Skip to main content
Download PDF

Nitrogen-containing bisphosphonate induces enhancement of OPG expression and inhibition of RANKL expression via inhibition of farnesyl pyrophosphate synthase to inhibit the osteogenic differentiation and calcification in vascular smooth muscle cells

BMC Cardiovascular Disorders volume 24, Article number: 494 (2024) Cite this article

Abstract

Background

Nitrogen-containing bisphosphonate(N-BP)had been found to inhibit the osteogenic differentiation and calcification in vascular smooth muscle cells (VSMCs), but the mechanism is not clear. We intend to verify that N-BP induces enhancement of OPG expression and inhibition of RANKL expression via inhibition of farnesyl pyrophosphate synthase(FPPS) to inhibit the osteogenic differentiation and calcification in VSMCs.

Methods

β-glycerophosphate (β-GP) was used to induce the osteogenic differentiation and calcification in VSMCs. VSMCs were treated with N-BP or pretreated with downstream products of farnesyl pyrophosphate synthase(FPPS) in mevalonate pathway, such as farnesol (FOH) or geranylgeraniol (GGOH). Alizarin red S staining and determination of calcium content were used to detect calcium deposition.Western Blotting were used to detect expressions of proteins(OPG and RANKL ) and osteogenic marker proteins (Runx2 and OPN).

Results

β-GP induced the osteogenic differentiation and calcification in VSMCs, increased RANKL protein expression and had no significant effect on OPG protein expression. With the treatment of N-BP, the expression of OPG protein was increased and expression of RANKL protein was decreased in VSMCs undergoing osteogenic differentiation and calcification. In addition, N-BP reduced the osteogenic marker proteins (Runx2 and OPN) expression and calcium deposition in VSMCs undergoing osteogenic differentiation and calcification. These effects of N-BP on the osteogenic differentiation and calcification in VSMCs were concentration-dependent, which could be reversed by the downstream products of FPPS, such as FOH or GGOH.

Conclusion

N-BP increases OPG expression and decreases RANKL expression via inhibition of FPPS to inhibit the osteogenic differentiation and calcification in VSMCs.

Peer Review reports

Background

Vascular calcification (VC) is defined as the deposition of calcium-phosphate complexes in the cardiovascular system, which increases the morbidity and mortality of cardiovascular diseases [1,2,3,4,5,6]. VC was initially thought to be a passive process, but in fact it is an active and tightly regulated process with complex mechanisms [7, 8]. The differentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells is considered to play a key role in the progression of VC [9, 10]. Therefore, it is important to explore signaling pathway to alleviate osteogenic differentiation in VSMCs, which can improve treatment options for VC. In recent years, nitrogen-containing bisphosphonate (N-BP) has been shown to have an inhibitory effect on the osteogenic differentiation and calcification in VSMCs [11, 12]. However, it is not clear how N-BP inhibits the osteogenic differentiation and calcification in VSMCs via specific regulatory mechanisms.

N-BP, such as zoledronic acid(ZOL), is the drug for the treatment of osteoporosis. Both epidemiological and clinical studies have shown that patients with low bone mineral density are at significantly increased risk of VC [13, 14]. Some studies also suggested that the drugs that are effective on bone metabolism could also be effective on VC [13, 15]. N-BP has a high affinity for bone tissue and inhibit the activity of farnesate pyrophosphate synthetase(FPPS), leading to osteoclast apoptosis [16]. FPPS is an important enzyme in mevalonate metabolic pathway, so inhibition of FPPS by N-BP can block mevalonate pathway (Fig. 1) [13, 17]. In the study of Tsubaki, N-BP increased OPG and inhibited RANKL in mouse bone marrow stromal cell [18]. At the same time, this effect of N-BP could be reversed by adding downstream products of FPPS, such as farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP) [18]. The study of Pan and Viereck also showed that N-BP increased OPG and inhibited RANKL in the human osteoblast cells [19, 20]. However, in different cells, different concentrations of N-BP can regulate OPG and RANKL in different directions [21,22,23,24]. Regarding the mechanism of osteogenic differentiation and calcification in VSMCs, RANKL can promote the osteogenic differentiation and calcification in VSMCs via the RANKL/RANK signaling pathway. OPG can bind RANKL competitively to block the RANKL/RANK signaling pathway, which plays a protective role in the osteogenic differentiation and calcification in VSMCs (Fig. 2) (26,27). If N-BP can increase OPG and inhibit RANKL via inhibition of FPPS in VSMCs, it may alleviate osteogenic differentiation and calcification in VSMCs. At present, the clinical studies did not show the protective effect of N-BP on VC [25, 26]. The current experimental studies showed the protective effect of N-BP on VC, but the mechanism is unclear [27, 28]. Therefore, we intend to verify that N-BP induces enhancement of OPG expression and inhibition of RANKL expression via inhibition of FPPS to inhibit the osteogenic differentiation and calcification in VSMCs.

