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(Circulation. 2004;110:2460-2466.)
© 2004 American Heart Association, Inc.

Molecular Cardiology

Broad-Spectrum CC-Chemokine Blockade by Gene Transfer Inhibits Macrophage Recruitment and Atherosclerotic Plaque Formation in Apolipoprotein E–Knockout Mice

Christina A. Bursill, PhD; Robin P. Choudhury, MD; Ziad Ali, MD; David R. Greaves, PhD; Keith M. Channon, MD, FRCP

From the Department of Cardiovascular Medicine (C.A.B., R.P.C., Z.A., K.M.C.), University of Oxford, John Radcliffe Hospital, and the Sir William Dunn School of Pathology (C.A.B., D.R.G.), University of Oxford, Oxford, England.

Correspondence to Prof Keith Channon, Department of Cardiovascular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK. E-mail keith.channon{at}cardiov.ox.ac.uk

Received June 18, 2004; revision received July 24, 2004; accepted July 29, 2004.

	Abstract

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Background— The CC-chemokines (CKs) recruit monocytes/macrophages to sites of inflammation; several different CC-CKs play a role in the pathogenesis of atherosclerosis. The vaccinia virus expresses a 35-kDa soluble protein (35K) that binds to and inactivates nearly all of the CC-CKs, providing a potentially useful therapeutic strategy for broad-spectrum CC-CK inhibition in atherosclerosis. A recombinant adenovirus encoding soluble 35K (Ad35K) was generated to investigate the effect of 35K gene transfer on atherosclerosis in Western diet–fed apolipoprotein E–knockout (ApoE KO) mice.

Methods and Results— ApoE KO mice received tail-vein injections of phosphate-buffered saline, Ad35K, or control adenovirus AdGFP encoding green fluorescence protein. Two weeks after Ad35K gene transfer, atherosclerotic lesion area was significantly reduced in aortic roots by 55% compared with PBS or AdGFP control mice (P<0.05). Furthermore, 35K gene transfer strikingly reduced the macrophage content in aortic root lesions by 85% (P<0.01) and reduced lipid deposition in descending aortas by more than half (P<0.05). By an in vitro chemotaxis assay, plasma and aortic homogenates from 35K gene transfer mice promoted significantly less CC-CK–induced cell migration than did PBS or AdGFP controls.

Conclusions— These findings show that a single intravenous injection of a recombinant adenovirus encoding the broad-spectrum CC-CK inhibitor 35K can reduce atherosclerosis by inhibiting CC-CK–induced macrophage recruitment in atherosclerotic ApoE KO mice. These experiments suggest that CC-CKs play an important role in atherogenesis and are a rational target for therapeutic intervention.

Key Words: gene therapy • inflammation • atherosclerosis • plaque • aorta

	Introduction

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Much evidence suggests that atherosclerosis is an inflammatory disease in which monocyte recruitment plays an important role in disease initiation, progression, and clinical events.^1–5 Chemokines (CKs) are chemoattractant cytokines that direct the migration of specific leukocytes to sites of inflammation or infection and are therefore implicated in atherosclerosis, in particular, members of the CC-CK class.⁶ A host of CC-CKs have been identified in human atherosclerotic lesions, eg, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1 (MIP-1), regulated upon activation, normal T-cell expressed and secreted (RANTES), eotaxin, macrophage-derived chemokine, pulmonary and activation-regulated chemokine, and thymus and activation-regulated chemokine.^7–10 Further evidence that individual CC-CKs are important in atherosclerosis is provided by knockout (KO) or transgenic mice and plasmid transfection studies. Atherosclerotic lesion size was decreased in apolipoprotein E (ApoE)–KO mice with either a targeted deletion of CCR2, the receptor for the CC-CK MCP-1,¹¹ or with targeted deletion of the MCP-1 gene.¹² Conversely, overexpression of MCP-1 accelerates atherosclerosis in ApoE^–/– mice.¹³ Studies with soluble decoy CKs have also found reductions in atherosclerotic lesion development. For example, an N-terminal–deletion mutant of MCP-1 that blocks the MCP-1/CCR2 signaling pathway^14,15 and a modified RANTES peptide (Met-RANTES) that blocks the RANTES/CCR5 signaling pathway attenuated lesion size in ApoE KO mice.¹⁶ These studies demonstrate convincing proof of principle that individual CC-CKs are important in atherosclerosis. However, targeting a single CK/CK receptor pathway is limited by potential redundancy in CK signaling; more broad-spectrum blockade of CC-CK activity may provide a more rational and effective therapeutic strategy in atherosclerosis. Such a strategy has evolved in viruses that encode proteins that bind and inactivate CKs, providing a mechanism to reduce host immunity.¹⁷ The vaccinia virus (strain Lister) expresses the 35-kDa protein 35K (also referred to as vCKBP¹⁸ and vCCI¹⁹) that binds with high affinity to almost all CC-CKs but not to other CK classes.^20,21 We and others have previously demonstrated that recombinant 35K potently inhibits CC-CK—induced cell migration and signaling.²²

