A Novel RANTES Antagonist Prevents Progression of Established Atherosclerotic Lesions in Mice
Background— Atherosclerosis is a chronic inflammatory disease that represents the primary cause of death through coronary disease and stroke. Chemokines are known to play a crucial role in this disease by recruiting inflammatory leukocytes to the endothelium. Recently, the chemokine variant [44AANA47]-RANTES was shown to impair inflammatory cell recruitment in vivo by interfering with heparin binding and oligomerization.
Methods and Results— In this study we report that curative treatment with [44AANA47]-RANTES limits atherosclerotic plaque formation in LDLr−/− mice. This was associated with reduced infiltration of T cells and macrophages and reduced production of matrix metalloproteinase (MMP)-9. By contrast, the relative smooth muscle cell and collagen content was increased, indicating a more stable plaque phenotype. In addition, we provide evidence for direct inhibition of leukocyte recruitment into aortic root lesions, attenuated leukocyte rolling and arrest in mesenteric vessels, as well as a reduced proinflammatory response following Con A stimulation in vitro.
Conclusions— Interference with chemokine oligomerization and chemokine/heparin interactions is a powerful novel approach that inhibits progression of established atherosclerosis in mice. By inhibiting leukocyte recruitment into plaques, [44AANA47]-RANTES mediates a less inflammatory plaque phenotype and thus reduced systemic inflammatory state.
It is now generally recognized that atherosclerosis is a chronic inflammatory disease that can lead to acute clinical events after plaque rupture and thrombosis.1,2 Prevention and current treatments for atherosclerosis are mainly based on 3-hydroxy-3-methylglutaryl (HMG) coenzyme A (CoA) reductase inhibitors, also known as statins. These drugs improve cardiovascular outcome by their lipid-lowering, plaque-stabilizing, and antiinflammatory effects.3 Nevertheless, atherosclerosis remains the primary cause of heart disease and stroke accounting for up to 50% of deaths in Western countries. Thus, a challenge for future research is the identification and development of promising novel antiinflammatory therapies.
Chemokines (chemotactic cytokines) are small proteins that direct the movement of circulating leukocytes to sites of inflammation or injury.4 They have been implicated in a wide range of inflammatory diseases. Consistently, the presence of chemokines and chemokine receptors within human and animal atherosclerotic lesions is well documented.4,5 The recruitment of inflammatory cells is mediated through interactions of chemokines with high-affinity cell surface receptors and a low affinity interaction with glycosaminoglycans (GAGs) of the extracellular matrix and endothelial cell surfaces. The immobilization of chemokines via binding to GAGs is thought to facilitate their retention on cell surfaces and enable localized gradients to form. It has been postulated that blocking the interaction of chemokines with GAGs may offer an alternative therapeutic strategy. In particular, the binding and oligomerization of chemokines by GAGs has been shown to be essential for the in vivo function of several chemokines such as CCL5/RANTES (regulated on activated normal T-cell expressed and secreted).6 Moreover, the role of CCL5/RANTES oligomerization has recently been shown to affect other activities such as apoptosis.7 Mutagenesis studies have led to the identification of specific chemokine binding sites for heparin, often used to predict binding to heparan sulfate, a commonly occurring cell surface GAG. The variant [44AANA47]-RANTES with specific mutations in the principal RANTES/GAG binding site has been shown to exhibit potent inhibitory capacity in murine models of inflammatory cell infiltration such as peritonitis, lung inflammation, and experimental autoimmune encephalomyelitis (EAE).8 This effect was attributed to impaired chemokine oligomerization, crucial for its in vivo function, whereas receptor activation was not abrogated.8 Thus, [44AANA47]-RANTES acts as a potent inhibitor of endogenous RANTES in vivo, but retains its ability to induce chemotaxis in vitro. By forming heterodimers with the wild-type protein, [44AANA47]-RANTES prevents it from oligomerization on the endothelial cell surface. Interestingly, this new mechanism accounting for [44AANA47]-RANTES-mediated inhibition seems to have an increased therapeutic potency over blocking the chemokine/receptor interaction via Met-RANTES, as observed in the EAE model.8,9 In a previous report, we have demonstrated that Met-RANTES efficiently reduced the onset of atherosclerotic plaque formation in mice.10 Here, we hypothesized that the chemokine antagonist [44AANA47]-RANTES interfering with oligomerization would attenuate progression of advanced atherosclerotic lesions and might therefore offer a new therapeutic approach.
