Decreased age-related cardiac dysfunction, myocardial nitrative stress, inflammatory gene expression, and apoptosis in mice lacking fatty acid amide hydrolase

Abstract

Recent studies have uncovered important cross talk between inflammation, generation of reactive oxygen and nitrogen species, and lipid metabolism in the pathogenesis of cardiovascular aging. Inhibition of the endocannabinoid anandamide metabolizing enzyme, the fatty acid amide hydrolase (FAAH), is emerging as a promising novel approach for the treatment of various inflammatory disorders. In this study, we have investigated the age-associated decline of cardiac function and changes in inflammatory gene expression, nitrative stress, and apoptosis in FAAH knockout (FAAH(-/-)) mice and their wild-type (FAAH(+/+)) littermates. Additionally, we have explored the effects of anandamide on TNF-alpha-induced ICAM-1 and VCAM-1 expression and monocyte-endothelial adhesion in human coronary artery endothelial cells (HCAECs). There was no difference in the cardiac function (measured by the pressure-volume conductance catheter system) between 2- to 3-mo-old (young) FAAH(-/-) and FAAH(+/+) mice. In contrast, the aging-associated decline in cardiac function and increased myocardial gene expression of TNF-alpha, gp91phox, matrix metalloproteinase (MMP)-2, MMP-9, caspase-3 and caspase-9, myocardial inducible nitric oxide synthase protein expression, nitrotyrosine formation, poly (ADP-ribose)polymerase cleavage and caspase-3/9 activity, observed in 28- to 31-mo-old (aging) FAAH(+/+) mice, were largely attenuated in knockouts. There was no difference in the myocardial cannabinoid CB(1) and CB(2) receptor gene expression between young and aging FAAH(-/-) and FAAH(+/+) mice. Anandamide dose dependently attenuated the TNF-alpha-induced ICAM-1 and VCAM-1 expression, NF-kappaB activation in HCAECs, and the adhesion of monocytes to HCAECs in a CB(1)- and CB(2)-dependent manner. These findings suggest that pharmacological inhibition of FAAH may represent a novel protective strategy against chronic inflammation, oxidative/nitrative stress, and apoptosis associated with cardiovascular aging and atherosclerosis.

Fig. 1

Hemodynamics in young (2- to 3-mo-old) and aging (28-to 31-mo-old) mice measured by the Millar pressure-volume conductance catheter system. Values are means ± SE of 7–11 experiments in each group. LVEDP, left ventricular end-diastolic pressure; LVSP, left ventricular systolic pressure; −dP/dt, diastolic decrement; tau (τ), relaxation time constant; FAAH, fatty acid amide hydrolase. *P < 0.05 vs. young mice; #P < 0.05 vs. aging FAAH^+/+ mice.

Fig. 2

Myocardial cannabinoid CB₁ and CB₂ receptors, gp91phox, and TNF-α gene expression. Values are means ± SE of 6–15 experiments in each group. *P < 0.05 vs. young mice; #P < 0.05 vs. aging FAAH^+/+ mice.

Fig. 3

Myocardial inducible nitric oxide synthase (iNOS) protein expression, nitrotyrosine formation, and matrix metalloproteinase (MMP)-2 and -9 gene expression. Values are means ± SE of 5–15 experiments in each group. *P < 0.05 vs. young mice; #P < 0.05 vs. aging FAAH^+/+ mice.

Fig. 4

Myocardial markers of apoptosis (PARP cleavage, caspase-3/7 activity, and caspase-3 and -9 gene expression). Values are means ± SE of 5–15 experiments in each group. *P < 0.05 vs. young mice; #P < 0.05 vs. aging FAAH^+/+ mice.

Fig. 5

Effect of anandamide (AEA) on TNF-α-induced ICAM-1 and VCAM-1 expression in human coronary artery endothelial cells (HCAECs). Values are means ± SE; n = 6. Cells were treated with either TNF-α (50 ng/ml) or AEA (15 μM) for 6 h or pretreated with AEA with the indicated concentrations followed by treatment with TNF-α for 6 h, and then cell surface ELISA was performed as described in MATERIALS AND METHODS (A and B). VC, vehicle control. A: ICAM-1 expression. *P < 0.05 vs. controls; #P < 0.05 vs. TNF-α. B: VCAM-1 expression. *P < 0.05 vs. controls; #P < 0.05 vs. TNF-α. Cells were pretreated with CB₁/CB₂ antagonists (1 μM) from 1 h before and during the treatment with TNF-α ± AEA (15 μM) as indicated for 6 h, and cell surface ELISA was performed (C and D). C: ICAM-1 expression. *P < 0.05 vs. control; #P < 0.05 vs. TNF-α. Paragraph symbol: Pg< 0.05 vs. AEA + TNF-α. D: VCAM-1 expression. *P < 0.05 vs. control; #P < 0.05 vs. TNF-α. Paragraph symbol: P < 0.05 vs. AEA + TNF-α.

Fig. 6

Effect of AEA on TNF-α-induced monocyte adhesion to HCAECs. HCAECs were treated as described in legend to Fig. 5. Top: representative images depicting monocytes adhered to the endothelial cells. Bottom: quantification of monocyte adhesion to endothelial cells. Values are means ± SE; n = 6. *P < 0.05 vs. control; #P < 0.05 vs. TNF-α. Paragraph symbol: P < 0.05 vs. AEA + TNF-α.

Fig. 7

Effect of AEA on TNF-α-induced NF-κB activation in HCAECs. Representative immunofluorescence images of NF-κB activation in endothelial cells. TNF-α markedly activated NF-κB (note the intense nuclear staining). AEA significantly inhibits TNF-α-induced activation of NF-κB. Images shown are representative of 3 independent experiments yielding identical results.