Activation of Endocannabinoid System Is Associated with Persistent Inflammation in Human Aortic Aneurysm

Abstract

Human aortic aneurysms have been associated with inflammation and vascular remodeling. Since the endocannabinoid system modulates inflammation and tissue remodeling, we investigated its components in human aortic aneurysms. We obtained anterior aortic wall samples from patients undergoing elective surgery for aortic aneurysm or coronary artery disease as controls. Histological and molecular analysis (RT-qPCR) was performed, and endocannabinoid concentration was determined using LC-MRM. Patient characteristics were comparable between the groups except for a higher incidence of arterial hypertension and diabetes in the control group. mRNA level of cannabinoid receptors was significantly higher in aneurysms than in controls. Concentration of the endocannabinoid 2-arachidonoylglycerol was significantly higher, while the second endocannabinoid anandamide and its metabolite arachidonic acid and palmitoylethanolamide were significantly lower in aneurysms. Histology revealed persistent infiltration of newly recruited leukocytes and significantly higher mononuclear cell density in adventitia of the aneurysms. Proinflammatory environment in aneurysms was shown by significant upregulation of M-CSF and PPARγ but associated with downregulation of chemokines. We found comparable collagen-stained area between the groups, significantly decreased mRNA level of CTGF, osteopontin-1, and MMP-2, and increased TIMP-4 expression in aneurysms. Our data provides evidence for endocannabinoid system activation in human aortic aneurysms, associated with persistent low-level inflammation and vascular remodeling.

Figure 1

Activation of the endocannabinoid system in aortic aneurysms. mRNA levels of cannabinoid receptors (a) CB1, (b) CB2, (c) TRPV1, and (d) GPR55 showed a significant increase in aneurysm of ascending aorta (AAA) as compared to the normal aorta of patients with coronary artery disease (CAD). Tissue concentration of the endocannabinoids (c) anandamide (AEA) and (d) 2-arachidonoyl glycerol (2-AG), as well as their degradation products (e) arachidonic acid (AA), and (f) palmitoylethanolamide (PEA) in comparison between the groups. n = 19 in the AAA group, n = 30 in the CAD RT-qPCR, and n = 25 in the CAD LC-MRM group. mRNA levels in RT-qPCR are related to controls and GAPDH using comparative ΔΔCt method. Bracket indicates p < 0.05.

Figure 2

Infiltration of aortic wall with inflammatory cells. Representative image of newly recruited leukocytes stained with MAC-387 antibody (a) in aorta from patients with coronary artery disease (CAD) and (b) from aneurysm of the ascending aorta (AAA). Arrows indicate the stained leukocytes and the dotted line delineates the adventitia (a) from the media (m). (c) Quantitative analysis of newly recruited leukocytes showed comparable cell density between the groups with significantly higher density in adventitia than in the media. Representative image of mononuclear cells stained using CD11b antibody in (d) aorta from CAD and (e) from AAA patient. (f) Quantitative analysis of mononuclear cells showed significantly higher cell density in adventitia of AAA samples than in CAD samples or respective media of the aortic wall. Scale bar in (a), (b), (d), and (e): 200 μm. n = 19 in the AAA group and n = 24 in the CAD histology group. Bracket indicates p < 0.05 between the groups; ∗ indicates p < 0.05 versus media.

Figure 3

mRNA expression of cytokines and chemokines in aortic wall. The mRNA expression of proinflammatory cytokines (a) TNFα, (b) IFNγ, and (c) IL-1β was comparable between ascending aortas from aneurysms (AAA) and patients with coronary artery disease (CAD). The mRNA expression of (d) IL-6 was lower, while (e) macrophage colony-stimulating factor (M-CSF) and (f) peroxisome proliferator-activated receptor (PPAR)γ were significantly higher in AAA than in CAD aortic tissue. The expression of (g) the anti-inflammatory cytokine IL-10 was comparable between the groups. (h) A significantly lower expression of potent mononuclear chemoattractants CCL2 and (i) nonsignificantly lower level of related chemokine CCL4 were found in AAA samples. n = 19 in the AAA group and n = 30 in the CAD RT-qPCR group. mRNA expression in RT-qPCR is related to controls and GAPDH using comparative ΔΔCt method. Bracket indicates p < 0.05.

Figure 4

Collagen area and expression of remodeling related factors in aortic aneurysm. Representative images of picrosirius red staining show comparable collagen-deposition pattern in media (m) and adventitia (a) of (a) aorta from patient with coronary artery disease (CAD) and (b) aneurysm of the ascending aorta (AAA). (c) The planimetric evaluation of the collagen-stained area in the inner and outer media showed comparable results between both groups. The mRNA expression of (d) the collagen-related connective tissue growth factor (CTGF) and (e) extracellular matrix remodeling related factor osteopontin-1 is significantly lower in AAA when compared to the CAD aortic samples. (f) The mRNA expressions of early remodeling factor tenascin C and (g) transforming growth factor- (TGF-) β1 are comparable between the groups. n = 19 in the AAA group, n = 24 in the CAD histology, and n = 30 in the CAD RT-qPCR group. mRNA expression in RT-qPCR is related to controls and GAPDH using comparative ΔΔCt method. Bracket indicates p < 0.05.

Figure 5

Expression of matrix metalloproteinases and their tissue inhibitors in aortic aneurysm. The mRNA expression of (a) matrix metalloproteinase- (MMP-) 1, (b) MMP-2, (c) MMP-9, and (d) MMP-14 was not significantly different in aorta from patients with aneurysm of the ascending aorta (AAA) compared to coronary artery disease (CAD) except for MMP-2. The mRNA expression of (e) tissue inhibitor of matrix metalloproteinase- (TIMP-) 1, (f) TIMP-2, and (g) TIMP-4 was not significantly different in aorta from patients with AAA compared to CAD except for TIMP-4. (h) Ratios of different MMP/TIMP showed only a significant difference for MMP-2/TIMP-4 between AAA and CAD group. n = 19 in the AAA group and n = 30 in the CAD RT-qPCR group. mRNA expression in RT-qPCR is related to controls and GAPDH using comparative ΔΔCt method. Bracket indicates p < 0.05.