Dapagliflozin targets SGLT2/SIRT1 signaling to attenuate the osteogenic transdifferentiation of vascular smooth muscle cells

Abstract

Vascular calcification is a complication that is frequently encountered in patients affected by atherosclerosis, diabetes, and chronic kidney disease (CKD), and that is characterized by the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs). At present, there remains a pressing lack of any effective therapies that can treat this condition. The sodium-glucose transporter 2 (SGLT2) inhibitor dapagliflozin (DAPA) has shown beneficial effects in cardiovascular disease. The role of this inhibitor in the context of vascular calcification, however, remains largely uncharacterized. Our findings revealed that DAPA treatment was sufficient to alleviate in vitro and in vivo osteogenic transdifferentiation and vascular calcification. Interestingly, our study demonstrated that DAPA exerts its anti-calcification effects on VSMCs by directly targeting SGLT2, with the overexpression of SGLT2 being sufficient to attenuate these beneficial effects. DAPA was also able to limit the glucose levels and NAD⁺/NADH ratio in calcified VSMCs, upregulating sirtuin 1 (SIRT1) in a caloric restriction (CR)-dependent manner. The SIRT1-specific siRNA and the SIRT1 inhibitor EX527 attenuated the anti-calcification effects of DAPA treatment. DAPA was also to drive SIRT1-mediated deacetylation and consequent degradation of hypoxia-inducible factor-1α (HIF-1α). The use of cobalt chloride and proteasome inhibitor MG132 to preserve HIF-1α stability mitigated the anti-calcification activity of DAPA. These analyses revealed that the DAPA/SGLT2/SIRT1 axis may therefore represent a viable novel approach to treating vascular calcification, offering new insights into how SGLT2 inhibitors may help prevent and treat vascular calcification.

Keywords: Calorie restriction; Dapagliflozin; SGLT2; SIRT1; Vascular calcification.

Dapagliflozin attenuated vascular smooth muscle cells osteogenic transdifferentiation and vascular calcification through the SIRT1-mediated deacetylation and degradation of HIF-1α in a manner dependent on caloric restriction.

Fig. 1

DAPA protects against vascular calcification in a mouse model of CKD. (a) Comparison of survival rates in different treatment groups (n = 20). (b) Whole-aorta Alizarin Red S staining (n = 3). Scale bars: 5 mm. (c) Alizarin Red S and Von Kossa staining of thoracic aortic sections (n = 6). Scale bars: 100 μm. (d) Detection of aortic RUNX2, BMP2, α-SMA, and SM22α protein levels by western immunoblotting (n = 6). (e) Double immunofluorescence staining was used to assess RUNX2 and α-SMA within the mouse thoracic aorta, with ImageJ being used for quantification (n = 6). Scale bars: 50 μm. Data were analyzed with one-way ANOVAs, and all are presented as means ± SEM

Fig. 2

DAPA abrogates rat aortic ring calcification. (a) Rat aortic ring calcification was analyzed via Von Kossa staining (n = 6). Scale bars: 200 μm. (b) ImageJ was used to quantify the calcification-positive areas (n = 6). (c) Quantitative analysis of the calcium content in rat aortic rings (n = 6). (d) Rat aortic rings were analyzed to measure ALP activity (n = 6). Data were analyzed with one-way ANOVAs, and all are presented as means ± SEM

Fig. 3

DAPA reduces the severity of high-phosphate-induced VSMCs calcification. (a) HASMCs Alizarin Red S staining following treatment with phosphate and various DAPA concentrations (n = 6). (b) HASMCs calcium content (n = 6). (c) HASMCs ALP activity (n = 6). (d) Representative Western immunoblotting results analyzing RUNX2, BMP2, α-SMA, and SM22α levels in HASMCs with quantification performed in ImageJ (n = 6). (e) Double immunofluorescence analysis of RUNX2 and α-SMA in HASMCs with quantification performed in ImageJ (n = 6). Scale bars: 50 μm. Data were analyzed with one-way ANOVAs, and all are presented as means ± SEM

