The Mechanism of Advanced Glycation End Products-Induced Morphological Changes of Tight Junction in Endothelial Cell

Aim To investigate the effect of advanced glycation end products modified human serum albumin(AGE-HSA) on morphological changes of tight junction associated protein ZO-1 in endothelial cell and the mechanism in this pathological procedure. Methods Human umbilical vein endothelial cell(hUVEC)-derived cell line(ECV304) were incubated with AGE-HSA in different concentrations and timing. To visualize the morphological changes of tight junction protein ZO-1,the treated cell were incubated with mouse anti-ZO-1 primary antibody and then FITCanti-mouse IgG secondary antibody.The morphological changes of ZO-1 were observed with confocal microscope.The cell were pre-administrated with PD98059,a specific inhibitor of MEK1(ERK upstream kinase)or SB203580,a specific inhibitor of p38 MAP kinase,respectively,before exposed to AGE-HSA,then the cell were rinsed with DMEM for three times and exposed to AGE-HSA. The cell were transfected with dominant negative MEK1 or MEK6b(p38 upstream kinase) mutant adenoviruse,then exposed to AGE-HSA.And the cell were transfected with constitutively active MEK1 or MEK6b mutant adenoviruse. Results In normal control group,ZO-1 staining appeared as a continuous and smooth line along the regions of cell cell contact.Under the stimulation of AGE HSA,morphology of ZO-1 in endothelial cell were changed greatly in a concentration and time-dependent manner.The changes were partially blocked by PD98059 and SB203580.The transfection of dominant negative MEK1 and MEK6b mutant adenoviruse had the similar effects.The transfection of constitutively active MEK1 and MEK6b disrupted the structure of ZO-1. Conclusion AGE modified proteins can induce morphological changes of ZO-1 in endothelial cell.Activations of ERK and p38 MAP kinase pathways play an important role in this procedure.