Role And Mechanisms Of Autophagy In Human Umbilical Vein Endothelial Cells Exposed To Oxidized Low Density Lipoprotein

Objective:To investigate the role and mechanisms of autophagy in the injury of human umbilical vein endothelial cells ( HUVECs ) induced by oxidative low density lipoprotein ( ox-LDL ).Methods:1. After ox-LDL exposure, the formation of autophagosomes was observed by transmission electron microscopy, monodansylcadaverine ( MDC ) staining autophagic vacuole ( AV ), immunofluorescence staining microtubule-associated protein light chain 3 ( MAP1-LC3 ) protein, and western-blot examining the ratio of LC3-II/LC3-I, beclin1 and lysosome associated membrane protein 2a ( lamp2a ) protein levels. The content of T-SOD and MDA in their culture medium were also assessed. The roles of oxidative stress and LOX-1 in the activation of autophagy induced by ox-LDL is investigated by using vitC, vitE and LOX-1mAb interference. 2. To investigate the role and mechanisms of autophagy in ox-LDL-induced injury of HUVECs, cultured HUVECs were randomly divided into four groups: the control, ox-LDL, ox-LDL+rapamycin and ox+3-MA. The cells were used to detect the ratio of LC3-II/LC3-I by western blot, while the proliferation and apoptosis of cells were measured by MTT and flow cytometry methods. Lactate dehydrogenase ( LDH ) and endothelin-1 ( ET-1 ) content in the supernatant were detected with enzyme linked immunosorbent assay. In addition, the aggregation of Dil labled ox-LDL in HUVECs and the cellular distribution of Dil-ox-LDL with MDC, MAP1-LC3 and lamp2a were observed. 3. The formation of autophagosomes, apoptotic bodies, and cell necrosis were observed by transmission electron microscopy after ox-LDL exposure. Real-time PCR, western blot, flow cytometry and MTT methods were used to study the apoptotic and autophagic mechanisms. The contribution of autophagic and apoptotic mechanisms to ox-LDL-induced upregulation of MAP1-LC3, beclin1, and P53 mRNA and protein levels were assessed by pretreatment with rapamycin, an autophagy inducer, the autophagic inhibitor, 3-MA, and z-vad-fmk, an apoptosis inhibitor.Results:1. Autophagy was induced in HUVECs as detected by MDC staining, immunofluorescence staining and TEM. The upregulation of the ratio of LC3-II/LC3-I and the beclin1 protein level induced by ox-LDL reached peaks at 0.5 h and 6 h point. ox-LDL also increased the lamp2a protein level at 0.5 h and 6 h point. The downregulation of the T-SOD level and the upregulation of the MDA level in the culture medium is also significant at 0.5 h and 6 h, which was inhibited by pretreatment with vitC and vitE. The increase in the ratio of LC3-II/LC3-I was reversed by vitC and vitE pretreatment, but not LOX-1mAb. LOX-1mAb decreased the increase of lamp2a protein level induced by ox-LDL, while, vitC and vitE only inhibited the increase of lamp2a at 6 h point, but not 0.5 h point. 2. ox-LDL aggregated in HUVECs brought about an increase in the ratio of LC3-II/LC3-I in HUVECs, increased the content of LDH and ET-1 in the supernatant, as well as induced the proliferation and apoptosis of cells. The autophagic inducer rapamycin decreased the aggregation of Dil-labled ox-LDL ( Dil-ox-LDL ), increased the upregulation of autophagic level induced by ox-LDL, decreased the content of LDH and ET-1, and inhibited the ox-LDL-induced proliferation of cells. Conversely, the autophagic inhibitor 3-MA increased the aggregation of Dil-ox-LDL, decreased the increases of LC3-II/LC3-I induced by ox-LDL, increased the cell apoptosis and death. In addition, Dil-ox-LDL colocalized with the autophagy marker MDC, MAP1-LC3, and the lysosomal ( lamp2a ). HUVECs treated with Dil-ox-LDL showed a much greater degree of overlap of MAP1-LC3 and Lamp2a than control. 3. ox-LDL brought about an increase in the formation of autophagosomes, autolysosomes, apoptotic bodies, and cell necrosis in HUVECs. The formation of apoptotic bodies was later than that of autophagosomes, then followed by necrosis. ox-LDL-induced increases of p53 mRNA and protein levels were somewhat later than the increases in MAP1-LC3 and Beclin1. 3-MA pretreatment not only decreased the upregulation of LC3, beclin1 mRNA, the ratio of LC3-II/LC3-1 and beclin1 protein levels induced by ox-LDL, but increased the ox-LDL-induced increase of annexin V-positive staining, prompted the cell death. z-vad-fmk decreased the upregulation of p53, LC3, beclin1 mRNA, the ratio of LC3-II/LC3-I and beclin1 protein levels induced by ox-LDL, inhibited the ox-LDL-induced cell proliferation. The cell proliferation but not apoptosis of HUVECs induced by ox-LDL was inhibited by rapamycin pretreatment.Conclusion:Ox-LDL treatment can not only induce autophagy, but cell proliferation, apoptosis and necrosis of HUVECs, thus playing a harmful effect on the survival of HUVECs. The aggregation of ox-LDL is involved in the injury of HUVECs induced by ox-LDL. The autophagic inducer rapamycin has a cytoprotective action in against ox-LDL-induced cell proliferation and cytokine secretion by promoting the degradation of ox-LDL, while, 3-MA, the autophagic inhibitor, can increase the ox-LDL-induce cell apoptosis and death by increasing the aggregation of ox-LDL.