| BACKGROUNDThe mechanism of the atherosclerosis (As), is now generally believed closely associated with the increased of lipoproteins level. The increase of endothelial permeability of the arterial vessel wall is favorite for lipoprotein deposit in subendothelial space. Studies has showed that the presence of oxidatively modified low-density lipoprotein (oxLDL) detected in the intima of atherosclerotic plaques and foam cells. It is believed that the oxLDL is closely related to As. But so far,it has not been clearly understood if the oxLDL promotes the endothelium permeability to LDL. Both desmoglein1(DSG1) and desmocollin2 (DSC2) are the member of cadherins. They function at the site of interface of the endothelial cells, and play a role of the vessel walls'physical barriers and maintaining the integrity of the cell to cell. The desmosomal junction of the cell-cell in the permeability regulation may play an important role. this study is to explore the effects of oxLDL on expression of DSG1and DSC2 in endothelial cells, the effect of permeability injury induced by oxidized lipoprotein, and possible mechanisms of these.Part I. Effects of oxLDL on DSG1,DSC2 expression in HUVECsObjective: To identify the effects of oxLDL on DSG1 and DSC2 expression in HUVECs.Methods: Replaced with fresh medium every 24 hours before treatments. The different concentrations of oxLDL (0 mg/L,10 mg/L,25 mg/L,50 mg/L) were incubated with HUVECs for 24h, or with 50mg/L of oxLDL for different times (0h, 6h,12h and 24h). RT-PCR and Western blotting were applied to determine the DSG1 and DSC2 expression in mRNA and protein, respectively.Results: We found that oxLDL decreased DSG1 and DSC2 in both protein and mRNA levels in dose-dependent and time-dependent manners (P<0.05)..Part II. Effects of oxLDL on monolayer permeability in HUVECsObjective: To investigate the effects of oxLDL on monolayer permeability in HUVECs.Methods: HUVECs were cultured in DMEM medium, and replaced with fresh medium every 24 hours before treatments. Microscope was used to observe the monolayer information. HUVECs cultured in upper compartment of transwell were treated by oxLDL(50mg/L) and/or SOD(50mg/L) for 24 hours. And then, The LDL was added in upper compartment for 4 hours until to test the content of the LDL in the lower compartment. This effect represented change of the monolayer permeability to LDL.Results: The oxidized lipoprotein promoted the endothelial monolayer permeability to LDL(P<0.05). Superoxide dismutase(SOD) partly inhibited the effects of oxLDL on monolayer barrier damage(P<0.05).Partâ…¢. ROS involved in oxLDL regulation of DSG1 and DSC2 expression in HUVECsObjective: To explore the role of ROS in the regulation of expression on DSG1 and DSC2 induced by oxLDL in HUVECs.Methods: HUVECs were incubated with oxLDL(50mg/L) and LDL(50mg/L), BSA(100mg/L), H2O(25mg/L), respectively, for 24h. control group was designed to PBS incubation. HUVECs were pretreated for 1h with 50mg/L superoxide dismutase(SOD), and then were incubated for another 24h with 50mg/l of oxLDL. Intracellular ROS production was detected by Dichlorofluorescin diacetate (DCFH-DA) dye. The mRNA and protein expression of DSG1and DSC2 was determined by RT- PCR and Western blotting, respectively. Immunoreactivity of DSG1 was detected by laser scanning confocal microscope (LSCM).Results: oxLDL and H2O2 decreased expression of DSG1, DSC2, both mRNA and protein, in HUVECs(P<0.05) , while detected effects were not observed in treatment groups by LDL and BSA. oxLDL increased generation of cellular reactive oxygen species, and reduced expression of DSG1, DSC2 mRNA and protein(P<0.05). Furthermore, oxLDL reduced immunostaining of DSG1 in HUVECs. The results also showed this effect was inhibited by SOD supplement.Conclusions :These findings suggest that oxLDL may interfere the expression of desmosome proteins, DSG1 and DSC2, by which endothelial permeability to LDL is increased. Production of reactive oxygen species(ROS) may be involved in the promotion of monolayer permeability by oxLDL. |
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