Abstract
This study was designed to explore the protective effect of D4F, an apolipoprotein A-I mimetic peptide, on nuclear factor-κB (NF-κB)-dependent Fas/Fas ligand (FasL) pathway-mediated apoptosis in macrophages induced by oxidized low-density lipoprotein (ox-LDL). Our results showed that ox-LDL induced apoptosis, NF-κB P65 nuclear translocation and the upregulation of Fas/FasL pathway-related proteins, including Fas, FasL, Fas-associated death domain proteins (FADD), caspase-8 and caspase-3 in RAW264.7 macrophages, whereas silencing of Fas blocked ox-LDL-induced macrophage apoptosis. Furthermore, silencing of P65 attenuated macrophage apoptosis and the upregulation of Fas caused by ox-LDL, whereas P65 expression was not significantly affected by treatment with Fas siRNA. D4F attenuated the reduction of cell viability and the increase in lactate dehydrogenase leakage and apoptosis. Additionally, D4F inhibited ox-LDL-induced P65 nuclear translocation and upregulation of Fas/FasL pathway-related proteins in RAW264.7 cells and in atherosclerotic lesions of apoE−/− mice. However, Jo2, a Fas-activating monoclonal antibody, reversed the inhibitory effect of D4F on ox-LDL-induced cell apoptosis and upregulation of Fas, FasL and FADD. These data indicate that NF-κB mediates Fas/FasL pathway activation and apoptosis in macrophages induced by ox-LDL and that D4F protects macrophages from ox-LDL-induced apoptosis by suppressing the activation of NF-κB and the Fas/FasL pathway.
Highlights
Atherosclerosis (AS) is a chronic inflammatory disease of the arterial wall
Oil red O staining and intracellular total cholesterol (TC) quantitative assay indicated that oxidized low-density lipoprotein (ox-low-density lipoprotein (LDL)), but not LDL, remarkably induced lipid accumulation, which is an important inducer of macrophage apoptosis (Supplementary Fig. S1)
Macrophage apoptosis occurs throughout all stages of atherosclerosis and plays an important role in plaque progression and instability, which may contribute to the majority of cardiovascular complications[33]
Summary
Atherosclerosis (AS) is a chronic inflammatory disease of the arterial wall. Macrophages ingest an excess amount of oxidized low-density lipoprotein (ox-LDL) and are converted into foam cells, which are the characteristic components of atherosclerotic plaques and are closely associated with the development and progression of AS1. It has been reported that ox-LDL-induced macrophage apoptosis is mediated by the Fas/FasL death receptor signaling pathway and may be blocked by antagonistic Fas antibody[14]. D4F has been demonstrated to have anti-atherogenic effects, such as improving reverse cholesterol transport (RCT) in macrophages from apoE−/− mice[17] and in RAW264.7 cells[18], preventing the oxidation of low-density lipoprotein (LDL), decreasing ox-LDL-induced monocyte chemotactic activity and increasing the anti-inflammatory properties of HDL. Our recent work has shown that D4F reduces ox-LDL-induced cytotoxicity of human umbilical vein endothelial cells (HUVECs) by preventing the downregulation of pigment epithelium-derived factor[25], and alleviates macrophage-derived foam cell apoptosis by inhibiting CD36 expression and the endoplasmic reticulum stress-C/ EBP homologous protein pathway[26]. We investigated the inhibitory effect of D4F on NF-κB activation and subsequent Fas/FasL death receptor pathway-mediated macrophage apoptosis
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