Cannabinoid Receptor 2 Signaling Does Not Modulate Atherogenesis in Mice

BACKGROUND:Strong evidence supports a protective role of the cannabinoid receptor 2 (CB(2)) in inflammation and atherosclerosis. However, direct proof of its involvement in lesion formation is lacking. Therefore, the present study aimed to characterize the role of the CB(2) receptor in Murine atherogenesis. METHODS AND FINDINGS:Low density lipoprotein receptor-deficient (LDLR(-/-)) mice subjected to intraperitoneal injections of the selective CB(2) receptor agonist JWH-133 or vehicle three times per week consumed high cholesterol diet (HCD) for 16 weeks. Surprisingly, intimal lesion size did not differ between both groups in sections of the aortic roots and arches, suggesting that CB(2) activation does not modulate atherogenesis in vivo. Plaque content of lipids, macrophages, smooth muscle cells, T cells, and collagen were also similar between both groups. Moreover, CB(2) (-/-)/LDLR(-/-) mice developed lesions of similar size containing more macrophages and lipids but similar amounts of smooth muscle cells and collagen fibers compared with CB(2) (+/+)/LDLR(-/-) controls. While JWH-133 treatment reduced intraperitoneal macrophage accumulation in thioglycollate-elicited peritonitis, neither genetic deficiency nor pharmacologic activation of the CB(2) receptor altered inflammatory cytokine expression in vivo or inflammatory cell adhesion in the flow chamber in vitro. CONCLUSION:Our study demonstrates that both activation and deletion of the CB(2) receptor do not relevantly modulate atherogenesis in mice. Our data do not challenge the multiple reports involving CB(2) in other inflammatory processes. However, in the context of atherosclerosis, CB(2) does not appear to be a suitable therapeutic target for reduction of the atherosclerotic plaque

Binding of CD40L to Mac-1's i-domain involves the EQLKKSKTL motif and mediates leukocyte recruitment and atherosclerosis-but does not affect immunity and thrombosis in mice

Rationale: CD40L figures prominently in chronic inflammatory diseases such as atherosclerosis. However, since CD40L potently regulates immune function and hemostasis by interaction with CD40 receptor and the platelet integrin GPIIb/IIIa, its global inhibition compromises host defense and generated thromboembolic complications in clinical trials. We recently reported that CD40L mediates atherogenesis independently of CD40 and proposed Mac-1 as an alternate receptor. Objective: Here, we molecularly characterized the CD40L-Mac-1 interaction and tested whether its selective inhibition by a small peptide modulates inflammation and atherogenesis in vivo. Methods and Results: CD40L concentration-dependently bound to Mac-1 I-domain in solid phase binding assays, and a high-affinity interaction was revealed by surface-plasmon-resonance analysis. We identified the motif EQLKKSKTL, an exposed loop between the α1 helix and the β-sheet B, on Mac-1 as binding site for CD40L. A linear peptide mimicking this sequence, M7, specifically inhibited the interaction of CD40L and Mac-1. A cyclisized version optimized for in vivo use, cM7, decreased peritoneal inflammation and inflammatory cell recruitment in vivo. Finally, LDLr -/- mice treated with intraperitoneal injections of cM7 developed smaller, less inflamed atherosclerotic lesions featuring characteristics of stability. However, cM7 did not interfere with CD40L-CD40 binding in vitro and CD40L-GPIIb/IIIa-mediated thrombus formation in vivo. Conclusions: We present the novel finding that CD40L binds to the EQLKKSKTL motif on Mac-1 mediating leukocyte recruitment and atherogenesis. Specific inhibition of CD40L-Mac-1 binding may represent an attractive anti-inflammatory treatment strategy for atherosclerosis and other inflammatory conditions, potentially avoiding the unwanted immunologic and thrombotic effects of global inhibition of CD40L.Fil: Wolf, Dennis. Albert-Ludwigs-Universität Freiburg; Alemania. Baker IDI Heart and Diabetes Institute; AustraliaFil: Hohmann, Jan David. Baker IDI Heart and Diabetes Institute; AustraliaFil: Wiedemann, Ansgar. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Bledzka, Kamila. Cleveland Clinic. Department of Molecular Cardiology; Estados UnidosFil: Blankenbach, Hermann. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Marchini, Timoteo Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Gutte, Katharina. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Zeschky, Katharina. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Bassler, Nicole. Baker IDI Heart and Diabetes Institute; AustraliaFil: Hoppe, Natalie. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Rodriguez, Alexandra Ortiz. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Herr, Nadine. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Hilgendorf, Ingo. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Stachon, Peter. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Willecke, Florian. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Duerschmied, Daniel. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: von zur Muhlen, Constantin. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Soloviev, Dmitry A.. Cleveland Clinic. Department of Molecular Cardiology; Estados UnidosFil: Zhang, Li. University of Maryland; Estados UnidosFil: Bode, Christoph. Albert-Ludwigs-Universität Freiburg; AlemaniaFil: Plow, Edward F.. Cleveland Clinic. Department of Molecular Cardiology; Estados UnidosFil: Libby, Peter. Harvard Medical School; Estados UnidosFil: Peter, Karlheinz. Baker IDI Heart and Diabetes Institute; AustraliaFil: Zirlik, Andreas. Albert-Ludwigs-Universität Freiburg; Alemani

