Pieter H.E. Groot

Chemokines and atherosclerosis.

Chemokines or chemotactic cytokines represent an expanding family of structurally related small molecular weight proteins, recognised as being responsible for leukocyte trafficking and activation. Soon after the discovery of this class of cytokines, about a decade ago, monocyte chemoattractant protein-1 (MCP-1) was found to be highly expressed in human atherosclerotic lesions and postulated to be central in monocyte recruitment into the arterial wall and developing lesions. In this review, we will discuss our present knowledge about MCP-1 and its receptor CCR2 and their role in atherogenesis. Although less well established, other chemokines such as RANTES, MIP-1alpha and MIP-1beta have also been implicated in atherosclerotic lesion formation as are a number of more recently discovered chemokines like MCP-4, ELC and PARC. The role of these chemokines in the progression of atherosclerosis will be discussed as well as the emerging role of IL-8, mostly know for its effects on neutrophils. Particular attention will be given not only to the involvement of chemokines in the inflammatory recruitment of monocytes/macrophages, but also to their role in the related local immune responses and vascular remodelling which occur during the formation of unstable atherosclerotic plaques.

Expression and Cellular Localization of the CC Chemokines PARC and ELC in Human Atherosclerotic Plaques

Local immune responses are thought to play an important role in the development of atherosclerosis. Histological studies have shown that human atherosclerotic lesions contain T lymphocytes throughout all stages of development, many of which are in an activated state. A number of novel CC chemokines have been described recently, which are potent chemoattractants for lymphocytes: PARC (pulmonary and activation-regulated chemokine), ELC (EBI1-ligand chemokine), LARC (liver and activation-regulated chemokine), and SLC (secondary lymphoid-tissue chemokine). Using reverse transcriptase-polymerase chain reaction and in situ hybridization, we have found gene expression for PARC and ELC but not for LARC or SLC in human atherosclerotic plaques. Immunohistochemical staining of serial plaque sections with specific cell markers revealed highly different expression patterns of PARC and ELC. PARC mRNA was restricted to CD68+ macrophages (n = 14 of 18), whereas ELC mRNA was widely expressed by macrophages and intimal smooth muscle cells (SMC) in nearly all of the lesions examined (n = 12 of 14). ELC mRNA was also found to be expressed in the medial SMC wall of highly calcified plaques (n = 4). Very low levels of ELC mRNA expression could also be detected in normal mammary arteries but no mRNA expression for PARC was detected in these vessels (n = 4). In vitro, ELC mRNA was found to be up-regulated in aortic SMC stimulated with tumor necrosis factor-a and interferon-gamma but not in SMC stimulated with serum. Both PARC and ELC mRNA were expressed by monocyte-derived macrophages but not monocytes. The expression patterns of PARC and ELC mRNA in human atherosclerotic lesions suggest a potential role for these two recently described CC chemokines in attracting T lymphocytes into atherosclerotic lesions.

Cloning, in vitro expression, and functional characterization of a novel human CC chemokine of the monocyte chemotactic protein (MCP) family (MCP-4) that binds and signals through the CC chemokine receptor 2B.

Here we describe the characterization of a novel human CC chemokine, tentatively named monocyte chemotactic protein (MCP-4). This chemokine was detected by random sequencing of expressed sequence tags in cDNA libraries. The full-length cDNA revealed an open reading frame for a 98-amino acid residue protein, and a sequence alignment with known CC chemokines showed high levels of similarity (59–62%) with MCP-1, MCP-3, and eotaxin. MCP-4 cDNA was cloned into Drosophila S2 cells, and the mature protein (residues 24–98) was purified from the conditioned medium. Recombinant MCP-4 induced a potent chemotactic response (EC50 = 2.88 ± 0.15 nm) and a transient rise in cytosolic calcium concentration in fresh human peripheral blood monocytes but not in neutrophils. Binding studies in monocytes showed that MCP-4 and MCP-3 were very potent in displacing high affinity binding of125I-MCP-1 (IC50 for MCP-4, MCP-3, and unlabeled MCP-1 of 2.1 ± 1.4, 0.85–1.6, and 0.7 ± 0.2 nm respectively), suggesting that all three chemokines interact with the CC chemokine receptor-2 (MCP-1 receptor). This was confirmed in binding studies with Chinese hamster ovary cells, stably transfected with the CC chemokine 2B receptor. Northern blot analysis in extracts of normal human tissues showed expression of mRNA for MCP-4 in small intestine, thymus, and colon, but the level of protein expression was too low to be detected in Western blot analysis. However, expression of MCP-4 protein was demonstrated by immunohistochemistry in human atherosclerotic lesion and found to be associated with endothelial cells and macrophages.

