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(Circulation. 2001;103:2096.)
© 2001 American Heart Association, Inc.

Basic Science Reports

New Anti–Monocyte Chemoattractant Protein-1 Gene Therapy Attenuates Atherosclerosis in Apolipoprotein E–Knockout Mice

Weihua Ni, MD; Kensuke Egashira, MD; Shiro Kitamoto, MD; Chu Kataoka, MD; Masamichi Koyanagi, MD; Shujiro Inoue, MD; Katsumi Imaizumi, PhD; Chiyuki Akiyama, MS; Ken-ichi Nishida, PhD; Akira Takeshita, MD

From the Department of Cardiovascular Medicine (W.N., K.E., S.K., C.K., M.K., S.I., A.T.), Graduate School of Medical Sciences, and the Laboratory of Nutrition Chemistry (K.I.), Division of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan; and New Product Research Laboratories (C.A., K.N.), Daiichi Pharmaceutical Co, Tokyo, Japan.

Correspondence to Kensuke Egashira, MD, Department of Cardiovascular Medicine, Graduate School of Medicine, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail egashira{at}cardiol.med.kyushu-u.ac.jp

	Abstract

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Background—Monocyte recruitment into the arterial wall and its activation may be the central event in atherogenesis. Monocyte chemoattractant protein-1 (MCP-1) is an important chemokine for monocyte recruitment, and its receptor (CCR2) may mediate such in vivo response. Although the importance of the MCP-1/CCR2 pathway in atherogenesis has been clarified, it remains unanswered whether postnatal blockade of the MCP-1 signals could be a unique site-specific gene therapy.

Methods and Results—We devised a new strategy for anti–MCP-1 gene therapy to treat atherosclerosis by transfecting an N-terminal deletion mutant of the human MCP-1 gene into a remote organ (skeletal muscle) in apolipoprotein E–knockout mice. This strategy effectively blocked MCP-1 activity and inhibited the formation of atherosclerotic lesions but had no effect on serum lipid concentrations. Furthermore, this strategy increased the lesional extracellular matrix content.

Conclusions—We conclude that this anti–MCP-1 gene therapy may serve not only to reduce atherogenesis but also to stabilize vulnerable atheromatous plaques. This strategy may be a useful and feasible form of gene therapy against atherosclerosis in humans.

Key Words: apolipoproteins • atherosclerosis • gene therapy

	Introduction

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The infiltration and activation of mononuclear cells into blood vessel walls are critical steps in the early stages of atherosclerosis^{1 2} and are key cellular components in unstable atheromas prone to rupture.³ Monocyte chemoattractant protein-1 (MCP-1) is a member of the C-C chemokine family and a potent chemotactic factor for monocytes.⁴ Recent studies have demonstrated that MCP-1 expression is increased in atherosclerotic lesions^{5 6} and that blocking the expression of MCP-1 or its receptor CCR2 decreases atheroma formation in hypercholesterolemic mice.^{7 8} These previous studies establish a central role for the MCP-1/CCR2 pathway in atherogenesis. However, because MCP-1/CCR2-deficient mice also have a markedly impaired Th2 and Th1 cytokine responses, respectively,^{9 10} it remains unclear whether the decreased atherogenesis in MCP-1/CCR2-deficient mice is due only to blockade of the MCP-1/CCR2 signaling pathway or to the combined blockade of MCP-1 and Th1/Th2 cytokine. Evidence suggests that a Th1 cytokine, interferon- , plays an important role in atherogenesis.¹¹ Therefore, it is unclear whether postnatal blockade of the MCP-1/CCR2 pathway may be a useful site-specific gene therapy against atherosclerosis.

We performed the present study to evaluate the use of gene therapy to block MCP-1 activity in vivo by using an N-terminal deletion mutant of MCP-1, called 7ND, which lacks the N-terminal amino acid 2 to 8. This mutant MCP-1 has been shown to bind to the receptor for MCP-1 (CCR2) and block MCP-1– mediated monocyte chemotaxis.^{12 13} We hypothesized that for this approach to work, the transfected cells must secrete 7ND protein into the circulating blood and that the 7ND protein must bind to the MCP-1 receptor on monocytes or target cells in remote organs, thereby blocking signaling of MCP-1. Such inhibition of MCP-1 activity would suppress MCP-1–mediated inflammation and thereby improve the function of the target organs. If this approach is successful, direct gene transfer into target organs would not be necessary. Apolipoprotein E–knockout (ApoE-KO) mice develop hypercholesterolemia and atherosclerotic lesions similar to those seen in humans and are widely used for studying the pathogenesis of atherosclerosis.^{14 15} Therefore, we tested the effectiveness of this new strategy in ApoE-KO mice.

