Wnt1 inhibits vascular smooth muscle cell calcification by promoting ANKH expression

doi:10.1016/j.yjmcc.2019.07.008

Journal of Molecular and Cellular Cardiology

Volume 135, October 2019, Pages 10-21

https://doi.org/10.1016/j.yjmcc.2019.07.008 Get rights and content

Highlights

•
Novel mechanism of Wnt1 protects against vascular calcification.
•
ANKH, a vascular calcification inhibitor, is a target gene of Wnt1/β-catenin signal.
•
Agonist of Wnt/β-catenin signal is a potential medicine for vascular calcification.

Abstract

Aims

Wnt signaling plays a critical role in vascular calcification (VC). Wnt factors induce different physiological and pathological effects on cardiovascular functions. Wnt1, a ligand of Wnt/β-catenin signaling, promotes pro-angiogenesis and reduces myocardial infarction. The role of Wnt1 on VC in chronic kidney disease (CKD) is not fully understood.

Methods and results

We used human vascular smooth muscle cells (VSMCs) and a rat model of chronic renal failure (CRF), and observed a native protective mechanism by which VC is reduced via the activation of Wnt1 and its transcriptional target ANKH inorganic pyrophosphate transport regulator (ANKH) gene. ANKH is an essential calcification inhibitor that effluxes inorganic pyrophosphate (PPi) from VSMCs to play an inhibitory role in VC. Vascular ANKH and plasma PPi were significantly downregulated in the rat model of CRF. The knockdown or inhibition of ANKH reversed the effect of Wnt1 on VC in VSMCs. Clinical analysis revealed low plasma levels of Wnt1 and PPi were associated with CKD in patients. Applying a Wnt/β-catenin signaling agonist can alleviate the progression of VC.

Conclusion

This work reveals the ANKH regulation of Wnt1 in VSMCs is essential for blocking VC. Our findings may contribute to the development of medications that target Wnt signaling and/or ANKH to inhibit VC.

Introduction

Cardiovascular disease (CVD) is a major cause of morbidity and mortality in patients with chronic kidney disease (CKD), especially those with end-stage renal disease (ESRD) [1,2]. Vascular calcification (VC) is the most apparent cardiovascular abnormality in patients with CKD [3]. Much attention has been focused on its etiology, mechanisms, and consequences [4,5].

The Wnt/β-catenin signaling pathway plays a critical role in CVD. Wnt ligands (Wnt2, Wnt3a, Wnt5a, Wnt7a, and Wnt7b) that are driven by Msx2 were shown to positively regulate VC in a diabetic mouse model [6,7]. However, Wnt/β-catenin signaling was suppressed in the aorta during the calcification process in uremic rats [8]. Clinical CKD-mineral and bone disorder (MBD) was attributed to higher circulating Wnt signaling inhibitor, Dickkopf-1 (Dkk1) [9]. Another Wnt inhibitor, sclerostin (SOST), has been reported increased in CKD patients and was an independent risk factor for VC [[10], [11], [12]]. Altogether, these studies indicate that different Wnt ligands and inhibitors may exert various effects on vascular cells in VC. Wnt1, a ligand of Wnt/β-catenin signaling, is reported to be a proangiogenic molecule [13] and can prevent myocardial infarction [14]. An autosomal-dominant mutation of the LRP6 gene (i.e., a co-receptor of Wnt1 and other Wnts) can lead to early coronary artery disease [15]. Nevertheless, the role of Wnt1 in VC is unknown.

Vascular calcification is a highly regulated vascular smooth muscle cell (VSMC)-mediated process. The etiology of VC involves the osteogenic differentiation of VSMCs [16] and the regulation of native protectors, including ANKH inorganic pyrophosphate transport regulator (ANKH), matrix Gla protein (MGP), ecto-nucleotide pyrophosphatase phosphodiesterase 1 (ENPP1), and Fetuin-A, among others [17]. Wnt1 regulates the proliferation of VSMCs [18]. Unknown, however, is whether Wnt1 regulates VC inhibition.

Among the above native protectors of VC is ANKH, which is a multipass transmembrane protein that mediates the intracellular-to-extracellular channeling of inorganic pyrophosphate (PPi). ANKH is positively regulated by Wnt1 and a direct Wnt transcriptional target that mediates the inhibitory effect of Wnt1 on VC. ANKH mutant mice exhibited pathological calcification in the tunica media of the aorta [19,20]. We previously found that ANKH mediates nuclear factor-κB signaling in VC [21], suggesting that ANKH is a central mediator of signaling pathways in VC. Furthermore, both in vitro and in vivo data indicated that Wnt signaling agonism may be therapeutically beneficial for counteracting the development of CKD-related VC.