Fig. 1
figure 1

Schematic diagram of the mevalonate pathway. FPPS, farnesyl pyrophosphate synthase; SQS, squalene synthase; FNT, farnesyltransferase; GGT-1, geranylgeranyltransferase-1; GGPPS, geranylgeranyl pyrophosphate synthase; N-BPs, nitrogen-containing bisphosphonatesand; FOH, farnesol; GGOH, geranylgeraniol

Full size image
Fig. 2
figure 2

The role of RANKL/OPG in vascular calcification

Full size image

Methods

Cells culture and interventions

The VSMCs (Rat aortic thoracic smooth muscle cells, A7r5) were bought from Cell Bank, Chinese Academy of Sciences. The VSMCs were cultured in DMEM medium (Gibco, USA) containing 10% FBS (ExCell, China) at 37 °C in a 5% CO2 incubator.The culture medium was updated every 2–3 days.VSMCs less than 10 passages were used for experiments. To establish a model of osteogenic differentiation and calcification in VSMCs, the VSMCs were induced in DMEM medium containing 10 mM β-glycerophosphate (β-GP) (Sigma-Aldrich, USA) for 72 h. In some experiments, the VSMCs were preincubated for 2 h with 1 µM or 5 µM ZOL (MedChemExpress, USA), then VSMCs were co-cultured with or without 10 mM β-GP for 72 h. Furthermore, in some other experiments, the VSMCs were preincubated for 2 h with 5 µM ZOL, 30 µM farnesol (FOH)(Sigma-Aldrich, USA) plus 5 µM ZOL or 30 µM geranylgeraniol (GGOH)(Sigma-Aldrich, USA) plus 5 µM ZOL, then the VSMCs were co-cultured with or without 10 mM β-GP for 72 h. In the control group, the VSMCs were not given any intervention.

Alizarin red S staining

Alizarin red S staining was performed to detect calcium deposition. The VSMCs were washed three times with Phosphate-Buffered Saline (PBS)(Beyotime, China), fixed in 4% paraformaldehyde for 30 min at room temperature, then stained with 1% Alizarin red S solution (Solarbio, China) for 5 min at room temperature. Subsequently, the VSMCs were washed with distilled water. The formation of calcified purple-red spots was quantified by microscopy(Olympus, Japan).

Determination of calcium content

The calcium contents were determined by Calcium Assay Kit (Beyotime, China) according to the manufacturer’s instructions. 100–200 µl sample lysate was added to each well of the 6-well plate. The VSMCs were fully lysed and the supernatant was separated by centrifugation. The o-cresolphthalein complexone and detection buffer were mixed 1:1 to prepare the detection working solution for use. Then,50 µl sample and 150 µl detection working solution were added to each well of the 96-well plate and mixed well. The absorbance was assessed at 575 nm using an enzyme-labeled instrument. The total protein concentration was determined by BCA Protein Assay. The relative calcium content normalized to the protein concentration was expressed as µg/mg protein.

Western blotting

Total protein was extracted from the VSMCs using RIPA lysis buffer (Beyotime, China) supplemented with protease inhibitor (Beyotime, China). The protein concentrations were detected using a BCA protein assay kit (Beyotime, China). Equal amounts of protein lysates were loaded and separated on a 10% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes (Beyotime, China). After blocking with 5% nonfat milk (diluted in Tris-buffered saline with Tween-20) for 2 h at room temperature, the membranes were incubated with primary antibodies at 4 °C overnight and incubated with secondary antibodies for 2 h at room temperature. The primary antibodies were as follows: Anti-RANKL (1:1000, Proteintech, China), Anti-OPG (1:1000, ABclonal, China), Anti-RUNX2 (1:1000, Proteintech, China), OPN (1:2000, Proteintech, China), GAPDH (1:2000, Proteintech, China). The secondary antibodies were as follows: HRP Goat Anti-Mouse IgG (1:5000, ABclonal, China) and HRP Goat Anti-Mouse IgG (1:5000, ABclonal, China). Proteins were detected by ECL chemiluminescence detection reagent (vazyme, China) and Amersham Imager 600 (GE Healthcare, UK). Western blotting results were quantitated using Image J software. Protein expression was normalized to GAPDH.

