Effects Of TNF-α On The Transcytosis Of Ldl Accross Vascular Endothelial Cells And The Underlying Mechanism

Atherosclerosis is the pathological basis of various cardio- and cerebrovascular diseases and accounts for the major causes of death in the elderly people. Recent survey shows that the incidence of AS is increasing and the initial age of incidence keeps going down. Thus, AS is a huge challenge to the health of human being.To date, it is widely known that the most important pathologicla feature of AS is the deposition of LDL, especially oxidized LDL (Ox-LDL), in the subendothelial,membrane, leading to foam cell formation, inflammation and a series of changes, ultimately contributing to the formation of atherosclerotic plaque. Although the pathogenesis of AS is far from fully understood,the role of hyperlipidemia and endothelial injury are of specifically concerned. Endothelial cell injury and dysfunction were once considered the initial step to the development of atherosclerosis.In this regard, we first studied the effects of LDL and Ox-LDL on the viability of endothelial cells and found that both LDL and Ox-LDL can promote the growth of endothelial cells at low concentrations. At very high concentrations that far above normal plasma levels, LDL and Ox-LDL can damage endothelial cell.Given that the concentrations of LDL and Ox-LDL to damage endothelial cell is far higher than normal levels and some patients with normal plasma lipid concentrations also develops atherosclerosis, we hypothesized that the initial step of atherosclerosis may not be the injury of endothelial cell, but the increased transcytosis of LDL and Ox-LDL across endothelial cells. Based on this, we established the in vitro model of transcytosis of LDL across vascular endothelial cells. .We first confirmed the transcytosis of LDL across vascular endothelial cells and found this process is highly dependent on cell membrane lipid rafts and ceramide structures. M-β-CD and Desipramine, two frequently used lipid raft disruptors, can effectively prevent the transcytosis of. lipoprotein. Taking into account that the inflammation plays important roles in the development of atherosclerosis, we investigated the effects of the inflammatory mediator, tumor necrosis factor (TNF-α) on this transcytosis process. It was found that TNF-αsignificantly increased the transcytosis of LDL across vascular endothelial cells. Mechanistically, we further investigated the changes of the lipoprotein receptor (LDL-R) and transcytosis associated proteins, caveolin-1 and clathrin and found TNF-αsignificantly increased the protein expression of the LDL-R, caveolin-1 and clathrin. . Hopefully, these studies could provide new insight into the pathogenensis of atherosclersis and novel strategies for the prevention or treatment of atherosclerosis.PartⅠEffects of LDL,Ox-LDL on the viability of HUVECs and the role of methyl-β-cyclodextrin and desipramineObjective: To explore the effects of LDL and ox-LDL on the vitality of human umbilical vein endothelial cells (HUVECs) and the effect of lipid raft disruptor, M-β-CD or Desipramine, on this process.Materials and Methods: HUVECs were cultured and then treated with different concentrations of LDL, Ox-LDL, M-β-CD or Desipramine. Using cell counting kit-8 to measure the viability of HUVECs.Results: 1. LDL and ox-LDL stimulated the viability of HUVECs in a dose-dependent manner. When <300μg/ml, LDL facilitated cell proliferation and 100μg/ml LDL had the most prominent effect, whereas, When >500μg /ml, LDL induced cell damage. ox-LDL facilitated cell proliferation at lower concentrations and the effect peaked at 100μg/ml, whereas, when >300μg /ml, Ox-LDL damaged the cells. 2. M-β-CD and Desipraminon reversed the damage of HUVECs induced by higher concentration of LDL (500μg /ml) and ox-LDL(300μg /ml).Conclusion : Both LDL and Ox-LDL in the low concentrations can facilitate HUVECs proliferation, but in higher concentrations can induce cell damage. Both M-β-CD and Desipramine reversed the damage of HUVECs induced by higher concentration of LDL and Ox-LDL.PartⅡEstablishment of the in vitro transcytosis model and effects of TNF-αon the transcytosis of LDL across endothelial cells and the underlying mechanismObjective: To investigate the effects of TNF-αon the LDL transcytosis process and the underlying mechanism.Materials and Methods: 1. In vitro labeling of LDL with FITC into FITC-LDL. 2. Using transwell to establish in vitro transcytosis model, Simulating the endothelial cell monolayer of the blood vessels. By measuring the fluorescencent intensity of FITC in the out chamber to reflect the changes of transcytosis of LDL. Based on this model, the effects of M-β-CD and Desipramine on the transcytosis of LDL across vascular endothelial cells were investigated. 3. The effects of TNF-α. on the transcytosis of LDL across vascular endothelial cells were also investigated. 4. TNF-α-stimulated changes in Protein expression of caveolin-1,clathrin and LDLR in HUVECs were determined by Western blot.Results: 1. transcytosis of LDL across endothelial cells can occur in the established in vitro model. 2.M-β-CD, Desipramine can reduce the transcytosis of LDL in human umbilical vein endothelial cells. 3. TNF-αstimulated the changes of cell vitality in a dose-dependent manner. When <100ng/ml, TNF-αcan facilitate cell proliferation and 100ng/ml LDL has the most prominent effect. With 30ng/ml TNF-αtreated cells 1 to 4 days, as the time increased, the vitality of HUVECs increased. 4. Inflammatory cytokines TNF-αcan promote the transcytosis of LDL in human umbilical vein endothelial cell. 5. With 30ng/ml TNF-αtreated cells from 1 to 4 days, as the time increased, the protein expression of caveolin-1,LDLR aslo increase. whereas, The expression of Clathrin increased from the first day to the third day, but declined at the fourth day. With 30ng/ml TNF-αtreated cells from 30min to 24h, as the time increased, the protein expression of caveolin-1,LDLR and clathrin aslo increased.Conclusion: 1. LDL deposited in the subendothelial space by transcytosis across vascular endothelial cells. 2. LDL transcytosis in human umbilical vein endothelial cells is related to lipid rafts. 3. TNF-αcan promote the vitality of HUVECs in dose and time-dependent manner. 4. TNF-αcan promote LDL transcytosis in human umbilical vein endothelial cells, and this promotion may be related to the increased expression of LDLR,caveolin-1 and clathrin.