	Methods

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Experimental Animals
C57BL/6J ApoE-KO mice,¹⁶ purchased from Jackson Laboratory, were bred and maintained in the Laboratory of Animal Experiments at Kyushu University.¹⁷ The present study protocol was reviewed and approved by the Committee on Ethics of Animal Experiments, Kyushu University School of Medical Sciences.

Expression Vector and Preparation of HVJ-Liposome Complexes
FLAG-tagged [3'(C terminus)] mutant MCP-1 (7ND) was constructed by recombinant polymerase chain reaction with a wild-type human MCP-1 cDNA (a generous gift from Dr T. Yoshimura, National Cancer Institute, Frederick, Md) as template and cloned into the BamHI (5') and NotI (3') sites of the pcDNA expression vector plasmid (Invitrogen Corp). All sequences were confirmed by double-stranded DNA sequencing. HVJ-liposome solution was prepared as previously described.¹⁸

Protocol 1: Effect of 7ND Gene Transfection on Intradermal Monocyte Recruitment Induced by Recombinant MCP-1
Either 5 µg 7ND plasmid DNA encapsulated in HVJ-liposome or PBS was injected into the femoral muscle of 8-week-old female C57BL mice. Three days later, recombinant human MCP-1 (100 ng/20 µL; Pepro Tech EC Ltd) or vehicle (20 µL PBS) was injected intradermally in the back of each mouse. After 24 more hours, the injected dermis (5 mmx5 mm) was excised en bloc, and histopathological sections were prepared for immunohistochemical staining of monocytes. Monocytes recruited into the dermis were counted as previously described.¹⁹

Protocol 2: Effects of 7ND Gene Transfection on Monocyte Recruitment and Atherosclerotic Lesion Formation in ApoE-KO Mice
ApoE-KO mice (7 or 8 weeks of age) were fed a Western-type diet containing 20% fat (wt/wt) and 0.15% cholesterol (wt/wt) (Oriental Yeast) and randomized into 2 groups of 8 mice. The PBS-treated group received an intramuscular injection of 50 µL of PBS into the femoral muscles at weeks 0 and 3 after the start of the Western-type diet. The 7ND gene–transfected group was injected with 5 µg 7ND plasmid DNA encapsulated in HVJ-liposome on the same time schedule. At week 6, mice were killed after collection of blood from the vena cava.

Tissue Preparation and Quantification of Atherosclerosis
After the mice were killed, the heart and aorta were removed rapidly after perfusion with PBS and then embedded in OCT compound and quick-frozen in liquid nitrogen. Approximately 200 serial cross sections (6 µm thick) of the aortic root were prepared according to the method described by Paigen et al,²⁰ with a slight modification. In brief, atherosclerotic lesions in the aortic sinus region were examined at 5 locations, each separated by 120 µm, with the most proximal site starting where the 3 aortic valves first appear. Seven serial sections prepared from each location were conventionally stained with oil red O, Masson’s trichrome, and orcein stains.^{17 21} Other sections were stained immunohistochemically with monoclonal antibodies against monocyte/macrophage (MOMA-2, 10 µg/mL, Serotec) or -smooth muscle (-SM) actin (4 µg/mL, DAKO). Some sections were also stained immunohistochemically for MCP-1 with a rabbit anti-rat polyclonal antibody.²² As a negative control, nonimmune IgG was used. After incubation with biotinylated, affinity-purified goat anti-rat IgG followed by avidin-biotin amplification, the slides were incubated with 3',3'-diaminobenzidine (DAB) and counterstained with hematoxylin.