Accordingly, we sought to evaluate the potential of broad-spectrum blockade of CC-CK activity as a strategy to reduce atherosclerotic plaque progression and alter composition in vivo by using adenovirally mediated 35K gene transfer in the ApoE KO mouse.

	Methods

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Generation of Adenovirus Expressing 35K
The adenoviral plasmid (Ad) containing the 750-bp fragment of 35K of the vaccinia virus (Lister strain) incorporating a carboxy-terminal hemagglutinating (HA) epitope tag was constructed as described previously.²² A recombinant adenovirus Ad35K was generated by transfection in 293 cells with the AdEasy system.²³ A control recombinant adenovirus AdGFP encoding enhanced green fluorescence protein (GFP; Clonetech) was used as a control for viral infection and was prepared as described earlier. Viruses were isolated and purified as described previously.²⁴

Assessment of Cell Migration
Specific CCR5- and CCR2-receptor–directed cell migration was assessed in transwell membranes (6.0-mm diameter, 8-µm pore size; Receptor Technologies) as previously described.²² In brief, 293 cells were grown to 50% confluence in Dulbecco’s modified Eagle’s medium with 10% (vol/vol) fetal calf serum and then cotransfected (Fugene6, Roche) with plasmids encoding either CCR5 or CCR2 plus GFP to facilitate visualization. Transfected cells were harvested and allowed to migrate overnight toward samples placed in the lower chamber. Migrated cells on the underside of membranes were fixed and quantified by computer analysis of GFP fluorescence in confocal microscope images. Each experimental sample was analyzed in duplicate, and 3 separate images were quantified for each membrane.

Animals and Gene Transfer
To induce atherosclerosis, 4-week-old ApoE KO C57BL6 mice were fed a Western-type diet (21% milk fat, 0.15% cholesterol; 100244 Dyets Inc) for 6 weeks (n=10 to 15/treatment group). Two weeks before euthanization, Ad35K or AdGFP (10¹¹ viral particles in 300 µL phosphate-buffered saline [PBS]) or PBS alone was administered by tail-vein injection. Mice were humanely killed with an overdose of isoflurane anesthetic, and plasma was collected by cardiac puncture. Mice were then perfusion-fixed through the left ventricle with 4% paraformaldehyde in PBS (5 mL). Hearts and descending aortas were excised. Hearts were fixed overnight in paraformaldehyde and embedded in paraffin. Serial transverse sections (5 µm) through the aortic root were stained with Masson-Goldner/elastin stain (Sigma). To evaluate aortic lesions en face, fixed descending aortas were opened longitudinally, pinned out, and stained with oil red O. Aortic root lesion area (mm²), cholesterol clefts (mm²), and oil red O staining in descending aortas were quantified from digitized microscopic images with Image Pro-Plus software (Media Cybernetics). All animal procedures were carried out in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986 and after local ethics board review.

Histology and Immunohistochemistry of Atherosclerotic Aortic Root Sections
Paraffin-embedded aortic root sections were stained for collagen with Sirius red, for smooth muscle -actin with an anti–-actin–alkaline phosphatase (AP) conjugate (Sigma), and for macrophages with mouse macrophage antigen (MOMA)-2 (Accurate Chemicals). For MOMA-2 immunostaining, sections were dewaxed, blocked for 8 hours with 1% horse serum, and incubated overnight at 4°C with MOMA-2 (1:100, 5 µg/mL). Sections were then incubated for 30 minutes with biotinylated anti-rat immunoglobulin followed by avidin-biotin–AP complex and visualized with Vector Red alkaline phosphatase substrate (Vector Laboratories). Sections were counterstained with methyl green.

For measurement of lipid in aortic root lesions, fresh aortic root sections (5 µm) from a separate cohort of mice administered PBS, AdGFP, or Ad35K (n=5/treatment group) were fixed with formalin, stained with oil red O, and counterstained with hematoxylin. Oil red O staining (mm²) was quantified from digitized microscopic images with Image Pro-Plus software (Media Cybernetics).

