Selective Inhibition Of PI3K/Akt/mTOR Signaling Pathway Regulates Autophagy Of Macrophage And Vulnerability Of Atherosclerotic Plaque

BackgroundArteriosclerosis related cardiovascular diseases are the most leading cause of mortality worldwide. They are usually formed by the accumulation of fatty materials, that causes the abundant number of macrophages and white blood cells that harden the arteries and form plaques. There are different biological markers that play very important role in its formation. Low Density lipoproteins pro-inflammatory cytokines along with other signaling pathways are of great importance. On the other hand autophagy (a Greek word which means "self eating") is an evolutionary cellular catabolic process that keeps the homeostasis which degrades the pathological events taking place in the beginning of the formation of foam cells. In our paper we did both vitro and vivo experiments to show that there are different molecular signaling pathways that contribute in recruiting pro-inflammatory cytokines that could drive condition such as atherosclerosis. But some drugs can inhibit the pathways that affect the autophagy and result in suppression of macrophage conversion of foam cells, thus preventing the formation of the plaque. Thus new direction has been pointed in treating atherosclerosis.Objective To test the hypothesis that selective inhibition of PI3K/Akt/mTOR signaling pathway may enhance the stability of atherosclerotic plaques by activation of macrophage autophagy.MethodsIn vitro study, selective inhibitors or siRNA of PI3K/Akt/mTOR pathwayswere used to treat the rabbit’s peritoneal primary macrophage cells. We used PI3K inhibitor LY294002(10μmol/L), Akt inhibitor triciribine (20μmol/L), mTOR inhibitor rapamycin (10nmol/L), mTOR-siRNA (30nmol/L) and PBS as control to treat rabbit primary macrophages, respectively. Inflammation related cytokines secreted by macrophage were measured by means of ELISA. Autophagosome was detected bytransmission electron microscopy.Protein expression levels ofAkt,mTOR, Atg5-Atg12conjugated form and autophagy related gene Beclinl were assayed bywestern blot. Protein1light chain3II dots (LC3-Ⅱ) were observed under a laerscanningconfocalmicroscope. In vivo study,forty New Zealand White rabbits underwent balloon-induced abdominal aortic wall injury were fed on a diet of1%cholesterol for8weeks and then were randomly allocated to triciribine group (1.0mg/kg/d, n=10),rapamycin group (0.5mg/kg/d, n=10), mTOR-siRNA group (siRNA dissolved in RNAse-free and endotoxin-free water at3μg/μl and then diluted in PBS,500μg/2ml per rabbit and were injected intraperitoneally (i.p.) once a week for8weeks, n=10) and control group (PBS1.0ml/kg/d,n=10). Eight weeks after drug administrated, IVUS was carried out to observe the plaque imaging and calculatinglumen area (LA), external elastic membrane area (EEMA), plaque area(PA) and plaque burden(PB%). At the end of week16, plaque rupture was induced in all rabbits by pharmacological triggers using Chinese Russell’s viper venom andhistamine. Twenty-four hours after pharmacological triggering, rabbits were sacrificed for pathological studies.Ultrastructural changes of plaque area, collagen area, macrophages,smooth muscle cells (α-SMC), mTOR and Atg5-Atg12conjugated form in the plaques were measured.Results In vitro study, IL-10increased in group treated withLY294002, and declined evidently in group treated with triciribine, rapamycin and mTOR-siRNA compared to the control group; IFN-y detection got the opposite result.Fewer typical autophagosomes were detected in LY294002treated cells while more were detected in triciribine, rapamycin and mTOR-siRNA-treated cells. The expression levels of Beclinl and Atg5-Atg12conjugated formdecreased in LY294002group, while increased in other three treatment groups. Expression levels of p-mTOR decreased significantly in the four treatment groups, however, p-AKT increased in rapamycin and mTOR-siRNA treated group while decreased in the other two compared to the control.In vivo study, the blood lipid profile was not statistically significant among the four groups after drug treatment or gene intervention.IVUS found that EEMA, PA and PB decreased significantly in the triciribine, rapamycin and mTOR-shRNA groups.The ratio of abdominal aortic plaque rupture was higher in the control group than that in the experimental group.Macrophage RAM-11and mTOR stainings were significantly reduced as compared with the control group but SMC changed little among the four groups. Atg5-Atg12conjugated form staining enhanced evidently in the treated group.ConclusionSelectively inhibition of Akt/mTOR pathway can inhibit the atherosclerosis progression and enhance the stability of atherosclerotic plaques by activation of macrophage autophagy.