Sitagliptin attenuates arterial calcification by downregulating oxidative stress-induced receptor for advanced glycation end products in LDLR knockout mice

Diabetes is a complex disease characterized by hyperglycemia, dyslipidemia, and insulin resistance. Plasma advanced glycation end products (AGEs) activated the receptor for advanced glycation end products (RAGE) and the activation of RAGE is implicated to be the pathogenesis of type 2 diabetic mellitus (T2DM) patient vascular complications. Sitagliptin, a dipeptidyl peptidase-4 (DPP4) inhibitor, is a new oral hypoglycemic agent for the treatment of T2DM. However, the beneficial effects on vascular calcification remain unclear. In this study, we used a high-fat diet (HFD)-fed low-density lipoprotein receptor deficiency (LDLR−/−) mice model to investigate the potential effects of sitagliptin on HFD-induced arterial calcification. Mice were randomly divided into 3 groups: (1) normal diet group, (2) HFD group and (3) HFD + sitagliptin group. After 24 weeks treatment, we collected the blood for chemistry parameters and DPP4 activity measurement, and harvested the aorta to evaluate calcification using immunohistochemistry and calcium content. To determine the effects of sitagliptin, tumor necrosis factor (TNF)-α combined with S100A12 was used to induce oxidative stress, activation of nicotinamide adenine dinucleotide phosphate (NADPH), up-regulation of bone markers and RAGE expression, and cell calcium deposition on human aortic smooth muscle cells (HASMCs). We found that sitagliptin effectively blunted the HFD-induced artery calcification and significantly lowered the levels of fasting serum glucose, triglyceride (TG), nitrotyrosine and TNF-α, decreased the calcium deposits, and reduced arterial calcification. In an in-vitro study, both S100A12 and TNF-α stimulated RAGE expression and cellular calcium deposits in HASMCs. The potency of S100A12 on HASMCs was amplified by the presence of TNF-α. Sitagliptin and Apocynin (APO), an NADPH oxidase inhibitor, inhibited the TNF-α + S100A12-induced NADPH oxidase and nuclear factor (NF)-κB activation, cellular oxidative stress, RAGE expression, osteo transcription factors expression and calcium deposition. In addition, treatment with sitagliptin, knockdown of RAGE or TNF-α receptor blunted the TNF-α + S100A12-induced RAGE expression. Our findings suggest that sitagliptin may suppress the initiation and progression of arterial calcification by inhibiting the activation of NADPH oxidase and NF-κB, followed by decreasing the expression of RAGE.

www.nature.com/scientificreports/ anti-atherosclerotic effects by reducing the reactive oxygen species (ROS) generation, preventing mitochondrial depolarization, improving endothelial functions, and reducing vascular inflammation 33 . It has been reported that TNF-α plays a crucial role in arterial calcification in diabetic LDLR −/− mice 13 . Our previous study showed that TNF-α and vascular tumor necrosis factor receptor (TNFR) signaling lead to human aortic smooth muscle cells (HASMCs) calcification and antioxidants blunt the TNF-α/TNFR signaling to retard the HASMCs calcification 34 .
To our knowledge, no one has investigated the effect of sitagliptin on the progression of T2DM patients' arterial calcification. In this study, we hypothesized that sitagliptin improved arterial calcification. We used a chow or high-fat diet (HFD)-fed LDLR −/− mice supplemented with vehicle or Sitagliptin to investigate the effect on animal arterial calcification and HASMCs model to determine how sitagliptin exerts its effect. We demonstrated that sitagliptin attenuated the levels of weight, aortic calcium content, DPP4 activity and arterial calcification in HFD-induced LDLR −/− mice. Medial calcification is mostly associated with hyperglycemia 15 . We used HASMCs in the in-vitro study to mimic VSMCs. The results showed that TNF-α and S100A12 increased the levels of RAGE expression and calcium deposition. Treatment with sitagliptin decreased the TNF-α-induced osteogenic gene expression including RAGE, RUNX2, MSX2 and BMP-2. Moreover, sitagliptin reduced the activity of NADPH oxidase by inhibiting the expression of p47, thereby reducing the production of superoxide and hydrogen peroxide and the activity of NF-κB.