Section snippets

Materials

Sodium phosphate (Na₂HPO₄ and NaH₂PO₄) and anti-transcription factor II B (TFIIB) antibody were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Minimum essential medium-α, Dulbecco's modified Eagle's medium, fetal bovine serum, and penicillin-streptomycin were obtained from Gibco (Grand Island, NE, USA). TRIzol was obtained from Invitrogen (Carlsbad, CA, USA). Alizarin red S, cetylpyridinium chloride, and adenine were purchased from Amresco (Cochran Solon, OH, USA). Probenecid, PCR

Wnt1 abrogated Pi-induced VSMC calcification in vitro

To study the role of Wnt1 on VC, we first established an in vitro calcifying model of VSMCs that mimicked systemic hyperphosphatemia in end-stage renal disease patients by employing inorganic phosphate (Pi) [24,25]. As shown in Fig. 1A, Pi dose-dependently intensified human VSMC calcification. A significant elevation of SOST expression was observed with Pi induction in both protein and mRNA level (Fig. 1B, C), which was consistent with previous studies [9,26]. To investigate whether Wnt1 also

Discussion

The present study sought to identify a mechanism that counteracts osteogenic differentiation in the regulation of pathological calcium deposition in the vasculature in CKD. We found that Wnt1, through Wnt/β-catenin signaling, attenuated VC in vitro and in vivo by regulating ANKH expression in human VSMCs.

Medial artery calcification, a major cause of vascular stiffness, has a poor prognosis for CKD patients and independently portends mortality in patients with end-stage renal disease [4,35]. To

Funding

This work was supported by research grants from the National Natural Science Foundation of China [grant number 81270854, 30900627, and 81700648]; Beijing Hospital Nova Project [grant number BJ-2016-032]; and the Beijing Municipal Commission of Science and Technology [grant number Z151100004015083].

Declaration of Competing Interest

This research was sponsored by the National Natural Science Foundation of China and may lead to the development of medications for VC that may be patented by Cun-Yu Wang and Gexin Zhao.

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¹: These authors contributed equally to this work.

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Original articleWnt1 inhibits vascular smooth muscle cell calcification by promoting ANKH expression

Highlights

Abstract

Aims

Methods and results

Conclusion

Introduction

Section snippets

Materials

Wnt1 abrogated Pi-induced VSMC calcification in vitro

Discussion

Funding

Declaration of Competing Interest

Kidney Int.

Atherosclerosis.

Kidney Int.

Regul. Pept.

Kidney Int.

Dev. Biol.

Cell.

Dev. Cell

J. Biol. Chem.

Lancet.

Bone.

Chronic kidney disease and the risk for cardiovascular disease, renal replacement, and death in the United States Medicare population, 1998 to 1999

J. Am. Soc. Nephrol.

Coronary-artery calcification in young adults with end-stage renal disease who are undergoing dialysis

N. Engl. J. Med.

Determinants of coronary vascular calcification in patients with chronic kidney disease and end-stage renal disease: a systematic review

J Nephrol.

Arterial media calcification in end-stage renal disease: impact on all-cause and cardiovascular mortality

Nephrol. Dial. Transplant.

Vascular calcification in chronic kidney disease: an update

Nephrol. Dial. Transplant.

Msx2 promotes cardiovascular calcification by activating paracrine Wnt signals

J. Clin. Invest.

Targeted reduction of vascular Msx1 and Msx2 mitigates arteriosclerotic calcification and aortic stiffness in LDLR-deficient mice fed diabetogenic diets

Diabetes.

Chondrocyte rather than osteoblast conversion of vascular cells underlies medial calcification in uremic rats

Arterioscler. Thromb. Vasc. Biol.

CKD-induced wingless/integration1 inhibitors and phosphorus cause the CKD-mineral and bone disorder

J. Am. Soc. Nephrol.

Independent association between serum sclerostin levels and carotid artery atherosclerosis in prevalent haemodialysis patients

Clin. Kidney J.

Original article
Wnt1 inhibits vascular smooth muscle cell calcification by promoting ANKH expression