Statistical analysis

All the data were continuous data and presented as mean ± SD. All results were obtained from 3 identical independent experiments. Differences between two groups were compared using Student’s t-test. The Student’s t-test was used to verify the induction of osteogenic differentiation and calcification model in VSMCs by β-GP. Differences among more than two groups were compared using one-way ANOVA. The one-way ANOVA was used to verify the mechanism and effect of N-BP on osteogenic differentiation and calcification in VSMCs. All statistical analyses were performed by use of SPSS 20.0 software. The graphs were plotted by GraphPad Prism 8.0 software. P < 0.05 was considered as statistically significant.

Results

Induction of osteogenic differentiation and calcification model in VSMCs by β-GP

To establish osteogenic differentiation and calcification model in VSMCs, we stimulated VSMCs with 10mM β-GP for 72 h. The calcium deposition(purple-red spots)was induced in VSMCs at 72 h (Fig. 3A). The calcium contents in the β-GP group were also higher than that in control group (Fig. 3B, p < 0.01). The expression levels of osteogenic marker proteins Runx2 and OPN in the β-GP groupwere elevated in comparison to the control group (Fig. 3C, D, p < 0.05). Meanwhile, the expression levels of RANKL in the β-GP group were elevated in comparison to the control group (Fig. 3C, 3D, p < 0.05), but the expression levels of OPG in the β-GP group were not significantly higher than that in the control group (Fig. 3C, 3D, p > 0.05).

Fig. 3
figure 3

Induction of osteogenic differentiation and calcification model in VSMCs. VSMCs were treated with or without 10 mM β-GP for 72 h. (A) Alizarin Red S Staining was used to assess VSMCs calcification. Representative images showed VSMCs calcification with purple-red spots. Scale bar = 10 μm. (B) Calcium content was detected by Calcium Assay Kit. (C, D) Representative western blotting for RANKL, OPG and calcification-related proteins( Runx2 and OPN). Statistical significance was analyzed by the t-test (*p < 0.05, **p < 0.01, ***p < 0.001). The data is represented as mean ± SD (n = 3)

Full size image

Effect of N-BP on VSMCs calcification

ZOL (a type of N-BP) reduced the purple-red calcium deposition induced by β-GP in VSMCs at 72 h. Compared with 1µM ZOL, 5µM ZOL had a more obvious effect on reducing the purple-red calcium deposition in calcified VSMCs (Fig. 4A). Meanwhile, ZOL reduced the calcium contents in calcified VSMCs at 72 h (Fig. 4B, p < 0.05).Compared with 1µM ZOL, 5µM ZOL had a more obvious effect on reducing the calcium contents in calcified VSMCs (Fig. 4B, p < 0.01).

Fig. 4
figure 4

Effect of N-BP on VSMCs calcification. VSMCs were preincubated for 2 h with 1 µM or 5 µM ZOL, then VSMCs were co-cultured with or without 10 mM β-GP for 72 h. (A) Alizarin Red S Staining was used to assess VSMCs calcification. Representative images showed VSMCs calcification with purple-red spots. Scale bar = 10 μm. (B) Calcium content was detected by Calcium Assay Kit. Statistical significance was analyzed by one-way ANOVA (*p < 0.05, **p < 0.01, ****p < 0.0001). The data is represented as mean ± SD (n = 3)

Full size image

Effect of N-BP on RANKL, OPG and osteogenic marker proteins (Runx2 and OPN) expression in VSMCs of osteogenic differentiation and calcification

ZOL reduced the protein expression levels of RANKL in VSMCs undergoing osteogenic differentiation and calcification at 72 h (Fig. 5A, B, p < 0.05). Compared with 1µM ZOL, 5µM ZOL had a more obvious effect in reducing the protein expression levels of RANKL (Fig. 5A, B, p < 0.01). The protein expression levels of OPG in the β-GP group were slightly higher than that in the control group at 72 h, but the difference was not statistically significant (Fig. 5A, C, p > 0.05). Compared with the control group, ZOL plus β-GP group had the higher protein expression levels of OPG at 72 h (Figs. 5A, 3C, p < 0.05). Compared with the β-GP group, 1µM ZOL plus β-GP group increased the protein expression levels of OPG at 72 h, but the difference was not statistically significant (Fig. 5A, C, p > 0.05). Compared with the β-GP group, 5µM ZOL plus β-GP group had the higher protein expression levels of OPG at 72 h (Fig. 5A, C, p < 0.05). In addition, ZOL reduced the osteogenic marker proteins Runx2 and OPN in VSMCs undergoing osteogenic differentiation and calcification at 72 h (Fig. 5A, D. 5E, p < 0.05). Compared with 1µM ZOL, 5µM ZOL had a more obvious effect in reducing the protein expression levels of Runx2 and OPN (Fig. 5A, D. 5E, p < 0.05).