Results
Effects of HFD and sitagliptin on HFD-induced LDLR −/− body weight gain, aortic calcification and atherosclerotic plaque formation. We found that the HFD-fed LDLR −/− mice had a significantly higher body weight than the regular chow-fed mice ( Fig. 1a; 33.8 ± 1.9 vs. 41.8 ± 2.3 g). Sitagliptin attenuated HFD-induced body weight gain ( Fig. 1a; 41.8 ± 2.3 vs. 36.8 ± 2.5 g). HFD-fed mice had a higher level of blood glucose, triglyceride (TG), cholesterol (CHL), low-density lipoproteins (LDL) and TNF-α than the regular chow-fed mice (Table 1). These findings suggest that HFD fed LDLR −/− mouse is a valid model as an obesityassociated T2DM animal model. HFD-fed LDLR −/− mice treated with sitagliptin drastically lowered the fasting Figure 1. HFD promoted calcification, which was attenuated by sitagliptin, in male LDLR −/− mice. LDLR −/− mice were fed with a chow diet or HFD and simultaneously treated with sitagliptin (Sita, 100 mg/kg/day) for 6 months. (a) LDLR −/− mouse body weight was recorded weekly. (b) HFD-induced artery atherosclerotic plaque formation was detected by H&E staining. HFD-induced artery calcification was detected by alizarin red staining. HFD promoted aortic calcification, which was dominant in the medial layer. The arrows point to the area of calcium deposition. The lumen showed blood cell remnants. L lumen, I intima, M media, and A adventitia. (ND = 6, HFD = 6, and sitagliptin = 6). LDLR −/− mice aorta were photographed at × 100 and × 400 magnifications. (c) Aortic calcium levels and (d) DPP4 activity were assayed. (e) Immunostaining of aortic RAGE showed that sitagliptin could significantly decrease the stimulatory effects of HFD on RAGE. Arrowhead indicates positive RAGE position. *P < 0.05 compared with the chow group, and # P < 0.05 compared with the HFD groups (Aortic calcium levels; Chow = 6, HFD + vehicle = 6, and HFD + sitagliptin = 6. DPP4 activity was assayed; Chow = 5, HFD + vehicle = 5, and HFD + sitagliptin = 5). Numerically, a moderate reduction in LDL was observed, but the level of reduction was not statistically significant. HFD-fed LDLR −/− mice had a remarkable increase in calcification in the medial layer of the upper descending aorta as indicated by alizarin red staining and aorta calcium content analysis (Fig. 1b,c) compared to the regular chow-fed mice. HFD also caused the LDLR −/− mice to have much higher levels of serum DPP4 activity (Fig. 1d). Sitagliptin treatment significantly blunted the HFD-induced calcium deposition in the media of the descending aorta (Fig. 1b), aortic acid-extracted calcium content (Fig. 1c) and the levels of serum DPP4 activity (Fig. 1d). In addition, we found that HFD-fed LDLR −/− mice had a higher level of RAGE in the medial layer of the upper descending aorta (Fig. 1e). Orally administration of sitagliptin drastically reduced the expression of RAGE in aorta (Fig. 1e).
Effects of TNF-α and S100A12 on RAGE expression and calcium deposition in HASMCs. T2DM patients had higher levels of TNF-α and S100A12 compared to non-diabetic subjects 35 and was associated with the severity of coronary artery diseases and vascular calcification 23,24 . We use the HASMC model to investigate (1) whether TNF-α + S100A12 induced HASMC calcification and (2) and how sitagliptin blunted the effects of TNF-α + S100A12 treatment. TNF-α (10 ng mL −1 ) treatment significantly increased the expression of S100A12 in HASMCs but did not significantly stimulate the expression of S100A12 with recombinant S100A12. (Fig. 2a). We demonstrated the expression of RAGE with S100A12 treatment in the presence or absence of TNF-α. S100A12 did not impair the cell viability of HASMCs (Fig. 2b). S100A12 produced a dose dependent increase on the expression of RAGE and the potency of S100A12 on RAGE expression appeared to be amplified by the presence of TNF-α (Fig. 2c,d). The calcium deposits on S100A12-treated HASMCs were much more pronounced in the presence of TNF-α (Fig. 2e).
