Empagliflozin prevents neointima formation by impairing smooth muscle cell proliferation and accelerating endothelial regeneration

Figures

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  FIGURE 1. Sodium glucose co-transporter 2 (SGLT2) is expressed in human vascular cells and the murine femoral artery. (A,B) Western blot analysis for SGLT2 protein expression and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) chosen as loading control in non-diabetic and diabetic human smooth muscle cells (SMCs, n = 3, A) and endothelial cells (ECs, n = 3, B). Full-length blots are presented in Supplementary Figure II. (C) Vascular SGLT2 expression in the native murine femoral artery as determined by immunohistochemical co-staining of α-smooth muscle actin (α-SMA, green) with SGLT-2 (red), and DAPI (blue; n = 6, upper scale bar 100 μm, lower scale bar 25 μm).

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  FIGURE 2. Empagliflozin improves diabetic endothelial cell (EC) function and prevents diabetic and non-diabetic smooth muscle cell (SMC) proliferation and migration. (A,B) Total cell count assessment of non-diabetic (black) and diabetic (red) SMCs (A) stimulated with 5% fetal calf serum (FCS) and indicated concentrations of empagliflozin and ECs with serum-free medium and indicated concentrations of empagliflozin (B) assessed by WST-1 cleavage to formazan at 24 h. (C–F) Determination of cell migration of non-diabetic (C) and diabetic (D) SMCs in serum free media (black), treated with 5% FCS (gray), and FCS 5% supplemented with 750 nM empagliflozin (red) as well as of non-diabetic (E) and diabetic ECs (F) in serum-free media (black), treated with 5% FCS (gray), and serum-free media supplemented with 750 nM empagliflozin (red) assessed by cell coverage at indicated time points in a scratch-wound assay (A–F: n = 3, n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

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  FIGURE 3. Empagliflozin improves vascular function in mice. (A,B) Evan’s blue staining at 3 days after electrical injury of the murine carotid artery in non-diabetic and diabetic mice treated with empagliflozin or standard diet (A, scale bar 1 mm) and quantification of non-re-endothelialized distance (B, n = 9, **P < 0.01). (C,D) Verhoeff–Van Gieson staining 21 days following wire-induced injury of non-diabetic and diabetic murine femoral arteries (C, scale bar 100 μm, arrowheads mark internal and external elastical laminae) and quantification of neointima/media ratio (D, n = 9, ***P < 0.0001). (E,F) Neointimal cell proliferation as determined by co-staining for Ki-67 (green), of α-smooth muscle actin (red), and DAPI (blue) at 10 days following wire-induced injury of non-diabetic and diabetic murine femoral arteries (E, scale bar 100 μm) and quantification (F, n = 6–8, *P < 0.05, ***P < 0.001).

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  FIGURE 4. Apelin is upregulated by empagliflozin, prevents smooth muscle cell (SMC) proliferation and augments endothelial cell (EC) proliferation. (A,B) Two out of 20 most regulated gene sets from RNA-sequencing data of human diabetic coronary artery SMCs (n = 3). (C,D) Apelin mRNA expression as assessed by qRT-PCR in diabetic SMCs incubated with 5% fetal calf serum (FCS) with and without 750 nM empagliflozin (C, n = 3, *P < 0.05) and in diabetic ECs incubated with serum-free medium with and without 750 nM empagliflozin (D, n = 3, *P < 0.05). (E,F) Total cell count assessment of diabetic SMCs (E) stimulated with 5% FCS with or without 600 nM recombinant apelin and ECs with serum-free medium with or without 600 nM recombinant apelin (F) assessed by WST-1 cleavage to formazan at 24 h (n = 3, *P < 0.05, **P < 0.01).