Angiotensin II-induced redox-sensitive SGLT1 and 2 expression promotes high glucose-induced endothelial cell senescence

Abstract

High glucose (HG)-induced endothelial senescence and dysfunction contribute to the increased cardiovascular risk in diabetes. Empagliflozin, a selective sodium glucose co-transporter2 (SGLT2) inhibitor, reduced the risk of cardiovascular mortality in type 2 diabetic patients but the protective mechanism remains unclear. This study examines the role of SGLT2 in HG-induced endothelial senescence and dysfunction. Porcine coronary artery cultured endothelial cells (ECs) or segments were exposed to HG (25 mmol/L) before determination of senescence-associated beta-galactosidase activity, protein level by Western blot and immunofluorescence staining, mRNA by RT-PCR, nitric oxide (NO) by electron paramagnetic resonance, oxidative stress using dihydroethidium and glucose uptake using 2-NBD-glucose. HG increased ECs senescence markers and oxidative stress, down-regulated eNOS expression and NO formation, and induced the expression of VCAM-1, tissue factor, and the local angiotensin system, all these effects were prevented by empagliflozin. Empagliflozin and LX-4211 (dual SGLT1/2 inhibitor) reduced glucose uptake stimulated by HG and H₂ O₂ in ECs. HG increased SGLT1 and 2 protein levels in cultured ECs and native endothelium. Inhibition of the angiotensin system prevented HG-induced ECs senescence and SGLT1 and 2 expression. Thus, HG-induced ECs ageing is driven by the local angiotensin system via the redox-sensitive up-regulation of SGLT1 and 2, and, in turn, enhanced glucotoxicity.

Keywords: SGLT2; angiotensin system; endothelial cells; high glucose; pro-atherothrombotic markers; senescence.

Figure 1

The selective SGLT2 inhibitor empagliflozin prevents the high glucose‐induced ECs senescence but not replicative senescence and H₂O₂‐induced senescence. A, ECs at P1 are exposed either to normal (5 mmol/L, NG) or high glucose (25 mmol/L, HG) in the absence or presence of increasing concentrations of empagliflozin for 96 h. B, ECs at P1 or P3 are exposed to increasing concentrations of empagliflozin for 48 h. C, ECs at P1 are exposed to N‐acetyl cysteine (NAC, an antioxidant, 1 mmol/L) or empagliflozin (EMPA, 100 nmol/L) for 30 min before being exposed to 100 μmol/L of H₂O₂ for 1 h. Thereafter, the medium is replaced and ECs are incubated for 48 h. SA‐β‐gal activity is determined by flow cytometry. D, ECs are exposed either to normal or high glucose for 96 h in the presence or absence of empagliflozin (100 nmol/L) before Western blot analysis of p53, p21, and p16. Results are shown as representative immunoblots (upper panels) and corresponding cumulative data (lower panels). Data are expressed as mean ± SEM of n = 3‐4. *P < 0.05 vs. control NG and ^# P < 0.05 vs. control HG

Figure 2

High glucose causes a redox‐sensitive induction of ECs senescence and promotes oxidative stress in ECs involving NADPH oxidase and cyclooxygenases, which is inhibited by empagliflozin. A, ECs are exposed to either normal or high glucose for 24 h in the presence or absence of empagliflozin (100 nmol/L), before dihydroethidium staining. Ethidium fluorescence is determined by confocal microscope. B, ECs are exposed either with N‐acetyl cysteine (NAC, 1 mmol/L), VAS‐2870 (VAS, a NADPH oxidase inhibitor, 5 μmol/L), or Indomethacin (INDO, a COX inhibitor, 30 μmol/L) for 30 min before the addition of high glucose for 96 h, and the subsequent determination of SA‐β‐gal activity by flow cytometry. C, ECs are exposed to normal or a high glucose for 96 h in the presence or absence of empagliflozin (100 nmol/L) before Western blot analysis of the NADPH oxidase subunits p22^phox, p47^phox, COX‐1 and COX‐2. Results are shown as representative immunoblots (upper panels) and corresponding cumulative data (lower panels). Data are expressed as mean ± SEM of n = 4‐5. *P < 0.05 vs. control normal glucose and ^# P < 0.05 vs. control high glucose

Figure 3

Empagliflozin prevents the high glucose‐induced endothelial dysfunction. ECs are exposed either to normal or high glucose for 96 h in the presence or absence of empagliflozin (100 nmol/L). A, Western blot analysis is performed to determine the expression level of eNOS, VCAM‐1, and tissue factor. Results are shown as representative immunoblots (upper panels) and corresponding cumulative data (lower panels). B, Electron paramagnetic resonance is used to determine the formation of NO under basal conditions and in response to bradykinin (100 nmol/L) for 30 min. C, Platelet aggregation experiments are performed to determine the inhibitory effect of ECs on U46619‐induced platelet aggregation. Representative platelet aggregation curves (upper panels) and corresponding cumulative data (lower panels). Data are expressed as mean ± SEM of n = 3‐4 (A), 3 (B), 4 (C). *P < 0.05 vs. respective control normal glucose and ^# P < 0.05 vs. control high glucose

