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.

Graphical abstract

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.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-024-05486-8.

Introduction

Vascular calcification is a pathological condition that is commonly detected in the context of aging, diabetes, and chronic kidney disease (CKD), with affected patients exhibiting abnormal phosphate and calcium deposition within the walls of the vascular endothelium [1–3]. In recent studies, vascular calcification has been established as a risk factor that is independently associated with the incidence of cardiovascular events (MACE) [4, 5]. The process of vascular calcification is tightly regulated and highly complex, with vascular smooth muscle cells (VSMCs) osteogenic transdifferentiation serving as the key driver of this process, much as is observed in the setting of osteogenesis [6–9]. While several studies have yielded strong insight into the molecular mechanisms governing vascular calcification, there remains a pressing lack of any effective therapies capable of treating this condition. Additional research focused on elucidating the pathogenesis of this condition is warranted in order to aid in the design of novel treatment strategies.

The sodium-glucose co-transporter (SGLT2) inhibitor dapagliflozin (DAPA) has been demonstrated to lower blood glucose levels through its ability to increase urinary glucose excretion while limiting the renal resorption of glucose [10]. In addition to exerting anti-hyperglycemic effects, SGLT2 inhibitors also exhibit antioxidant, anti-inflammatory, and autophagy-inducing effects [11]. There is a wealth of evidence suggesting that DAPA is capable of alleviating cardiovascular risk and improving prognostic outcomes in patients with CKD [12, 13]. Recent studies also indicate that the SGLT2 inhibitors empagliflozin and canagliflozin are capable of inhibiting osteogenic VSMCs transformation and delaying vascular calcification through their ability to inhibit inflammatory signaling activity [14–16]. Caloric restriction (CR) refers to the chronic reduction of total calorie intake without malnutrition. As SGLT2 inhibitors can mimic CR, thereby engaging nutrient deprivation signaling mechanisms, they can ultimately reduce the incidence of cardiovascular events [17–20]. Recent studies suggest that CR can also help preserve renal function [21, 22]. Notably, SGLT2 is not expressed within all cells, but it has been reported that VSMCs express SGLT2 [23]. The precise role that DAPA plays in the context of vascular calcification, however, remains unknown.

Sirtuins (SIRTs) comprise a family of nicotinamide adenine dinucleotide (NAD⁺)-dependent histone deacetylases that serve as central regulators of vascular calcification [24], with SIRT1 playing a pronounced protective role in the context of vascular calcification [25]. Activating SIRT1 signaling may thus represent an attractive approach to the treatment of vascular calcification. In addition, SIRT1 signaling is a key response of cells to conditions of glucose deprivation, and SGLT2 inhibitors can activate this pathway [20]. Several studies point to the ability of DAPA to activate downstream SIRT1 signaling, thereby preventing cardiovascular damage [26, 27]. Hypoxia-inducible factor-1 alpha (HIF-1α) is the central cellular regulator of hypoxia homeostasis, and it can directly drive VSMCs to undergo osteogenic transformation while also directly influencing vascular calcification [28, 29]. In past studies, SIRT1 was found to regulate HIF-1α by binding to this protein at specific lysine residues whereupon it promotes HIF-1α deacetylation and degradation [30]. Whether this SIRT1/HIF-1α signaling axis plays a role in the anti-calcifying effects of DAPA, however, remains to be established.

Here, the effects of the SGLT2 inhibitor DAPA on vascular calcification and the underlying mechanisms were explored at length. This study utilized two murine models of vascular calcification, cell-based in vitro assays, and ex vivo assays using rat aortic rings. Through these analyses, DAPA was found to significantly inhibit osteogenic VSMCs transformation via targeting the SIRT1/HIF-1α signaling axis, thus delaying the onset of vascular calcification. Strikingly, DAPA was able to directly target SGLT2 and regulate SIRT1 signaling activity in a CR-dependent manner. This study is the first report detailing the mechanistic importance of the DAPA-mediated CR pathway in the context of vascular calcification, offering new insights into how SGLT2 inhibitors may help prevent and treat vascular calcification.