Sitagliptin, N-acetyl cysteine (NAC), TNFR-1 small interfering RNA (siRNA) and RAGE siRNA affected the expression levels of RAGE and calcium deposits on TNF-α/S100A12-treated HASMCs. The DPP4 inhibitor had anti-atherosclerotic effects by reducing the production of ROS and inflammatory cytokines in adipocytes and aorta 33 . RAGE signaling was at least in part mediated by the oxidative stress from NADPH oxidase 21 . We used TNF-α + S100A12-treated HASMCs to investigate whether sitagliptin neutralized the oxidative stress and inhibited TNF-α-induced activation of NADPH oxidase. We incubated HASMCs with various doses of sitagliptin and found that sitagliptin did not inhibit the cell proliferation rate (Fig. 3a). The siRNA for RAGE significantly lowered the TNF-α + S100A12 induced expression of RAGE (Fig. 3b) and calcification (Fig. 3c) without affecting the expression of TNFR. The siRNA for TNFR-1 was very effective in blunting the TNF-α + S100A12 induced up-regulation of the RAGE and TNFR-1 expression (Fig. 3b). Sitagliptin treatment appeared to exert a dose-dependent effect on suppressing the TNF-α + S100A12-induced RAGE expression (Fig. 3b) and calcium deposits (Fig. 3c). Moreover, sitagliptin was as effective as NAC, in blunting the TNF-α + S100A12-induced calcium deposits (Fig. 3c).
Sitagliptin blunts TNF-α + S100A12-induced HASMC oxidative stress, osteogenic marker expression, and calcification. Activated RAGE promoted the activation of NADPH oxidase 36 , which led to the generation of intracellular H 2 O 2 in VSMCs 37 . To determine whether sitagliptin interrupted the RAGE signaling pathway by inhibiting NADPH oxidase activity, the measurements of intracellular H2O2 and NADPH oxidase activity were assayed.

Sitagliptin blunts TNF-α + S100A12-induced expression of osteogenic markers and calcification.
To determine whether sitagliptin inhibited osteogenic marker expression and cell calcium deposition.

Discussion
Hyperglycemia and dyslipidemia are the traditional risk factors of arterial calcification 38 . In this study, HFD-fed LDLR −/− mice were used as a model system to investigate the potential effect of sitagliptin on T2DM patients' initiation and progression of artery calcification. We found that HFD feeding caused obesity, hyperglycemia, hyperlipidemia and severe artery calcification on LDLR −/− mice. HFD feeding also increased inflammation and oxidative stress as indicated by serum TNF-α and nitrotyrosin levels. Orally administration of sitagliptin (100 mg kg −1 day −1 ) moderately decreased the HFD-induced serum TNF-α (45% reduction) and nitrotyrosin (70% reduction), improved the levels of blood glucose and triglyceride, reduced the body weight, aortic calcium content and DPP4 activity, and significantly blunted the artery calcification. In-vitro studies showed that sitagliptin attenuated TNF-α and S100A12-induced calcium deposition and attenuated the expression of RAGE and osteogenic markers such as MSX2, BMP-2, and RUNX2. Sitagliptin reduced RAGE accumulation by Figure 2. Effect of the combination of TNF-α with S100A12 on induced RAGE accumulation and calcium deposition in HASMCs. (a) The accumulation of S100A12 by TNF-α but not recombinant S100A12 in HASMCs was assayed. (b) The cytotoxicity effect of S100A12 was determined by MTT assay. The induction of RAGE by (c) S100A12 combined with TNF-α or (d) S100A12 alone in HASMCs was determined by Western blotting assay. The accumulation of RAGE by combination of S100A12 with TNF-α in HASMCs was assayed. S100A12 enhanced TNF-α-induced RAGE protein accumulation in HASMCs. (e) HASMCs were cultured in osteogenic differentiation medium treatment with S100A12 for 4 days in the presence or absence of TNF-α. Calcium deposition was induced dose-dependent by TNF-α for 4 days. N = 6 for each set of experiments. *P < 0.05 compared with the control group.