For expanded materials and methods, please see http://atvb. ahajournals.org.
As a model of in vivo atherosclerosis, we used 10-week-old male LDLr−/− C57BL/6 mice. For histological and atherosclerotic plaque development analysis as well as proliferation and cytokine analysis, littermate mice were fed with high-cholesterol diet (1.25% cholesterol; Research Diets) for 22 weeks. [44AANA47]-RANTES (10 μg/ mouse) was administered daily by i.p. injection beginning 11 weeks after initiating the high cholesterol diet (n=10). Control mice (11 weeks diet: n=3; 22 weeks diet: n=10) were injected in parallel with PBS. All animal studies have been approved by the local Ethical Committee.
Atherosclerotic Lesion Size Quantification
Atherosclerotic lesions within the thoraco-abdominal aorta and aortic sinus were analyzed by Sudan IV staining for lipid deposition. We calculated for each aortic root an average of lipid deposition from 6 sections (5 μm) separated by 50 μm from each other. Quantification of lipid deposition was performed by computer image analysis using the MetaMorph6 software (Zeiss).
Immunostaining was performed on cryosections of mouse aortic roots as previously described.10 Collagen staining was performed with Sirius red. Data are expressed as relative stained areas of total plaque sizes (n=10 per group).
For measurements of leukocyte counts and cholesterol content, blood samples were collected at end of diet. Cholesterol was measured in the serum with the Infinity kit (Thermo Electron Corporation) according to the manufacturer’s guidelines.
Splenocytes were isolated from [44AANA47]-RANTES- or PBS-treated mice after 22 weeks of diet (n=10 per group) and were stimulated in triplicates with varying concentrations of Con A (Sigma). After 72 hour, cell proliferation was determined using a nonradioactive MTS cell proliferation assay (Promega) according to the manufacturer’s guidelines.
For cytokine analysis, splenocytes were cultured under the same conditions as described for the proliferation assay and stimulated with 2 μg ml−1 Con A. Supernatants were recovered after 48 hour (for IFN-γ, interleukin (IL)-12 and transforming growth factor (TGF)-β measurement) and 72 hour (for IL-4, IL-5, IL-10 and IL-13). Murine IFN-γ, IL-12 (p70), IL-4, IL-10 and TGF-β were assayed by ELISA using paired antibodies according to the manufacturer’s instructions (R&D Systems).
Real-Time RT Polymerase Chain Reaction (PCR)
Total RNA from mouse abdominal aortas and spleens was extracted from [44AANA47]-RANTES- or PBS-treated mice after 22 weeks of diet using TRI Reagent (MRC Inc., Cincinnati, Ohio) and reverse transcription was performed using the Quantitect kit (Qiagen). Real-time PCR (RT PCR) was performed with the ABI Prism 7000 Sequence Detection System (Applied Biosystems; n=10 per group).11
Western Blot Analysis
Proteins from spleen were harvested in ice-cold radioimmunoprecipitation (RIPA) lysis buffer. Total protein concentrations were determined using the bicinchoninic acid (BCA) quantification assay (Pierce) and Western blotting was performed as previously described.12
LDLr−/− mice were fed with a high cholesterol diet for 17 weeks, and [44AANA47]-RANTES (n=9 per group) or PBS (n=8 per group) was administered daily as described above during the last 6 weeks of diet. At the end of the experiment, intravital microscopy of the microvasculature of the mesentery was performed as previously described.8
Adoptive Transfer of Labeled Peritoneal Cavity Cells
Adoptive transfer was performed as previously described.13 Total leukocytes isolated from thioglycollate stimulated donor mice were injected i.v. in recipient LDLr−/− mice. In parallel, recipient mice were treated with [44AANA47]-RANTES or PBS i.p. After 3 days, fluorescent-labeled leukocytes into the atherosclerotic lesions of recipient mice were examined by fluorescent microscopy and counted.
