
Male apoE knockout mice were purchased from Jackson Laboratory (Bar Harbor, Me). apoE–/– CCR2–/– and apoE–/– CCR2+/+ mice with the same genetic background (C57BL/6J and 129/svjae hybrids) were supplied by Dr Charo.12
Experimental Protocol
The study protocol was reviewed and approved by the Committee on the Ethics of Animal Experiments, Kyushu University Graduate School of Medical Sciences. A part of this study was performed at the Kyushu University Station for Collaborative Research and the Morphology Core Unit, Kyushu University Faculty of Medical Sciences.
To determine the specific role of CCR2 on leukocytes, we used the BMT technique to create mice with and without a leukocyte-selective CCR2 deficiency. At 8 weeks of age, BMT was performed as described previously.21 Bone marrow cells were harvested from femurs and tibias of either test (apoE–/–CCR2–/–) or control (apoE–/–CCR2+/+) donor mice. The recipient apoE–/–CCR2+/+ mice received 1x107 bone marrow cells (0.3 mL) 4 hours after whole body irradiation with 7 Gy of X-rays (200-KVp, 20-mA, 0.3-mm Cu filter) at 1 Gy/min. These 2 groups of mice are referred to as BMT-apoE–/–CCR2–/– and BMT-apoE–/–CCR2+/+, respectively. At 14 weeks of age, BMT-apoE–/–CCR2–/– and BMT-apoE–/–CCR2+/+ mice were infused with Ang II (1.9 mg/kg per day) or phosphate-buffered saline via osmotic mini-pump (Alzet, Cupertino, Calif).21
In all experiments, mice were euthanized on day 7 or 28 of treatment for morphometric, immunohistochemical, and biochemical analysis. Peripheral arterial blood was collected immediately before the mice were euthanized. The aortas were isolated and either fixed in 10% buffered formalin for histological and immunohistochemical analysis or snap-frozen in liquid nitrogen (LN2) and stored at –80°C for biochemical analysis. Systolic blood pressure was measured by the tail-cuff method before and 28 days after treatment.
Histology and Immunohistochemistry
To quantify the extent of the atherosclerotic lesions, the aortic arch and the thoracic aorta was opened longitudinally, stained with oil red O, and pinned out on a black wax surface. The percentage of the plaque area stained by oil red O to the total luminal surface area was determined.
To further quantify the atherosclerotic lesions in the aortic root, serial cryostat sections (6 μm) of the aortic root were prepared as described.14 In brief, atherosclerotic lesions in the aortic root were examined at 5 locations, each separated by 120 μm, 4 to 5 serial sections were prepared from each location. Some of these sections were stained with Elastica van Gieson and oil red O (for lipid staining). Elastica van Gieson staining was used to delineate the internal elastic lamina for determination of the intimal area. The lipid composition of the lesion was determined by calculating the percent of the oil red O positive area to the total cross-sectional vessel wall area. The remaining sections were used for immunohistochemical analysis. Air-dried cryostat sections were fixed in acetone and stained with the respective antibody: antimouse macrophage antibodies (Mac-3; Serotec Inc, Raleigh, NC) and antihuman MCP-1 antibodies (Santa Cruz Biotechnology Inc, Santa Cruz, Calif). The sections were then counterstained with hematoxylin. Respective nonimmune IgGs (Dako) were used as negative controls. Similarly, the number of macrophage accumulated into the aortic root lesion was estimated.
A single observer blinded to the experiment protocol performed quantitative analysis of atherosclerotic lesions. All images were captured with a Nikon microscope equipped with a video camera and analyzed using Adobe Photoshop 6.0 and National Institutes of Health Image Software. In each case, the average value for 4 to 5 locations or sections for each animal was used for analysis.
Real-Time Reverse-Transcription Polymerase Chain Reaction Analysis
Real-time polymerase chain reaction amplification was performed with the mouse cDNA using the ABI PRISM 7000 Sequence Detection System (Applied Biosystems).21 The respective polymerase chain reaction primers and TaqMan probes were designed from GenBank databases using a software program (Table I, available online at http://atvb/ahajournals.org).
