Effects Of Continuous And Intermittent High Glucose On Endothelial Cells And The Mechanism Analysis

Objective:The effect of constant and intermittent hyperglycemia on the membrane deformation of endothelial cells and the internal mechanism were investigated in the study.Methods:The human umbilical vein endothelial cells were cultured in vitro and randomly divided into eight groups before exposure to the different cultured medium for 7 days:â…°continuous normal glucose medium (DMEM of low glucose,5mM);â…±continuous high-glucose medium (DMEM of high glucose,25mM);â…²alternating normal and high-glucose media (25/5mM) every 24h; The medium of these groups fromâ…³toâ…µwere continuous normal glucose medium with different concentration L-NAME, nonspecific NOS inhibitor, including 1mM,5mM and 10mM; Groupâ…¶andâ…·were formed by incubating cells in DMEM of low glucose with 20mM mannitol, both continuously or in alternating fashion.1. NO was determined as nitrite concentration in culture supernatants by the Gris reaction.2. Through western blotting analysis with specific antibodies against eNOS, eNOS expression of endothelial cells from different groups was evaluated.3. From the analysis of the force-distance curves measured by atomic force microscope, the membrane average stiffness for different groups, as well as different region of endothelial cells each group was acquired.4. F-actin cytoskeleton labeled with fluorescence each group was viewed and quantified by laser scanning confocal microscope.Result:1. Endothelial cells response to constant and intermittent high glucose pronouncedly released less NO into the culture supernatants, decreased the eNOS expression and the elasticity of endothelial cells' membrane, when compared to the control group. It was found that the membrane of cellular periphery was stiffer than that of cellular centre in the condition of both continuous and fluctuating hyperglycemia. The fluorescent intensity of F-actin staining for the two groups rose remarkably. However, except eNOS expression, there was no variance when the comparison of the other results between constant and intermittent high glucose was performed.2. The increasing concentration of L-NAME from 1mM to 10mM declined NO production, doweregulated eNOS expression, together with the enhancement in the membrane stiffness and cytoskeleton F-actin staining in a concentration-dependent manner. Compared to the cellular membrane in the central region, the stiffness of the peripheral membrane exposed to different concentration of L-NAME increased significantly. The fluorescent imaging for the cytoskeleton obviously displayed that these F-actin filaments response to the rising concentration L-NAME, especially 5mM and 10mM L-NAME, were more pronounced than those seen in control group.3. The constant and intermittent hyperosmosis has no effect on NO synthesis, the membrane stiffness and F-actin staining of endothelial cells. No difference of the membrane stiffness was viewed between in the cellular central and peripheral area for both groups. While endothelial cells in the stable and shift hyperosmosis descended eNOS expression compared with the control one.Conclusion:It was clarified that constant and intermittent high glucose stiffened the endothelial membrane mediated by the mechanism that the dysfunction of NO synthesis redistributed the ctyoskeleton F-actin.