All results are expressed as mean (±SEM). Differences between the probability values below 0.05 were considered significant using the 2-tailed Student t test or the Mann–Whitney Rank Sum Test.
[44AANA47]-RANTES Inhibits Atherosclerotic Plaque Progression in Mice
To assess the therapeutic potential of [44AANA47]-RANTES treatment on established atherosclerosis, low-density lipoprotein receptor–deficient (LDLr−/−) mice were fed a high-cholesterol diet for 11 weeks before beginning of treatment. Then, [44AANA47]-RANTES was administered by daily injection under continuous diet feeding for another 11 weeks, whereas control mice were injected in parallel with PBS. Weight, total cholesterol, and the number of circulating leukocytes were comparable between controls and treated animals (supplemental Table I, available online at http://atvb.ahajournals.org). After a total of 22 weeks of diet, the extent of atherosclerotic lesions within thoraco-abdominal aortas of the PBS-injected mice was significantly increased as compared to controls euthanized after 11 weeks of diet (Figure 1a). In mice treated with [44AANA47]-RANTES, progression of atherosclerotic lesions was significantly attenuated. The same pattern was observed in the aortic roots (Figure 1b).
[44AANA47]-RANTES Induces a More Stable Plaque Phenotype
The immunohistochemical analysis of atherosclerotic lesions revealed a significant reduction of inflammatory leukocyte infiltration in [44AANA47]-RANTES-treated mice, as observed by reduced relative T cell and macrophage content (Figure 2a and 2b). Consistently, we found reduced production of MMP-9, a major extracellular matrix-degrading enzyme (Figure 2c) mainly expressed by macrophages within atherosclerotic lesions. The relative smooth muscle cell content, however, was significantly increased (Figure 2d). This was associated with a higher amount of collagen within lesions of [44AANA47]-RANTES-treated mice (Figure 2e). In conclusion, our findings indicate that [44AANA47]-RANTES treatment leads to reduced lesion size development and a more stable plaque phenotype.
The Inflammatory Response Is Reduced in [44AANA47]-RANTES Treated Mice
We next investigated whether the observed antiatherosclerotic effect of [44AANA47]-RANTES treatment was associated with a modulation of pro- or antiinflammatory cytokine secretion. For this purpose, spleen-isolated cells from PBS-injected controls or mice treated with [44AANA47]-RANTES were collected after 22 weeks of high-cholesterol diet and analyzed for their proliferative response and capacity to produce various cytokines following stimulation with concanavalin A (Con A). We observed a reduced proliferation and secretion of the proinflammatory Th1 cytokines IFN-γ and tumor necrosis factor (TNF)-α in [44AANA47]-RANTES–treated mice as compared to control mice, whereas no difference in Th2 cytokine profiles (IL-4, IL-5, IL-6, IL-13) was observed (Figure 3a and 3b, and data not shown). In addition, no difference in TGF-β and IL-10 cytokine levels, markers of regulatory T lymphocytes or alternatively activated macrophages,14 was found. Similarly, we did not observe significant differences in mRNA levels of the Th2 response-linked transcription factor GATA-3 and the regulatory T cell marker FoxP3 induced by [44AANA47]-RANTES-treatment. Conversely, the mRNA levels of the Th1 marker Tim-3 were reduced (Figure 3c). In support of these findings, we found that [44AANA47]-RANTES suppressed the activation (phosphorylation) of signal transducer and activator of transcription (STAT) 4, which plays a key role in IL-12–dependent Th1 lineage commitment15 (Figure 3d).