Fig. 5
figure 5

Effect of N-BP on RANKL, OPG and osteogenic marker proteins (Runx2 and OPN) expression in VSMCs of osteogenic differentiation and calcification. VSMCs were preincubated for 2 h with 1 µM or 5 µM ZOL, then VSMCs were co-cultured with or without 10 mM β-GP for 72 h. Statistical significance was analyzed by the one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). The data is represented as mean ± SD (n = 3)

Full size image

Effect of N-BP on VSMCs calcification due to inhibition of FPPS

In order to reverse the inhibition effect of N-BP on VSMCs calcification due to inhibition of FPPS, we added the downstream products of FPPS in mevalonate pathway, such as FOH or GGOH. ZOL reduced the purple-red calcium deposition induced by β-GP in VSMCs at 72 h, which was reversed by FOH or GGOH (Fig. 6A). Meanwhile, ZOL reduced the calcium contents in calcified VSMCs at 72 h (Fig. 6B, p < 0.0001), which was also reversed by FOH or GGOH (Fig. 4B, p < 0.01).

Fig. 6
figure 6

Effect of N-BP on VSMCs calcification due to inhibition of FPPS. VSMCs were preincubated for 2 h with 5 µM ZOL, 30 µM FOH plus 5 µM ZOL or 30 µM GGOH plus 5 µM ZOL, then VSMCs were co-cultured with or without 10 mM β-GP for 72 h. FOH and GGOH were the downstream products of FPPS in mevalonate pathway. (A) Alizarin Red S Staining was used to assess VSMCs calcification. Representative images showed VSMCs calcification with purple-red spots. Scale bar = 10 μm. (B) Calcium content was detected by Calcium Assay Kit. Statistical significance was analyzed by one-way ANOVA (*p < 0.05, **p < 0.01, ****p < 0.0001). The data is represented as mean ± SD (n = 3)

Full size image

Effect of N-BP on RANKL, OPG and osteogenic marker proteins( Runx2 and OPN) expression in VSMCs undergoing osteogenic differentiation and calcification due to inhibition of FPPS

In order to reverse the regulatory effect on RANKL, OPG and osteogenic marker proteins( Runx2 and OPN) expression in VSMCs of osteogenic differentiation and calcification due to inhibition of FPPS, we added the downstream products of FPPS in mevalonate pathway such as FOH or GGOH. 5µM ZOL induced enhancement of OPG protein expression and inhibition of RANKL protein expression in VSMCs undergoing osteogenic differentiation and calcification at 72 h (Fig. 7A, B, C, p < 0.001), which was reversed by FOH or GGOH (Fig. 7A, B, C, p < 0.05). In addition, 5µM ZOL reduced the osteogenic marker proteins Runx2 and OPN in VSMCs undergoing osteogenic differentiation and calcification at 72 h (Fig. 7A, D, E, p < 0.0001), which was reversed by FOH or GGOH (Fig. 7A, D, E, p < 0.01).

Fig. 7
figure 7

Effect of N-BP on RANKL, OPG and osteogenic marker proteins( Runx2 and OPN) expression in VSMCs of osteogenic differentiation and calcification due to inhibition of FPPS. VSMCs were preincubated for 2 h with 5 µM ZOL, 30 µM FOH plus 5 µM ZOL or 30 µM GGOH plus 5 µM ZOL, then VSMCs were co-cultured with or without 10 mM β-GP for 72 h. FOH and GGOH were the downstream products of FPPS in mevalonate pathway. Statistical significance was analyzed by the one-way ANOVA (*p < 0.01, **p < 0.01, ***p < 0.001, ****p < 0.0001). The data is represented as mean ± SD (n = 3)

Full size image

Discussion

In this study, we investigated the effect and mechanism of N-BP on the osteogenic differentiation and calcification in VSMCs. In terms of the osteogenic differentiation and calcification in VSMCs (Figure 2), previous studies showed that Runx2 was a key marker to drive this phenotypic differentiation. Runx2 is usually up-regulated during the osteogenic differentiation in VSMCs, promotes the expression of downstream osteogenic marker such as OPN, promotes the secretion of calcified vesicles, and finally leads to the deposition of calcium-phosphate complexes in the vascular wall [29, 30]. In adition, previous studies showed RANKL could promote the osteogenic differentiation and calcification via the RANKL/RANK signaling pathway in VSMCs, which also could up-regulate the expression of Runx2 and OPN [31, 32]. OPG can bind RANKL competitively to block the osteogenic differentiation and calcification in VSMCs [31, 32].