Scientific Reports | (2021) 11:17851 | https://doi.org/10.1038/s41598-021-97361-w www.nature.com/scientificreports/ downregulating the activated NF-κB translocation and attenuated ROS generation by reducing NADPH oxidase p47 subunit translocation. Data strongly suggest that the protective effect of sitagliptin may not be a consequence of its glucose lowering effect via the inhibition of DPP4. Instead, the protective effect of sitagliptin against calcification was provided by 1) blocking the activation of cell membrane surface NADPH, and 2) down-regulation of TNF-α and RAGE (Fig. 6). Sitagliptin, a DPP4 inhibitor was the first commercialized approved for treatment of patients with T2DM in Oct 2006 in USA and in 2007 by the European Medicines Agency (EMA) at a dosage of 100 mg daily 39 . Sitagliptin is suggested to have a cardiovascular protective effect and glucose-lowering effect similar to saxagliptin and alogliptin 39 . Zheng et al. showed that treatment with 100 mg kg −1 day −1 of sitagliptin increased the mass and function of pancreatic β-cells, reduced the levels of fasting blood glucose, postprandial blood glucose and HbA1c in T2DM mice 40 . Previous studies reported that significance of sitagliptin on vascular protection in Zucker diabetic fatty rats. Sitagliptin decreased blood glucose concentrations and increased plasma insulin concentrations, augmented acetylcholine-induced vascular relaxation reduced the DPP4 activity, malondialdehyde levels, the expressions of p22 phox and monocyte chemoattractant protein-1 41 . Sitagliptin attenuated the progress of atherosclerosis in ApoE knockout mice via AMP-activated protein kinase (AMPK) and Mitogen-activated protein kinase (MAPK)-dependent mechanisms followed by reducing leukocyte-endothelial cell interaction and inflammation reactions. These vascular protection and anti-atherosclerotic effects of sitagliptin were mediated by attenuating oxidative stress and NF-κB signaling 32,42 .
The proinflammatory cytokine S100A12, a RAGE agonist, is associated with coronary atherosclerotic plaque rupture. S100A12 increased the atherosclerotic plaque size, expression of bone morphogenic protein and other osteoblastic genes in aorta and cultured vascular smooth muscle 23 , and promoted the VSMC osteochondrogenic mineralization in transgenic mice. This finding provided a strong evidence that the S100/RAGE axis enhanced vascular calcification 43 . The activities of S100A12 including chemotactic activity and activation of intracellular signaling cascades led to induction of cytokine production and oxidative stress 44 . In addition, Nox inhibition Sitagliptin and NAC attenuated the calcium deposition mediated by TNF-α combined with S100A12. The induction of RAGE protein accumulation by TNF-α combined with S100A12 in HASMCs was attenuated by sitagliptin. Western blotting assay showed the effects of the knockdown of TNFR-1 and RAGE proteins by these siRNAs. Compared with the TNF-α combined with S100A12-stimulated cells in the presence of scrambled siRNAs, any combination of TNFR-1 or RAGE siRNAs dramatically abolished TNF-α-stimulated calcification. *P < 0.05 compared with the control group, and # P < 0.05 compared with the TNF-α combined with S100A12 groups. N = 6 for each set of experiments. (c) Sitagliptin attenuated the calcium deposition mediated by TNF-α combined with S100A12. Suppressed RAGE accumulation in HASMCs and siRNAs against RAGE-downregulated calcium deposition (NAC antioxidant agent, SC scramble, TNF-R1 TNF-α receptor 1). www.nature.com/scientificreports/ reduced osteogenic programming and calcification 23 . The signaling of S100A12 was controlled by the presence of RAGE and oxidative stress signaling. In this study, HFD-induced the mice hyperglycemia and dyslipidemia