Materials and methods

Reagents and antibodies

Sodium phosphate (Cat#342483), cobalt chloride (CoCl₂) (Cat#769495), CaCl₂ (Cat#G5670), and β-glycerophosphate (β-GP) (Cat#G9422) were from Sigma-Aldrich (MO, USA). Alizarin red S (Cat#G8550) was from Solarbio (Beijing, China). Dapagliflozin (Cat#HY-10450), EX527 (Cat#HY-15452), and Vitamin D3 (Cat#HY-15398) were from MedChemExpress (NJ, USA). Cetylpyridinium chloride was from Aladdin (Shanghai, China). Trypsin, penicillin, streptomycin, fetal bovine serum (FBS), and Dulbecco’s Modified Eagle’s Medium (DMEM) were from GIBCO. A glucose kit (Cat#A154-1-1), a calcium assay kit (Cat#C004-2-1), and an ALP assay kit (Cat#A059-2-1) were from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). A NAD⁺/NADH assay kit (Cat#S0175) was from Beyotime (Shanghai, China). A Von Kossa staining kit (Cat#G1034) from Servicebio (Wuhan, China). Anti-α-SMA (Cat#67735-1-Ig, 1:10000 for WB, 1:200 for IF), anti-RUNX2 (Cat#20700-1-AP, 1:500 for WB, 1:200 for IF), anti-BMP2 antibody (Cat#66383-1-Ig, 1:1000 for WB), anti-HIF-1α (Cat#66730-1-Ig, 1:2000 for WB, 1:200 for IF), and anti-GAPDH (Cat#10494-1-AP, 1:10000 for WB) were from Proteintech (Wuhan, China). Anti-SM22α (Cat# ab14106, 1:2000 for WB) was from Abcam. Anti-RUNX2 (Cat#12556, 1:1000 for WB), anti-SIRT1 (Cat#9474, 1:1000 for WB, 1:200 for IF), and anti-HIF-1α (Cat#36169, 1:1000 for WB) were from Cell Signaling Technology (MA, USA). Anti-acetyl-HIF-1α (Cat#HY0140, 1:2000 for WB) was from Immunoway.

Cell culture

The thoracic aorta of a 6-week-old C57BL/6 mouse was used to harvest primary VSMCs as in a prior report [5]. Briefly, these mice were euthanized using sodium pentobarbital (150 mg/kg, ip), after which the thoracic aorta was removed and separated from the adventitia and endothelium, with the remaining tissue being cut into 1 mm² tissue blocks. These aortic tissue blocks were then cultured for 1–2 weeks in complete media supplemented with 20% FBS. After the VSMCs migrated from the explants, the tissue blocks were removed and the VSMCs were cultured with DMEM containing 10% FBS. All subsequent experiments were conducted with primary VSMCs between passages 5 and 8. Human aortic smooth muscle cells (HASMCs) were obtained from the American Type Culture Collection (ATCC, USA). DMEM supplemented with 10% FBS and penicillin/streptomycin was used to culture all cells in a 5% CO2 incubator at 37ºC. To induce VSMCs calcification, cells were cultured for 7 days in DMEM supplemented with 1% FBS and 3 mmol/L sodium phosphate (Pi) for 7 days, replacing the media every other day. To determine the role that DAPA plays in VSMCs calcification, these cells were treated with various DAPA concentrations (2.5µM, 5µM, 10µM).

Animal experiments

The Ethics Committee for Animal Experiments of the Second Hospital of Shanxi Medical University provided approval for all animal studies (Approval No. DW2023045), which were conducted as per the NIH Guide for the Care and Use of Laboratory Animals. For these assays, C57BL/6 mice (males, 6 weeks old) were obtained from the Laboratory Animal Centre of Shanxi Medical University. A 5/6 nephrectomy model of CKD-associated vascular calcification was established as reported previously [5]. Briefly, isoflurane (2%) was used to anesthetize these mice, followed by the removal of two-thirds of the left kidney, with the entirety of the right kidney then being removed after a one-week interval. These mice were separated into the following groups: a sham control group fed a normal diet, a DAPA group in which sham control mice fed a normal diet were administered 5 mg/kg DAPA per day via gastric perfusion, a CKD model group in which mice were fed a 1.8% high phosphorus diet, and a CKD + DAPA model group in which mice were fed a 1.8% high phosphorus diet and administered 5 mg/kg DAPA per day via gastric perfusion. Animal treatments were maintained for an 8-week period, followed by the euthanasia of these mice with sodium pentobarbital (150 mg/kg, ip). The aorta was then isolated and murine serum was harvested for further experimentation. A separate model of vascular calcification was established through vitamin D3 (VitD3) overload [4, 15]. At 8 weeks of age, mice were randomly divided into three groups (n = 10/group): control, VitD3, and VitD3 + DAPA groups. Mice in the appropriate groups were subcutaneously injected with VitD3 (5 × 10⁵ IU/kg) on three consecutive days, with mice in the VitD3 + DAPA group additionally receiving 5 mg/kg DAPA daily.

Rat arterial ring organ culture

Sprague-Dawley rats (6 weeks old, males) were euthanized with sodium pentobarbital (150 mg/kg, i.p.), followed by the isolation of the thoracic aorta. The resected aortas were then cut to produce 2–3 mm arterial rings that were subsequently cultured for 7 days in DMEM supplemented with 10 mM β-GP, 3 mM CaCl2, and 10% FBS, exchanging media every other day. Calcification of the aortic rings was detected by Von Kossa staining.

