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

Treatment with the CB₂ agonist JWH-133 does not modulate atherosclerosis in mice.

<p>A and B, LDLR^−/− mice consuming high cholesterol diet for 16 weeks (HCD) received intraperitoneal injections of 5 mg/kg JWH-133 (N = 10) or vehicle control (Tocris, N = 8) three times a week. Intimal lesion area in the aortic root (A) and arch (B) are diplayed as pooled data ± SEM; representative images stained for lipid deposition (Oil-red-O) are shown below the corresponding graph. C, The abdominal aortas of mice treated as described above underwent <i>en face</i> analysis of lipid deposition. Oil-red-O-positive staining in relation to total wall area was quantified and is displayed as pooled data ± SEM (N = 8 and 10); 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 given as mean ± SEM (N = 8 and 10).</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

Characteristics of study animals before and after feeding.

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

Baseline study characteristics.

<p>Data are presented as mean ± SEM, ND not determined, LFD low fat diet, HFD high fat diet, WT wildtype, PC Phosphatidylcholine, BW body weight.</p

Genetic absence of CD40L reduces MCP-1 gene expression in adipose tissue and MCP-1 plasma levels.

<p>Diet-induced obesity was induced by feeding with a high fat diet for 20 weeks. MCP-1 gene transcripts in adipose tissue were quantified by quantitative PCR after whole tissue RNA preparation and normalization for GAPDH expression (A). Levels of circulating MCP-1 were determined in plasma by cytometric bead array. Data are presented as mean ± SEM of at least 6 animals per group.</p

Genetic deficiency of CD40L does not protect from diet-induced obesity.

<p>WT and CD40L^−/− mice consumed a high fat diet (HFD) for 20 weeks. Relative increase of body weight (shown as % of body weight at week 0) and total body fat as assessed by MRI-based body composition analysis are shown for the indicated time points (A, B). Weight of epididymidal (EFP), peri-renal (RFP), and cardiac fat pads (CFP) fat are displayed as percentage of total body weight (BW, C). Data are presented as mean ± SEM of at least 15 mice per group.</p

Absence of CD40L does not ameliorate insulin sensitivity in diet-induced obesity.

<p>Plasma glucose (A) and insulin levels (B) were determined in animals fasted overnight (A) and for 6 hours (B) overnight. Insulin tolerance (ITT) and glucose tolerance testing (GTT) were performed after intraperitoneal insulin (0.5 U/kg lean body mass) or glucose (1 g/kg lean body mass) injection (C–F). Inlays represent area under the curve calculation (AUC) of the indicated glucose curve.</p