Fig. 4

DAPA targets SGLT2 to attenuate the calcification of VSMCs. (a) Murine aortic SGLT2 levels were analyzed by Western immunoblotting (n = 6). (b) SGLT2 levels in murine aorta sections were analyzed with immunofluorescence staining. Scale bars: 100 μm. (c) SGLT2 levels in HASMCs were analyzed via Western immunoblotting (n = 6). (d) SGLT2 levels in HASMCs were analyzed with immunofluorescence staining. Scale bars: 50 μm. (e) Relative SGLT2 protein levels were analyzed in HASMCs following SGLT2 plasmid transfection (n = 6). (f) Alizarin Red S staining was used to analyze calcium deposition in HASMCs overexpressing SGLT2 (n = 6). (g) RUNX2, BMP2, α-SMA, and SM22α levels in HASMCs overexpressing SGLT2 were analyzed via Western immunoblotting (n = 6). Data were analyzed with two-tailed t-tests (a, c, and e) and one-way ANOVAs, and are presented as means ± SEM

Fig. 5

DAPA promotes caloric restriction-mediated SIRT1 expression to protect against vascular calcification. (a, b) The NAD⁺/NADH ratio was measured in (a) murine aortas (n = 6) and (b) HASMCs (n = 6). (c) SIRTs family mRNA levels were analyzed in DAPA-treated HASMCs (n = 3). (d, e) Western immunoblotting was used to detect SIRT1 protein levels in (d) murine aortas (n = 6) and (e) following the overexpression of SGLT2 (n = 6). (f) Alizarin Red S staining analyses of HASMCs following SIRT1 knockdown (n = 6). (g) Western immunoblotting was used to analyze the expression of SIRT1, RUNX2, BMP2, α-SMA, and SM22α following SIRT1 knockdown (n = 6). (h) Alizarin Red S staining of EX527-treated HASMCs (n = 6). (i) Western immunoblotting was used to analyze the expression of SIRT1, RUNX2, BMP2, α-SMA, and SM22α in EX527-treated HASMCs (n = 6). (j) Von Kossa staining of EX527-treated rat aortic rings (n = 6). Scale bars: 200 μm. Data were analyzed with one-way ANOVAs, and all are presented as means ± SEM

Fig. 6

DAPA promotes SIRT1-mediated HIF-1α deacetylation and degradation. (a, b) HIF-1α levels in (a) murine aortas (n = 6) and (b) HASMCs (n = 6) as measured by Western immunoblotting. (c) SIRT1 and HIF-1αwithin murine aortas were assessed via double immunofluorescence staining. Scale bars: 50 μm. (d, e) Western immunoblotting was used to assess the expression of HIF-1α in HASMCs following (d) siSIRT1-mediated SIRT1 knockdown (n = 6) and (e) EX527 treatment (n = 6). (f) SIRT1 and HIF-1α within HASMCs were detected via double immunofluorescence staining. Scale bars: 50 μm. (g) Western immunoblotting was used to detect acetyl-HIF-1α levels following SIRT1 knockdown in HASMCs (n = 6). (h) HIF-1α protein degradation was analyzed under the indicated different conditions (n = 3). (i) Analysis of the degradation of HIF-1α in HASMCs treated with DAPA with or without MG132 (n = 3). (j) Alizarin Red S staining of HASMCs following culture in the presence of DAPA, CoCl2, and high-phosphate medium (n = 6). (k) Western immunoblotting was used to detect HIF-1α, RUNX2, BMP2, α-SMA, and SM22α protein levels, with ImageJ for quantification (n = 6). Data were analyzed with one-way ANOVAs, and all are presented as means ± SEM

Fig. 7

DAPA protects against aortic calcification in non-CKD mice. To establish a non-CKD model of vascular calcification, mice received subcutaneous injections of VitD3 (5 × 10⁵ IU/kg) daily for 3 days, with DAPA (5 mg/kg) being administered orally to appropriate animals in an effort to protect against aortic calcification. (a) Whole aorta Alizarin Red S staining results from VitD3-overloaded mice. Scale bars: 5 mm. (b, c) Levels of (b) calcium content and (c) ALP activity in aortic tissues from VitD3-overloaded mice (n = 6). (d-g) RUNX2, BMP2, α-SMA, and SM22α mRNA levels were analyzed in aortic tissues from VitD3-overloaded mice (n = 6). (h) SIRT1, HIF-1α, RUNX2, BMP2, α-SMA, and SM22α protein levels in the aortic tissues from VitD3-overloaded mice were analyzed via Western immunoblotting (n = 6). Data were analyzed with one-way ANOVAs, and all are presented as means ± SEM