Flow Cytometry Analysis
Flow cytometry analysis was performed as described previously.21 To determine CCR2 expression in monocytes, antibodies against phycoerythrin-conjugated antimouse monocyte (CD80) (Becton Dickinson Biosciences, San Jose, Calif), goat antimouse CCR2 (Santa Cruz Biotechnology Inc), and fluorescein isothiocyanate-conjugated mouse antigoat IgG (Santa Cruz Biotechnology Inc) were used. To determine CCR2 fluorescence intensity in lymphocytes and neutrophils, leukocytes were also stained using antibodies against phycoerythrin-conjugated antimouse CD11b (Mac-1), cy-chrome–conjugated antimouse T-cell receptor ? chain monoclonal antibody (Becton Dickinson Biosciences). In control experiments, fluorescein isothiocyanate-conjugated nonspecific goat IgG was used to measure nonspecific binding. Stained cells were analyzed by fluorescence-activated cell sorter Calibur (Becton Dickinson Biosciences).
Plasma Measurements
Commercially available enzyme-linked immunosorbent assay kits (Biosource International, Camarillo, Calif) were used to measure plasma total cholesterol, triglyceride, low-density lipoprotein cholesterol, and mouse MCP-1 according to the manufacturer’s instructions.
Statistical Analysis
Data were expressed as mean±SEM. Statistical analysis of differences was compared by analysis of variance using Bonferroni correction for multiple comparisons. P<0.05 was considered statistically significant.
Results
Bone Marrow-Derived CCR2 Is Critical for Ang II-Induced Acceleration of Atherosclerosis
To determine the role of BM-derived CCR2, Ang II was infused in BMT-apoE–/–CCR2+/+ and BMT-apoE–/–CCR2–/– mice. As reported previously,11 Ang II infusion accelerates atherosclerotic process in BMT-apoE–/–CCR2+/+ mice. In contrast, Ang II-induced acceleration of atherosclerosis was suppressed in BMT-apoE–/–CCR2–/– mice (Figure 1A and 1B). In addition, Ang II-induced aortic inflammatory changes as well as lipid accumulation were markedly attenuated in BMT-apoE–/–CCR2–/– mice (Figure 1A and 1B).
Figure 1. Suppressed Ang II-induced atherosclerotic lesions in BMT-apoE–/– CCR2–/– mice. A, Histopathologic and immunohistochemical pictures of atherosclerotic lesions. From the top to bottom panels, photomicrographs of the intraluminal surface of the aortic arch and thoracic aorta stained with oil red O, those of atherosclerotic lesions in the aortic root stained with oil red O, and those of atherosclerotic lesions in the aortic root immunostained with antimurine macrophage antibody (Mac-3). Bar indicates 100 μm. B, Quantitative comparison of atherosclerotic lesion size (% of oil red O stained area) in BMT-apoE–/–CCR2+/+,BMT-apoE–/–CCR2–/–, BMT-apoE–/–CCR2+/+ + Ang II, and BMT-apoE–/–CCR2–/– + Ang II groups. Data are reported as mean±SEM. N=6 to 8. *P<0.05, **P<0.01 versus BMT-apoE–/–CCR2+/+ group. P<0.05, P<0.01 versus BMT-apoE–/–CCR2+/+ + Ang II group.
Ang II-induced gene and protein expression of MCP-1 was examined 7 days after Ang II-infusion. Ang II infusion increased MCP-1 mRNA and immunoreactive MCP-1 levels in the aortic root, which were similar between BMT- apoE–/–CCR2+/+ and BMT-apoE–/–CCR2–/– mice (Figure 2A and 2B). Aortic CCR2 gene expression was also suppressed in BMT- apoE–/–CCR2–/– mice infused with and without Ang II (Figure 2C).