Quantification of atherosclerotic lesions was performed by a single observer blinded to the experimental protocol. All images were captured and analyzed by National Institutes of Health Image software. Lipid lesion formation was analyzed by determining the percent area of oil red O stained to the total cross-sectional vessel wall area. The average value for the 5 locations for each animal was used for analysis. To assess the quality of the lesion, the areas containing collagen (aniline blue on Masson’s trichrome stain), elastin (dark-brown on orcein stain), macrophage accumulation (MOMA-2–positive area), and smooth muscle (-SM actin–positive area) were estimated. The plaque stability score was calculated by the formula plaque stabilization score=(-SM actin–positive area+collagen area)/(macrophage area+oil red O area).

To stain -SM actin with a mouse anti-human -SM actin monoclonal antibody, the primary antibody was first incubated with a goat anti-mouse immunoglobulin conjugated to peroxidase-labeled dextran polymer (EnVision+^TM, DAKO); the nonreacting binding site on the secondary antibody was blocked with normal mouse serum (DAKO). The sections were incubated with the primary-secondary complex for 1 hour and visualized after incubation with DAB.

Western Blots
After immunoprecipitation, a FLAG Western detection kit (Stratagene) was used to detect FLAG-conjugated 7ND MCP-1 in the serum.

Serum Lipid Analyses
Serum total and HDL cholesterol and triacylglycerol concentrations were determined by commercially available kits (Wako Pure Chemicals).

Statistical Analysis
All data are expressed as mean±SEM. Mean values were compared by means of ANOVA and Bonferroni’s multiple comparison tests. A value of P<0.05 was considered statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
7ND Gene Transfection Inhibited Intradermal Monocyte Recruitment Induced by Recombinant MCP-1
We asked whether the intramuscular expression of 7ND could reduce the monocyte recruitment into the dermis induced by recombinant MCP-1. Wild-type C57BL mice were anesthetized, and their femoral muscle was exposed. Either an HVJ-liposome solution (5 µg of encapsulated human 7ND plasmid DNA) or PBS was injected into the muscle. We examined 7ND protein production after intramuscular injection of a C-terminal FLAG epitope-tagged 7ND gene. Western blot analysis showed that FLAG/7ND protein was secreted into the serum (Figure 1a). Three days after mice were injected with the 7ND gene or PBS, recombinant human MCP-1 or vehicle was injected into the dermis. Twenty-four hours after the intradermal injection, immunohistochemical sections of the injection sites were prepared and the number of MOMA-2–positive monocytes present in the injection site was determined. In the mice receiving PBS, the number of monocytes recruited into the dermis was significantly greater in the area of MCP-1 injection than in the area of vehicle injection (Figure 1b). This increase in MOMA-2–positive monocytes was blocked by the intramuscular injection of the 7ND gene. Such blockade of monocyte recruitment into the dermis also occurred on days 7 and 21 (data not shown), suggesting that inhibition of MCP-1–mediated chemotaxis persists for 3 weeks after single intramuscular injection of 7ND gene. It is unlikely that an immune response against human 7ND or FLAG protein might have reduced the efficacy of 7ND gene therapy.

View larger version (32K): [in this window] [in a new window]   Figure 1. Blockade of MCP-1 activity in remote organ (dermis) after transfection of 7ND gene into femoral muscle. a, Secretion of 7ND protein into systemic circulation. Western blot analysis of 7ND protein in serum after intramuscular injection of either C-terminal FLAG-tagged 7ND gene or PBS. Blots are representative of 5 separate experiments. FLAG-7ND protein contents were assessed at indicated times. b, Quantification of monocyte infiltration into dermis induced by intradermal injection of recombinant human MCP-1. *P<0.01 vs vehicle; P<0.01 vs PBS-injected mice. Data are reported as mean±SEM, n=6 to 7.

7ND Gene Transfection Attenuated Monocyte Recruitment and Atherosclerotic Lesion Formation in ApoE-KO Mice
We next asked whether this strategy could be a useful form of gene therapy to treat atherosclerosis. ApoE-KO mice were fed a Western-type diet for 6 weeks. These mice were injected with either the 7ND vector plasmid (5 µg) encapsulated in HVJ-liposome or PBS on days 0 and 21. After 6 weeks on the diet, the PBS-injected mice showed typical fatty atherosclerotic lesions in the aortic root stained with oil red O (Figure 2a). Macrophages were the predominant cell type in atherosclerotic lesions (Figure 2a).