contributed to arterial calcification, whereas these were significantly downregulated after 24 weeks-treated sitagliptin (Fig. 1b, Table 1). A previous study showed that sitagliptin attenuated body weights and reduced local inflammation in adipose tissue of HFD-induced obese mice 45 . We suggested that sitagliptin decreased body weight gain by attenuating the levels of TNF-α and TG (Fig. 1a, Table 1). Moreover, DPP4 induced valvular calcification and promoted calcific aortic valve disease progression by inhibiting autocrine insulin-like growth factor-1 (IGF-1) signaling. Treatment with sitagliptin reduced the calcium deposits and increased plasma IGF-1 levels 46 . In our study, sitagliptin significantly reduced HFD-induced DPP4 activity consequently attenuated arterial calcification in LDLR −/− mice (Fig. 1c,d). GLP-1 was known to be involved in anti-inflammation 47 . Sitagliptin dietary www.nature.com/scientificreports/ supplementation reduced adiposity and improved glucose metabolism in obese and diabetic mice, which was associated with GLP-1 elevation 48 . Sitagliptin boosted the circulatory GLP-1 levels by retarding the degradation of GLP-1 49 . However, GLP-1 did not reduced the TNF-α-induced calcification in HASMCs (Fig. S2). We suggested that GLP-1 played a minor role in blunting of arterial calcification on HFD-fed LDLR −/− . Furthermore, sitagliptin attenuated the accumulation of RAGE and arterial calcification on HFD-induced LDLR −/− mice (Fig. 1e). These results suggested that sitagliptin attenuated HFD-induced arterial calcification by improvement lipid profile, anti-inflammation, and reducing the expression of RAGE on LDLR −/− mice. We next employed HASMCs to clarify the underlying mechanisms of HFD-induced arterial calcification. It has been known that TNF-α plays crucial role in HFD-induced arterial calcification 13 . HFD increased the accumulation of circulatory S100A12 in vivo. In addition, stress-mediated VSMC expressed S100A12 and overexpression of S100A12 accelerated arterial calcification in ApoE −/− mice 23 . The accumulation of S100A12 could be induced by TNF-α in HASMCs, whereas there was no effects with recombinant S100A12 treatment alone (Fig. 2a,b). Previously study suggested S100/calgranulins were endogenously expressed in granulocytes and myeloid cells, and were induced in VSMCs of the atherosclerotic vessel 50 . S100A12 augmented the atherosclerosis-triggered osteogenesis and TNF-α increased the expression of RAGE. Similar to the previous studies, treatment of TNF-α or S100A12 significantly increased the accumulation of RAGE and calcium deposition in HASMCs (Fig. 2c-e). RAGE activated the proinflammatory transcription factor NF-κB. Interestingly, activated NF-κB caused sustained RAGE expression and created a positive-feedback loop 21 . ApoE −/− /S100A12 transgenic mice-isolated VSMCs treated with the condition medium (contain macrophages and serum isolated from hyperlipidemic ApoE −/− mice) increased oxidative stress, the expression of osteogenic marker and calcification 23 . These findings suggest that the ROS or inflammatory cytokines from the macrophages in hyperlipidemic ApoE −/− mice might activate a mechanism which synergistically interacts with the RAGE/S100A12 signaling in stimulation of arterial atherosclerosis and calcification. We employed a combined strategy to mimic the in-vivo condition. There was no cell toxicity at the dosage of 100 μM (Fig. 3a). Treatment with TNF-α + S100A12 significantly induced the expression of RAGE and calcium deposition (Fig. 3b,c). Sitagliptin was found to exert a dose response on depressing the stimulation of TNF-α + S100A12. These results suggested that combination of TNF-α with S100A12 had synergetic effects to trigger calcium deposition in HASMCs.

