陆军军医大学学报 (Feb 2023)
Store-operated calcium entry involves in regulating the hyperpermeability of endothelial cells induced by ox-LDL
Abstract
Objective To explore the effects of store-operated calcium entry(SOCE) on oxidized low-density lipoprotein (ox-LDL)-induced hyperpermeability in human umbilical vein endothelial cells(HUVECs). Methods HUVECs at passage 3 were divided into the control group and the ox-LDL(10, 25, 50 and 100 μg/mL) intervention groups (n=3) for the examination of transendothelial electrical resistance (TER). HUVECs cultured in slides were randomly divided into the control group, the ox-LDL 24 h group and the ox-LDL 48 h group. The distribution of F-actin and VE-cadherin were detected by immunofluorescence, and the protein content of VE-cadherin was detected by Western blotting. Incubated with calcium fluorescence probe (Fluo-3/AM), intracellular free Ca2+ concentration (control group, ox-LDLgroup, ox-LDL+2-APB group) and SOCE (control group, ox-LDL 24 h group, ox-LDL 48 h group) were detected by laser confocal microscopy. Results Low dose of ox-LDL(10, 25 μg/mL) exerted no effect on TER. High dose of ox-LDL (50, 100 μg/mL) also had no effect on TER within 6 h, but after 6 h showed no effect on TER. However, high dose of ox-LDL(50, 100 μg/mL) reduced the decrease of TER in 12 h (P < 0.05), and the decrease was more significant in 24 h and 48 h. After the incubation with ox-LDL(100 μg/mL) 24 h and 48 h, the expression level of VE-cadherin decreased(P < 0.05), while the form and distribution of F-action changed; Intracellular free Ca2+ concentration increased obviously which was interrupted by 2-APB the SOCE inhibitor (P < 0.05), and the 2-APB can decrease the effect of ox-LDL on TER. After incubation with ox-LDL(100 μg/mL) 48 h, the amplitude of SOCE decreased (P < 0.05). The descending artery flow velocity of SOCE showed a slightly decreased trend after incubation with ox-LDL 24 h and a more significant decrease in 48 h(P < 0.05). Conclusion Ox-LDL up-regulate the intracellular free Ca2+ concentration of vascular endothelial cells by extending the duration of SOCE to effect the arrangement of cytoskeleton and cell junction, which increases the cell permeability.
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