Western blot analysis

VSMCs were lysed on ice with RIPA buffer (Bosterbio) containing protease and phosphatase inhibitors for 15 min, while aortic tissue samples were added to lysis buffer and then lysed using a tissue homogenizer. Lysates were then centrifuged (12,000 xg, 20 min, 4 °C), and supernatant protein content was analyzed via BCA assay (Bosterbio). After combining proteins with loading buffer (Bosterbio), they were boiled for 5 min at 100 °C, separated via SDS-PAGE, and transferred onto PVDF membranes (Millipore). These blots were then probed with primary antibodies and HRP-conjugated secondary antibodies (Bosterbio, 1:10000), after which Image Lab (Bio-Rad) was used for ECL chemiluminescent signal detection, and ImageJ was used for densitometric quantification.

Glucose, Calcium, and ALP activity assay

Glucose levels, calcium content, and ALP activity were all analyzed with kits from commercial sources. Briefly, appropriate buffers were used to homogenize VSMCs and aortic tissue samples, followed by centrifugation and collection of the corresponding supernatants. Based on the provided instructions, the extracts were colorimetric analyzed at optical densities of 505 nm, 610 nm and 520 nm, and then glucose content, calcium content and ALP activity were calculated. Finally, the protein content of the extract was measured with a BCA assay and used to normalize those results.

NAD+/NADH ratio assay

The total amount of NAD⁺ and NADH were extracted from aortic or VSMCs samples as directed using appropriate extraction reagents, and then the extracts were subjected to colorimetric analysis at an optical density of 450 nm, with the total concentration was calculated. Next, a part of the extract was heated in 60℃ for 30 min to promote the degradation of NAD⁺, and the NADH content was obtained. Finally, the amount of NAD⁺ was obtained by subtracting the amount of NADH from the total amount of NAD⁺ and NADH. Based on the results, the NDA⁺/NADH ratio could then be calculated.

Alizarin Red S staining

VSMCs staining was performed by fixing these cells for 30 min using 4% paraformaldehyde, followed by adding Alizarin Red S stain (1%, pH 4.2) at room temperature for 3–5 min. Calcified cells appeared red in color following three washes with ddH₂O. And then samples were dissolved with 500µL cetylpyridinium chloride solution (10%) and compared by colorimetric analysis of the eluated alizarin red at an optical density (OD) of 526 nm. When performing whole aorta staining, excessive connective tissue surrounding the aorta was separated, the aorta was fixed using 4% paraformaldehyde, followed by dehydration overnight in 95% alcohol. Aortas were then stained overnight in 0.003% Alizarin Red S stain with 1% KOH. Then, 2% KOH was used to wash away the staining solution, with the calcified aortic areas appearing purplish-red in color. Paraffin-embedded thoracic aorta sections were prepared by fixing the aorta with 4% paraformaldehyde prior to paraffin embedding and cutting into 5 μm posterior sections. These sections underwent standard deparaffinization and were stained for 2 min with 2% Alizarin Red S stain.

Von Kossa staining

Thoracic aortas were routinely deparaffinized, rehydrated, stained using silver nitrate, and illuminated for 4 h using an ultraviolet lamp. Sections were then rinsed with running water, stained in turn with hematoxylin and eosin, and imaged, with calcified areas appearing dark brown in color.

Immunofluorescence (IF) staining

VSMCs were initially fixed using 4% paraformaldehyde for 30 min, followed by permeabilization with 1% Triton-X. Samples were then blocked for 1 h with 5% BSA, rinsed thrice, and incubated overnight at 4 °C with primary antibodies. Cells were then incubated for 1 h at room temperature with a secondary antibody (Servicebio) in the dak. DAPI (Servicebio) staining solution was used to treat cells for 5 min to stain nuclei in the dark. Prepared 5-µm sections were routinely deparaffinized, rehydrated, and placed in an EDTA solution (Servicebio) for antigen repair in the microwave. After soaking sections for 1 h at room temperature using 5% BSA following 0.1% Triton X-100 treatment, sections were incubated with primary antibody at 4℃ overnight. Next day, sections were stained with secondary antibodies (Servicebio) at room temperature for 1 h in the dark. After staining with DAPI staining solution, cells were imaged via fluorescence microscopy.

Quantitative real-time PCR (qPCR)

Trizol (Takara) was used to isolate RNA from VSMCs and aortic tissue samples as directed, after which the PrimeScript RT Reagent Kit (Takara) was employed for reverse transcription and qPCR. The 2ΔΔCt method was employed to analyze relative gene expression, with GAPDH as a normalization control. All primers were from Sangon Biotech (Shanghai, China), and are compiled in Supplementary Table S1.

