Table 1.
Clinical parameters were assessed at the end of the feeding period. Body weight, arterial blood pressure and heart rate were similar in both groups. Data are presented as mean ± standard error of the mean; n = 14–16; p-values as stated, assessed by student’s t-test. DMSO, dimethyl sulfoxide; JZL184, inhibitor of monoacylglycerol lipase.
Table 2.
Assessment of cholesterol and phytosterols.
Cholesterol and phytosterol plasma levels were assessed at the end of the feeding period. There were no differences between the two treatment groups. Data are presented as mean ± standard error of the mean; n = 9–10; p-values as stated, assessed by student’s t-test. DMSO, dimethyl sulfoxide; JZL184, inhibitor of monoacylglycerol lipase.
Fig 1.
Assessment of atherosclerotic plaque size by oil red O staining.
Frozen sections through the aortic sinus of both DMSO-treated control mice and JZL184-treated mice with elevated 2-AG levels were stained using oil red O to visualize atherosclerotic plaques (A, B). Cumulative plaque area was divided by the area of the vessel wall to obtain the relative plaque burden (C). Cumulative plaque areas are depicted in panel D. Data are presented as mean ± standard error of the mean; n = 14; *, p < 0.05, assessed by student’s t-test. Scale bar, 500 μm. DMSO, dimethyl sulfoxide; JZL184, inhibitor of monoacylglycerol lipase.
Fig 2.
Quantification of CD68-positive macrophages and monocytes.
Frozen sections through the aortic sinus of both DMSO-treated control mice and JZL184-treated mice with elevated 2-AG levels were stained for CD68 to visualize macrophages and monocytes (A, B). Positively stained areas were divided by the area of the vessel wall to obtain relative vessel infiltration (C). Data are presented as mean ± standard error of the mean; n = 13–14; **, p < 0.01, assessed by student’s t-test. Scale bar, 500 μm. DMSO, dimethyl sulfoxide; JZL184, inhibitor of monoacylglycerol lipase.
Fig 3.
Quantification of smooth muscle cells within atherosclerotic plaques.
Smooth muscle cells were stained in the aortic sinus using an immunofluorescent antibody targeting alpha smooth muscle actin. Significant amounts of smooth muscle cells were detected in 7 DMSO-treated animals and in 8 JZL184-treated animals (A, B; white arrows). The proportion of smooth muscle cells to the plaque area was unaffected by JZL184 (C). Data are presented as mean ± standard error of the mean; n = 7–8; p-value as indicated, assessed by student’s t-test. Scale bar, 200 μm. α-SMA, alpha smooth muscle actin; Cy3, cyanine 3; DAPI, 4',6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; JZL184, inhibitor of monoacylglycerol lipase.
Fig 4.
Collagen fibers were visualized in the aortic sinus by Picro Sirius staining. Significant amounts of collagen fibers were detected in 8 DMSO-treated animals and in 9 JZL184-treated animals (A, B). Quantity and distribution pattern of collagen fibers did not differ between the two groups (C). Data are presented as mean ± standard error of the mean; n = 8–9; p-value as indicated, assessed by student’s t-test. Scale bar, 200 μm. DMSO, dimethyl sulfoxide; JZL184, inhibitor of monoacylglycerol lipase.
Table 3.
Flow cytometry-based analysis of bone marrow and peripheral blood leukocytes.
Leukocytes were stained for CD11b, CD3, CD19, Ly6C, and Ly6G. Prevalence of these surface markers within a pre-specified leukocyte gate was determined after measuring 50,000 counts. Data are presented as mean ± standard error of the mean; n = 11–13; p-values as stated, assessed by student’s t-test. CD, cluster of differentiation; DMSO, dimethyl sulfoxide; JZL184, inhibitor of monoacylglycerol lipase; Ly6C, lymphocyte antigen 6C; Ly6G, lymphocyte antigen 6G.
Fig 5.
2-AG promotes B6MCL macrophage migration as assessed by modified Boyden chamber experiments.
B6MCL macrophage migration was assessed by modified Boyden chamber experiments. 350,000 cells were seeded per well and allowed to migrate for 90 minutes. Cells adherent to the lower side of the polycarbonate membrane were counted automatically using Image J 1.48v software following DAPI staining. Addition of 2-AG [1 μM] to the lower chamber resulted in an increased migration of B6MCL macrophages (B) compared to DMSO (A). This effect was blunted by addition of CB1-inhibitor AM281 [1 μM] and by CB2-inhibitor AM630 [1 μM] (C). Data are presented as mean ± standard error of the mean; n = 4–6; *, p < 0.05, assessed by ANOVA and Bonferroni correction. Scale bar, 300 μm. 2-AG, 2-arachidonoylglycerol; AM281, selective inhibitor of CB1; AM630, selective inhibitor of CB2; DMSO, dimethyl sulfoxide.
Fig 6.
2-AG does not influence proliferation of B6MCL macrophages.
B6MCL macrophage proliferation was assessed using a flow cytometry-based BrdU assay. There were no changes in cell cycle distribution following treatment with 2-AG [1μM] for 60 minutes. Data are presented as mean ± standard error of the mean; n = 3; p > 0.05, assessed by ANOVA and Bonferroni correction. 2-AG, 2-arachidonoylglycerol; 7-AAD, 7-Aminoactinomycin D; BrdU, bromodeoxyuridine; DMSO, dimethyl sulfoxide.