Figure 4

Up‐regulation of the local angiotensin system mediates the high glucose‐induced ECs senescence, an effect prevented by empagliflozin. A, ECs are exposed to either an angiotensin‐converting enzyme inhibitor (Perindoprilat, 0.1 μmol/L) or an AT1R antagonist (Losartan, 30 μmol/L) for 30 min before the addition of high glucose for 96 h, and the subsequent determination of SA‐β‐gal activity by flow cytometry. B, ECs are exposed to normal or a high glucose for 96 h in the presence or absence of empagliflozin (100 nmol/L) before Western blot analysis of angiotensin‐conversion enzyme (ACE), and AT1R. Results are shown as representative immunoblots (upper panels) and corresponding cumulative data (lower panels). Data are expressed as mean ± SEM of n = 3‐4. *P < 0.05 vs. control normal glucose and ^# P < 0.05 vs. control high glucose

Figure 5

LX‐4211 but not empagliflozin inhibits basal glucose entry into ECs whereas both SGLT inhibitors prevent the increased glucose entry into H₂O₂‐ and high glucose‐treated ECs. After a 6‐h incubation period in serum‐free medium without glucose, ECs are incubated with either empagliflozin, LX‐4211 (a dual SGLT1 and 2 inhibitor) or high glucose (25 mmol/L) for 30 min in the presence (A) or absence of sodium replaced by choline chloride (B) before the addition of 2‐NBD‐glucose for 1 h. C,D, ECs are either untreated or exposed to H₂O₂ for 24 h, and high glucose for 48 h. After a 6‐h incubation period in serum‐free medium without glucose, ECs are exposed to either empagliflozin or LX‐4211 for 30 min before the addition of 2‐NBD‐glucose for 1 h. Thereafter, the ECs‐associated 2‐NBD‐glucose signal was determined by flow cytometry. Results are shown as cumulative data (left panels) and representative flow cytometry overlay histograms (right panels). Data are expressed as mean ± SEM of n = 3‐5. *P < 0.05 vs. respective control and ^# P < 0.05 vs control H₂O₂ or high glucose

Figure 6

Effect of H₂O₂ and high glucose on SGLT1 and SGLT2 mRNA and protein expression in ECs, and role of AT1R, NADPH oxidase and COXs. A,C) ECs are exposed to H₂O₂ (100 μmol/L) and, thereafter, the expression level of SGLT1 and 2 mRNA was determined after a 1‐h incubation period by RT‐PCR (A), and SGLT1 and 2 protein after a 24‐h period by Western blot analysis (C). B,D) ECs are exposed to high glucose and, thereafter, the expression level of SGLT1 and 2 mRNA was determined after a 4‐h incubation period by RT‐PCR (B), and SGLT1 and 2 protein after a 96‐h period by Western blot analysis (D). E,F) ECs are exposed to either an AT1R antagonist (Losartan, 1 μmol/L) VAS‐2870 (VAS, a NADPH oxidase inhibitor, 1 μmol/L), or indomethacin (INDO, a COX inhibitor, 30 μmol/L) for 30 min before the addition of high glucose for 96 h and, thereafter, the expression level of SGLT1 and SGLT2 was assessed by Western blot analysis. Results are shown as representative immunoblots (upper panels) and corresponding cumulative data (lower panels). Data are expressed as mean ± SEM of n = 3‐6. *P < 0.05 vs respective control and ^# P < 0.05 vs high glucose

Figure 7

Empagliflozin and LX‐4211 prevent the high glucose‐induced up‐regulation of SGLT1, SGLT2, and VCAM‐1 and down‐regulation of eNOS immunofluorescence signals in the endothelium of coronary artery segments. Porcine coronary artery segments were either untreated, or exposed to empagliflozin (100 nmol/L) or LX‐4211 (100 nmol/L) for 30 min before being incubated in either normo or high glucose containing RPMI for 24 h. Thereafter, segments were embedded in FSC22, frozen in liquid nitrogen and subsequently cryosectioned. Representative confocal immunofluorescence images showing SGLT1 (A), SGLT2 (B), eNOS (C), and VCAM‐1 (D) staining in red and autofluorescence in green (upper panels), and corresponding cumulative data of the endothelial signal (lower panels). Data are expressed as mean ± SEM of n = 3 (A), 4 (B), 8 (C) and 5 (D). *P < 0.05 vs respective control and ^# P < 0.05 vs high glucose. Original magnification, 20x