Western Immunoblotting
To evaluate recombinant 35K in mouse plasma, 150 µL of plasma was incubated for 2 hours with monoclonal anti-HA agarose–conjugated beads (Sigma). The beads were then washed and diluted 1:1 in 2x sodium dodecyl sulfate (SDS) sample buffer, and proteins were denatured by heating at 95°C for 3 minutes. Beads were pelleted by centrifugation, and the supernatant was separated on 14% SDS–polyacrylamide gel electrophoresis (PAGE) gels. After transfer to polyvinylidene difluoride membranes, 35K protein was detected with a rat monoclonal anti-HA high-affinity antibody diluted 1:2000 (Roche), followed by an anti-rat secondary antibody conjugated to horseradish peroxidase diluted 1:1500.

Plasma Lipids
Plasma lipids were measured by enzymatic assay (Roche) on heparinized blood plasma with a Cobas Mira Plus automated analyzer (Roche).

Statistical Analysis
All values are expressed as mean±SEM. Data were analyzed by 1-way ANOVA and Tukey’s post hoc test of significance for comparison of controls with the treatment groups. A value of P<0.05 was the criterion of significance.

	Results

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35K Protein Is Expressed in ApoE KO Mice After Adenoviral Gene Transfer
To evaluate the efficacy of Ad35K gene transfer for expression of 35K in ApoE KO mice in vivo, mice were infected with Ad35K, AdGFP, or PBS through a single tail-vein injection. Efficient liver-directed gene transfer was confirmed by widespread GFP fluorescence in liver sections from animals receiving AdGFP. Analysis of mouse plasma by Western immunoblotting to detect HA-tagged proteins revealed a single protein of molecular mass 35 kDa 14 days after Ad35K gene transfer (Figure 1). No HA-tagged proteins were detectable in plasma from mice injected either with PBS or with the control adenovirus AdGFP. No changes were observed in plasma total or HDL cholesterol concentrations between treatment groups (Table).

View larger version (15K): [in this window] [in a new window]   Figure 1. Detection of recombinant, soluble, 35K protein in ApoE KO mouse plasma. Control and mouse plasma samples were incubated with anti-HA agarose-conjugated beads and then fractionated by SDS-PAGE. Analysis of mouse plasma by Western immunoblotting with antibody to HA tag epitope revealed a single protein of molecular mass 35 kDa 14 days after Ad35K gene transfer. No HA-tagged proteins were detectable in plasma from mice injected either with PBS or with control adenovirus AdGFP. Abbreviations are as defined in text.

View this table: [in this window] [in a new window]   Aortic Root Lesion Data and Plasma Lipids

35K Gene Transfer Reduces Atherosclerosis in ApoE KO Mice
To determine the effect of Ad35K gene transfer on atherosclerosis, 4-week-old ApoE KO mice were fed a Western diet for 6 weeks. Two weeks before euthanization, PBS, AdGFP, or Ad35K was delivered by tail-vein injection. Paraffin-embedded aortic root sections were analyzed for atherosclerotic lesion size (Figure 2). Six weeks of Western diet feeding resulted in American Heart Association–designated type II or III lesions,²⁵ consisting of accumulated foam cells and lipid-laden smooth muscle cells. In more advanced lesions, there were collections of extracellular lipid (cholesterol clefts). Female mice had 25% larger lesions than did male mice after 6 weeks of Western diet feeding. Ad35K treatment significantly reduced atherosclerotic lesion size by more than half (55%) for both males and females in aortic roots (P<0.05) compared with either PBS or AdGFP control mice.

View larger version (77K): [in this window] [in a new window]   Figure 2. In vivo gene transfer of 35K reduces atherosclerotic lesion size in aortic roots of ApoE KO mice. Four-week-old ApoE KO mice were fed Western diet for 6 weeks. Two weeks before euthanization, mice were infected with PBS, AdGFP, or Ad35K via single tail-vein injection. Total lesion area (mm2) was determined on paraffin-embedded aortic root sections stained with Masson-Goldner/elastin stain. Upper panels are representative lesions of aortic root sections in treatment groups. Closed shapes are males, open shapes are females. **Denotes significant difference between PBS and AdGFP control groups for males and females, P<0.05, n=10 to 15 mice/treatment group. Abbreviations are as defined in text.

