The Acid Sphingomyelinase Inhibitor Amitriptyline Ameliorates TNF-α-Induced Endothelial Dysfunction

Abstract

Purpose Inflammation associated endothelial cell (EC) dysfunction is key to atherosclerotic disease. Recent studies have demonstrated a protective role of amitriptyline in cardiomyocytes induced by hypoxia/reoxygenation. However, the mechanism by which amitriptyline regulates the inflammatory reaction in ECs remains unknown. Thus, the aim of this study was to investigate whether amitriptyline protects against inflammation in TNF-α-treated ECs. Methods HUVECs were incubated with amitriptyline (2.5 μM) or TNF-α (20 ng/ml) for 24 h. EdU, tube formation, transwell, DHE fluorescence staining, and monocyte adhesion assays were performed to investigate endothelial function. Thoracic aortas were isolated from mice, and vascular tone was measured with a wire myograph system. The levels of ICAM-1, VCAM-1, MCP-1, phosphorylated MAPK and NF-κB were detected using western blotting. Results Amitriptyline increased the phosphorylation of nitric oxide synthase (eNOS) and the release of NO. Amitriptyline significantly inhibited TNF-α-induced increases in ASMase activity and the release of ceramide and downregulated TNF-α-induced expression of proinflammatory proteins, including ICAM-1, VCAM-1, and MCP-1 in ECs, as well as the secretion of sICAM-1 and sVCAM-1. TNF-α treatment obviously increased monocyte adhesion and ROS production and impaired HUVEC proliferation, migration and tube formation, while amitriptyline rescued proliferation, migration, and tube formation and decreased monocyte adhesion and ROS production. Additionally, we demonstrated that amitriptyline suppressed TNF-α-induced MAPK phosphorylation as well as the activity of NF-κB in HUVECs. The results showed that the relaxation response of aortic rings to acetylcholine in the WT-TNF-α group was much lower than that in the WT group, and the sensitivity of aortic rings to acetylcholine in the WT-TNF-α group and WT-AMI-TNF-α group was significantly higher than that in the WT-TNF-α group. Conclusion These results suggest that amitriptyline reduces endothelial inflammation, consequently improving vascular endothelial function. Thus, the identification of amitriptyline as a potential strategy to improve endothelial function is important for preventing vascular diseases.

A Near‐Infrared Fluorogenic Probe for Rapid, Specific, and Ultrasensitive Detection of Sphingosine in Living Cells and In Vivo

“…Since the overexpression of A𝛽 protein induces mitochondrial oxidative stress and activates the intrinsic apoptotic cascade, and previous studies have illustrated that H 2 O 2 could induce neurotoxicity in PC12 cells, [18a,19] Since changes in Sph levels are associated with the abnormal sphingolipid metabolism in cells, to investigate the possible mechanisms of A𝛽 42 oligomers and H 2 O 2 -induced changes in Sph levels, we further investigated intracellular sphingomyelinase and ceramidase activities in A𝛽 42 oligomers and H 2 O 2 treated cells. PC12 cells were preincubated for 2 h in the presence of 10 μm amitriptyline hydrochloride (AMI, an acid sph-ingomyelinase inhibitor [20] ), 20 μm GW4869 (a neutral sphingomyelinase inhibitor [21] ), 10 μm LCL-521 (an acid ceramidase inhibitor [22] ), 500 μm N-acetyl-cysteine (NAC, an efficient antioxidant [23] ) and 1 mm Trolox (a reactive oxygen scavenger [24] ), and then treated with A𝛽 42 oligomers (10 μm, 12 h) or H 2 O 2 (50 μm, 4 h), respectively. As shown in Figure 4a,c, the fluorescence of the cells preincubated with the inhibitors prior to the treatment with A𝛽 42 oligomers was lower than that of the control group.…”

GALNT4 promotes the endothelial cell inflammatory response via the NF-κB signaling pathway

“…The NF-κB pathways play an important role in ICAM-1, VCAM-1, and MCP-1 expression in HUVECs [31]. In this study, we used a speci c NF-κB inhibitor, PDTC, to examine the role of NF-κB in GALNT4-mediated cell adhesion molecule expression.…”

Acid sphingomyelinase promotes diabetic cardiomyopathy via NADPH oxidase 4 mediated apoptosis

“…Previous work has shown some mechanistic insight into the role of ASMase in mediating cardiovascular diseases. ASMase mediated TNF-α-induced endothelial dysfunction through inhibiting eNOS phosphorylation and activating of MAPK signaling, which were restored by its inhibitor, amitriptyline [18]. Moreover, targeting the ASMase pathway is protected from hypertensive vascular damage [11].…”