Figure 2. MCP-1, CCR2, and cytokine expression. A, Photomicrographs of cross-sections of the aortic root stained immunohistochemically for MCP-1 on day 28. Bar indicates 100 μm. B, MCP-1 gene expression by real-time reverse-transcription polymerase chain reaction in the aorta on day 7. Data are expressed as the ratio of MCP-1 mRNA to GAPDH mRNA. **P<0.01 versus BMT-apoE–/–CCR2+/+ group (n=6 each). C, CCR2 antigen expression on peripheral blood circulating monocytes by flow cytometry analysis. CCR2 gene expression of the aorta by real-time reverse-transcription polymerase chain reaction on day 7. Data are expressed as the ratio of MCP-1 mRNA to GAPDH mRNA. *P<0.05, **P<0.01 versus BMT-apoE–/–CCR2+/+ group; P<0.05 versus BMT-apoE–/–CCR2+/+ + Ang II group (n=6 each). D, IL-1? and IL-6 gene expression by real-time reverse-transcription polymerase chain reaction in the aorta on day 7. Data are expressed as the ratio of MCP-1 mRNA to GAPDH mRNA. *P<0.05, **P<0.01 versus BMT-apoE–/– CCR2+/+ group; P<0.05 versus BMT-apoE–/–CCR2+/+ + Ang II group (n=6 each).
Ang II-induced changes in CCR2 antigen on circulating leukocytes were examined by flow cytometric analysis on day 7. Ang II infusion increased CCR2 antigen on monocytes in BMT-apoE–/–CCR2+/+ mice, which was blunted in BMT-apoE–/–C CR2–/– mice (Figure 2C). No CCR2 antigen was detected on lymphocytes or neutrophils in the presence or absence of Ang II infusion (data not shown). These cytometric data indicate that CCR2 antigen was expressed mainly on circulating monocytes whether Ang II was infused.
In BMT- apoE–/–CCR2+/+ mice, Ang II infusion enhanced gene expression of IL-6 and IL-1? in the aorta on day 7 (Figure 2D). The Ang II-induced increases in IL-6 and IL-1? gene expression were reduced in BMT-apoE–/–CCR2–/– mice.
There were no significant differences in plasma MCP-1 levels between BMT-CCR2+/+ mice infused with or without Ang II on day 28 (Table I). There were no significant differences in Ang II-induced changes in systolic blood pressure or serum lipid levels (Table II, available online at http://atvb.ahajournals.org), suggesting that the observed effects of leukocyte-derived CCR2 deficiency cannot be explained by the effects on plasma lipids or Ang II-induced arterial hypertension. There were no significant differences in plasma MCP-1 levels between untreated BMT-apoE–/–CCR2+/+ and BMT-apoE–/–CCR2–/– mice on day 28 (Table II). In contrast, the plasma MCP-1 level dramatically increased in BMT-apoE–/–CCR2–/– mice infused with Ang II, compared with that in BMT-apoE–/–CCR2+/+ mice infused with Ang II.
Ang II-Induced AAA Formation Is Suppressed in BMT-ApoE–/–CCR2–/– Mice
As reported by others,6,7 Ang II infusion induced AAA formation associated with the recruitment and activation of monocytes/macrophages into the adventitia and media and the degradation of elastin and collagen layers (Figure 3A). In separate experiments, to determine the effect of BM-derived CCR2 deficiency on Ang II-induced aneurysm formation, we quantified the incidence and measured the diameter of AAA. Mice that displayed 10% increase in abdominal aortic diameter were defined to have AAA. Compared with BMT-apoE–/–CCR2+/+ mice, BMT-apoE–/–CCR2–/– mice showed a significant reduction in the incidence of AAA formation (9 of 10 BMT-apoE–/–CCR2+/+ mice had AAAs versus only 1 of 10 for BMT-apoE–/–CCR2–/– mice; P<0.01). Furthermore, the Ang II-induced increase in maximum diameter of the abdominal aorta was not observed in BMT-apoE–/–CCR2–/– mice (Figure 3B).