Immunostaining revealed that macrophage content in aortic root lesions was strikingly reduced by 85% (P<0.01) after 35K gene transfer in both males and females compared with PBS and AdGFP control mice (Figure 3). In addition, there was significantly less cholesterol cleft formation (40%, P<0.05) and oil red O staining (55%, P<0.05) in aortic root lesions between Ad35K-infected mice and PBS or AdGFP controls (Table). Collagen and -actin contents were both very low in these early lesions. No changes were detected in collagen content or -actin immunostaining between groups (Table).

View larger version (66K): [in this window] [in a new window]   Figure 3. In vivo gene transfer of 35K reduces macrophage recruitment in aortic roots of ApoE KO mice. Four-week-old ApoE KO mice were fed Western diet for 6 weeks. Two weeks before euthanization, mice were infected with PBS, AdGFP, or Ad35K. MOMA-2 antibody was used to detect total macrophages (mm2) in lesions of paraffin-embedded aortic root sections. Upper panels are representative images of MOMA-2 staining in lesions of aortic root sections for different treatment groups. Closed shapes are males, open shapes are females. **Denotes significant difference between PBS and AdGFP control groups for males and females, P<0.05, n=10 to 15 mice/treatment group. Abbreviations are as defined in text.

To determine whether Ad35K gene transfer also reduced lipid deposition in the descending aortas, excised aortas were stained with oil red O (Figure 4). Lipid deposition in Ad35K-treated mice was significantly reduced (P<0.05) by half compared with PBS- and AdGFP-treated control mice.

View larger version (43K): [in this window] [in a new window]   Figure 4. In vivo gene transfer of 35K reduces lipid deposition in descending thoracic aortas. Four-week-old ApoE KO mice were fed Western diet for 6 weeks. Two weeks before euthanization, mice were infected with PBS, AdGFP, or Ad35K. Percentage lipid deposition in descending aortas was determined with oil red O. Lower panels are representative images of oil red O staining in descending aortas for different treatment groups. **Denotes significant difference between PBS and AdGFP control groups, P<0.05, n=10 to 15 mice/treatment group. Abbreviations are as defined in text.

35K Protein Inhibits CC-CK Activity in Both Plasma and Aortas of ApoE KO Mice
To investigate the effects of in vivo 35K gene transfer on CC-CK activity, CCR5- and CCR2-dependent cell migration was assessed in response to plasma and aortic extracts from both wild-type C57BL6 mice and ApoE KO mice that had been injected with PBS, AdGFP, or Ad35K (Figure 5). Plasma from ApoE KO mice induced significantly higher levels of cell migration than did that from age-matched wild-type animals for CCR5 (2.5-fold, P<0.05) and CCR2 (1.7-fold, P<0.05)-directed cell migration. Consistent with this, homogenized aortas from ApoE KO mice also induced significantly higher levels of cell migration than did aortas from age-matched wild-type animals for CCR5-directed cell migration (2.5-fold, P<0.05), demonstrating that atherosclerosis increases CCR5- and CCR2-dependent chemotactic activity. However, both CCR5- and CCR2-directed cell migration induced by plasma from Ad35K gene transfer ApoE KO mice was reduced by 80% (P<0.01) compared with plasma from PBS or AdGFP control mice. Furthermore, homogenized aortas from Ad35K gene transfer ApoE KO mice also induced significantly less cell migration than did homogenized aortas from PBS or AdGFP control mice (Figure 5). These results suggest adenovirally mediated delivery of soluble 35K protein reduces atherosclerosis by inhibiting CC-CK activity in both the plasma and aortas of ApoE KO mice.

