Original articleWnt1 inhibits vascular smooth muscle cell calcification by promoting ANKH expression
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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2020, Acta HistochemicaCitation Excerpt :Furthermore, we elucidated the effects of miR-155-5p on the development of VC in vivo. Chen et al (Chen et al., 2019a) used human VSMCs and a rat model of chronic renal failure to find a native protective mechanism that VC was suppressed through the activation of Wnt1. Zheng et al (Zheng et al., 2016) found that miR-297a was decreased in VC rats and miR-297a could bind with fibroblast growth factor 23 to regulate VC.
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2020, BoneCitation Excerpt :ANK may also contribute to the ePPi levels needed to prevent AMC however evidence suggests it is less important than NPP1 [14,82]. Interestingly, a recent study reported that Wnt1 can inhibit AMC by upregulating Ank and increasing ePPi levels [84]. Mutations in the Enpp1 gene are associated with a very rare autosomal recessive condition called generalised arterial calcification of infancy (GACI) [85,86].
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These authors contributed equally to this work.