SGLT2 Inhibitors May Restore Endothelial Barrier Interrupted by 25-Hydroxycholesterol

Abstract

SGLT2 (Sodium-glucose Cotransporter-2) inhibitors are newer glucose-lowering drugs with many cardiovascular benefits that are not fully understood yet. Endothelial integrity plays a key role in cardiovascular homeostasis. 25-hydroxycholesterol (25-OHC), which is a proatherogenic stimuli that impairs endothelial barrier functions. VE-cadherin is an endothelial-specific protein crucial in maintaining endothelial integrity. The aim of this study was to assess the influence of SGLT2i on the integrity of endothelial cells interrupted by 25-OHC. We also aimed to evaluate whether this effect is associated with changes in the levels of VE-cadherin. We pre-incubated HUVECs with 10 μg/mL of 25-hydroxycholesterol (25-OHC) for 4 h and then removed it and incubated endothelial cells with 1 μM of empagliflozin, 1 μM canagliflozin, or 1 μM dapagliflozin for 24 h. The control group included HUVECs cultured with the medium or with 25-OHC 10 μg/mL. The integrity of endothelial cells was measured by the RTCA-DP xCELLigence system, and VE-cadherin was assessed in confocal microscopy. Our results show that SGLT2 inhibitors significantly increase endothelial integrity in comparison to medium controls, and they improve endothelial cell integrity interrupted by 25-OHC. This effect is associated with significant improvements in VE-cadherin levels. SGLT2i: empagliflozin, canagliflozin, and dapagliflozin have a beneficial effect on the endothelial cell integrity and VE-cadherin levels reduced by 25-OHC.

Keywords: 25-hydroxycholesterol; SGLT2i; VE-cadherin; atherosclerosis; canagliflozin; dapagliflozin; empagliflozin; endothelial barrier; endothelial integrity.

Figure 1

(A) The effect of SGLT2 inhibitors empagliflozin (1 µM), canagliflozin (1 µM), and dapagliflozin (1 µM) on the integrity of HUVECs. Data were collected by real-time cell electric impedance sensing system. (B) Representative plot from the real-time cell electric impedance sensing system, each line represents the mean of three wells of each condition.

Figure 2

(A) The effect of SGLT2 inhibitors empagliflozin (1 µM), canagliflozin (1 µM), and dapagliflozin (1 µM) on the integrity of HUVECs that are pre-stimulated with 25-OHC for 4 h. Data collected by real-time cell electric impedance sensing system. (B) Representative plot from the real-time cell electric impedance sensing system, each line represents the mean of three wells of each condition.

Figure 3

The expression and distribution of VE-cadherin in endothelial cells after treatment with 25-OHC and selected flozins. HUVEC cells were treated with medium (control), 10 μg/mL 25-OHC (4 h), followed by 24 h incubation with or without 1 μM dapagliflozin, empagliflozin, or canagliflozin. (A) Cells were stained for VE-cadherin (green) and DAPI (blue). (B) Quantification of VE-cadherin protein levels measured as mean fluorescence intensity in cell membrane (n = 30–40 cells). (C) Visualization by color mask (red) of all fluorescence points with intensity ≥1000. (D) The selected enlargements of a mature cell–cell junction (red frames). White scale bars represent 20 μm. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, and **** p < 0.0001.

Figure 4

Study protocol.