View larger version (18K): [in this window] [in a new window]   Figure 5. In vivo 35K gene transfer reduces CC-CK activity in ApoE KO Mice. A, In transwell membranes, 293 cells transfected with either CCR5 or CCR2 were allowed to migrate toward plasma (10 µL) from chow-fed wild-type (WT) and ApoE KO mice fed Western diet for 25 weeks that were euthanized 3 days after infection with PBS, AdGFP, or Ad35K. B, In transwell membranes, 293 cells transfected with CCR5 were allowed to migrate toward homogenized descending aortas (50 µL) from WT and ApoE KO mice euthanized 3 days after infection with PBS, AdGFP, or Ad35K. All values are mean±SEM, n=3 mice/treatment, presented as total green cell pixel count (duplicates, 3 scans/membrane). Denotes significant difference between ApoE KO (black bars) and WT (white bars) mice. *Denotes significant difference between PBS and AdGFP controls for both WT and ApoE KO mice, P<0.05. All other abbreviations are as defined in text.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In this article, we describe the use of adenovirally mediated gene transfer to express a soluble, broad-spectrum CC-CK inhibitor, 35K. To investigate the importance of CC-CKs in atherosclerosis, we delivered 35K by adenovirally mediated gene transfer into ApoE KO mice fed a Western diet through a single intravenous injection. We report the following important findings. First, adenovirally mediated expression of 35K significantly reduces atherosclerotic lesion size in the aortic roots of ApoE KO mice. Second, 35K gene transfer greatly reduces both macrophage recruitment and lipid deposition in atherosclerotic lesions. Finally, Ad35K gene transfer strikingly inhibits CCR5- and CCR2-mediated chemotactic activity in plasma and CCR5-mediated chemotactic activity in aortas from ApoE KO mice.

These findings provide important insights into the role of CC-CKs in atherosclerotic lesion development, demonstrating that the activation of CC-CKs is crucial for macrophage recruitment. CC-CKs act through G protein–coupled receptor-signaling pathways. Previous animal-KO and transgenic studies, as well as soluble decoy CK studies, have shown convincing proof of principle that individual CC-CK/CCR signaling pathways are important in atherosclerosis, particularly the MCP-1/CCR2 and the RANTES/CCR5 pathways. However, these studies targeted only a single CC-CK or CC-CK receptor, which is limited by redundancy in CC-CK signaling. In contrast, we used adenovirally mediated delivery of soluble 35K protein to provide broad-spectrum blockade of CC-CK activity, which resulted in a striking 85% reduction in macrophage recruitment and a 55% reduction in aortic root lesion size compared with controls. These reductions in total atherosclerotic lesions size and macrophage recruitment are at least comparable with other studies that have targeted CK/CK receptor signaling. For example, the ApoE/CCR2 double-KO mouse¹¹ had a 55% reduction in aortic root lesion size and a 60% reduction in macrophage recruitment. Targeted deletion of the MCP-1 gene in the ApoB transgenic mouse¹² also produced a 60% reduction in aortic root lesion size and a significant but not defined reduction in macrophage recruitment. The use of receptor antagonists also alters atherosclerosis. For example, plasmid transfection of an N-terminal–deletion mutant of MCP-1, which acts as a competitive inhibitor of MCP-1/CCR2 signaling, reduced macrophage recruitment by 21% and lesion size by 33%.¹⁴ Similarly, Met-RANTES, a competitive inhibitor of CCR5/RANTES signaling, required twice-weekly intraperitoneal injection into LDL receptor^–/– mice for 14 weeks in parallel with a high-fat diet to reduce macrophage recruitment by 45% and lesion size by 50%.¹⁶ Taken together, these previous studies suggest that reductions in atherosclerotic lesion size and macrophage recruitment can be achieved by manipulating a single CK/CK receptor signaling pathway. 35K gene delivery appears to be at least as effective in reducing lesion size and macrophage recruitment and exerts these effects within only 2 weeks after a single intravenous delivery. It is difficult to directly compare a 2-week gene-transfer study with monotargeted KOs that have constitutive deletion of the gene of interest. Future studies with long-term CC-CK inhibition combined with specific CK/CK receptor KOs will be required to more completely assess the potential importance of the CC-CK class in atherosclerosis and the relative merits of broad-spectrum verse monotargeting as a therapeutic strategy.

Despite the apparent importance of the CC-CK class in atherosclerosis, recent studies have also provided strong evidence that other CK classes such as the CX₃C and the CXC CKs also play important roles in atherosclerosis. CX₃CL1, also known as fractalkine, and its receptor CX₃CR1 are the only members of the CX₃C CK class. Fractalkine is present in human atherosclerotic lesions,^26,27 and smooth muscle cells in the neointima of human atherosclerotic plaques express its receptor, CX₃CR1.²⁷ Furthermore, 2 studies have shown that ApoE/CX₃CR1 double-KO mice had significant reductions in lesion size in the proximal aorta and in the thoracic and abdominal descending aortas compared with ApoE KO control mice.^28,29 There is also evidence that the CXC class of CK, responsible for neutrophil chemotaxis, is important in atherosclerosis. Adoptive transfer of CXCR2 KO bone marrow was protective in mice with myocardial ischemia-reperfusion injury, leading to reductions in inflammatory cell infiltration and infarct size.³⁰ These potentially important contributions by other CK classes may limit the effectiveness of CC-CK blockade alone in atherosclerosis. Indeed, our previous work confirmed that 35K has no effect on fracktalkine/CX3CR1 signaling.²² Future studies need to assess the relative importance of both CC and CXC CKs in atherosclerosis to identify the most promising therapeutic strategies.