Interruption of classic CD40L-CD40 signalling but not of the novel CD40L-Mac-1 interaction limits arterial neointima formation in mice

The co-stimulatory immune molecule CD40L figures prominently in a variety of inflammatory conditions including arterial disease. Recently, we made the surprising finding that CD40L mediates atherogenesis independently of its classic receptor CD40 via a novel interaction with the leukocyte integrin Mac-1. Here, we hypothesised that selective blockade of the CD40L-Mac-1 interaction may also retard restenosis. We induced neointima formation in C57/BL6 mice by ligation of the left carotid artery. Mice were randomised to daily intraperitoneal injections of either cM7, a small peptide selectively inhibiting the CD40L-Mac-1 interaction, scM7, a scrambled control peptide, or saline for 28 days. Interestingly, cM7-treated mice developed neointima of similar size compared with mice receiving the control peptide or saline as assessed by computer-assisted analysis of histological cross sections. These data demonstrate that the CD40L-Mac-1 interaction is not required for the development of restenosis. In contrast, CD40-deficient mice subjected to carotid ligation in parallel, developed significantly reduced neointimal lesions compared with respective wild-type controls (2872 ± 843 μm2 vs 35469 ± 11870 μm2). Flow cytometry in CD40-deficient mice revealed reduced formation of platelet-granulocyte and platelet-inflammatory monocyte- aggregates. In vitro, supernatants of CD40-deficient platelet-leukocyte aggregates attenuated proliferation and increased apoptosis of smooth muscle cells. Unlike in the setting of atherosclerosis, CD40L mediates neointima formation via its classic receptor CD40 rather than via its recently described novel interaction with Mac-1. Therefore, selective targeting of CD40L-Mac-1 binding does not appear to be a favorable strategy to fight restenosis. © Schattauer 2014.Fil: Willecke, Florian. University Of Freiburg; AlemaniaFil: Tiwari, Shilpa. University Of Freiburg; AlemaniaFil: Rupprecht, Benjamin. University Of Freiburg; AlemaniaFil: Wolf, Dennis. University Of Freiburg; AlemaniaFil: Hergeth, Sonja. University Of Freiburg; AlemaniaFil: Hoppe, Natalie. University Of Freiburg; AlemaniaFil: Dufner, Bianca. University Of Freiburg; AlemaniaFil: Schulte, Lisa. University Of Freiburg; AlemaniaFil: Anto Michel, Nathaly. University Of Freiburg; AlemaniaFil: Bukosza, Nora. University Of Freiburg; AlemaniaFil: Marchini, Timoteo Oscar. University Of Freiburg; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Jäekel, Markus. University Of Freiburg; AlemaniaFil: Stachon, Peter. University Of Freiburg; AlemaniaFil: Hilgendorf, Ingo. University Of Freiburg; AlemaniaFil: Zeschky, Katharina. University Of Freiburg; AlemaniaFil: Schleicher, Rebecca. University Of Tübingen; AlemaniaFil: Langer, Harald. University Of Tübingen; AlemaniaFil: von zur Muhlen, Constantin. University Of Freiburg; AlemaniaFil: Bode, Christoph. University Of Freiburg; AlemaniaFil: Karlheinz, Peter. Baker Idi Heart And Diabetes Institute; AustraliaFil: Zirlik, Andreas. University Of Freiburg; Alemani

Characteristics of study animals before and after feeding.

<p>Characteristics of study animals before and after feeding.</p

Inflammatory cell recruitment is differentially affected by CB₂ receptor stimulation.