β-GP is often used to induce the osteogenic differentiation and calcification in VSMCs. As shown in Fig. 3 and 10mM β-GP could induce the osteogenic differentiation and calcification in VSMCs at 72 h, which was consistent with the result of Huqiang He [33]. Meanwhile, the expression of RANKL protein was up-regulated in VSMCs undergoing the osteogenic differentiation and calcification induced by β-GP, which was consistent with the result of Jinmi Lee and Liu [34, 35]. RANKL is a molecule associated with inflammation and previous study had found that expression of RANKL was increased in calcified arteries [36]. The expression of OPG protein was slight up-regulated in VSMCs undergoing osteogenic differentiation and calcification induced by β-GP, but the expression of OPG protein in the β-GP group had no statistical significance compared with that in the control group. In terms of the OPG expression during the osteogenic differentiation and calcification in VSMCs, the previous studies had no consensus. In the study of Huqiang He, the expression of OPG protein was up-regulated during the osteogenic differentiation and calcification induced by β-GP in VSMCs [33]. In the study of Yang Ho Kang, the expression of OPG protein was down-regulated slightly during the osteogenic differentiation and calcification induced by β-GP at 4 weeks in VSMCs [37]. It’s important to note that the expression of OPG protein was up-regulated slightly during the osteogenic differentiation and calcification induced by β-GP at 2 weeks in VSMCs in the study of Yang Ho Kang [37]. Previous study also found that patients with VC had elevated serum OPG [38]. Some scholars speculated that the expression of OPG in VSMCs was elevated as a defensive manner against VC for short-term stimulation in calcification medium [37, 38]. However, but the expression of OPG was down-regulated after long term stimulation with high phosphate, which could reduce the binding of OPG to RANKL and accelerate VC [38]. In our study, the VSMCs were only co-cultured with by β-GP for 72 h, so the stimulation of the VSMCs by the high phosphorus was short-term and the expression of OPG protein was up-regulated slightly.

In our study, ZOL, a kind of N-BP, inhibited the osteogenic differentiation and calcification in a concentration-dependent manner in VSMCs. The concentrations of ZOL we chosed were 1 and 5 µM, because previous study showed that a peak serum concentration of ZOL in human body following a 4 mg dose administration was only from 1 to 5 µM [39]. In our preliminary experiment, we found that the number of VSMCs co-cultured with 10–100 µM ZOL for 72 h was significantly reduced, so we did not choose ZOL > 5 µM to explore the effect on osteogenic differentiation and calcification in VSMCs. Meanwhile, we found ZOL increased the expression of OPG protein and decreased the expression of RANKL protein in VSMCs undergoing osteogenic differentiation and calcification, which could down-regulate the expression of osteogenic marker protein Runx2 and OPN to inhibit the osteogenic differentiation and calcification in VSMCs. The previous studies showed that other kind of N-BP could also inhibit the osteogenic differentiation and calcification in VSMCs [11, 12], but the researchers did not find that N-BP inhibited this phenotypic differentiation via enhancement of OPG expression and inhibition of RANKL expression. The previous study also found N-BP induced enhancement of OPG expression and inhibition of RANKL expression in mouse bone marrow stromal cells [18]. However, some studies found that the regulatory effects of different concentrations of N-BP on OPG and RANKL in different cells are unclear [21,22,23,24]. Our experiment confirmed that 1–5 µM N-BP could increased the expression of OPG and decreased the expression of RANKL in VSMCs, which could inhibit osteogenic differentiation and calcification in VSMCs finally. N-BP itself has an inhibitory effect on FPPS.Thus, by the addition of FPPS downstream products such as FOH or GGOH in mevalonate pathway, we further verified in reverse that N-BP regulated OPG and RANKL via inhibiting FPPS. The previous study showed that the involvement of FPPS downstream products in prenylation of small GTPases (Ras and Rho) was clarified and these mall GTPases might play a major role in regulated OPG and RANKL [18]. In addition, most clinical studies found that N-BP did not inhibit vascular calcification in human [13, 40], which might be because the serum concentration of N-BP cannot be maintained consistently at 1–5 μm.