Cholesterol clefts in atherosclerotic plaque reflect the precipitation of cholesterol monohydrate crystals when cellular membranes and oily cholesteryl ester–rich droplets can accommodate no more cholesterol³¹ Because the primary atherogenic event of lipid deposition in the subendothelial space is not believed to be macrophage dependent,³² it does not necessarily follow that inhibition of macrophage recruitment through CC-CK blockade would decrease plaque cholesterol content. We observed reductions in both neutral lipid deposition by oil red O staining and in cholesterol clefts after 35K gene transfer. Our finding that the cholesterol cleft area in plaque was reduced by 40% is consistent with studies in which macrophage function disruption (through deletion of genes for macrophage scavenger receptor-A³³ or macrophage colony-stimulating factor [MCSF]³⁴) reduced plaque size^33,34 and qualitatively less advanced lesion stage.³⁴ In the latter case, lesion progression was retarded in MCSF-deficient mice despite significantly higher plasma cholesterol in that group. Taken together, these findings suggest that modification of atherogenic lipoproteins in the arterial wall and subsequent uptake by macrophages are more important in plaque progression and lipid deposition than the more "passive" influence of plasma cholesterol concentration.

Despite the striking changes in macrophage and lipid content, we observed no significant changes in smooth muscle cell -actin or collagen staining in aortic root lesions between treatment groups. Six weeks of Western diet feeding induces American Heart Association type II or III lesions²⁵ that contain very low absolute levels of -actin or collagen. In these lesions, it is likely too early in the atherosclerotic process for significant smooth muscle and/or collagen remodeling to have occurred after 35K gene transfer, making it difficult for differences to be detected.

To explore the potential mechanism underlying the action of 35K in atherosclerosis, we established an in vitro chemotaxis bioassay with either CCR5- or CCR2-transfected cells. We have demonstrated that Ad35K gene transfer significantly reduced CC-CK activity in both ApoE KO mouse plasma and aortas. These data indicate that 35K inhibits systemic CC-CK activity and explains the reduction in lesion size through inhibition of macrophage recruitment. Indeed, CC-CK activity is important in the initiation and progression of atherosclerosis. For CKs to recruit monocytes to the endothelium and trigger migration into the vessel wall, they must be bound to glycosaminoglycans (GAGs) on the endothelial cell surface. The exact mechanism of CC-CK inhibition by 35K has not yet been elucidated in vivo, although based on structural evidence from in vitro studies, it is thought to compete for binding with their cognate G protein–coupled receptors by interacting with specific residues conserved among many of the CC-CKs.¹⁹ Systemic soluble 35K protein may be acting via a number of mechanisms to inhibit CC-CK–induced macrophage recruitment in atherosclerosis. First, 35K protein may bind to circulating CC-CKs and prevent them from binding to the endothelium. Second, 35K may bind to CC-CKs on the endothelial cell surface and interfere with GAG–CC-CK interaction, effectively "stripping" the CC-CKs from the endothelial cell surface. Third, 35K may bind to the CC-CKs on the endothelial cell surface and remain bound, thereby preventing interaction with circulating monocytes. In previous work, we found that an increased viral dose of Ad35K resulted in an increase of CC-CK RANTES and MIP-1 in plasma,²² suggesting that the first 2 mechanisms are more likely where recombinant 35K is binding CC-CKs, sequestering them into the circulation and preventing them from binding to the endothelial GAGs.

In conclusion, we demonstrate that with a single intravenous injection, adenovirally mediated gene transfer of the soluble, broad-spectrum CC-CK inhibitor 35K effectively inhibited CCR5-mediated chemotaxis in both plasma and aortas and strikingly reduced macrophage recruitment and aortic root lesion size in the ApoE KO, Western diet–fed model of atherosclerosis. These findings highlight the importance of CC-CKs in macrophage recruitment in atherosclerosis and raise the prospect of broad-spectrum CC-CK inhibition as a rational therapeutic target in atherosclerosis.

	Acknowledgments

 
This work was supported by the British Heart Foundation. We thank Martina McAteer and Nicholas Alp for helpful advice.

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