Figure 5. Sitagliptin modulated ROS generation and the expression of RAGE. (a)
HASMCs were cultured in an osteogenic differentiation medium for 1 day in the presence or absence of TNF-α combined with S100A12 and concomitantly with sitagliptin. Sitagliptin blocked the induction of MSX2, BMP-2, and RUNX2 accumulation induced by TNF-α combined with S100A12. (b) Antioxidant agents (NAC and APO) attenuated the TNF-α combined with S100A12-induced bone marker MSX2 and (c) RAGE accumulation in HASMCs (sitagliptin; NAC: ROS scavenger; APO: Nox inhibitor). *P < 0.05 compared with the control group, and # P < 0.05 compared with the TNF-α combined with S100A12 groups. N = 6 for each set of experiments.  34 . ROS products were generated by mitochondrial oxidases, Nox, and nitric oxide synthases 16 . The NADPH oxidase is composed of Nox, p22 phox and p47 phox . It has been reported that activation of RAGE induced the expression of Nox-1, Nox-4, p22 phox and p47 phox and increase ROS production in VSMCs 51 . Generally, superoxide generated by NADPH oxidase was short-lived and was rapidly transferred to H 2 O 2 by superoxide dismutase 52 . To investigate whether sitagliptin attenuated ROS productions such as H 2 O 2 with the combination of TNF-α and S100A12. We measured the TNF-α + S100A12-induced H 2 O 2 production and NADPH oxidase activity after NAC, APO and sitagliptin treatment. As a result, combination of TNF-α and S100A12 increased ROS generation, which were significantly decreased by sitagliptin (Fig. 4a,b). We suspected that sitagliptin contributed its protective against HASMCs calcification by inhibiting NADPH oxidase activity.
Translocation of p47 from HASMCs cytosol to membrane indicates activation of NADPH oxidase and the migration of p65 from cytosol into nucleus indicates activation of NF-κB. It is known that suppression of the membrane translocation of p47phox was shown to inhibit the assembly of NADPH oxidase by alpha tocopherol 53 . We further investigated the p47 and p65 migration in HASMCs with TNF-α + S100A12, TNF-α + S100A12 + sitagliptin and TNF-α + S100A12 + APO treatment. Combination of TNF-α and S100A12 increased p47 translocation to the cell membrane, whereas p47 translocation was decreased by sitagliptin treatment (Fig. 4c,d). Previous studies have suggested RUNX2 plays a crucial role in oxidative stress-induced VSMC calcification and SMC-specific RUNX2 deficiency inhibited vascular calcification 54,55 . Another study demonstrated that sitagliptin attenuated oxidative stress in diabetic rats 56 . We found that sitagliptin attenuated TNF-α + S100A12-induced calcium deposition and the expression of osteogenic markers protein accumulation via anti-oxidation (Fig. 5a-c).
There were several study limitations. First, we demonstrated that inhibition of RAGE decreased the expression of osteogenic markers and subsequently inhibited HASMCs calcification. We also found that inhibition of the TNF-R1 decreased the expression of RAGE. These findings suggested that TNF-R1 might be the upstream regulator of RAGE in TNF-α/NF-κB pathway. The mechanism of TNF-R1 silence-suppressed accumulation of S100A12-induced RAGE and the relationship between sitagliptin and TNF-R1 were still unknown. Second, this study could not rule out the protection effect of sitagliptin by elevating GLP-1. It has been known DPP4 inhibitors improved glucose metabolism in diabetic patient, which was associated with GLP-1 elevation. Third, we demonstrated that sitagliptin attenuated HFD-induced calcification in LDLR −/− mice. The effects of sitagliptin treatment on HFD-supplemented wild-type mice should be further investigated.
In summary, in addition to the known roles of DPP4 inhibitors in influencing glucose levels and other pleiotropic effects, such as anti-inflammatory and anti-oxidative effects, we demonstrated that sitagliptin acts via a novel mechanism, independent of its blood glucose-regulating effect, to prevent arterial calcification. Figure 6. The proposed scheme of sitagliptin attenuates TNF-α + S100A12-induced VSMC calcification through inhibition of RAGE-Nox pathways. TNF-α enhances RAGE accumulation and activation by binding to the RAGE ligand, S100A12. S100A12 interacts with Nox1 NADPH oxidase to promote osteochondrogenic genes expression and calcium deposition in VSMCs via oxidative stress signaling. Sitagliptin reduces osteogenic mineralization by inhibiting the activation of NADPH oxidase and ROS production.

Conclusion
In addition to the known roles of DPP4 inhibitors in influencing glucose levels and other pleiotropic effects, such as anti-inflammatory and anti-oxidative effects, we demonstrated that sitagliptin acts via a novel mechanism, independent of its blood glucose-regulating effect, to prevent arterial calcification.
