Sodium Glucose Co-Transporter 2 Inhibitors Ameliorate Endothelium Barrier Dysfunction Induced by Cyclic Stretch through Inhibition of Reactive Oxygen Species

Figures

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  Figure A1. Study design Cells were starved for 18 h by serum reduction before a 2 h pre-incubation with either vehicle (0.06% DMSO) or one of the SGLT-2i’s (1 µM EMPA, 1 µM DAPA and 3 µM CANA) under starving conditions, followed by stimulation with 10% stretch for 24 h. HCAECs exposed to 5% stretch with vehicle were considered as control. Cell permeability, ROS levels, cell morphology, VE-cadherin expression, interleukin production and LDH activity were measured after 24 h.

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  Figure 1. SGLT-2i’s inhibit barrier leakage and ROS production of HCAECs exposed to 10% stretch. Cells were pre-incubated for 2 h with vehicle or one of the SGLT-2i’s (1 µM EMPA, 1 µM DAPA and 3 µM CANA), and were subsequently exposed to 5% stretch plus vehicle, 10% stretch with vehicle or SGLT-2i’s for 24 h. Cell permeability was measured with live cell imaging, and 6 images were made from each condition in each individual experiment (a) n = 5 individual experiments). ROS levels were measured at 24 h, and six images were taken from each condition in each individual experiment (b) n = 5 individual experiments). Representative images are shown in the lower panel and data are presented as mean ± SD. * p < 0.05 vs. 10% stretch, *** p < 0.001 vs. 10% stretch.

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  Figure 2. SGLT-2i’s restore VE-cadherin loss of HCAECs exposed to 10% stretch. Cells were treated with 5% stretch plus vehicle, 10% stretch plus either vehicle or one of the SGLT-2i’s (1 µM EMPA, 1 µM DAPA and 3 µM CANA). VE-cadherin and F-actin were stained after 24 h, and representative images of staining are presented (a) n = 3. VE-cadherin levels were quantified with western blot for EMPA treatment (b) n = 6, DAPA treatment (c) n = 6 and CANA treatment (d) n = 6. Representative bands of VE-cadherin and α-tubulin (internal reference) are shown in the lower panels of each bar graph (b–d) and data are presented as mean ± SD. * p < 0.05 vs. 10% stretch, ** p < 0.01 vs. 10% stretch, *** p < 0.001 vs. 10% stretch. Whole (uncropped) western blots can be found as supplemental information).

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  Figure 3. Alleviated stretch induced barrier dysfunction by EMPA might be mediated through ROS inhibition. ROS measurement and immunofluorescence staining were carried out in cells treated with 5% stretch plus vehicle, 10% stretch plus either vehicle or 5 mM NAC. After 24 h, 6 images per condition were taken from each individual experiment for ROS measurement (a) n = 5 individual experiments. Representative images of VE-cadherin and F-actin staining are presented in (b) (n = 3). Cell permeability was measured in cells treated with 5% stretch plus vehicle, 10% stretch plus vehicle, 1 µM EMPA, 5 mM NAC or combination of EMPA and NAC. Six images per condition were taken from each individual experiment (c, n = 5 individual experiments). Representative images of ROS and cell permeability are shown in the lower panel of each bar graph (a,c) and data are presented as mean ± SD. *** p < 0.001 vs. 10% stretch. ns: not significant.

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  Figure 4. ROS inhibition by SGLT-2i’s might be mediated through inhibition of NHE1 and NOXs. ROS levels were measured in cells treated with 5%. stretch, 10% stretch plus vehicle, 1 µM EMPA, 10 µM cariporide/1 µM GKT136901 and combination of EMPA and cariporide/GKT136901 (a,b). Six images were taken from each individual experiment (a) n = 5 individual experiments; (b) n = 5 individual experiments. Cell permeability was measured in HCAECs treated with 5% stretch plus vehicle, 10% stretch plus either vehicle, 10 µM cariporide or 1 µM GKT136901 (c,d). Six images of each condition were taken from each individual experiment (c, n = 5 individual experiments; (d) n = 5 individual experiments. Representative images are shown in the lower panels and data are presented as mean ± SD. ** p < 0.01 vs. 10% + Vehicle, *** p < 0.001 vs. 10% + Vehicle. ns: not significant.

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  Figure 5. SGLT-2i’s do not inhibit interleukin secretion of endothelial cells exposed to 10% stretch. Cells were pre-incubated for 2 h with vehicle or one of SGLT-2i’s (1 µM EMPA, 1 µM DAPA and 3 µM CANA), and were subsequently exposed to 5% stretch plus vehicle, 10% stretch with vehicle or SGLT-2i’s for 24 h. IL-6 and IL-8 were measured by ELISA (a–f) n = 6. Data are presented as mean ± SD. * p < 0.05 vs. 10% stretch. ns: not significant.