A major barrier in hematopoietic gene function studies is posed by the laborious and time-consuming generation of knockout mice with an appropriate genetic background. Here we present a novel lentivirus-based strategy for the in situ generation of hematopoietic knockdowns. A short hairpin RNA (shRNA) was designed targeting murine CC-chemokine receptor 2 (CCR2), which was able to specifically blunt CCR2 expression at the mRNA, protein, and functional levels in vitro. Reconstitution of irradiated recipient mice with autologous bone marrow that had been ex vivo transduced with shRNA lentivirus led to persistent down-regulation of CCR2 expression, which translated into a 70% reduction in CCR2-dependent recruitment of macrophages to an inflamed peritoneal cavity without noticeable side effects on related chemokine receptors or general inflammation status. These findings clearly demonstrate the potential of shRNA lentivirus-infected bone marrow transplantation as a rapid and effective method to generate hematopoietic knockdowns for leukocyte gene function studies.

Liver X receptor ( LXR) nuclear receptors regulate the expression of genes involved in whole body cholesterol trafficking, including absorption, excretion, catabolism, and cellular efflux, and possess both anti-inflammatory and antidiabetic actions. Accordingly, LXR is considered an appealing drug target for multiple indications. Synthetic LXR agonists demonstrated inhibition of atherosclerosis progression in murine genetic models; however, these and other studies indicated that their major undesired side effect is an increase of plasma and hepatic triglycerides. A significant impediment to extrapolating results with LXR agonists from mouse to humans is the absence in mice of cholesteryl ester transfer protein, a known LXR target gene, and the upregulation in mice but not humans of cholesterol 7 alpha-hydroxylase. To better predict the human response to LXR agonism, two synthetic LXR agonists were examined in hamsters and cynomolgus monkeys. In contrast to previously published results in mice, neither LXR agonist increased HDL-cholesterol in hamsters, and similar results were obtained in cynomolgus monkeys. Importantly, in both species, LXR agonists increased LDL-cholesterol, an unfavorable effect not apparent from earlier murine studies. These results reveal additional problems associated with current synthetic LXR agonists and emphasize the importance of profiling compounds in preclinical species with a more human-like LXR response and lipoprotein metabolism.

Objective: To selectively determine the role of leukocyte CC-chemokine receptor 2 (CCR2) in atherogenesis.Methods and results: Bone marrow progenitor cells harvested from CCR2(+/+) mice were transplanted into irradiated CCR2(-/-) mice, representing the whole-body absence of CCR2 except in leukocytes. Transplantation of CCR2(-/-) bone marrow into CCR2(-/-) mice served as control. Eight weeks after bone marrow transplantation, the diet of regular chow was switched to a high-cholesterol diet for another 10 weeks in order to induce atherosclerosis. No significant differences in serum cholesterol and triglyceride levels were observed between the two groups. However, the mean cross-sectional aortic root lesion area of CCR2(+/+)-> CCR2(-/-) mice amounted up to 12.28 +/- 3.28 x 10(4) mu m(2), compared with only 3.08 +/- 0.74 X 10(4) mu m(2) observed in the CCR2(-/-)-> CCR2(-/-.) group. Thus, the presence of CCR2 exclusively on leukocytes induces a fourfold increase in aortic lesion area. This extent of lesion development was comparable to C57B1/6 mice receiving CCR2(+/+) bone marrow (10.08 +/- 3.30 x 10(4) mu m(2)).Conclusion: These results point at a dominant role of leukocyte CCR2 in atherogenesis, implying that CCR2 from nonleukocyte sources, like endothelial cells or smooth muscle cells, is less critical in the initiation of atherosclerosis. Pharmacological inhibition of leukocyte CCR2 function might be a promising strategy to prevent atherosclerosis. (c) 2004 Elsevier B.V. All rights reserved.