Blood pressure analysis

A non-invasive tail-cuff method (Visitech Systems BP-2000, NC, USA) was used for measurements of murine blood pressure. Briefly, following instrument calibration, animals were secured, with blood pressure measurements being taken following murine acclimatization to the test environment.

Cellular transfection

A SIRT1 siRNA (Cat# s223591) and SGLT2 siRNA (Cat# 289404) were obtained from Thermo Fisher Scientific (MA, USA), while an SGLT2 plasmid (Cat#HG19530-G) was from SinoBiological (Beijing, China). VSMCs were seeded in 6-well plates, and were transfected with these constructs using Lipofectamine 3000 (Thermo, MA, USA) as directed when cells were 60% confluent, utilizing serum-free medium (OPTI-MEM, GIBCO). At 6 h post-transfection, media was replaced. Western blotting was used to detect the efficiency of protein overexpression or knockdown.

Statistical analysis

All analyses were repeated a minimum of three times. All data were analyzed with GraphPad Prism 9.0 (CA, USA), are reported as means ± SEM, and were compared with Student’s t-tests or one-way ANOVAs. P < 0.05 was used to define statistical significance.

Results

DAPA alleviates vascular calcification in mouse models of CKD

Initially, a murine model of CKD was established through a combination of 5/6 nephrectomy combined with a high phosphorus diet as a means of evaluating the ability of DAPA to protect against vascular calcification. After an 8-week feeding period, 60% of mice in the CKD group remained alive as compared to 80% of mice in the CKD + DAPA group, a significant increase, while all mice in the control and DAPA groups remained alive (Fig. 1a). DAPA also improved the blood pressure and renal function of these CKD model animals, without any differences among groups in terms of blood glucose levels (Fig.S1a-j). To establish whether DAPA was capable of delaying CKD-associated vascular calcification, calcified aortic areas were analyzed through Alizarin Red S and Von Kossa staining, revealing a significant reduction in aortic calcification in the DAPA-treated animals relative to CKD model mice (Fig. 1b, c). Dramatically increased arterial calcification and ALP activity were also apparent in samples of tissue collected from these CKD mice, and DAPA treatment alleviated this (Fig.S1k, l). A qPCR approach was also employed to analyze calcification-related marker genes in aortic tissue samples from these animals, revealing that DAPA treatment was associated with the downregulation of osteogenic markers (Runx2, Bmp2) together with the upregulation of key contractile markers (Acta2, Sm22a) (Fig.S2). DAPA was also able to profoundly suppress RUNX2 and BMP2 protein expression while increasing α-SMA and SM22α protein levels in the aortas of these mice (Fig. 1d). RUNX2 and α-SMA were also evaluated through double immunofluorescence staining, revealing results in line with the above findings (Fig. 1e). Together, these data provide support for the successful establishment of a murine CKD model and for the ability of DAPA to attenuate vascular calcification.

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

DAPA abrogates rat aortic ring calcification

To better understand how DAPA influences vascular calcification, rat aortic rings were incubated for 7 days in the presence of β-GP and treated with a range of DAPA concentrations (2.5µM, 5µM, or 10µM) during this period. Von Kossa staining confirmed the ability of DAPA to attenuate rat aortic ring calcification in a dose-dependent fashion (Fig. 2a, b). DAPA treatment also significantly reduced the calcium levels and ALP activity evident in these rat aortic rings (Fig. 2c, d). Together, these findings clearly demonstrated the ability of DAPA to prevent the calcification of rat aortic rings induced by β-GP.

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

DAPA reduces the severity of high-phosphate-induced VSMCs calcification

Next, HASMCs were incubated in the presence of sodium phosphate and various DAPA concentrations (2.5, 5, and 10 µM) as a means of evaluating the impact of DAPA on the calcification of these cells induced by sodium phosphate. Alizarin Red S staining analyses similarly indicated that DAPA is capable of inhibiting calcium salt deposition in these high-phosphate-treated HASMCs in a dose-dependent fashion (Fig. 3a). HASMCs ALP activity and calcium content analyses provided further support for this effect (Fig. 3b, c). Western immunoblotting was also conducted as an approach to analyzing osteogenic transformation-related protein levels. Through these analyses, DAPA was found to reduce RUNX2 and BMP2 levels while increasing the expression of α-SMA and SM22α, consistent with the suppression of high-phosphate-induced calcification of these cells (Fig. 3d). Double immunofluorescence staining was additionally used to assess RUNX2 and α-SMA within HASMCs (Fig. 3e), demonstrating that DAPA was able to inhibit high-phosphorus-induced osteogenic transformation.