Reduced Expression of Chemokine Receptors CCR2 and CCR5 as Well as Chemokine CCL2
The chemoattraction and subsequent migration of inflammatory cells in the intima in response to endothelial injury is a major process during atherogenesis. To assess whether [44AANA47]-RANTES might modulate the expression patterns of major chemokine receptors involved in leukocyte chemoattraction, we quantified the mRNA levels of the RANTES receptors CCR1, CCR3, and CCR5 as well as CCR2. The mRNA analysis revealed reduced levels of CCR2 and CCR5 within atherosclerotic aortas, whereas the mRNA level of CCR1 and CCR3 was not modulated by [44AANA47]-RANTES administration (Figure 4a). Moreover, we observed less expression of the proatherogenic chemokine CCL2/MCP-1 mRNA within the aortic tissue in mice treated with [44AANA47]-RANTES (Figure 4b).
[44AANA47]-RANTES Reduces Inflammatory Leukocyte Rolling and Arrest in Microvessels and Atherosclerotic Lesion Infiltration
To investigate a potential direct effect of [44AANA47]-RANTES on circulating leukocyte recruitment in our model, we used 2 different approaches. First, mice were fed on high cholesterol diet for 11 weeks before initiating the treatment with the chemokine variant under continuous diet feeding for another 6 weeks. We found increased numbers of rolling and arrested leukocytes within mesenteric microvessels of mice fed with high cholesterol diet as compared to normal chow controls, which correlates well with a recently published study.16 Using a quantitative assay to track labeled populations of blood monocytes, the authors show that the monocyte accumulation in aortas directly correlates with the increase of atherosclerotic lesion sizes in mice.16 As expected, treatment with [44AANA47]-RANTES efficiently reduced the numbers of rolling and arrested leukocytes within mesenteric microvessels (Figure 5a and 5b). To further investigate the [44AANA47]-RANTES effect on leukocyte recruitment at more plaque-prone arterial sites, we performed adoptive transfer experiments. Recipient LDLr−/− mice fed on high-cholesterol diet for 16 weeks were injected with fluorescent-labeled leukocytes and treated with [44AANA47]-RANTES or PBS. After 3 days, the number of fluorescent leukocytes recruited into aortic root lesions was significantly lower in [44AANA47]-RANTES-treated mice as compared to control mice (Figure 5c and 5d).
The recruitment of inflammatory cells in the intima is an essential step in the development and progression of atherosclerosis. This process is triggered by local production of chemokines and chemokine receptors from activated endothelial and inflammatory cells. The biological activity of chemokines has been shown to be critically influenced by their association with glycosaminoglycans (GAGs),6 tethered to proteoglycans on cell surfaces and in the extracellular matrix. GAGs such as heparin or heparin sulfate are highly sulfated oligosaccharides, which are posttranslationally sulfated by membrane-bound specific enzymes. The GAG interaction is thought to be responsible for the establishment of chemokine gradients over endothelial cells under vascular flow conditions, which is an important step for initial leukocyte recruitment and subsequent migration through the tissue.17 The chemokine variant [44AANA47]-RANTES was shown to impair inflammatory cell recruitment in vivo by interfering with heparin binding and oligomerization.8 By forming heterodimers with the wild-type RANTES, [44AANA47]-RANTES prevents it from oligomerization on the endothelial cell surface.
In this study, we report that curative treatment with [44AANA47]-RANTES limits atherosclerotic plaque progression of advanced atherosclerosis and inhibits leukocyte recruitment into atherosclerotic lesions. In addition, the treatment induced the development of a more stable plaque phenotype, as shown by reduced inflammatory leukocyte infiltration and reduced MMP-9 expression, whereas the relative SMC and collagen content was increased.
To investigate underlying mechanisms of the antiatherosclerotic effect, we studied the inflammatory state in [44AANA47]-RANTES–treated animals. Previous studies have demonstrated the presence of a predominantly proinflammatory Th1-type T cell response in atherosclerosis.18,19 We found that the proinflammatory immune response was reduced by [44AANA47]-RANTES treatment, as determined by reduced proliferative capacity and IFN-γ and TNF-α secretion in response to Con A, inhibition of transcription factor STAT4 activation, and reduced TIM3 mRNA levels. Although significant, the observed changes on Th1 cytokine/marker expression were only moderate and thus probably not the major cause for the observed antiatherosclerotic effects. More likely, the reduced proliferation and Th1 cytokine expression in response to Con A stimulation was a secondary effect of reduced leukocyte recruitment into atherosclerotic lesions. The presence of fewer inflammatory cells within the plaques probably leads to less proinflammatory cytokine and chemokine expression within the lesion, which consequently leads to a less systemic inflammatory state.