Dietary antioxidants are reported to suppress cellular expression of chemokines and adhesion molecules that recruit monocytes to the artery wall during atherosclerosis. In the present study we measured the effect of feeding apoE*3 Leiden mice or their non-transgenic (C57BL) littermates with atherogenic diets either deficient in, or supplemented with, dietary antioxidants (vitamin E, vitamin C and beta-carotene) for 12 weeks, on serum levels of CC (JE/MCP-1) and CXC (KC) chemokines and soluble adhesion molecules (sVCAM-1, sICAM-1) and atherosclerotic lesion size. ApoE*3 Leiden mice developed gross hypercholesterolaemia, and markedly accelerated (10-20 fold; P < 0.0001) atherogenesis, compared with non-transgenic animals. Antioxidant consumption reduced lesion area in non-transgenic, but not apoE*3 Leiden, mice. Serum sVCAM-1 and sICAM-1 levels were significantly (P<0.0001) increased (sVCAM-1 up to 3.9 fold; sICAM-1 up to 2.4 fold) by 4-8 weeks in all groups, and then declined. The initial increase in the concentration of adhesion molecules was reduced by 38%-61% (P < 0.05) by antioxidant consumption, particularly in non-transgenic mice. By contrast, serum chemokine levels tended to increase more rapidly from baseline in apoE*3 Leiden mice, compared with non-transgenic animals, but were unaffected by dietary antioxidants. We conclude that dietary antioxidants reduce circulating soluble adhesion molecules and atherosclerosis in C57BL mice.

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 x 10(5) mu m(2). At 9 weeks after transplantation, apoE(-/-) --> apoE(-/-) and apoE(-/-)/ CCR2(-/-) --> apoE(-/-) mice had developed significantly larger atherosclerotic lesions (4.49 +/- 0.92 x 10(5) mu m(2), P < 0.02 and 4.15 +/- 0.62 x 10(5) mu m(2), 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.

Objectives-CC and CXC chemokines are implicated in leukocyte recruitment during development of atherosclerotic lesions, suggesting circulating levels of chemokines may be useful serum markers of atherogenesis. Serum chemokine concentrations were measured in apolipoprotein (apo) E*3 Leiden mice and their nontransgenic littermates and related to the differing rates of atherogenesis in these animals.Methods and Results-Mice were fed a high-fat, high-cholesterol/ cholate (HFC/C) diet for 18 weeks. Circulating levels of JE/monocyte chemotactic protein-1 increased (P<0.05) after 2 to 4 weeks, coincident with development of diet-induced hypercholesterolemia, and remained elevated throughout the study. Circulating KC concentrations increased (P<0.05) after consumption of HFC/C diet; however, unlike JE, serum KC concentrations increased more rapidly in apoE*3 Leiden mice than their nontransgenic littermates. Hepatic expression of JE and KC mRNA were detected by in situ hybridization in all mice fed HFC/C diet. Aortic expression of JE mRNA was seen only in apoE*3 Leiden mice within macrophage-rich atherosclerotic lesions. By contrast, no aortic expression of KC mRNA was detected by in situ hybridization.Conclusions-Increases in serum chemokine concentrations did not reflect temporal aortic production of these molecules and proved less predictive than serum cholesterol of the markedly different extent of atheroma in apoE*3 Leiden and nontransgenic mice.

We describe here a classical molecular modeling exercise that was carried out to provide a basis for the design of novel antagonist ligands of the CCR2 receptor. Using a theoretical model of the CCR2 receptor, docking studies were carried out to define plausible binding modes for the various known antagonist ligands, including our own series of indole piperidine compounds. On the basis of these results, a number of site-directed mutations (SDM) were designed that were intended to verify the proposed docking models. From these it was clear that further refinements would be necessary in the model. This was aided by the publication of a crystal structure of bovine rhodopsin, and a new receptor model was built by homology to this structure. This latest model enabled us to define ligand-docking hypotheses that were in complete agreement with the results of the SDM experiments.