<p>A, Wild-type mice received intraperitoneal injections of 4% thioglycollate after pre-treatment with JWH-133 or vehicle control. Leukocyte recruitment into the peritoneal cavity was quantified after 72 and 4 h. Data represent mean ± SEM. Asterisks indicate significant change, defined as p<0,05. B, In parallel, thioglycollate-elicited accumulation of leukocytes in the peritoneal cavity was quantified in CB₂^−/−/LDLR^−/− mice and CB₂^+/+/LDLR^−/− control animals. Data for both 72 and 4 h stimulation are expressed as mean ± SEM. C, PMA-activated thioglycollate-elicited peritoneal leukocytes obtained from wild-type (Bl6) mice were allowed to adhere on TNFα-activated endothelial cells (EC) isolated by magnetic bead separation from wild-type mice in the presence or absence of 40 µM JWH-133. Adhering leukocytes were quantified under microscope after the indicated time points in the flow chamber (N = 3 each). In parallel experiments PMA-activated thioglycollate-elicited peritoneal leukocytes from CB₂^−/−/LDLR^−/− mice were allowed to adhere on TNFα-activated EC isolated from CB₂^−/−/LDLR^−/− mice. Adhesion was quantified and compared with the interaction of peritoneal leukocytes and EC isolated from CB₂^+/+/LDLR^−/− (N = 5 each). Pooled data represent mean ± SEM.</p

CB₂ receptor deficiency does not influence atherosclerosis in mice.

<p>A and B, CB₂^+/+/LDLR^−/− (N = 13) and CB₂^−/−/LDLR^−/− (N = 12) mice consumed HCD for 16 weeks and underwent analysis of intimal lesion area in the aortic root (A) and arch (B). Pooled data ± SEM are shown on the left; representative images stained for lipid deposition (Oil-red-O) are displayed below the corresponding graph. C, The abdominal aortas of mice treated as described above underwent <i>en face</i> analysis of the lipid deposition. Oil-red-O-positive staining in relation to total wall area was quantified and is dispayed as pooled data ± SEM (N = 13 and 12); representative images are shown below. D, Sections of aortic roots of mice treated as described above were analyzed for lipid-, macrophage-, collagen-, T cell-, smooth muscle cell- and apoptotic cell content. Oil-red-O-, Mac-3-, picosirius red-, CD4-, α-actin- and TUNEL-positive staining in relation to total wall area is described as mean ± SEM (N = 13 and 12). Asterisks indicate a significant change, defined as p<0,05.</p

Viability and ICAM-1 expression on murine endothelial cells is unaffected by CB₂ receptor signaling.

<p>A, Murine EC isolated from LDLR^−/− mice were stimulated with or without TNFα (20 ng/ml) and JWH-133 (4 µM and 40 µM, N = 4). In parallel, experiments, EC isolated from CB₂^−/−/LDLR^−/− mice and CB₂^+/+/LDLR^−/− control animals were stimulated with or without TNFα (20 ng/ml, N = 6). Cell lysates were analyzed for ICAM-1 by Western blotting. Western blots were analyzed densitometrically and adjusted for GAP-DH. Pooled data are given as mean ± SEM and representative blots are shown. B, Similarly, Murine EC isolated from wild-type mice where stimulated with indicated concentrations of JWH-133 and with or without TNFα (20 ng/ml). The cells were then analyzed for ICAM-1 expression using flow cytometric assays. Data is shown as mean ± SEM (N = 6). Asterisks indicate significant change, defined as p<0,05. C, In supernatants of EC treated as described above MCP-1 was quantified by ELISA. Data is shown as mean ± SEM. D and E, Murine EC isolated from wild-type mice were stimulated with indicated concentrations of JWH-133 and then the rate of apoptosis was determined using the Apo-ONE® Assay (D). Data is shown as the mean ± SEM (N = 5). The supernatant of cells treated in a similar manner were used to examine cytotoxicity with the CytoTox-ONE™ Assay (E). Data is shown as the percent of control (N = 6).</p

Pharmacokinetics of JWH-133 using mass spectrometry.

<p>Mice were subjected to intraperitoneal injection of JWH-133 (5 mg/kg body weight) on day 1, 3, 6, 8, and 10. On day ten, the serum levels of JWH-133 were determined at the indicated time points using mass spectrometry. The concentration of JWH-133 is given as the mean ± SEM (N = 6).</p