View larger version (73K): [in this window] [in a new window]   Figure 2. Inhibition of atherosclerosis formation and lesional macrophage accumulation by 7ND gene transfer. a, Photomicrographs of aortic atherosclerotic lesions stained with oil red O and MOMA-2 (monoclonal antibody against mouse monocyte/macrophage) from PBS-injected and 7ND-transfected ApoE-KO mice. b, Photomicrographs of aorta from PBS-injected ApoE-KO mouse stained with antibody against MCP-1 (left) or nonimmune IgG (negative control) (right). Bars represent 200 µm. c, Quantitative comparison of atherosclerotic lesion size (oil red O–stained area) in PBS-injected and 7ND-transfected ApoE-KO mice. *P<0.05 vs PBS-injected group. Data are reported as mean±SEM, n=8. d, Quantitative analysis of monocyte/macrophage area in atherosclerotic lesions from PBS-injected and 7ND-transfected ApoE-KO mice. *P<0.05 vs PBS-injected group. Data are reported as mean±SEM, n=7 to 8.

Immunoreactivity for MCP-1 was localized to the intimal cells of atherosclerotic lesions (Figure 2b), whereas no such immunostaining was evident in the aortas of wild-type mice (data not shown). The atherosclerotic lesions were significantly smaller in 7ND-transfected mice than in PBS-injected mice (Figure 2c). In keeping with the reduction in the severity of atherosclerotic lesions by 7ND gene transfection, monocyte/macrophage infiltration was less in 7ND gene-transfected mice than in PBS-injected mice (Figure 2d).

7ND Gene Transfection Increased Atheromatous Plaque Stability
Because an increase in the extracellular matrix content is a characteristic feature of plaque stabilization,³ we examined whether 7ND gene therapy could affect the amount and distribution of extracellular matrix proteins, such as collagen and elastin in atherosclerotic lesions. Quantitative analysis demonstrated that lesional extracellular matrix deposition was greater in 7ND-transfected mice (Figure 3b). The increased lesional extracellular matrix content was present mainly in the capsule and shoulder area of atheromatous lesions. We further examined the lesional composition of vascular smooth muscle cells by using -SM actin immunostaining and found that 7ND transfection increased the amount of -SM actin in both the media and the intima (Figure 3b). The plaque stability score, which assesses the collagen, macrophage, and lipid composition of lesions, was greater in 7ND-transfected mice than in PBS-injected mice (Figure 3c).

View larger version (67K): [in this window] [in a new window]   Figure 3. Stabilization of atheroma after 7ND gene transfer. a, Photomicrographs of aortic root stained for -SM actin or stained with Masson’s trichrome and orcein. Bars represent 200 µm. b, Quantitative comparison of lesional composition in PBS-injected and 7ND-transfected ApoE-KO mice. *P<0.05 vs PBS-injected group. Data are reported as mean±SEM, n=7 to 8. c, Plaque stability score of lesions from PBS-injected and 7ND-transfected ApoE-KO mice. *P<0.05 vs PBS-injected group. Data are reported as mean±SEM, n=7 to 8.

We finally asked whether the beneficial effects of 7ND gene transfer could be due to changes in serum lipid concentrations or peripheral leukocyte counts. There were no significant differences in serum lipid concentrations and in the peripheral white blood cell count between the PBS- and 7ND-injected mice (Table).

View this table: [in this window] [in a new window]   Table 1. Serum Lipid Concentrations and White Blood Cell Count in ApoE-KO Mice

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We have demonstrated for the first time that postnatal blockade of the MCP-1/CCR2 signaling pathway by the intramuscular transfer of a mutant gene significantly reduces the formation of atherosclerotic plaques in ApoE-KO mice. Recently, it was reported that 7ND forms inactive heterodimers with wild-type MCP-1 and inhibits monocyte chemotaxis in vitro.^{12 13} This present study revealed that in vivo transfection of the 7ND gene into skeletal muscle can effectively block MCP-1 activity in remote organs. Most importantly, we found that 7ND transfection reduced the formation of atherosclerotic lesions. Our data strongly suggest that activation of the MCP-1/CCR2 pathway plays an essential role in the formation of atherosclerotic lesions in ApoE-KO mice. We also found that the beneficial effects of 7ND gene therapy are independent of serum lipid concentrations. Therefore, therapeutic inhibition of the MCP-1/CCR2 pathway in addition to lipid-lowering therapy is likely to lead to more lipid-poor stable atherosclerotic plaques.