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.

Hyperlipidaemia may accelerate the development of atherosclerosis by enhancing the expression of chemokines by cells within the arterial wall. Chemokines of the CC subfamily are clearly implicated in atherogenesis; however, recent reports Suggest that CXC chemokines may play a hitherto Unrecognised role in monocyte recruitment into atheromatous lesions expressing these molecules. Here, we examine whether circulating levels of CXC chemokines may reflect the pathogenic changes Occurring during early atherogenesis. ApoE*3 Leiden mice developed marked hypercholesterolaemia, and early Type 1 'fatty streak' lesions, following consumption of an atherogenic diet high in saturated fat and cholesterol, and containing sodium cholate, for Lip to 4 weeks. By contrast, their non-transgenic littermates (C57BL/6J) exhibited a much less pronounced hypercholesterolaemia and did not develop fatty streak lesions, when fed the same diet. Under these conditions, serum concentrations of CXC chemokines, KC and Macrophage Inflammatory Protein-2 (MIP-2) were significantly (P < 0.0005) elevated in apoE*3 Leiden mice Consuming HFC/C diet, compared with apoE*3 Leiden mice consuming a chow diet. Further, serum concentrations of KC were significantly (P < 0.02) higher at 4 weeks in apoE*3 Leiden mice fed HFC/C diet compared with their non-transgenic littermates consuming the same diet. Expression of KC mRNA was detected in both aortic and hepatic tissues of apoE*3 Leiden mice, and the non-transgenic controls. Thus. hypercholesterolaemia is associated with elevated serum concentrations of CXC chemokines, KC and MIP-2 that probably reflect chronic inflammation during atherogenesis. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

OBJECTIVE:Studies in mice have shown that genetic disruption of monocyte chemotactic protein-1 or its receptor, the C-C chemokine receptor 2 (CCR2), inhibits atherosclerosis, but few data exist in humans to suggest that the monocyte chemotactic protein-1-CCR2 interaction is important in atherogenesis. A common polymorphism in the human CCR2 gene resulting in a substitution of isoleucine for valine (Val64Ile) has been associated with other disease phenotypes in humans.METHODS AND RESULTS:A cohort of first-degree relatives of persons with premature coronary artery disease was recruited and quantitatively phenotyped for the extent of CAC, a marker of coronary atherosclerosis, by using electron beam CT. The extent of CAC was significantly lower in subjects with the CCR2-Ile64 variant (Val/Ile and Ile/Ile genotypes) than in subjects carrying 2 Val64 alleles, even after adjustment for traditional risk factors.CONCLUSIONS:This study provides genetic evidence linking CCR2 with coronary atherosclerosis in humans.

Apolipoprotein E (apoE) is a high affinity ligand for several receptor systems in the liver, including the low-density lipoprotein (LDL) receptor, and non-LDL receptor sites, like the LDL receptor-related protein (LRP), the putative remnant receptor and/or proteoglycans. Although the liver is the major source of apoE synthesis, apoE is also produced by a wide variety of other cell types, including macrophages. In the present study, the role of the LDL receptor in the removal of lipoprotein remnants, enriched with macrophage-derived apoE from the circulation, was determined using the technique of bone marrow transplantation (BMT). Reconstitution of macrophage apoE production in apoE-deficient mice resulted in a serum apoE concentration of only 2% of the concentration in wild-type C57Bl/6 mice. This low level of apoE nevertheless reduced VLDL and LDL cholesterol 12-fold (P<0.001) and fourfold (P<0.001), respectively, thereby reducing serum cholesterol levels and the susceptibility to atherosclerosis. In contrast, reconstitution of macrophage apoE synthesis in mice lacking both apoE and the LDL receptor induced only a twofold (P<0.001) reduction in VLDL cholesterol and had no significant effect on atherosclerotic lesion development, although serum apoE levels were 93% of the concentration in normal C57Bl/6 mice. In conclusion, a functional (hepatic) LDL receptor is essential for the efficient removal of macrophage apoE-enriched lipoprotein remnants from the circulation and thus for normalization of serum cholesterol levels and protection against atherosclerotic lesion development in apoE-deficient mice.