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

As a means of gaining greater insight into how DAPA shapes vascular calcification, isolated primary murine VSMCs were subjected to high-phosphate treatment, with immunofluorescent α-SMA and SM22α staining being used to detect these cells (Fig. S3a). DAPA-mediated reductions in VSMCs calcification were confirmed through Alizarin Red S staining, analyses of ALP activity, and measures of calcium content (Fig. S3b-d). Western blotting similarly confirmed the ability of DAPA to abrogate this VSMCs osteogenic transformation under high-phosphate treatment conditions (Fig. S3e). Overall, these analyses demonstrate that DAPA is capable of attenuating high-phosphate-induced VSMCs osteogenic transformation and calcification in a dose-dependent manner, and reaching the maximum protective effect at 5µM. Therefore, we selected 5µM DAPA as the optimal concentration for subsequent experiments.

DAPA targets SGLT2 to attenuate the calcification of VSMCs

The mechanisms through which DAPA is capable of inhibiting VSMCs osteogenic transformation and calcification under high-phosphate conditions were next probed. VSMCs have been reported to express SGLT2 [15, 31–33]. SGLT2 expression was therefore analyzed in murine aortas via Western immunoblotting, revealing a slight increase in SGLT2 expression in the CKD model group relative to controls (Fig. 4a), with immunofluorescence yielding similar findings (Fig. 4b). SGLT2 expression levels in HASMCs were also assessed under normal and high-phosphate culture conditions, with the results being consistent with those from aortic tissues (Fig. 4c, d). These findings support a potential role for SGLT2 in the process of vascular calcification.

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

To clarify how SGLT2 shapes this process of vascular calcification, this gene was next overexpressed successfully in HASMCs through plasmid transfection (Fig. 4e). Alizarin Red S staining demonstrated that SGLT2 overexpression drove HASMCs calcium deposition and abrogated the ability of DAPA to protect against calcification (Fig. 4f). Consistently, the overexpression of SGLT2 promoted the upregulation of osteogenic markers (RUNX2, BMP2) together with the downregulation of contractile markers (α-SMA, SM22α), while weakening the ability of DAPA to protect against calcification (Fig. 4g). Furthermore, down-regulation of SGLT2 by siRNA attenuated HASMCs calcification, while DAPA addition did not show better effects (Fig. S4a-c). Overall, these findings provide evidence that DAPA is capable of attenuating high-phosphate-induced VSMCs osteogenic transformation by targeting SGLT2.

DAPA promotes caloric restriction-mediated SIRT1 expression to protect against vascular calcification

DAPA is an SGLT2 inhibitor, and it therefore functions by reducing the uptake of glucose into cells. As such, glucose levels were next examined in HASMCs (Fig. S5a). Consistent with such a model, HASMCs stimulated with phosphate exhibited increased glucose content, while DAPA inhibited this increase. SGLT2 inhibitors also function as CR mimetics, exerting their cardiovascular benefits through NAD⁺/NADH ratio upregulation [17, 20]. Accordingly, an analysis of the NAD⁺/NADH ratio revealed that DAPA was capable of markedly increasing this ratio in calcified murine aortas and HASMCs (Fig. 5a, b). Given that SIRTs family proteins are NAD⁺-dependent histone deacetylases and the key cellular factors that respond to glucose deprivation, the expression of SIRTs at the mRNA level was next analyzed in high-phosphate-treated HASMCs in the presence or absence of DAPA treatment. Only SIRT1 was found to be substantially upregulated following DAPA administration (Fig. 5c). Consistently, SIRT1 was upregulated at the protein level in both the aortas of CKD model mice and in calcified HASMCs (Fig. 5d, Fig. S5b). This ability of DAPA to promote SIRT1 overexpression was also eliminated when SGLT2 was overexpressed (Fig. 5e), supporting the ability of DAPA to promote SIRT1 upregulation in an SGLT2-dependent fashion.

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

To determine whether SIRT1 is necessary for the DAPA-mediated prevention of calcification, siSIRT1 was used to successfully knock down the expression of this protein in HASMCs (Fig.S6a). Alizarin Red S staining (Fig. 5f), calcium content (Fig.S6b), and ALP activity assays (Fig. S6c) demonstrated that knocking down SIRT1 attenuated these anti-calcifying effects of DAPA. Western immunoblotting and qPCR also demonstrated that the loss of SIRT1 expression attenuated the ability of DAPA to promote the downregulation of BMP2 and RUNX2 as well as the upregulation of α-SMA and SM22α (Fig. 5g, Fig. S6d-g).