Unless otherwise specified, all other chemicals and reagents obtained from Sigma-Aldrich (St. Louis, MO). All methods in this study were reported in accordance with ARRIVE guidelines. Histology and immunohistochemistry. Aorta samples were cut into 4 sections and processed for histological staining, as described in our previous study 34 . Paraffin sections (5 µm) from the dissenting aorta were stained using various agents for semi-quantification of atherosclerotic lesion size and severity (hematoxylin and eosin (H&E) staining) and aortic calcium deposition (alizarin red S staining). Immunohistochemical (IHC) staining of RAGE and VSMC actin (SM α-actin) was performed as previously described 57 .
Cell culture and cell viability assay. HASMCs were purchased from Life Technology (Grand Island, NY; Catalog number C0075C). The cells were grown and passaged as described previously 34 . Briefly, the HASMCs were grown in M231 medium containing SMC growth supplements and a 1% antibiotic-antimycotic mixture in an atmosphere of 95% air and 5% CO 2 at 37 °C in plastic flasks. At confluence, the cells were subcultured at a ratio of 1:3, and passages 3 through 8 were used. The cytotoxicity of S100A12 protein and sitagliptin on HASMC cell viability were measured with the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay.
Soluble DPP-4 activity measurement. Serum  www.nature.com/scientificreports/ The aortic segments from experimental mice were extracted using 0.6 N HCl for 24 h and the calcium content of the extracts was determined using a BioChain Calcium Kit (BioChain, Hayward, CA, USA) as previously described 34 . The results were expressed as µg mg −1 of wet aortic tissues.
Nox activity assay and hydrogen peroxide determination. Nox activity was determined with superoxide-dependent lucigenin chemiluminescence, as previously described 34 . Confluent HASMCs in 6-well plates were pretreated with various concentrations of antioxidant reagents followed by treatment with TNF-α + S100A12 with or without sitagliptin for 1 day. Cell membrane extract (40 µg) and 5 µM dark-adapted lucigenin were added to a 96-well luminometer plate and adjusted to a final volume of 250 µL with oxidase assay buffer before 100 µM NADPH was added. Relative light units (RLUs) were measured with a luminometer (Dynatech ML2250, Dynatech Laboratories Inc., VA). Light emission was recorded every 3 min for a total of 30 min and expressed as mean RLUs min −1 .
The ROS production in HASMCs was determined by fluorometric assay using dichloro-dihydro-fluorescein diacetate (DCFH-DA) as the probe. This method was based on the oxidation by H 2 O 2 of nonfluorescent DCFH-DA to fluorescent 2' , 7'-dichlorofluorescin. Confluent HASMCs in 24-well plates were pretreated with various concentrations of antioxidant reagents followed by treatment with TNF-α + S100A12 with or without sitagliptin for 1 day. The cells were washed with PBS, and then 250 μL of serum-free M231 containing 10 µM DCFH-DA was added to the well for 30 min. The fluorescence intensity (relative fluorescence units) was measured at 485 nm excitation and 530 nm emission using a fluorescence microplate reader after the plates were incubated for 45 min at 37 °C. siRNA transfection. siRNA oligonucleotides against RAGE were suspended in RNase-free water at a concentration of 10 µM. Cells were seeded one day before transfection to ensure HASMCs were 85%-95% confluent on the day of transfection. For transfection, the regular cell culture medium was replaced with a serum-free medium without antibiotics. The cells were transfected with siRNA using oligofectamine at a ratio of 1 siRNA: 2 oligofectamine (µg:µL) at a final concentration of 25-50 nM siRNA. The cells were incubated with the siRNAoligofectamine complex for 5 h. Then, the serum-free medium was replaced with a normal medium (containing 10% FBS) without antibiotics, and the cells were incubated for 48 h before further analysis.
Statistical analyses. Data were expressed as mean ± standard deviation (SD). Statistical evaluation was performed using Student's t-test or one-way analysis of variance, followed by Dunnett's test. A P value of < 0.05 was considered significant.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.