TNF-α Promotes Atherosclerotic Lesion Formation By Increasing Transcytosis Of LDL Across Endothelial Cells:Crosstalk Between NF-κB And PPAR-γ

Objective:Hyperlipidemia is one of the most important causes of atherosclerosis (AS). Numerous reviews have suggested that subendothelial retention of low density lipoprotein (LDL) is the initial steps of AS and the retention of LDL is achieved by the transcytosis of LDL across the vascular endothelial cells. Tumor necrosis factor-a (TNF-α) is an established pro-atherosclerotic factor, but the mechanism is not completely understood. Here we first established the model of the transcytosis in vitro and explored whether or not TNF-a could promote atherosclerosis by directly increasing the transcytosis of LDL particles across human umbilical vein endothelial cells (HUVECs), and further explore the roles of NF-κB and PPAR-γ related with TNF-α in this process.Methods:In the present study, we developed an in vitro model to investigate the transcytosis of LDL across a tight monolayer of HUVEC cultured in trans well-inserts by using LDL labeled with Fluorescein isothiocyanate (FITC) in the absence or presence of6-fold excess of unlabeled LDL. The effects of TNF-α and inhibitor on the transcytosis of LDL across vascular endothelial cells were also investigated by established the model of the transcytosis; FITC-LDL fluorescence intensity in HUVECs and human umbilical vein was measured by confocal microscope. Aortic roots were stained for lipids with Oil-red O for evaluation of aortic lesions in ApoE-/-mice. CD154signal were used by immunohistochemical analyses. By an ELISA-based transcription activity assay, the TNF-α-stimulated NF-κB and PPAR-γ activities in HUVECs were determined. TNF-a-stimulated and inhibitor changes in protein expression of LDLR, Caveolin-land Caveolin-2in HUVECs were measured by Western blot.Results:With this model the concentration dependent transcytosis of LDL across endothelial cells were characterized in experiments for the first time. When the concentration of LDL increased from50μg/ml to100μg/ml, the amount of LDL transcytosis also increased significantly, exhibiting an apparent concentration dependent manner. By establishing an in-vitro model to assay the transcytosis of LDL across vascular endothelial cells, we first demonstrated that TNF-a could significantly stimulate the transcytosis of LDL across endothelial cells and this effect can blocked not only by the inhibitors of transcytosis, NEM and M-β-CD (MCD), but also by NF-kB inhibitors Bay-11-7082(Bay) and PDTC and PPAR-y inhibitors GW9662and T0070907(T007). Similarly, in the confocal laser experiments we found that the uptake and retention of TNF-a-stimulated LDL particles were inhibited by transcytosis inhibitors NEM and MCD and the inhibitor of NF-kB and PPAR-γ. In ApoE-/-mice, we found TNF-a injection indeed accelerates the formation of atherosclerotic plaque in the arteries, further supporting the long standing view of TNF-a as a pro-atherogenesis factor. Blockade of the transcytosis of LDL by NEM and MCD substantially prevents the early atherosclerosis changes in artery walls, suggesting a critical role of LDL transcytosis in the initiation or development of AS. Meanwhile, consistent with above in-vitro findings, the TNF-a-promoted early atherosclerotic changes in artery walls were not only reversed by NF-kB inhibitors, PDTC, but also attenuated by PPAR-y inhibitors, GW9662. In addition, we studied the expression of the CD40ligand, CD154, which is involved in the early atherogenesis and contributes to the initial recruitment of inflammatory cells to damaged endothelium. Our results showed both NF-kB and PPAR-y inhibitors lowered the CD154expression in plaque which was substantially elevated by TNF-a in ApoE-/-mice. By using an ELISA-based transcriptional factor-DNA binding activity assay, we found TNF-a could significantly activate NF-kB, howeve, the activity of PPAR-y was also up-regulated by TNF-a. NF-kB inhibitors, Bay and PDTC primarily prevented the TNF-a-stimulated NF-kB activation, but also reversed the TNF-a-stimulated PPAR-y activity to some extent. Likewise, PPAR-y inhibitors, GW9662and T007almost completely abolished the TNF-a-stimulated PPAR-y activation, but also blocked the TNF-a-stimulated NF-kB activity. To further illustrate the relationship of NF-kB and PPAR-y transcription factor, we found that NF-kB and PPAR-y could form an active transcriptional compound which include NF-kB P65subunit and PPAR-y and bind with NF-kB response elements (KBRE) and PPAR-y response elements (PPRE) respectively, and promote each other’s activation after TNF-a stimulation by cross-binding tests. We found TNF-a could significantly up-regulate the expression of low density lipoprotein protein receptor (LDLR) and caveolae protein caveolin-1, caveolin-2, and this effect was reduced by both NF-kB and PPAR-y inhibitors. Inhibition of one of the activation of two transcription factors, can play the role of prevention and treatment of the development of AS.Conclusion:The transcytosis of LDL exhibits a concentration dependent manner and is the theoretical basis of the increased risk of hyperlipidemia in AS. TNF-a promotes AS by directly increasing the LDL transcytosis across endothelial cells and LDL retention in the vascular wall. In this process, NF-kB and PPAR-y are activated, enhancing each other’s activation and form the active complexes to up-regulate the expression of proteins associated with LDL transcytosis, including LDLR, Caveolin-1and-2, thereby promoting the transcytosis of LDL and progression of AS in vascular walls.