The Mechanism Of Advanced Glycation End Products-Induced Morphological Changes Of Tight Junction In Endothelial Cells

Object:This study was performed to investigate the effect of advanced glycation end products modified human serum albumin (AGE-HSA) on morphological changes of tight junction associated protein zona occludens-1 (ZO-1) and in endothelial cells and the mechanism in this pathological procedure. Methods:AGE-modified human serum albumin (AGE-HSA) was prepared by incubation of 1.75 mg/ml human serum albumin with 100 mmol/L of D-glucose for 8 weeks. Human umbilical vein endothelial cells (HUVEC)-derived cell line (ECV304) grown on gelatin-coated glass bottom microwell Petri dishes were allowed to reach confluence and subjected to AGE-HSA treatments. Endothelial cells (ECs) were incubated with AGE-HSA in different concentrations and timing. The cells were pre-administrated with Y-27632, a specific inhibitor of Rho kinase before exposed to AGE-HSA. PD98059, a specific inhibitor of MEK1 (ERK upstream kinase) or SB203580, a specific inhibitor of p38 MAP kinase, were pre-administrated to the cells respectively before AGE-HSA administration. The cells were pre-infected with recombinant adenovirus of dominant negative mutants of MEK1 or MEK6b (p38 upstream kinase) and then subjected to AGE-HSA. The cells were transfected with recombinant adenoviruse of constitutively active mutants of MEK1 and MEK6b for 12 h. To visualize the morphological changes of tight junction protein ZO-1, the treated cells were incubated with mouse anti-ZO-1 primary antibody and then FITC-anti-mouse IgG secondary antibody. The morphological changes of ZO-1 were observed with confocal microscope. Phosphorylation of p38 MAPK was detected with anti-phospho p38 antibody by SDS-PAGE and Western blot.Results:1. Immunofluorescent detection of ZO-1 revealed a continuous localization at cell-cell contacts under normal condition. Exposure of ECs to AGE-HSA led to disruption of cortical ZO-1 distribution while more intraceUular ZO-1 staining was observed. Cells subjected to higher-concentration and longer-time AGE-HSA exposure showed more and more remarkable discrete ZO-1 distributions.2. The Pre-treatment of Y-27632 to ECs could preserve the normal morphological characteristics of ZO-1 after AGE-HSA stimulation.3. The pre-administration of PD98059 or SB203580 to ECs could attenuate the disrupting effects on ZO-1 induced by AGE-HSA.4. Infection of the cells with recombinant adenovirus of dominant negative mutants of MEK1 or MEK6b partly abolished the morphology changes elicited by AGEs; the cells infected with recombinant adenovirus of constitutively active MEK1 or MEK6b showed the changes similar to that of the cells exposed to AGEs.5. An increase of p38 phosphorylation was detected by western blot in AGE-HSA treated cells, and this increase was blocked by the presence of Y-27632 before AGE-HSA application.Conclusions:1. AGE-HSA could induce morphology changes of ZO-1 in endothelial cells in time- and dose- dependent manners. 2. Y-27632 could attenuate the ZO-1 morphology changes induced by AGEs, which implys that Rho kinase plays an important role in signal transduction evoked by AGEs.3. ERK and p38 MAPK are involved in the signaling mechanisms in AGE-induced dysfunction of endothelial cells.4. AGE-HSA could induce an increase in p38 MAPK phosphorylation, which could be blocked by the presence of Y-27632. This rusult suggests that Rho kinase acts on the upstream of p38 MAPK.