There were some limitations in our study. Firstly, this study was conducted in vitro. We still need to conduct experiments in vivo to more fully verify protective role and mechanism of N-BP on VC. Secondly, in this experiment, by adding downstream products of FPPS, N-BP was reversely verified to regulate RANKL and OPG by inhibiting FPPS. We hope to use more direct methods to explore the regulation of OPG and RANKL by FPPS in VSMCs in subsequent studies.

In conclusion, our findings demonstrated the protective role and mechanism of N-BP on the osteogenic differentiation and calcification in VSMCs. N-BP could induce enhancement of OPG expression and inhibition of RANKL expression via inhibition of FPPS, which could inhibit the osteogenic differentiation and calcification in VSMCs. Although the current clinical study did not show the protective effect of N-BP on VC, our experiment found that the regulation of N-BP on RANKL and OPG through inhibiting FPPS had reference value for future clinical treatment of VC. Perhaps, in the future, we can explore the treatment of VC in terms of N-BP drug dose, N-BP drug affinity to VSMCs, and FPPS enzyme downstream products.

Data availability

Data is provided within the manuscript or supplementary information files.

Abbreviations

VC:

Vascular calcification

VSMCs:

Vascular smooth muscle cells

N-BP:

Nitrogen-containing bisphosphonate

ZOL:

Zoledronic acid

FPPS:

Farnesate pyrophosphate synthetase

FPP:

Farnesyl pyrophosphate

GGPP:

Geranylgeranyl pyrophosphate

FOH:

Farnesol

GGOH:

Geranylgeraniol

β-GP:

β-glycerophosphate

References

  1. Feng S, Qi Y, Xiao Z, et al. CircHIPK3 relieves vascular calcification via mediating SIRT1/PGC-1α/MFN2 pathway by interacting with FUS. BMC Cardiovasc Disord. 2023;23(1):583.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Tsai S, Li Y, Wu X. Serum magnesium level and cardiac valve calcification in hemodialysis patients. BMC Cardiovasc Disord. 2023;23(1):610.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Urbain F, Ponnaiah M, Ichou F, et al. Impaired metabolism predicts coronary artery calcification in women with systemic lupus erythematosus. EBioMedicine. 2023;96:104802.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Sun M, Fang Y, Zheng J, et al. Role of symbiotic microbiota dysbiosis in the progression of chronic kidney disease accompanied with vascular calcification. Front Pharmacol. 2024;14:1306125.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Tian XX, Luo JY, Liu F, et al. Prognostic value of fibrinogen-to-albumin ratio combined with coronary calcification score in patients with suspected coronary artery disease. BMC Cardiovasc Disord. 2023;23(1):181.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Huang EP, Lin HS, Chen YC, et al. Lower attenuation and higher kurtosis of coronary artery calcification associated with vulnerable plaque - an agatston score propensity-matched CT radiomics study. BMC Cardiovasc Disord. 2023;23(1):158.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Lee SJ, Lee IK, Jeon JH. Vascular calcification-new insights into its mechanism. Int J Mol Sci. 2020;21(8):2685.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Ye D, Liu Y, Pan H, et al. Insights into bone morphogenetic proteins in cardiovascular diseases. Front Pharmacol. 2023;14:1125642.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Li J, Li C, Huang Z, et al. Empagliflozin alleviates atherosclerotic calcification by inhibiting osteogenic differentiation of vascular smooth muscle cells. Front Pharmacol. 2023;14:1295463.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Jaminon A, Reesink K, Kroon A, Schurgers L. The role of vascular smooth muscle cells in arterial remodeling: focus on osteogenic marker processes. Int J Mol Sci. 2019;20(22):5694.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Cutini PH, Rauschemberger MB, Sandoval MJ, Massheimer VL. Vascular action of bisphosphonates: in vitro effect of alendronate on the regulation of cellular events involved in vessel pathogenesis. J Mol Cell Cardiol. 2016;100:83–92.

    Article  PubMed  CAS  Google Scholar 

  12. Zhou S, Fang X, Xin H, Guan S. Effects of alendronate on the Notch1–RBP–Jκ signaling pathway in the osteogenic differentiation and mineralization of vascular smooth muscle cells. Mol Med Rep. 2013;8(1):89–94.

    Article  PubMed  CAS  Google Scholar 

  13. Xu W, Lu G, Gong L, et al. Non-nitrogen-containing bisphosphonates and nitrogen-containing bisphosphonates for the treatment of atherosclerosis and vascular calcification: a meta-analysis. Med (Baltim). 2024;103(23):e38404.