HASMCs were also treated with the SIRT1 inhibitor EX527 (10µM), with Alizarin Red S staining consistently revealing that DAPA was able to attenuate calcium deposition within HASMCs, while EX527 eliminated this beneficial effect (Fig. 5h). These findings were further supported by analyses of both calcium content and ALP activity (Fig. S6h, i). Western immunoblotting and qPCR also demonstrated that EX527 was capable of abrogating the ability of DAPA to inhibit the osteogenic transformation of HASMCs (Fig. 5i, Fig. S6j-m). These same experiments were repeated using primary murine VSMCs, with Alizarin Red S staining, calcium content, ALP activity, and Western immunoblotting assays all providing support for the ability of EX527 to attenuate the anti-calcifying effects of DAPA in vitro (Fig. S7). To further better confirm the functional importance of SIRT1 in this setting, rat aortic rings were treated with EX527. Von Kossa staining demonstrated that DAPA was able to prevent the calcification of these aortic rings, while EX527 disrupted this protective effect (Fig. 5j). Together, these findings emphasized the key role that SIRT1 plays in the anti-calcifying activity of DAPA.

DAPA promotes SIRT1-mediated HIF-1α deacetylation and degradation

HIF-1α has recently been found to serve as a central regulator of high-phosphate-induced VSMCs calcification [28, 29]. HIF-1α-mediated signaling has also been linked to the cardioprotective and renoprotective effects of SGLT2 inhibitors [17, 34, 35], and SIRT1 serves as a key HIF-1αregulator [30]. To validate the role of the SIRT1/HIF-1α signaling axis as a mediator of anti-calcifying effects of DAPA, HIF-1α levels were next analyzed in murine aortas by Western immunoblotting. While CKD was associated with increased HIF-1α expression, DAPA reduced this induction (Fig. 6a). Comparable findings were also obtained in vitro (Fig. 6b). Dual-immunofluorescent staining also revealed the potential ability of DAPA to regulate SIRT1/HIF-1α signaling in murine aortas (Fig. 6c). To better clarify the association between SIRT1 and HIF1α, SIRT1 was knocked down in HASMCs using a siRNA construct. Western immunoblotting additionally indicated that the ability of DAPA to inhibit HIF-1α was eliminated following SIRT1 silencing (Fig. 6d), and similar results were observed following EX527 treatment (Fig. 6e). Double immunofluorescence staining of SIRT1 and HIF-1α in HASMCs also supported a role for SIRT1/HIF-1α signaling in the anti-calcifying effects of DAPA (Fig. 6f).

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

SIRT1 has been demonstrated to deacetylate HIF-1α, thereby modulating its stability and degradation [30, 36]. Initially, acetyl-HIF-1α levels were analyzed via Western immunoblotting (Fig. 6g), revealing that DAPA treatment reduced acetyl-HIF-1α levels whereas the silencing of SIRT1 increased these levels. These data suggest that DAPA induces the SIRT1-mediated deacetylation of HIF-1α. Next, the ability of DAPA treatment to influence HIF-1α degradation was assessed by utilizing cycloheximide (CHX) to suppress HIF-1α protein synthesis, with the HIF-1α degradation rate then being analyzed in calcified HASMCs. DAPA-treated cells exhibited significantly more rapid HIF-1α degradation, but the knockdown of SIRT1 was sufficient to attenuate this enhanced degradation (Fig. 6h). This thus indicated that DAPA is capable of accelerating HIF-1α degradation in a manner mediated by SIRT1. To better clarify how HIF-1α is degraded in this setting, cultured HASMCs were treated with the proteasome inhibitor MG132 and DAPA together with high-phosphate medium. MG132 was able to preserve HIF-1α protein stability, delaying its degradation in response to DAPA treatment (Fig. 6i). HIF-1α thus undergoes proteasomal degradation in this experimental setting.

Next, experiments were performed to determine whether preserving HIF-1α stability was sufficient to mitigate the anti-calcifying effects of DAPA. Cobalt chloride (CoCl2, 100µM), which can promote HIF-1α stabilization, was able to markedly attenuate the anti-calcifying effects of DAPA as analyzed by Alizarin Red S staining (Fig. 6J), calcium content, and ALP activity assays in HASMCs (Fig. S8a, b). CoCl2 was also confirmed to suppress the anti-calcifying effects of DAPA as determined through qPCR analyses of osteogenesis-related marker genes (Fig. S8c-f), with Western blotting demonstrating the CoCl2-mediated stabilization of HIF-1α and the abolishment of the anti-calcifying effects of DAPA (Fig. 6k). Consistently, CoCl2 was able to prevent the anti-calcifying effects of DAPA on primary murine VSMCs (Fig. S9). Together, these findings suggested that DAPA was capable of exerting anti-calcifying effects through its ability to drive SIRT1-mediated HIF-1α deacetylation and degradation.

