Renin-angiotensin system inhibitor attenuates oxidative stress induced human coronary artery endothelial cell dysfunction via the PI3K/AKT/mTOR pathway

Figures

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  Figure 1. Effect of RAS inhibitor on HCAECs with H2O2 treatment. A – Protective effect of RAS inhibitor on cell apoptosis in HCAECs induced by H2O2. B – Preventive effects of captopril on cell viability against H2O2-induced injury determined by MTT assay. *p < 0.05 compared with the control group. C – Effect of RAS inhibitor on H2O2-induced inducible nitric oxide (NO) synthase expression. D – Effect of RAS inhibitor on H2O2-induced expression of p53, ICAM-1, p-eNOS, p-AKT and p-mTOR. E – Inhibitory effect of RAS inhibitor on production of intracellular ROS The data are mean values from 3 independent experiments. *p < 0.05, relative to the control group.

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  Figure 2. RAS inhibitor attenuated H2O2-induced viability losses and apoptosis in HCAECs via the AKT/mTOR pathway. HCAECs were pre-treated with captopril or combined with AKT inhibitor or activator, then exposed to H2O2 for 48 h. A – Inhibition of apoptosis in HCAECs co-treated with captopril and AKT inhibitor and then exposure to H2O2. B – Preventive effects of captopril and AKT inhibitor on cell viability induced by H2O2, determined by MTT assay *p < 0.05, when compared with the control group; #p < 0.05, when compared with the H2O2 alone group.

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  Figure 3. RAS inhibitor attenuated the H2O2-induced increases in expressions of p53, ICAM-1, p-eNOS, p-AKT and p-mTOR via AKT/mTOR pathway. A – HCAECs were pre-treated with captopril or combined with AKT inhibitor or activator, then exposed to H2O2 for 48 h. Cell lysate was analyzed for p53, ICAM-1, p-eNOS, p-Akt and p-mTOR protein levels by immunoblotting. B – Shows a column bar graph analysis *p < 0.05, when compared with the control group; #p < 0.05, when compared with the H2O2 alone group.

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  Figure 4. Involvement of the AKT/mTOR signaling pathway in RAS inhibitor-mediated protection against H2O2-induced inducible nitric oxide (NO) synthase expression. HCAECs were pre-treated with captopril or combined with AKT inhibitor or activator, then exposed to H2O2 for 48 h. This effect of captopril on H2O2-induced NO expression was reversed by AKT activator but not by AKT inhibitor *p < 0.05, when compared with the control group; #p < 0.05, when compared with the H2O2 alone group.

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  Figure 5. Involvement of the AKT/mTOR signaling pathway in inhibitory effect of RAS inhibitor on production of intracellular ROS. The intracellular ROS accumulation was determined by measuring the DCF-derived fluorescence after incubation of cells with DCFH-DA for 30 min. The data are mean values from 3 independent experiments *p < 0.05, when compared with the control group; #p < 0.05, when compared with the H2O2 alone group.

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  Figure 6. Inhibitory effect of RAS inhibitor against H2O2-induced cell proliferation through the AKT/mTOR signaling pathway. HCAECs were pre-treated with captopril or combined with AKT inhibitor or activator, then exposed to H2O2 for 48 h. Representative images and quantitative analysis illustrate that BrdU expression was significantly increased in the H2O2 alone and captopril + AKT activator group. Captopril + AKT inhibitor reversed the effect of H2O2-induced cell proliferation *p < 0.05, when compared with the control group; #p < 0.05, when compared with the H2O2 alone group.

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  Figure 7. Effect of RAS inhibitor on H2O2-modulated cell migration through the AKT/mTOR signaling pathway. Confluent monolayer of HCAECs was wounded by scratching in the presence of captopril or combined with AKT inhibitor or activator to stimulate cell migration toward the wound area. Images were captured immediately (0 h), and at 48 h after the wound scratch

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  Figure 8. RAS inhibitor reduced H2O2-induced inhibition of HCAEC motility in a transwell migration assay via the AKT/mTOR signaling pathway. HCAEC migration rate was examined after H2O2 stimulation with or without AKT inhibitor or activator. Captopril alone or in co-treatment with AKT inhibitor increased the rate of HCAEC migration *p < 0.05, when compared with the control group; #p < 0.05, when compared with the H2O2 alone group.