Effect And Mechanism Of Apelin-13 Products On Macrophage Foam Cell Cholesterol Efflux Via Autophagy

Atherosclerotic cardio-cerebral vascular disease is serious harm to human health and life. There is a lot of pathogenesis which is involved in inflammation, damage response, abnormal lipid metabolism. A key event in the progression of atherosclerosis is the differentiation of monocytes to macrophages that take up excess lipoprotein-derived cholesterol to form foam cells within the walls of blood vessels. Autophagy is an evolutionarily conserved catabolic process that acts as a major survival mechanism by degrading and recycling damaged organelles and aggregated proteins in cytosol via lysosomal system. Recently, autophagy has been shown to participate in AS. A special kind of autophagy, termed as“lipophagy”, which can deliver cholesterol ester to lysosome, followed by the hydrolyzation and release of cholesterol from cholesterol ester,promoting cholesterol egress from lipid-laden cells to high density lipoprotein（HDL） through ATP-binding cassette A1（ABCA1）. Thus,activation of autophagy can decrease, whereas inhibition of autophagy increases, the intracellular lipid deposits. Therefore, autophagy is a potential target to promote cholesterol efflux and prevent atherosclerotic cardiovascular disease.Apelin is a recently discovered adipocytokine, which mainly secreted by adipose tissue and characterized bioactive peptide hormone. It is expressed in a diverse range of tissues, especially in the cardiovascularsystem. Apelin and APJ constitute a signaling pathway, which is involved in a wide range of physiological and pathological functions including dilatation of arteries, systolic effect and so on. As a kind of vascular oxidative stress adjustment, it has a pivotal role of cardiovascular function. Apelin isoforms have at least four bioactive forms, including apelin-12,-13,-17, and-36. In patients with cardiovascular disease（CVD） the plasma apelin levels decreased significantly than normal, and it is closely related to the development and stability of the artery atherosclerotic plaque. These findings suggest an anti-atherosclerotic role of apelin. However, the exact mechanism involved has not been fully elucidated.The process of autophagy and its mechanism are complicated, which are regulated by the many signaling pathways, including phosphatidyl inositol 3- kinase（phosphoinositide 3- kinases, PI3Ks） that is the key of the autophagy pathway regulating molecules. The Class I PI3 K and Class III PI3 K signaling pathways regulate autophagy in different periods. Recent studies have found that apelin induced autophagy in the cell protection by PI3 Ks signaling pathways, suggesting antiatherosclerotic role of apelin is closely related to the autophagy.This study is focus on autophagy, which is a key factor of mediated cholesterol efflux and investigates the possible role and mechanism of apelin-13 in atherosclerosis. Using of agonist, antagonist or si RNA to investigate effect of apelin-13 and signaling transduction mechansim on autophagy. It will provide new experimental evidence for exploring the role of apelin-13 in atherosclerosis and pathogenesis of atherosclerosis.Part I: Effect of Apelin-13 Promotes on Cholesterol Efflux in THP-1 Macrophage-derived Foam Cells through inducing autophagyAims: In order to observe effect of apelin-13 on cholesterol efflux in THP-1 macrophage-derived foam cells and detect the change of autophagy.Methods: Human THP-1 monocytes were preincubated with Phorbol-12- myristate-13-acetate（PMA） and oxidized low density lipoprotein（ox-LDL） to form foam cells. Cells was treated by apelin-13 and observed the concentration-dependent or time-dependent effect. Cellular cholesterol content and lipid droplet were measured by High Performance Liquid Chromatography（HPLC） and Oil Red O stain, respectively. The cholesterol efflux was assessed by liquid scintillation counting. Autophagy was evaluated in cells by fluorescence microscopy, or western immunoblotting assays. In fluorescence microscopy experiments, autophagy was evaluated by examining the punctate forms（type II） of the autophagy marker LC3. Experiments examined either GFP-LC3 or endogenous LC3 stained with the LC3 antibody. Our experiment also used PTX or rapamycin or vinblastine to incubate together with apelin-13 for 24 h. The levels of autophagy activity and cholesterol efflux were examined.Results: THP-1 macrophage-derived foam cells were treated with apelin-13（0,1,10,100 n M）for 24 hours and cholesterol efflux was increased by liquid scintillation counting assays. Cellular cholesterol content and lipid droplet, which was determinated by HPLC and Oil Red O stain respectively, were reduced by apelin-13 treated. Also, apelin-13 significantly increased LC3 and decreased the expression of p62. This effect of apelin-13 was appeared with concentration-dependent manner. Furthermore, cells were incubated with 100 n M apelin-13 for 0, 6, 12, 24 hours, respectively. Datawas shown that cellular cholesterol efflux was