Furthermore, we found that the lesions in 7ND-transfected mice were not only smaller but exhibited a greater amount of extracellular matrix protein, including collagen and elastin. Lesional vascular smooth muscle cells (VSMC) also were abundant in the 7ND-transfected mice. Taken together, these data suggest that the MCP-1/CCR2 pathway plays an important role in the regulation of the lesional content of extracellular matrix proteins as well as in the regulation of VSMC migration. Interestingly, MCP-1 has been shown to stimulate VSMC migration²³ and dedifferentiation²⁴ and to mediate collagen synthesis.^{25 26} Stable plaques are characterized by increased extracellular matrix content, increased content of differentiated VSMC, and decreased lipid and macrophage accumulation.³ Because the plaque stability score was greater in 7ND-transfected mice than in PBS-injected mice (Figure 3c), 7ND gene transfer may serve to stabilize vulnerable atheromatous plaques prone to rupture.

It is important to note that the inhibitory effects of 7ND gene therapy on hypercholesterolemia-induced atherosclerosis (30% reduction) were partial and less than those demonstrated in MCP-1–deficient or CCR2-deficient mice (50% reduction).^{7 8} These data imply that in addition to the MCP-1/CCR2 pathway, other chemokines (eg, IL-8 or MCP-2) or cytokines (eg, interferon- or tumor necrosis factor) may be involved in atherogenesis. Indeed, double-knockout mice (interferon- plus ApoE) exhibit a significant reduction (60%) in atherosclerotic lesion size.¹¹

Conclusions
Our strategy represents a promising form of gene therapy for the treatment of human vascular disease without apparent side effects. Future study should require careful observation over a long period of time to establish the true risk-benefit ratio.

	Acknowledgments

 
This study was supported by Grants-in-Aid for Scientific Research (11470164, 11158216, 11557056, 10307019, and 10177226) from the Ministry of Education, Science, and Culture, Tokyo, Japan.

Received August 18, 2000; revision received November 8, 2000; accepted November 14, 2000.

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				A. Schober, A. Zernecke, E. A. Liehn, P. von Hundelshausen, S. Knarren, W. A. Kuziel, and C. Weber Crucial Role of the CCL2/CCR2 Axis in Neointimal Hyperplasia After Arterial Injury in Hyperlipidemic Mice Involves Early Monocyte Recruitment and CCL2 Presentation on Platelets Circ. Res., November 26, 2004; 95(11): 1125 - 1133. [Abstract] [Full Text] [PDF]

				C. Weber, A. Schober, and A. Zernecke Chemokines: Key Regulators of Mononuclear Cell Recruitment in Atherosclerotic Vascular Disease Arterioscler. Thromb. Vasc. Biol., November 1, 2004; 24(11): 1997 - 2008. [Abstract] [Full Text] [PDF]

				M. Ishibashi, K. Egashira, Q. Zhao, K.-i. Hiasa, K. Ohtani, Y. Ihara, I. F. Charo, S. Kura, T. Tsuzuki, A. Takeshita, et al. Bone Marrow-Derived Monocyte Chemoattractant Protein-1 Receptor CCR2 Is Critical in Angiotensin II-Induced Acceleration of Atherosclerosis and Aneurysm Formation in Hypercholesterolemic Mice Arterioscler. Thromb. Vasc. Biol., November 1, 2004; 24(11): e174 - e178. [Abstract] [Full Text] [PDF]

				C. A. Bursill, R. P. Choudhury, Z. Ali, D. R. Greaves, and K. M. Channon Broad-Spectrum CC-Chemokine Blockade by Gene Transfer Inhibits Macrophage Recruitment and Atherosclerotic Plaque Formation in Apolipoprotein E-Knockout Mice Circulation, October 19, 2004; 110(16): 2460 - 2466. [Abstract] [Full Text] [PDF]