We next addressed the possible modulation of chemokine receptors in our experimental model. Although [44AANA47]-RANTES does not function as a receptor antagonist, it is conceivable that the interaction of RANTES with the antagonist leads to reduced chemokine receptor binding, thus triggering modulation of receptor expression. Furthermore, it is possible that altered RANTES signaling not only directly affects the expression of RANTES chemokine receptors, but may indirectly modulate other chemokine receptors as well. It has been previously shown that [44AANA47] RANTES has a comparable affinity as wild-type RANTES to the CCR5 receptor (IC50 of 6.8±2.8 nmol/L versus wild-type IC50 of 2.15±2.11 nmol/L), whereas the affinity of [44AANA47] to CCR1 is 50- to 100-fold reduced (IC50 of 380±147 nmol/L versus wild-type IC50 of 4.9±2.5 nmol/L).33 Importantly, it has been demonstrated that [44AANA47] RANTES does not downregulate CCR5 on monocytes, suggesting that the inhibitory effect mediated by [44AANA47] RANTES is not a direct consequence of leukocyte desensitization.8 Experimental evidence further suggests that [44AANA47] RANTES-mediated inhibition of oligomerization is likely to interfere with CCR1 mediated leukocyte arrest in flow conditions, but not CCR5-mediated transmigration.20 In a previous in vitro study using selective receptor antagonists, Weber and colleagues identified distinct functional specializations of the chemokine receptors CCR1 and CCR5 in monocyte recruitment.21 RANTES-induced arrest was predominantly mediated via CCR1, whereas CCR5 is implicated in cell spreading along the endothelium. Both CCR1 and CCR5 contribute to transendothelial migration induced by RANTES.
In the present study, we determined the mRNA levels of major chemokine receptors known to be expressed in atherosclerotic lesions. We found reduced levels of CCR2 and CCR5 within atherosclerotic aortas, whereas the mRNA level of CCR1 and CCR3 was not modulated by [44AANA47]-RANTES administration. CCR2 and CCR5 are expressed by inflammatory cells (mainly macrophages and T lymphocytes) present within atherosclerotic lesions.1 The reduced expression of the 2 receptors might therefore be explained, at least in part, by the lower percentage of these inflammatory cells in lesions of [44AANA47]-RANTES mice. In addition, given the redundancy of the chemokine system, it is conceivable that modulating one chemokine-receptor pair might also affect other ligands or receptors as well.
Studies in mice in which the chemokine receptor CCR2 was genetically deleted revealed its fundamental role in atherogenesis.22 Deletion of CCR2 prevented the accumulation of macrophages and the formation of high-fat diet–induced atherosclerotic lesions in apolipoprotein E–deficient (Apoe−/−) mice. Regarding the role of CCR5 and CCR1 in atherosclerosis, we and others recently found that CCR5 deficiency resulted in reduced atherosclerotic lesion formation and inflammation, whereas CCR1 deficiency had the opposite effect.23–25 Little is known about the specific role of CCR3 in atherosclerotic lesion development. Increased CCR3 expression has been described in human atherosclerotic lesions, mainly present on macrophages.26 In a recent study, CCL11/eotaxin, the ligand for CCR3, was shown to induce MMP-2 in human arterial smooth muscle cells.27 Matrix metalloproteinases have been implicated in the pathogenesis of acute coronary syndromes, not only via degradation of fibrous caps which can lead to plaque rupture, but also by modulation of vascular smooth muscle cell (VSMC) function.28
Interestingly, latest findings suggest the existence of distinct monocyte subsets with differential chemokine receptor usage in atherosclerosis. Tacke and colleagues studied the migration and differentiation properties of the recently described CCR2(+)CX3CR1(+)Ly-6C(hi) and CCR2(−)CX3CR1(++)Ly-6C(lo) monocyte subsets29 in Apoe−/− mice.30 CCR2+ monocytes efficiently accumulated in atherosclerotic plaques, whereas CCR2− monocytes were poorly recruited. The 2 subsets were shown to depend on different chemokine receptors to enter atherosclerotic plaques: CCR5 was selectively upregulated in CCR2− monocytes, whereas CX3CR1 was not required for plaque entry. By contrast, CCR2+ monocytes used CX3CR1 together with CCR2 and CCR5.