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.

Macrophage-derived foam cells play an important role in the initiation and progression of atherosclerosis. To examine the role of the macrophage low density lipoprotein receptor (LDLr) in atherosclerotic lesion formation, bone marrow from LDLr knockout [LDLr(-/-)] mice was transplanted into irradiated wild-type C57Bl/6 [LDLr(+/+)] mice. After 3 months on an atherogenic diet, C57Bl/6 mice, reconstituted with LDLr(-/-) bone marrow, showed a mean lesion area of 34.7 x 10(3)+/-22.4 x 10(3) microm(2) compared with 100. 8 x 10(3)+/-33.0 x 10(3) microm(2) (P<0.001) in control C57Bl/6 mice that were transplanted with LDLr(+/+) bone marrow. There were no significant differences in total serum cholesterol, triglyceride levels, and lipoprotein profiles between the 2 groups. Histochemical analysis of macrophage LDLr expression in the atherosclerotic lesions indicated that C57Bl/6 mice, reconstituted with LDLr(+/+) bone marrow, showed extensive staining of the foam cells in the atherosclerotic lesions, whereas mice reconstituted with LDLr(-/-) bone marrow showed only a few LDLr-positive foam cells. In vitro, peritoneal macrophages isolated from wild-type C57Bl/6 mice were, respectively, 4.7- and 10.7-fold more effective in cell association and degradation of atherogenic (125)I-beta-very low density lipoprotein than were LDLr(-/-) peritoneal macrophages, establishing that the LDLr on macrophages is important for the interaction of macrophages with beta-very low density lipoprotein. It is concluded that the LDLr on macrophages can facilitate the development of atherosclerosis, possibly by mediating the uptake of atherogenic lipoproteins.

Lipoprotein lipase (LPL) synthesis by macrophages is upregulated in early atherogenesis, implicating the possible involvement of LPL in plaque formation. However, it is still unclear whether macrophage-derived LPL displays a proatherosclerotic or an antiatherosclerotic role in atherosclerotic lesion development. In this study, the role of macrophage-derived LPL on lipid metabolism and atherosclerosis was assessed in vivo by transplantation of LPL-deficient (LPL−/−) and wild-type (LPL+/+) bone marrow into C57BL/6 mice. Eight weeks after bone marrow transplantation (BMT), serum cholesterol levels in LPL−/−→C57BL/6 mice were reduced by 8% compared with those in LPL+/+→C57BL/6 mice (P <0.05, n=16), whereas triglycerides were increased by 33% (P <0.05, n=16). Feeding the mice a high-cholesterol diet increased serum cholesterol levels in LPL−/−→C57BL/6 and LPL+/+→C57BL/6 mice 5-fold and 9-fold, respectively, resulting in a difference of ≈50% (P <0.01) after 3 months on the diet. No effects on triglyceride levels were observed under these conditions. Furthermore, serum apolipoprotein E levels were reduced by 50% in the LPL−/−→C57BL/6 mice compared with controls under both dietary conditions. After 3 months on a high-cholesterol diet, the atherosclerotic lesion area in LPL−/−→C57BL/6 mice was reduced by 52% compared with controls. It can be concluded that macrophage-derived LPL plays a significant role in the regulation of serum cholesterol, apolipoprotein E, and atherogenesis, suggesting that specific blockade of macrophage LPL production may be beneficial for decreasing atherosclerotic lesion development. (Arterioscler Thromb Vasc Biol. 2000;20:e53-e62.)

A weak HTS hit at the CCR2B receptor has been converted into a potent antagonist by array SAR studies. Selectivity over the closely related CCR5 receptor is also achieved.