				C. Alonso-Villaverde, B. Coll, S. Parra, M. Montero, N. Calvo, M. Tous, J. Joven, and L. Masana Atherosclerosis in Patients Infected With HIV Is Influenced by a Mutant Monocyte Chemoattractant Protein-1 Allele Circulation, October 12, 2004; 110(15): 2204 - 2209. [Abstract] [Full Text] [PDF]

				A. Saiura, M. Sata, K.-i. Hiasa, S. Kitamoto, M. Washida, K. Egashira, R. Nagai, and M. Makuuchi Antimonocyte Chemoattractant Protein-1 Gene Therapy Attenuates Graft Vasculopathy Arterioscler. Thromb. Vasc. Biol., October 1, 2004; 24(10): 1886 - 1890. [Abstract] [Full Text] [PDF]

				S. Shimizu, H. Nakashima, K. Masutani, Y. Inoue, K. Miyake, M. Akahoshi, Y. Tanaka, K. Egashira, H. Hirakata, T. Otsuka, et al. Anti-monocyte chemoattractant protein-1 gene therapy attenuates nephritis in MRL/lpr mice Rheumatology, September 1, 2004; 43(9): 1121 - 1128. [Abstract] [Full Text] [PDF]

				H. Niiyama, H. Kai, T. Yamamoto, T. Shimada, K.-I. Sasaki, T. Murohara, K. Egashira, and T. Imaizumi Roles of endogenous monocyte chemoattractant protein-1 in ischemia-induced neovascularization J. Am. Coll. Cardiol., August 4, 2004; 44(3): 661 - 666. [Abstract] [Full Text] [PDF]

				S. Kitamoto, K. Nakano, Y. Hirouchi, Y. Kohjimoto, S. Kitajima, M. Usui, S. Inoue, and K. Egashira Cholesterol-Lowering Independent Regression and Stabilization of Atherosclerotic Lesions by Pravastatin and by Antimonocyte Chemoattractant Protein-1 Therapy in Nonhuman Primates Arterioscler. Thromb. Vasc. Biol., August 1, 2004; 24(8): 1522 - 1528. [Abstract] [Full Text] [PDF]

				Y.-M. Chen, W.-C. Chiang, S.-L. Lin, K.-D. Wu, T.-J. Tsai, and B.-S. Hsieh Dual Regulation of Tumor Necrosis Factor-{alpha}-Induced CCL2/Monocyte Chemoattractant Protein-1 Expression in Vascular Smooth Muscle Cells by Nuclear Factor-{kappa}B and Activator Protein-1: Modulation by Type III Phosphodiesterase Inhibition J. Pharmacol. Exp. Ther., June 1, 2004; 309(3): 978 - 986. [Abstract] [Full Text] [PDF]

				M. Ishibashi, K.-i. Hiasa, Q. Zhao, S. Inoue, K. Ohtani, S. Kitamoto, M. Tsuchihashi, T. Sugaya, I. F. Charo, S. Kura, et al. Critical Role of Monocyte Chemoattractant Protein-1 Receptor CCR2 on Monocytes in Hypertension-Induced Vascular Inflammation and Remodeling Circ. Res., May 14, 2004; 94(9): 1203 - 1210. [Abstract] [Full Text] [PDF]

				T. Wada, K. Furuichi, N. Sakai, Y. Iwata, K. Kitagawa, Y. Ishida, T. Kondo, H. Hashimoto, Y. Ishiwata, N. Mukaida, et al. Gene Therapy via Blockade of Monocyte Chemoattractant Protein-1 for Renal Fibrosis J. Am. Soc. Nephrol., April 1, 2004; 15(4): 940 - 948. [Abstract] [Full Text] [PDF]

				W. Ni, S. Kitamoto, M. Ishibashi, M. Usui, S. Inoue, K.-i. Hiasa, Q. Zhao, K.-i. Nishida, A. Takeshita, and K. Egashira Monocyte Chemoattractant Protein-1 Is an Essential Inflammatory Mediator in Angiotensin II-Induced Progression of Established Atherosclerosis in Hypercholesterolemic Mice Arterioscler. Thromb. Vasc. Biol., March 1, 2004; 24(3): 534 - 539. [Abstract] [Full Text]

R.P. Brande