AGEs-Induced Activation Of NF-κB And Expression Of COX-2 In Endothelial Cells: Mechanism And Intracellular Pathway

BackgroundAGEs are heterogeneous compounds of the end products of nonenzymatical glycation and oxidation of proteins and lipids. AGEs increase in various tissues as a function of age and hyperglycemia. The formation of AGEs is accelerated in given pathological processes and the accumulation of AGEs has been implicated in the pathogenesis of numerous disorders, including diabetes, dialysis related amyloidosis, atherosclerosis and Alzheimer's disease. AGEs can be recognized by several cell surface receptors including scavenger receptors, p60/p90, and RAGE. RAGE is a specific receptor recognizing AGEs and has been found to be present in many cells. Interaction between RAGE and AGEs has been shown to generate an oxidative stress which trigger the activation of nuclear factor (NF)-Kfi. The activation of NF-KB may upregulate the expression of its target genes. In endothelial cells, AGEs could induce a series of gene expression such as internal cell adhesion molecular-1 (1CAM-1), vascular cell adhesion molecule-1(VCAM-1), monocyte chemoattractant protein-1 (MCP-1), and E-selectin. Those induced factors may contribute to the increase of endothelial permeability and adhesion of inflammatory cells, which lead to dysfunction of endothelium. Therefor, activation of NF-KB and subsequent down-stream biological events might be involved in the pathogenesis ofAGEs-related diseases. Elucidating the signal transduction involved in the activation of NF-Kfi will be profitable for understanding the pathogenesis of diseases as well as for the development of novel therapeutic strategies. Oxidative stress induced by AGEs have been shown to contribute to the activation of NF-KB. Activation of NADPH oxidase has been found to be the main source of oxidative stress induced by AGEs. NADPH oxidase could be activated via two pathways. The activation of NADPH oxidase by phorbol ester (PMA) is through PKC while that by lysophosphatidylcholine (LPC) through PTk But which pathway AGEs induce the activation of NADPH oxidase via is unclear until now. In monocyte, the activation of p38 MAPK is required for the activation of NF-кB induced by AGEs. Whether p38 MAPK participates in the activation of NF-кB in endothelial cells is unknown.The activation of NF-кB could enhance expression of a series of genes relating to immunity and inflammation. Cyclooxygenase 2 is one of the important proteins regulated by NF-кB. COX-2 could be induced in endothelial cells, vascular smooth muscle cells, and some other cells. Expression of COX-2 could cause inflammation and oxidative stress which might contribute to the pathological processes. However, whether COX-2 could be induced by AGEs in endothelial cells has not been reported.The aim of the study was to investigate the intracellular mechanisms of AGEs-induced activation of NF-кB and expression of COX-2 in endothelial cells.MethodsDRA joint synovium was used as material to investigate the activationof NF-кB in vivo and human umbilical vein endothelial cells (HUVECs) and HUVEC-derived cell line (ECV304) were was used as endothelial cell models to study AGE-induced activation of NF-кB and expression of COX-2 in vitro.1. NF-кB activation induced by AGE in endothelial cells.(1) Immunohistochemical staining was used to detect the translocation of NF-кB in joint synovium of DRA patients.(2) The activation of NF-кB induced by AGEs on HUVEC was measured by immunohistochemical staining and evaluated by the ratio of optical density between nuclear and cytoplasm.(3) The degradation of IкB a and the activation of NF-кB induced by AGEs were detected by Western blot and EMSA, respectively.2. The mechanisms for AGE-induced NF-кB activation in endothelial cells.(1) Cellular O2- was measured by cytochrom C-Fe3+ reduction and GSH by ThioGlo-1 reagent. ECV304 were stimulated with different concentration of AGE, and cellular (V- and GSH were measured. To observe the role of ROS on AGE-induced NF-кB activation, ECV304 were pretreated with antioxidant PDTC, and AGE-in...