Apolipoprotein E (apoE), a high affinity ligand for lipoprotein receptors, is synthesized by the liver and extrahepatic tissues, including cells of the monocyte/macrophage cell lineage. The role of monocyte/macrophage-derived apoE in atherogenesis was assessed by transplantation of apoE-deficient (apoE−/−) bone marrow into normolipidemic C57Bl/6 mice. No significant effect could be demonstrated on serum apoE levels in C57Bl/6 mice, transplanted with apoE-deficient bone marrow compared with control transplanted mice. Furthermore, no consistent effect on serum cholesteryl esters and triglyceride concentrations could be demonstrated on either a standard chow diet or a high cholesterol diet. Quantitative analysis of atherosclerosis in mice transplanted with apoE-deficient bone marrow, after two months on a high cholesterol diet, revealed a 4-fold increase in the atherosclerotic lesion area as compared to animals transplanted with apoE+/+ bone marrow. Analysis of the ability of apoE-deficient macrophages to release cholesterol after loading with acetylated LDL revealed that the release of cholesterol from apoE-deficient macrophages was impaired as compared to wild-type macrophages in the absence and the presence of specific cholesterol acceptors. In conclusion, apoE production by macrophages retards the formation of atherosclerotic plaques, possibly by mediating cholesterol efflux. We anticipate that pharmacological approaches to increase apoE synthesis and/or secretion by macrophages might be beneficial for the treatment of atherosclerosis.

The effect of monocyte/macrophage-derived wild-type mouse apolipoprotein E (apoE), human apoE3-Leiden, and human apoE2 on serum cholesterol levels and the development of atherosclerosis in apoE-deficient (apoe-/-) mice was investigated by using bone marrow transplantation (BMT). At 4 weeks after BMT, murine apoe+/+ bone marrow reduced serum cholesterol levels by 87% in apoe-/- mice, whereas macrophage-derived human apoE3-Leiden and human apoE2 induced a maximal, transient reduction of 35% and 48%, respectively. At 4 months after BMT, atherosclerosis was 23-fold (P<0.001) reduced in apoe+/+-->apoe-/- mice, whereas no significant reduction in apoE3-Leiden.apoe-/--->apoe-/- and apoE2.apoe-/--->apoe-/- mice could be demonstrated. A highly significant decrease in serum cholesterol levels (78% reduction) and atherosclerosis (21-fold, P<0. 001) was found in apoE3-Leiden.apoe-/- animals expressing high levels of apoE in multiple tissues, whereas apoE2 was ineffective even at high concentrations. Furthermore, in contrast to apoE-deficient macrophages, cholesterol efflux from apoE2 or apoE3-Leiden macrophages was not impaired. In conclusion, apoE3-Leiden as well as apoE2 are less effective in reducing cholesterol levels and atherosclerosis in apoe-/- animals, compared with apoe+/+, with apoE2

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.

Human CC chemokine receptor 1 (CCR1) has been proposed as a receptor for CKβ8. To obtain conclusive evidence, binding–displacement studies of 125I-CKβ8 (25–99) were performed on membranes of Chinese hamster ovary cells expressing human CCR1. The ic50 for displacement of 125I-CKβ8 (25–99) with CKβ8 (25–99) was 0.22 nM. The longer forms of CKβ8 (24–99 and 1–99) also displaced 125I-CKβ8, with ic50 values of 6.5 and 16 nM, respectively. Displacement profiles of 125I-CKβ8 (25–99) on freshly prepared human monocytes indicated that CCR1 was the major receptor for CKβ8. We conclude that CCR1 is a receptor for different-length CKβ8 and that CKβ8 (25–99) has a similar affinity for CCR1 as macrophage inflammatory protein-1α (MIP-1α). The longer variants of CKβ8 are significantly less potent than CKβ8 (25–99) and MIP-1α on CCR1 and monocytes (P < 0.05).