increased by apelin-13. The cellular cholesterol content and lipid droplet was decreased in apelin-13 treatment groups. LC3 expression was upregulated, p62 expression was downregulated after apelin-13 treated cells for different time. The effect of apelin-13 was appeared with time-dependent manner. Consistently, apelin-13 obviously enhanced autophagy, as manifested by GFP-labeled autophagosomes. Furthermore, PTX abolished the enhancement of cholesterol efflux and autophagy activities induced by apelin-13. rapamycin,an inhibitor of the mammalian target of rapamycin（m TOR）, induced autophagy as expected, enhanced apelin-13-induced autophagy. Whereas vinblasine, a microtubule depolymerizing agent that attenuated the effects of apelin-13, as manifested by the changes of LC3 and GFP-labeled autophagosomes, Consistently, both cholesterol efflux activity and total cholesterol quantification showed that rapamycin promoted cell cholesterol efflux apelin-13. Conversely, the inhibition of autophagy by vinblasine exerted the opposite effect.Conclusions:（1） apelin-13 promotes cellular cholesterol efflux and discreases accumulation of cellular cholesterol in THP-1macrophage-derived foam cells,with concentration-dependent and time-dependent manner.（2） The LC3 expression is upregulated, whereas p62 expression is downregulated by apelin-13 with concentration-dependent and time-dependent manner respectively.（3）apelin-13 promotes cholesterol efflux from THP-1 macrophage-derived foam cells through autophagy pathway.Part II: Mechanism of Apelin-13 Products on THP-1 Macrophage-derived Foam Cells Cholesterol Efflux Via AutophagyAims: To investigate the possible mechanism of Autophagy and cellular cholesterol efflux were induced by Apelin-13 in THP-1 macrophage-derived foam cells.Methods: Human THP-1 monocytes were preincubated with Phorbol-12- myristate-13-acetate（PMA） and oxidized low density lipoprotein（ox-LDL） to form foam cells. Cells were treated with Apelin-13and（or） intervention, such as antagonist or si RNA of some key factors of IP3 Ks signaling pathway. Cellular cholesterol efflux was assessed by liquid scintillation counting. The protein was examined by western immunoblotting assays.Results: THP-1 macrophage-derived foam cells were treated with apelin-13（0,1,10,100 n M）for 24 hours,decreased the levels of class I PI3 K as well as phosphorylated m TOR and Akt,whereas increased the expression of PTEN and ULK1 by apelin-13 in dose-dependent respectively. THP-1 macrophage-derived foam cells were treated with PTEN agonist SF1670. As demonstrated, LC3 expression was significantly down-regulated, p62 expression was significantly up-regulated in cells treated by SF1670 alone compared with control group. LC3 expression was significantly up-regulated, p62 expression was significantly down-regulated in cells treated by combination of SF1670 and apelin-13 compared with apelin-13 alone group. At the same time, cellular cholesterol efflux in cells treated by combination of SF1670 and apelin-13 was siginificantly decreased as compared with these treated by apelin-13 alone. THP-1macrophage-derived foam cells were treated with ULK1. As demonstrated,LC3 expression was significantly up-regulated, p62 expression was significantly down-regulated in cells treated by combination of Control-si RNA and apelin-13 compared with Control-si RNA group. LC3 expression was significantly down-regulated, p62 expression was significantly up-regulated in cells treated by the combination of ULK1-si RNA and apelin-13 compared with combination of Control-si RNA and apelin-13 group. At the same time, cellular cholesterol efflux in cells treated by combination of Control-si RNA and apelin-13 was siginificantly decreased as compared with the combination of ULK1-si RNA and apelin-13. In addition, apelin-13 enhanced the expression of Class III PI3 K and Becin-1 in dose-dependent respectively. THP-1 macrophage-derived foam cells were treated with Class III PI3 K agonist 3-MA. As demonstrated,LC3 expression was significantly down-regulated, p62 expression was significantly up-regulated in cells treated by 3-MA alone compared with control group. LC3 expression was significantly up-regulated, p62 expression was significantly down-regulated in cells treated by combination of 3-MA and apelin-13 compared with apelin-13 alone group. At the same time, cellular cholesterol efflux in cells treated by combination of 3-MA and apelin-13 was siginificantly decreased as compared with these treated by apelin-13 alone. THP-1 macrophage-derived foam cells were treated with Beclin-1 si RNA. As demonstrated, LC3 expression was significantly up-regulated, p62 expression was significantly down-regulated in cells treated by combination of Control-si RNA and apelin-13 compared with Control-si RNA group. LC3 expression was significantly down-regulated,p62 expression was significantly up-regulated in cells treated by the combination of Beclin-1-si RNA and apelin-13 compared with combination of Control-si RNA and apelin-13 group. At the same time, cellularcholesterol efflux in cells treated by combination of Control-si RNA and apelin-13 was siginificantly decreased as compared with the combination of Beclin-1-si RNA and apelin-13.Conclusions:（1）apelin-13 promotes cholesterol efflux from THP-1macrophage-derived macrophage foam cells by inhibiting Class I PI3K-mediated autophagic pathway.