Figure 3. Suppression of Ang II-induced abdominal aortic aneurysm formation in BMT-apoE–/–CCR2–/– mice. A, Photomicrographs of the gross appearance of the ascending aorta to the abdominal aorta. There was significant abdominal aortic aneurysm formation in BMT-apoE–/– CCR2+/+ mice infused with Ang II, whereas there was no aneurysm formation in BMT-apoE–/–CCR2–/– mice. B, Diameters of the abdominal aorta. N=10 each. *P<0.01 versus BMT-apoE–/–CCR2+/+ mice with no Ang II infusion. P<0.01 versus BMT-apoE–/–CCR2+/+ mice with Ang II infusion.
Discussion
The important and novel finding of this study was suppressed Ang II-induced acceleration of atherosclerotic process in BMT-apoE–/–CCR2–/– mice. The present study, therefore, represents the first direct evidence for the critical role of CCR2 on leukocytes, especially on monocytes, in Ang II-induced acceleration of atherosclerosis.
BMT from apoE–/–CCR2–/– to apoE–/–CCR2+/+ mice was associated with blunted expression of CCR2 antigen on circulating leukocytes, especially on circulating monocytes. This BMT-apoE–/–CCR2–/– mice displayed suppressed monocyte/macrophage infiltration and lipid accumulation into the atherosclerotic lesions induced by Ang II infusion. To elucidate the mechanism of suppressed Ang II-induced inflammation in BMT-apoE–/–CCR2–/– mice, we examined local and systemic expression of MCP-1. The lack of a detectable difference in Ang II-induced local MCP-1 expression between BMT-apoE–/–CCR2+/+ and BMT-apoE–/–CCR2–/– mice suggests that suppression of Ang II-induced inflammation might result from the lack of CCR2 on monocytes, but not the result of reduced expression of MCP-1. The increase in plasma MCP-1 concentrations in BMT-apoE–/–CCR2+/+ and BMT-apoE–/–CCR2–/– mice infused with Ang II might reflect compensatory local overproduction of MCP-1 in any tissue, including vascular tissues. The present data of attenuated gene expression of inflammatory cytokines in the aorta from BMT-apoE–/–CCR2–/– mice support the notion that activated lesional leukocytes might produce inflammatory and growth-promoting signals, which in turn lead to further acceleration of atherosclerosis. Our present data suggest that anti-inflammation caused by blockade of CCR2-mediated monocyte infiltration can interrupt the positive feedback cycle between inflammation and atherogenesis.
We previously reported that CCR2 expression and function are enhanced in circulating monocytes in hypertensive animals and humans through an AT1 receptor-mediated mechanism; increased CCR2 on monocytes is an important predictor of the presence of hypertension; and monocyte CCR2 is critical for monocyte-mediated inflammation and remodeling in Ang II-induced hypertension in mice.21 Our present data extend our previous study by showing the decisive role of CCR2 on monocyte in Ang II-induced acceleration of atherosclerosis. Overall, these findings rule out the possibility that expression of CCR2 by resident arterial cells is involved primarily in the mechanism of Ang II-induced vascular remodeling and atherosclerosis. However, Sata22 and other investigators have reported that BM-derived progenitor cells can migrate to atherosclerotic lesions, differentiate into vascular wall cells, and thus contribute to the development of vascular remodeling and atherosclerosis. Although the degrees to which BM-derived progenitors contribute to the mechanism of vascular disease remain unclear, we do not completely exclude the possibility that expression of CCR2 by BM-derived progenitor cells other than circulating monocytes contributed to the present results.
In conclusion, the present study provides first evidence that CCR2 expressed on monocytes has a critical role in Ang II-induced acceleration of the atherosclerotic process. This finding might also apply to the vascular pathology of atherosclerosis caused by other stimuli such as hypercholesterolemia and/or hypertension, because enhanced CCR2 expression on circulating monocytes has been demonstrated in animals and humans with hypercholesterolemia23 or hypertension.21
Acknowledgments
This study was supported by grants-in-aid for Scientific Research (14657172, 14207036) from the Ministry of Education, Culture, Sports, Science, and Technology, Tokyo, Japan; Health Science Research grants (Comprehensive Research on Aging and Health, and Research on Translational Research) from the Ministry of Health, Labor, and Welfare, Tokyo, Japan; and by the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research, Tokyo, Japan.
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