Transplantation of Monocyte CC-Chemokine Receptor 2-Deficient Bone Marrow Into ApoE3-Leiden Mice Inhibits Atherogenesis

Objective—To determine the role of leukocyte CC-chemokine receptor 2 (CCR2) in the early development of atherosclerosis Methods and Results—Bone marrow cells harvested from CCR2 (−/−) and CCR2 (+/+) mice were transplanted into ApoE3–Leiden mice, a mouse strain susceptible for diet-induced atherosclerosis. Eight weeks after bone marrow transplantation, the diet of regular chow was switched to a high-cholesterol diet (1% cholesterol, 15% fat, 0.5% cholate) for another 8 weeks to induce atherosclerosis. No significant differences in serum cholesterol and triglyceride levels were observed between the CCR2 (+/+) → ApoE3–Leiden and CCR2 (−/−) → ApoE3–Leiden mice. However, the mean cross-sectional aortic root lesion area of CCR2 (−/−) → ApoE3–Leiden mice was only 2.94±1.94×104 &mgr;m2 compared with 20.94±12.71×104 &mgr;m2, for CCR2 (+/+) → ApoE3–Leiden mice. Thus, the absence of CCR2 on leukocytes induces an 86% reduction of aortic lesion area as compared with controls (n=10, P <0.01). Conclusion—These results provide direct evidence that CCR2 expressed by leukocytes plays a critical role in the initiation of early atherosclerosis and that pharmacological intervention in CCR2 function represents an attractive target to inhibit atherogenesis.

Localisation of mRNA for JE/MCP-1 and its receptor CCR2 in atherosclerotic lesions of the ApoE knockout mouse.

MCP-1 has potent chemotactic activity for monocytes and is strongly implicated in the pathogenesis of atherosclerosis. In the present study, we have used in situ hybridisation to examine the gene expression of JE, the murine homologue of MCP-1, and its receptor, CCR2, during the development of atherosclerotic lesions in the ApoE knockout mouse. Interestingly, the earliest expression of JE detected during lesion development was found to be localised in mesenchymal cells in the adventitia and not in the intima. Macrophages were subsequently found to accumulate in these affected regions of the adventitia and these cells were found to express high levels of JE. At this stage, early macrophage-rich lesions with high expression of JE were also seen in the intima, but expression of mRNA for the receptor for JE (CCR2) was only found on adventitial macrophages and not in the intima. This sequence of events suggests that adventitial inflammation may be an important early event in lesion development and responsible for the subsequent accumulation of macrophages in the intima possibly by recruitment from the adventitia as well as via the vessel lumen.

Conformationally restricted indolopiperidine derivatives as potent CCR2B receptor antagonists

The preparation and biological evaluation of a series of indolopiperidine CCR2B receptor antagonists possessing a conformationally restricted C-5 linker chain in combination with a restricted piperidine ring are described. Compared to the parent compound 1, analogue 8 shows a dramatic improvement in selectivity against a range of 5-HT and dopaminergic receptors.