（2）apelin-13 promotes cholesterol efflux from THP-1 macrophage-derived macrophage foam cells by activating Class III PI3K/Beclin-1-mediated autophagic pathwayPart III: Effect of apelin-13 on RCT, aortic lipid deposition and atherosclerotic lesion in apolipoprotein E knockout mice through autophagyAims: To observe the effect of apeli N-13 on RCT, plasma lipid profile,aortic lipid deposition and atherosclerotic lesion and ATG expression in apo E knockout(apo E^-/-) miceMethods: Six-week-old male apo E^-/- mice were fed with Western diet and randomly divided into 4 groups（n=15）: control（Con） group, apelin-13 group, apelin-13+PTX group, PTX group. control group only was fed with high-fat/ high-cholesterol diet; apelin-13 group received intraperitoneal injections of 5mg/kg apelin-13 once every other day; pelin-13+PTX group received intraperitoneal injections of the same volume of pelin-13 and PTX once every other day; PTX group was received PTX once every other day.The control group was received intravenous injections the same volume of PBS at the same time. MPM was radiolabeled with 3H-cholesterol and intraperitoneally injected into the apo E^-/- mice at 1 day before sacrifice. After8 weeks, all animals were killed. Blood, feces and hepatic tissue werecollected to detected RCT efficiency with liquid scintillation counting. Lipid deposition in blood vessel wall was observed by stereoscopic microscope.Triglyceride（TG）, total cholesterol（TC）, HDL-C and LDL-C were determined by commercially enzymatic methods. Lipid accumulation in aorta and aortic sinus were evaluated by Oil Red O stain. Collagen levels were evaluated by Masson’s staining. Atherosclerotic lesions in aortic sinus was tested by HE stain. Protein expression of ATG in aorta was detected by western blot.Results:（1）Stereoscopic microscope results showed that aortic plaques were decreased significantly in apelin-13 group compared with Control group. However, aortic plaques were increased significantly in apelin-13+PTX group compared with apelin-13 group.（2）Oil red O staining showed that lipid accumulation in aortic sinus in apelin-13 group was reduced compared with control group. Compared with the apelin-13 group,lipid accumulation in aortic sinus in apelin-13+PTX group was increased significantly.（3） Masson trichromatic staining results showed that collagen fiber content in the aortic sinus was decreased in apelin-13 group compared with control group. the collagen fiber content was increased significantly in apelin-13+PTX group compared with apelin-13 group.（4） compared with control group,The excretion of 3H-cholesterol originating from cholesterol-loaded MPM into plasma, liver, gallbladder, and feces significantly was increased in apelin-13 group; however, The excretion of3H-cholesterol was reduced significantly in apelin-13+PTX group compared with apelin-13 group.（5）TG and TC levels have not changed, HDL-C were increased and LDL-C levels were decreased in pelin-13 group compared with pelin-13+PTX group. HDL-C levels were decreased and LDL-C levelswere increased in apelin-13+PTX group compared with apelin-13 group.（6）HPLC results showed that lipid levels in peritoneal macrophages were significantly reduced in apelin-13 group compared with control group.Compared with the apelin-13 group, lipid levels in peritoneal macrophages in mice were increased significantly in apelin-13+PTX group.（7） Western blotting results showed that the expression of LC3 obviously increased and p62 reduced in pelin-13 group compared with control group in peritoneal macrophages of the apo E^-/- mice. Inhibition of apelin-13 could obviously increase the protein expression of p62 and reduce LC3 in peritoneal macrophages of the apo E^-/- mice.（8）Western blotting results showed that the expression of PTEN was significantly increased and class 1 PI3 K was decreased in pelin-13 group compared with control group, Compared with the pelin-13 group, the expression of PTEN was significantly decreased and class1PI3 K was increased in pelin-13+PTX group. whereas the phosphorylation of Ak and m TOR were decreased in apelin-13 group compared with control group, Inhibition of apelin-13 could obviously increase the phosphorylation of Ak and m TOR（9） Western blotting results showed that apelin-13 upregulated the expression of class III PI3 K and Beclin-1,whereas PTX nearly abolished the effect of apelin-13 on peritoneal macrophages of the apo E^-/- mice.Conclusions:（1） apelin-13 has the inhibition effect on As lesion.（2）apelin-13 reduced lipid accumulation in the aortic wall, plasma lipid level,and enhanced RCT in apo E^-/- mice.（3） class1PI3 K and class III PI3 K signaling pathway are involved in the effect of apelin-13 on apo E^-/- mice.