    Article  CAS  Google Scholar 

  14. Sharif N, Gilani SZ, Suter D, et al. Machine learning for abdominal aortic calcification assessment from bone density machine-derived lateral spine images. EBioMedicine. 2023;94:104676.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Suzuki S, Suzuki M, Hanafusa N, Tsuchiya K, Nitta K. Denosumab recovers aortic Arch Calcification during Long-Term Hemodialysis. Kidney Int Rep. 2020;6(3):605–12.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Ebetino FH, Sun S, Cherian P, et al. Bisphosphonates: the role of chemistry in understanding their biological actions and structure-activity relationships, and new directions for their therapeutic use. Bone. 2022;156:116289.

    Article  PubMed  CAS  Google Scholar 

  17. Manaswiyoungkul P, de Araujo ED, Gunning PT. Targeting prenylation inhibition through the mevalonate pathway. RSC Med Chem. 2019;11(1):51–71.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Tsubaki M, Satou T, Itoh T, et al. Bisphosphonate- and statin-induced enhancement of OPG expression and inhibition of CD9, M-CSF, and RANKL expressions via inhibition of the Ras/MEK/ERK pathway and activation of p38MAPK in mouse bone marrow stromal cell line ST2. Mol Cell Endocrinol. 2012;361(1–2):219–31.

    Article  PubMed  CAS  Google Scholar 

  19. Viereck V, Emons G, Lauck V, et al. Bisphosphonates pamidronate and zoledronic acid stimulate osteoprotegerin production by primary human osteoblasts. Biochem Biophys Res Commun. 2002;291(3):680–6.

    Article  PubMed  CAS  Google Scholar 

  20. Pan B, Farrugia AN, To LB, et al. The nitrogen-containing bisphosphonate, zoledronic acid, influences RANKL expression in human osteoblast-like cells by activating TNF-alpha converting enzyme (TACE). J Bone Min Res. 2004;19(1):147–54.

    Article  CAS  Google Scholar 

  21. Kim HJ, Kim HJ, Choi Y, et al. Zoledronate enhances osteocyte-mediated osteoclast differentiation by IL-6/RANKL Axis. Int J Mol Sci. 2019;20(6):1467.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Walter C, Klein MO, Pabst A, et al. Influence of bisphosphonates on endothelial cells, fibroblasts, and osteogenic cells. Clin Oral Investig. 2010;14(1):35–41.

    Article  PubMed  CAS  Google Scholar 

  23. Maruotti N, Corrado A, Neve A, Cantatore FP. Bisphosphonates: effects on osteoblast. Eur J Clin Pharmacol. 2012;68(7):1013–8.

    Article  PubMed  CAS  Google Scholar 

  24. Rachner TD, Singh SK, Schoppet M, et al. Zoledronic acid induces apoptosis and changes the TRAIL/OPG ratio in breast cancer cells. Cancer Lett. 2010;287(1):109–16.

    Article  PubMed  CAS  Google Scholar 

  25. Torregrosa JV, Fuster D, Gentil MA, et al. Open-label trial: effect of weekly risedronate immediately after transplantation in kidney recipients. Transplantation. 2010;89(12):1476–81.

    Article  PubMed  CAS  Google Scholar 

  26. Okamoto M, Yamanaka S, Yoshimoto W, et al. Alendronate as an effective treatment for bone loss and vascular calcification in kidney transplant recipients. J Transpl. 2014;2014:269613.

    Google Scholar 

  27. Synetos A, Toutouzas K, Benetos G, et al. Catheter based inhibition of arterial calcification by bisphosphonates in an experimental atherosclerotic rabbit animal model. Int J Cardiol. 2014;176(1):177–81.

    Article  PubMed  Google Scholar 

  28. Synetos A, Toutouzas K, Drakopoulou M, et al. Inhibition of aortic valve calcification by local delivery of Zoledronic Acid-an experimental study. J Cardiovasc Transl Res. 2018;11(3):192–200.

    Article  PubMed  Google Scholar 

  29. Quaglino D, Boraldi F, Lofaro FD. The biology of vascular calcification. Int Rev Cell Mol Biol. 2020;354:261–353.

    Article  PubMed  Google Scholar 

  30. Durham AL, Speer MY, Scatena M, et al. Role of smooth muscle cells in vascular calcification: implications in atherosclerosis and arterial stiffness. Cardiovasc Res. 2018;114(4):590–600.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Liu H, Ru NY, Cai Y, et al. The OPG/RANKL/RANK system modulates calcification of common carotid artery in simulated microgravity rats by regulating NF-κB pathway. Can J Physiol Pharmacol. 2022;100(4):324–33. https://doi.org/10.1139/cjpp-2021-0329.

