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A novel CCR2 antagonist inhibits atherogenesis in apoE deficient mice by achieving high receptor occupancy.

ABSTRACT: CC Chemokine Receptor 2 (CCR2) and its endogenous ligand CCL2 are involved in a number of diseases, including atherosclerosis. Several CCR2 antagonists have been developed as potential therapeutic agents, however their in vivo clinical efficacy was limited. In this report, we aimed to determine whether 15a, an antagonist with a long residence time on the human CCR2, is effective in inhibiting the development of atherosclerosis in a mouse disease model. First, radioligand binding assays were performed to determine affinity and binding kinetics of 15a on murine CCR2. To assess the in vivo efficacy, western-type diet fed apoE^-/- mice were treated daily with 15a or vehicle as control. Treatment with 15a reduced the amount of circulating CCR2⁺ monocytes and the size of the atherosclerotic plaques in both the carotid artery and the aortic root. We then showed that the long pharmacokinetic half-life of 15a combined with the high drug concentrations ensured prolonged CCR2 occupancy. These data render 15a a promising compound for drug development and confirms high receptor occupancy as a key parameter when targeting chemokine receptors.

SUBMITTER: Bot I

PROVIDER: S-EPMC5427923 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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A novel CCR2 antagonist inhibits atherogenesis in apoE deficient mice by achieving high receptor occupancy.

Bot Ilze I Ortiz Zacarías Natalia V NV de Witte Wilhelmus E A WE de Vries Henk H van Santbrink Peter J PJ van der Velden Daniël D Kröner Mara J MJ van der Berg Dirk-Jan DJ Stamos Dean D de Lange Elizabeth C M EC Kuiper Johan J IJzerman Adriaan P AP Heitman Laura H LH

Scientific reports 20170303 1

CC Chemokine Receptor 2 (CCR2) and its endogenous ligand CCL2 are involved in a number of diseases, including atherosclerosis. Several CCR2 antagonists have been developed as potential therapeutic agents, however their in vivo clinical efficacy was limited. In this report, we aimed to determine whether 15a, an antagonist with a long residence time on the human CCR2, is effective in inhibiting the development of atherosclerosis in a mouse disease model. First, radioligand binding assays were perf ...[more]

PMID: 28246398

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Project description:Mesenteric branch arteries isolated from cannabinoid type 1 receptor knockout (CB(1)(-/-)) mice, their wild-type littermates (CB(1)(+/+) mice), and C57BL/J wild-type mice were studied to test the hypothesis that murine arteries undergo high sensitivity Ca(2+)-induced relaxation that is CB(1) receptor dependent. Confocal microscope analysis of mesenteric branch arteries from wild-type mice showed the presence of Ca(2+) receptor-positive periadventitial nerves. Arterial segments of C57 control mice mounted on wire myographs contracted in response to 5 micromol/L norepinephrine and responded to the cumulative addition of extracellular Ca(2+) with a concentration-dependent relaxation that reached a maximum of 72.0 +/- 6.3% of the prerelaxation tone and had an EC(50) for Ca(2+) of 2.90 +/- 0.54 mmol/L. The relaxation was antagonized by precontraction in buffer containing 100 mmol/L K(+) and by pretreatment with 10 mmol/L tetraethylammonium. Arteries from CB(1)(-/-) and CB(1)(+/+) mice also relaxed in response to extracellular Ca(2+) with no differences being detected between the knockout and their littermate controls. SR141716A, a selective CB(1) antagonist, caused concentration-dependent inhibition of Ca(2+)-induced relaxation in both the knockout and wild-type strains (60% inhibition at 1 micromol/L). O-1918, a cannabidiol analog, had a similar blocking effect in arteries of both wild-type and CB(1)(-/-) mice at 10 micromol/L. In contrast, 1 micromol/L SR144538, a cannabinoid type 2 receptor antagonist, or 50 micromol/L 18alpha-glycyrrhetinic acid, a gap junction blocker, were without effect. SR141716A (1 to 30 micromol/L) was also assessed for nonspecific actions on whole-cell K(+) currents in isolated vascular smooth muscle cells. SR141716A inhibited macroscopic K(+) currents at concentrations higher than those required to inhibit Ca(2+)-induced relaxation, and appeared to have little effect on currents through large conductance Ca(2+)-activated K(+) channels. These data indicate that arteries of the mouse relax in response to cumulative addition of extracellular Ca(2+) in a hyperpolarization-dependent manner and rule out a role for CB(1) or CB(2) receptors in this effect. The possible role of a nonclassical cannabinoid receptor is discussed.

Project description:BackgroundThe endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) is a known modulator of inflammation and ligand to both, pro-inflammatory cannabinoid receptor 1 (CB1) and anti-inflammatory CB2. While the role of both receptors in atherogenesis has been studied extensively, the significance of 2-AG for atherogenesis is less well characterized.MethodsThe impact of 2-AG on atherogenesis was studied in two treatment groups of ApoE-/- mice. One group received the monoacylglycerol lipase (MAGL)-inhibitor JZL184 [5 mg/kg i.p.], which impairs 2-AG degradation and thus causes elevated 2-AG levels, the other group received vehicle for four weeks. Simultaneously, both groups were fed a high-cholesterol diet. The atherosclerotic plaque burden was assessed in frozen sections through the aortic sinus following oil red O staining and infiltrating macrophages were detected by immunofluorescence targeting CD68. In vitro, the effect of 2-AG on B6MCL macrophage migration was assessed by Boyden chamber experiments. Transcription of adhesion molecules and chemokine receptors in macrophages was assessed by qPCR.ResultsAs expected, application of the MAGL-inhibitor JZL184 resulted in a significant increase in 2-AG levels in vascular tissue (98.2 ± 16.1 nmol/g vs. 27.3 ± 4.5 nmol/g; n = 14-16; p < 0.001). ApoE-/- mice with elevated 2-AG levels displayed a significantly increased plaque burden compared to vehicle treated controls (0.44 ± 0.03 vs. 0.31 ± 0.04; n = 14; p = 0.0117). This was accompanied by a significant increase in infiltrating macrophages within the atherosclerotic vessel wall (0.33 ± 0.02 vs. 0.27 ± 0.01; n = 13-14; p = 0.0076). While there was no alteration to the white blood counts of JZL184-treated animals, 2-AG enhanced macrophage migration in vitro by 1.8 ± 0.2 -fold (n = 4-6; p = 0.0393) compared to vehicle, which was completely abolished by co-administration of either CB1- or CB2-receptor-antagonists. qPCR analyses of 2-AG-stimulated macrophages showed an enhanced transcription of the chemokine CCL5 (1.59 ± 0.23 -fold; n = 5-6; p = 0.0589) and its corresponding receptors CCR1 (2.04 ± 0.46 -fold; n = 10-11; p = 0.0472) and CCR5 (2.45 ± 0.62 -fold; n = 5-6; p = 0.0554).ConclusionTaken together, elevated 2-AG levels appear to promote atherogenesis in vivo. Our data suggest that 2-AG promotes macrophage migration, possibly by the CCL5-CCR5/CCR1 axis, and thereby contributes to vascular inflammation. Thus, decreasing vascular 2-AG levels might represent a promising therapeutic strategy in patients suffering from atherosclerosis and coronary heart disease.

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A novel CCR2 antagonist inhibits atherogenesis in apoE deficient mice by achieving high receptor occupancy.

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A novel CCR2 antagonist inhibits atherogenesis in apoE deficient mice by achieving high receptor occupancy.

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