A previous study testing the antiatherogenic effect of the HIV entry inhibitor TAK-779, an antagonist of CCR5 and CXCR3, demonstrated dramatic reduction of lesion size and T cell plaque content in mice, whereas the macrophage influx did not change.31 Conversely, the plaques of [44AANA47]-RANTES–treated mice had decreased macrophage and T cell contents, which may be explained by the different chemokine receptor expression patterns on these 2 cell types. Whereas CCR5 and CXCR3 are expressed at high levels on T cells, CCR1 is strongly expressed on macrophages. This suggests that the antiatherogenic effects of [44AANA47]-RANTES are attributable to interference with both CCR1 and CCR5.
In addition to modulation of CCR2 and CCR5 expression, we observed less aortic CCL2 chemokine expression in [44AANA47]-RANTES–treated mice. CCL2 is a powerful monocyte chemoattractant that is principally secreted by macrophages and, to a lesser extent, by activated endothelial cells and SMCs and known to play a proatherogenic role.32 The reduction of CCL2 expression by [44AANA47]-RANTES correlates with the reduced infiltration of monocytes within the atherosclerotic lesions. The reduced leukocyte influx attributable to [44AANA47]-RANTES inhibition probably results in reduced CCL2 secretion, which further decreases leukocyte recruitment.
To visualize the direct effect of [44AANA47]-RANTES on circulating leukocytes, we performed intravital microscopy examinations of treated animals. Leukocyte rolling and arrest on endothelium are characteristic events of chemotactic leukocyte recruitment. We examined mesenteric microvessels and found increased numbers of rolling and arrested leukocytes in mice fed with high cholesterol diet as compared to normal chow controls, whereas treatment with [44AANA47]-RANTES efficiently blocked the diet-induced effects.
Although reduced leukocyte arrest is a direct consequence of [44AANA47]-RANTES inhibition, as RANTES is known to trigger arrest, the reduced rolling might be a secondary consequence of reduced local inflammation. After their recruitment to inflammatory sites, leukocytes further trigger the ongoing local inflammatory response by secreting more inflammatory molecules. Blocking the recruitment by inhibiting local chemokine gradient formation will consequently reduce the expression of inflammatory molecules and thus expression of adhesion molecules, such as selectins.
We further confirmed the effect of [44AANA47]-RANTES on leukocyte infiltration into atherosclerotic plaques by performing adoptive transfer experiments with labeled leukocytes. As previously reported, the population of peritoneal cavity cells isolated after 3 days of thioglycollate injection is mainly composed of monocyte/macrophages, whereas only very few neutrophils are found.34 Thus we assume that the observed effect of [44AANA47]-RANTES in our adoptive transfer experiments is mainly an inhibition of monocyte/macrophage recruitment.
In conclusion, we have shown that [44AANA47]-RANTES, via direct inhibition of leukocyte recruitment, reduces progression of established atherosclerosis. The mechanism of interference of [44AANA47]-RANTES with the wild-type chemokine is thought to depend mainly on impaired GAG oligomerization attributable to heterodimer formation of wild-type and mutant RANTES, because the heterodimer was shown to be inactive in vivo.3 Importantly, this potent inhibitory effect was achieved in mice treated at advanced stages of the disease, which is of particular interest in view of a potential therapeutic drug development.
Sources of Funding
This work was supported by grants from the Swiss National Science Foundation to F.M. The authors (V.B., S.S., C.A., G.P., F.B., F.M.) belong to the European Vascular Genomics Network (http://www. evgn.org) a Network of Excellence supported by the European Community’s sixth Framework Program for Research Priority 1 “Life sciences, genomics and biotechnology for health”.
Original received July 5, 2007; final version accepted March 21, 2008.
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