Repopulation of Apolipoprotein E Knockout Mice With CCR2-Deficient Bone Marrow Progenitor Cells Does Not Inhibit Ongoing Atherosclerotic Lesion Development

Objective—Using bone marrow transplantation, we have previously demonstrated the critical role that hematopoietic CCR2 plays in early atherogenesis. Reconstitution of irradiated apolipoprotein (apo) E3–Leiden mice with CCR2-deficient bone marrow progenitor cells resulted in 86% reduction on overall atherosclerotic lesion development. However, no data on CCR2 in the cause of established atherosclerosis have been reported so far. Methods and Results—To study the role of CCR2 in established atherosclerotic lesions, bone marrow progenitor cells harvested from apoE−/− and apoE−/−/CCR2−/− mice were transplanted into lethally irradiated 16-week-old apoE−/− mice with established atherosclerotic lesions. No significant differences were found in serum total cholesterol and triglycerides levels at different time points after transplantation. At age 16 weeks, lesion size in control apoE−/− mice was 3.28±1.06×105 &mgr;m2. At 9 weeks after transplantation, apoE−/−→ apoE−/− and apoE−/−/CCR2−/−→ apoE−/− mice had developed significantly larger atherosclerotic lesions (4.49±0.92×105 &mgr;m2, P<0.02 and 4.15±0.62×105 &mgr;m2, P<0.04 compared with controls, respectively). However, no significant effect of disruption of hematopoietic CCR2 was observed on the progression of lesions. Furthermore, the macrophage positive area (78±4% versus 72±9%) and collagen content (11±6% versus 15±3%) of the lesions were similar as well. Conclusion—In contrast to the critical role of CCR2 in the initiation of atherogenesis, bone marrow progenitor cell-derived CCR2 does not influence the progression of established atherosclerotic lesions, pointing to additional mechanisms for recruitment of monocytes at later stages of lesion development.

Leucocyte cathepsin K affects atherosclerotic lesion composition and bone mineral density in low-density lipoprotein receptor deficient mice

AIMS Cathepsin K (CatK), an established drug target for osteoporosis, has been reported to be upregulated in atherosclerotic lesions. Due to its proteolytic activity, CatK may influence the atherosclerotic lesion composition and stability. In this study, we investigated the potential role of leucocyte CatK in atherosclerotic plaque remodelling. METHODS AND RESULTS To assess the biological role of leucocyte CatK, we used the technique of bone marrow transplantation to selectively disrupt CatK in the haematopoietic system. Total bone marrow progenitor cells from CatK(+/+), CatK(+/-), and CatK(-/-) mice were transplanted into X-ray irradiated low-density lipoprotein receptor knockout (LDLr(-/-)) mice. The selective silencing of leucocyte CatK resulted in phenotypic changes in bone formation with an increased total bone mineral density in the CatK(-/-) chimeras and an effect of gene dosage. The absence of leucocyte CatK resulted in dramatically decreased collagen and increased macrophage content of the atherosclerotic lesions while lesion size was not affected. The atherosclerotic lesions also demonstrated less elastic lamina fragmentation and a significant increase in the apoptotic and necrotic area in plaques of mice transplanted with CatK(-/-) bone marrow. CONCLUSION Leucocyte CatK is an important determinant of atherosclerotic plaque composition, vulnerability, and bone remodelling, rendering CatK an attractive target for pharmaceutical modulation in atherosclerosis and osteoporosis.

CCR2 and coronary artery disease: a woscops substudy

BackgroundSeveral lines of evidence support a role for CCL2 (monocyte chemotactic protein-1) and its receptor CCR2 in the development of atherosclerosis. The aim of the present study was to determine the association of the CCR2 Val64Ile polymorphism with the development of coronary artery disease in the WOSCOPS study sample set.FindingsA total of 443 cases and 1003 controls from the West of Scotland Coronary Prevention Study (WOSCOPS) were genotyped for the Val64Ile polymorphism in the CCR2 gene. Genotype frequencies were compared between cases and controls. The CCR2 Val64Ile polymorphism was found not to be associated with coronary events in this study population (odds ratio 1.15, 95% CI 0.82-1.61, p = 0.41).ConclusionsThis case-control study does not support an association of the CCR2 Val64Ile polymorphism with coronary artery disease in the WOSCOPS sample set and does not confirm a possible protective role for CCR2 Val64Ile in the development of coronary artery disease.