| Background and objectiveVascular endothclial cells (VECs) comprise a single cell layer of endothelium forming the interior surface of blood vessels. Endothelial dysfunction is a major cause of cardiovascular diseases, and basal autophagy can protect VECs against stressful circumstances. Consequently, manipulation of the signaling pathways regulating autophagy in VECs may provide novel therapeutic strategies to prevent or treat cardiovascular diseases such as atherosclerosisDifferent from most cell types whose autophagy can be induced by serum starvation, VECs have constant contact with serum in vivo, hence deprivation of serum usually induces apoptosis instead of autophagy in VECs So it is difficult to investigate the mechanism of VEC autophagy by scrum starvation, which makes it necessary to find inducers of VKC autophagy in the presence of serum.In previous studies, we identified a novel benzoxazine derivative:6-amino-2,3-dihydro-3-hydroxymethyl-l,4-benzoxazine (ABO) ABO could avert VEC apoptosis induced by nutrient deprivation and oxidized low-density lipoprotein. In addition, ABO could increase the protein level of ANXA7, promote the co-localization of ANXA7and LC3or GCA, and ultimately promote autophagy In conclusion, ABO may act as a protector of VECs or an inducer of VEC autophagy. However, the mechanisms how ABO regulate autophagy are unknown. ANXA7is a Ca-dependent GTPase, while phosphorylation has a significant positive effect on ANXA7GTPase activity and is one of the important protein modifications of LC3and GCA. So, whether ABO directly binds to ANXA7and affects the phosphorylation of ANXA7and its binding partners, whether ABO has beneficial effects by inducing autophagy in VECs and whether ANXA7inhibits atherosclerosis progression in vivo need further elucidation.Accumulating evidence implicates phosphatidylcholine-specific phospholipase C (PC-PLC) in endothelial dysfunction and the progression of atherosclerosis. Phosphatidylethanolamine binding protein1(PEBP1), as a PC-PLC binding partner, is an important secretion protein. Whether ABO affects the activity and level of PC-PLC, or the secretion of PEBP1needs further investigations.Based on the backgrounds mentioned above, the objective of this study was as follows: to investigate potentially underlying mechanism(s) how ABO regulate autophagy as well as the effects ABO on the development of atherosclerosis. The researches would provide us a brand-new target for atherosclerosis therapy and a potential tool to intervene in the development of atherosclerosis.1. The mechanism how ABO promotes VEC autophagy.1.1ABO directly binds to ANXA7and inhibits the phosphorylation of ANXA7. To investigate the mechanism how ABO regulates ANXA7and ultimately promotes autophagy, we first examined the interaction of ABO and ANXA7by immunoprecipitation and western blot. The results showed that ABO blocks the binding of ANXA7with this antibody in a dose-dependent way, but we could not see this effect when we used an anti-mCherry antibody to immunoprecipitate mCherry-ANXA7fusion protein expressed in HUVECs, suggesting that ABO might bind to ANXA7directly. Furthermore, we found that ABO could inhibit the phosphorylation of ANXA7at the threonine residues. By site-directed mutagenesis, we found that ABO directly bound to Thr286of ANXA7and inhibited its phosphorylation.1.2ABO inhibits the phosphorylation of the binding partners of ANXA7, such as GCA and LC3.As described previously, ANXA interacts with GCA and LC3, and ABO could promote the interaction of ANXA7and LC3. In this study, we found that ABO could promote the interaction of ANXA7and GCA in vitro and in vivo by double immunohistochemical staining. Furthermore, we found ABO could inhibite the phosphorylation of GCA and LC3by immunoprecipitation and western blot.2. The underlying mechanisms however ABO restricts atherosclerosis development and stabilizes established atherosclerotic lesion in apoE-/-mice. 2.1ABO restricts atherosclerosis development and stabilizes established atherosclerotic lesion in apoE-/-Mice.Male apoE-/-mice (8weeks old) were fed an atherogenic high-fat diet (21%fat,0.15%cholesterol) for14weeks Animals were randomized to4groups (n=6mice/group) for treatment. The Hist group was killed to determine the extent of established lesions Groups2and3received8weekly intraperitoneal injections of ABO (low-dosage,50mg/kg per day or high-dosage,100mg/kg per day). Control group4was injected with the same volume of DMSO and PBS. At the end of treatment, all mice were killed by intravenous injection of ketamine/xylazine (Sigma Aldrich, St. Louis, MO), and blood and tissue were collected for analysis. Morphometric assessment revealed significantly lower plaque burden in whole aorta, brachiocephalic artery and aortic root with both high-and low-dosage ABO than control treatment in apoE-mice As well, ABO preserved a stable plaque phenotype, with reduced lipid deposition and macrophage content and increased collagen content and smooth muscle cells. In situ zymography detected high activity of MMP-2/9in atherosclerotic lesions with control treatment, the addition of ABO significantly reduced their activity. Then, we detected and quantified apoptosis and necrosis in the endothelium and in the plaques by TUNEL, staining and H&E staining. We found that both types of cell death were decreased in ABO-treated groups. Further, we performed toxicity experiments to demonstrate whether there is potential toxicity of ABO to the mice by evaluating the influence of ABO on the weight of body and organ coefficients. The results showed that there were no significant differences in body weight and organ coefficients between the control group and ABO-treated groups.2.2The underlying mechanisms however ABO inhibits the development of atherosclerosis.First, ABO treatment did not affect plasma lipid levels in mice, suggesting that the anti-atherogenic effect of ABO was not related to lipid metabolism in apoE-/-mice Then, we assessed autophagy and the protein levels of p62and ANXA7in aortic ECs by transmission electron microscopy (TEM) and immunofluorescence analysis, respectively The results suggested that ABO promoted autophagy in the endothelium via elevating the protein level of ANXA7and the autophagic flux was intact. TUNEL staining and H&E staining showed that apoptosis and necrosis in the endothelium were decreased by ABO treatment.Furthermore, low-dosage ABO slightly and nonsignificantly decreased the protein level of PC-PLC in the endothelium, but high-dosage ABO significantly reduced the level as compared with controls. As well, the mean serum PC-PLC activity was lower with ABO treatment than controls. Enzyme-linked immunosorbent assay (ELISA) assay showed that treatment of HUVECs with ABO blunted the increase of IL-6and IL-8secretion induced by oxLDL. In vitro experiment showed that ABO Suppressed pxLDL-induced PC-PLC activity in an ANXA7-dependent manner in HUVECs. PEBP1Level in the endothelium and serum were significantly elevated during development of atherosclerosis. ABO suppressed the high levels of PEBP1in the endothelium and serum of apoE-/-mice... |
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