The Effects And Mechanisms Of Gas6 On Ox-LDL-stimulated Endothelial Tube Formation And Senescence

Background The neovascularization of atherosclerotic plaques is a key factor in increasing plaque instability,causing plaque hematoma or even rupture.Oxidized low density lipoprotein(ox-LDL)is a pathogenic factor of atherosclerosis.It has been shown that low concentration of ox-LDL directly promotes the angiogenesis and senescence of endothelial cells,but its specific mechanisms and effective regulatory targets have not yet been elucidated.Aim We conducted this project to detect the effects of different concentrations of ox-LDL on human umbilical vein endothelial cells(HUVECs)tube formation in vitro,senescence and the expression of growth arrest-specific gene 6(Gas6).Moreover,by manipulating the level of Gas6 in HUVECs,we aim to investigate the effects and mechanisms of Gas6 on HUVECs angiogenesis and senescence induced by ox-LDL.Methods Part 1 ?The concentrations of ox-LDL chosen for the following experiments were 0,10,20,40,80 and 160 ?g/ml,and the HUVECs were incubated with above levels of ox-LDL for 24h.Matrigel assay and MTT assay were used to evaluate HUVECs tube formation in vitro and cell viability.? P-galactosidase staining and DNA-labeling cell proliferation assay were performed to observe the effects of different concentrations of ox-LDL on the senescence and proliferation of HUVECs.? When the optimal concentrations of ox-LDL were identified with previous experiments,the levels of Gas6 were detected with Western blotting.Part 2 ?Lentiviruses carrying shRNA targeting human Gas6 gene(Gas6-shRNA),and recombinant human Gas6 protein(rhGas6)were applied to manipulate Gas6 levels respectively.The cells were grouped as follows:the non-targeting control group,Gas6-shRNA infected group,rhGas6 0 ng/ml group,rhGas625 ng/m group,rhGas6 50 ng/ml group and rhGas6 100 ng/ml group.? Matrigel assay and ?-galactosidase staining were conducted to evaluate the tube formation and cellular senescence of HUVECs under above stimulations.Part 3 ? While the optimum concentration of rhGas6 had been identified in Part2 experiment,Akt,Src,p70S6K and Erk protein and each of their phosphorylated protein were detected with Western blotting.?Based on the signal pathways identified in the previous step,the regulation mechanisms of Gas6 on the tube formation and cell senescence of HUVECs induced by ox-LDL were further explored with the effective inhibitors of the corresponding signal pathways.Results Part 1 Ox-LDL showed a biphasic effects on the tube formation of HUVECs in vitro,that is,lower concentration of ox-LDL(40?g/ml)promoted the tube formation of cells,while higher concentrations(160?g/ml)exerted inhibitory effect in comparison of control.The cytotoxic effect of ox-LDL on the endothelial cells was not involved in the process(P>0.05).Higher concentrations of ox-LDL significantly decreased the HUVECs proliferation(P<0.05)but increased the cellular staining positive rate of(3-galactosidase(P<0.05).The level of Gas6 in the cells treated with lower concentration of ox-LDL was significantly higher than that of the control group(P<0.01),meantime the higher concentration of ox-LDL reduced the level of Gas6(P<0.01).Part 2 The levels of intracellular Gas6 were significantly decreased after HUVECs being infected with lentivirus carrying shRNA targeting human Gas6 gene(P<0.01 in comparison with the non-targeting control group).The number of junctions per field and tube length in Gas6-shRNA group were declined(P<0.01 in comparison with the non-targeting control group),suggesting that the capacity of HUVECs angiogenesis in Gas6-shRNA group were weakened.Cellular positive staining rate of ?-galactosidase was significantly higher than that of the non-targeting control group(P<0.05).Exogenous administration of rhGas6 to HUVECs could reverse the inhibitory effect of high concentration ox-LDL on tube formation in vitro.When the concentration of rhGas6 reached 100ng/ml,the number of junctions was significantly higher than that of control group(P<0.01),and so was the length of tube(P<0.01).The ?-galactosidase staining assay showed that the positive staining rate decreased with the augment of rhGas6 concentrations,and the difference was statistically significant when the concentration reached 100ng/ml.Part 3 Western blotting showed that the levels of p-Akt and p-p70S6K in the Gas6-shRNA group were down-regulated but were significantly up-regulated in the 100ng/ml rhGas6 group under the corresponding ox-LDL concentration,compared with the control group(P<0.01 or P<0.05)respectively.The expression of p-Akt and p-p70S6K in 100 ng/ml rhGas6 group declined remarkably after the pretreatment with LY294002 and Rapamycin,which were the PI3K-Akt-p70S6K signaling pathway inhibitors.Furthermore,the effects of 100ng/ml rhGas6 on the tube formation and ?-galactosidase staining were also inhibited by pretreatment of LY294002 and Rapamycin,and the differences were statistically significant.Conclusion Ox-LDL has biphasic effects on the HUVECs tube formation in vitro,that is,low-concentration ox-LDL promotes tube formation,but high concentration inhibits it,and its inhibitory effect is closely related to cell senescence.Gas6 can reverse the effect of ox-LDL-induced HUVECs tube formation and cell senescence via PI3K-Akt-p70S6K pathways,suggesting that Gas6 can be a potential target for the control of angiogenesis in atherosclerotic plaques,which provides new insights into stabilization arterial plaque and reducing the incidence of adverse cardiovascular and cerebrovascular events.