DAPA protects against aortic calcification in non-CKD mice

The final goal of this study was to establish whether DAPA is capable of preventing aortic calcification in other murine models of vascular calcification not related to CKD through its ability to modulate CR-dependent SIRT1 signaling. To that end, a vitamin D3 overload model of vascular calcification was established. In line with the above results, treatment with DAPA alleviated aortic calcification in these VitD3-challenged mice (Fig. 7a). DAPA also reversed VitD3-induced increases in aortic calcium levels and ALP activity (Fig. 7b, c), while suppressing the VitD3-associated upregulation of calcification-related gene expression (Bmp2, Runx2) and promoting Acta2 and Sm22a upregulation (Fig. 7d-g). These qPCR results supported the ability of DAPA to protect against aortic calcification in these VitD3-overloaded mice. Consistent with these findings, DAPA was able to downregulate BMP2 and RUNX2 at the protein level while promoting aortic α-SMA and SM22α upregulation in these VitD3 model mice (Fig. 7h). Strikingly, DAPA was also able to promote SIRT1/HIF-1α signaling through the upregulation of the NAD⁺/NADH ratio (Fig.S10). Together, these results emphasize the ability of DAPA to protect against vascular calcification in a non-CKD mouse model, potentially through the regulation of SIRT1 signaling.

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

Discussion

Vascular calcification is one of the most prominent risk factors associated with the incidence of cardiovascular events, and it is frequently detected in patients suffering from diabetes or CKD [37]. To date, no effective therapeutic strategies have been established to manage vascular calcification. In the present study, DAPA was found to effectively inhibit VSMCs osteogenic transformation in vitro while also protecting against ex vivo rat aortic ring calcification and mitigating vascular calcification in vivo in mouse models of both CKD and VitD3 overload. From a mechanistic perspective, DAPA was found to interact with SGLT2 and thereby promote SIRT1 upregulation via the CR-dependent modulation of the NAD⁺/NADH ratio, ultimately triggering HIF-1α deacetylation and degradation. This is the first study to our knowledge demonstrating the ability of DAPA to activate SIRT1 signaling in a CR-dependent fashion as a means of attenuating vascular calcification. Based on these findings, DAPA appears to hold promise as a new approach to managing or preventing vascular calcification.

DAPA is a hypoglycemic SGLT2 inhibitor that is mainly employed as a treatment for type 2 diabetes patients. In addition to their hypoglycemic activity, however, SGLT2 inhibitors are also known to exert an array of beneficial cardiovascular effects through the suppression of oxidative stress and inflammation together with the regulation of ketone body metabolism and inhibiting inflammation and oxidative stress [38]. While there have been several studies exploring the mechanisms underlying such activity in other forms of cardiovascular disease, there has been little research on the exact mechanisms whereby SGLT2 inhibitors influence vascular calcification. A growing body of evidence has underscored the complex pathological nature of vascular calcification, revealing that this process is related to oxidative stress endoplasmic reticulum stress, inflammatory activity, apoptotic cell death, and aging [8]. These factors ultimately promote the osteoblast-like transformation of VSMCs. SGLT2 inhibitors including canagliflozin and empagliflozin have recently been found to prevent this osteogenic VSMCs transformation through their ability to inhibit inflammatory pathway activation [14–16]. The present data align well with these past reports and suggest that DAPA is capable of attenuating the high-phosphate-induced osteogenic transformation of VSMCs and preventing vascular calcification. A prior report revealed that DAPA or siRNA-mediated SGLT2 inhibition in a model of ischemia/reperfusion injury was sufficient to alleviate endothelial dysfunction and cardiac microvascular damage [39]. In this study, increased SGLT2 protein levels were detected in both calcified VSMCs and calcified aortic tissues, and the ability of DAPA to protect against such calcification was abrogated when SGLT2 was overexpressed. This interaction between DAPA and SGLT2 thus appears to be central for the maintenance of VSMCs phenotypes in this experimental context.

Recent reports suggest that CR can slow the aging process and protect against cardiovascular metabolic disease [40, 41]. CR diets can effectively inhibit atherosclerotic development and vascular calcification in apoE-deficient mice [42]. High-calorie diets are also believed to promote progressive vascular calcification, although how CR affects vascular calcification remains poorly understood [22, 43]. SGLT2 inhibitors function as CR mimetics, reducing intracellular glucose availability and thereby triggering nutrient deprivation signaling [17, 19]. However, whether DAPA protects against calcification through this CR pathway was not clear. Analyses conducted in this study revealed that DAPA was able to reduce the levels of glucose in calcified VSMCs while promoting an increase in the NAD⁺/NADH ratio within calcified VSMCs and aortas. SIRTs are NAD⁺-dependent deacetylases, the activity of which is directly regulated by NAD⁺ levels or the NAD⁺/NADH ratio [44], and SIRT1 is the primary member of this protein factor involved in the regulation of CR [45]. SIRT1 also plays a specific role in regulating vascular calcification [25]. Here, all SIRTs family members other than SIRT4 were found to be expressed in the context of high-phosphate-induced VSMCs calcification, but only SIRT1 was significantly upregulated following treatment with DAPA. This suggested that the DAPA-induced activation of SIRT1 signaling is mediated by the CR pathway. Consistent with this model, the suppression of SIRT1 expression using siSIRT1 or EX527 weakened the benefits associated with DAPA administration. This suggests that SIRT1 is a central regulator of the beneficial anti-calcification effects of DAPA treatment.