    Article  PubMed  CAS  Google Scholar 

  32. Zhao L, Wang S, Liu H, et al. The pharmacological effect and mechanism of Lanthanum Hydroxide on vascular calcification caused by chronic renal failure hyperphosphatemia. Front Cell Dev Biol. 2021;9:639127.

    Article  PubMed  PubMed Central  Google Scholar 

  33. He HQ, Law BYK, Zhang N, et al. Bavachin Protects Human Aortic Smooth Muscle Cells against β-Glycerophosphate-Mediated Vascular Calcification and apoptosis via activation of mTOR-Dependent autophagy and suppression of β-Catenin signaling. Front Pharmacol. 2019;10:1427.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Lee J, Hong SW, Kim MJ, et al. Metformin, resveratrol, and exendin-4 inhibit high phosphate-induced vascular calcification via AMPK-RANKL signaling. Biochem Biophys Res Commun. 2020;530(2):374–80.

    Article  PubMed  CAS  Google Scholar 

  35. Liu SY, Meng XF, Liu SW, Hao CL, Li LF, Zhang N. Effect of Bushen Huoxue decoction on inhibiting osteogenic differentiation of vascular smooth cells by regulating OPG/RANK/RANKL system in vascular calcification. Ann Transl Med. 2019;7(6):125.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Kawakami R, Nakagami H, Noma T, Ohmori K, Kohno M, Morishita R. RANKL system in vascular and valve calcification with aging. Inflamm Regen. 2016;36:10.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Kang YH, Jin JS, Son SM. Long term effect of high glucose and phosphate levels on the OPG/RANK/RANKL/TRAIL system in the Progression of Vascular Calcification in rat aortic smooth muscle cells. Korean J Physiol Pharmacol. 2015;19(2):111–8.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Makarović S, Makarović Z, Steiner R, Mihaljević I, Milas-Ahić J. Osteoprotegerin and vascular calcification: clinical and prognostic relevance. Coll Antropol. 2015;39(2):461–8.

    PubMed  Google Scholar 

  39. Wu L, Zhu L, Shi WH, Zhang J, Ma D, Yu B. Zoledronate inhibits the proliferation, adhesion and migration of vascular smooth muscle cells. Eur J Pharmacol. 2009;602(1):124–31.

    Article  PubMed  CAS  Google Scholar 

  40. Cai G, Keen HI, Host LV, et al. Once-yearly zoledronic acid and change in abdominal aortic calcification over 3 years in postmenopausal women with osteoporosis: results from the HORIZON Pivotal Fracture Trial. Osteoporos Int. 2020;31(9):1741–7.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

The research was supported by Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy.

Funding

The research was supported by the Open Research Project of Key Laboratories in Jiangsu Province Universities (No.XZSYSKF2023022), the Young Talent Development Plan of Changzhou Health Commission (No.CZQM2021026) and the Guiding planning Project of Qinghai Health Commission.

Author information

Authors and Affiliations

  1. Department of Nephrology, First Affiliated Hospital of Soochow University, Suzhou, 215006, Jiangsu, China

    Wei Xu & Guoyuan Lu

  2. Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, Xuzhou, 221004, Jiangsu, China

    Wei Xu, Lifeng Gong & Weigang Tang

  3. Department of Nephrology, Wujin Hospital Affiliated with Jiangsu University, Changzhou, 213000, Jiangsu, China

    Wei Xu, Lifeng Gong & Weigang Tang

  4. Department of Nephrology, People’s Hospital of Hainan Tibetan Autonomous Prefecture, Hainan Tibetan Autonomous Prefecture, Qinghai, 813099, China

    Wei Xu

Authors
  1. Wei Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lifeng Gong
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Weigang Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Guoyuan Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Wei Xu and Guoyuan Lu contributed to the conceptualization. Wei Xu and Lifeng Gong contributed to the all cell experiments. Wei Xu and Weigang Tang contributed to the analysis of the data and production of figures and tables. Wei Xu and Guoyuan Lu contributed to the writing. All authors approved final manuscript.

Corresponding author

Correspondence to Guoyuan Lu.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Xu, W., Gong, L., Tang, W. et al. Nitrogen-containing bisphosphonate induces enhancement of OPG expression and inhibition of RANKL expression via inhibition of farnesyl pyrophosphate synthase to inhibit the osteogenic differentiation and calcification in vascular smooth muscle cells. BMC Cardiovasc Disord 24, 494 (2024). https://doi.org/10.1186/s12872-024-04048-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12872-024-04048-x

Keywords

BMC Cardiovascular Disorders

ISSN: 1471-2261

Contact us