Hypoxic signaling has been suggested to be central to the regulation of bone development and vascular calcification [46, 47]. HIF-1α functions as a major driver of calcification capable of promoting osteogenic VSMCs transformation through the induction of osteogenic factor expression [29]. In more recent years, HIF-1α was identified as a key mediator of vascular calcification [28]. These present results support these past findings and offer novel evidence in support of the ability of DAPA to attenuate the expression of both HIF-1α and osteogenic factors in VSMCs subjected to high-phosphorus conditions and in tissue-based models of vascular calcification. There is increasingly strong evidence that the stability of the HIF-1α protein is regulated at both the transcriptional and post-translational levels [48]. SIRT1 is known to deacetylate HIF-1α, thereby decreasing its stability [49]. Prior work from our group also found that DAPA was capable of attenuating myocardial hypertrophy through the activation of downstream SIRT1/HIF-1α signaling [27]. Here, SIRT1 was found to promote HIF-1α deacetylation and proteasomal degradation following DAPA treatment. This suggests that this SIRT1/HIF-1α signaling axis is vital for the DAPA-mediated inhibition of VSMCs transformation.

There are some limitations to these analyses. For one, SGLT2 inhibitors are reportedly capable of directly binding to Na/H exchanger 1 (NHE1) and thereby protecting cardiomyocytes [50, 51]. NHE1 also exhibits important functions in VSMCs [52, 53], and it may therefore play a role in the effects that DAPA has on vascular calcification, highlighting an important focus for further research. Moreover, SGLT2 inhibitors have been found to control SIRT1 activity through the AMPK pathway, while SIRT1 is also capable of promoting AMPK pathway activation [17]. Given the complex regulatory relationship between SGLT2 inhibitors and SIRT1, it is possible that cross-regulatory interactions between AMPK and SIRT1 in VSMCs may influence the ability of DAPA to combat calcification. Lastly, SIRT1 deficiency has been shown to drive more severe DNA damage and VSMCs senescence, thereby driving osteogenic transformation and vascular calcification [54]. Additional signaling mechanisms downstream may therefore play a role in the ability of DAPA/SIRT1 to protect against calcification, emphasizing a need for further research to definitively clarify the mechanisms through which these SGLT2 inhibitors prevent vascular calcification.

In conclusion, these analyses revealed a previously undiscovered mechanism through which DAPA is capable of inhibiting VSMCs osteogenic transformation and vascular calcification through a CR-dependent mode of action. Mechanistically, DAPA can activate SIRT1, in turn promoting HIF-1α deacetylation and degradation. Based on these results, this DAPA/SGLT2/SIRT1 axis may represent a viable therapeutic strategy, offering a new approach to managing vascular calcification.

Electronic Supplementary Material

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Abbreviations

CKD

chronic kidney disease

MACE

major adverse cardiovascular events

VSMCs

vascular smooth muscle cells

HASMCs

human aortic smooth muscle cells

SGLT2

sodium-glucose transporter 2

DAPA

dapagliflozin

CR

caloric restriction

NAD⁺

nicotinamide adenine dinucleotide

ALP

alkaline phosphatase

AMPK

adenosine 5‘-monophosphate (AMP)-activated protein kinase

SIRT1

sirtuin 1

HIF-1α

hypoxia-inducible factor-1 alpha

RUNX2

runt-related transcription factor 2

BMP2

bone morphogenetic protein 2

α-SMA

alpha smooth muscle actin (Acta2)

SM22α

smooth muscle protein 22 alpha (Sm22a)

β-GP

β-glycerophosphate

CoCl₂

cobalt chloride

VitD3

Vitamin D3

Author contributions

ZL, CG, and HL conceived the study and designed the experiments; LL, HL, and QC conducted the experiments and analyzed the data; JW, YQ, JY, HeL, and JQ conducted experiments; LL wrote the draft; ZL and HL revised the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (81800171, 81900275, 81302768, 82270435), the postdoctoral research project of Zhejiang Province (ZJ2023046), the Health Commission scientific research of Shanxi Province (2022001), and the Science and Technology Foundation of Shanghai Province (21ZR1441300, 172R1418300).

Data availability

All data are available in the main text or the Supplementary Material. Derived research data supporting our conclusion are available from the corresponding author on request.

Declarations

Ethics approval and consent to participate

All animal experiments followed the NIH Guide for the Care and Use of Laboratory Animals and carried out with the approval of the Ethics Committee for Animal Experiments of the Second Hospital of Shanxi Medical University.

Conflict of interest

All the authors declared no competing interests.