**1. Introduction**

Physiological calcification is a multifactorial metabolic process normally restricted to the bones and teeth. Calcification and mineralization (we will use these terms interchangeably thereon) primarily designate the formation of apatite crystals. Apatite is made of phosphate and calcium ions (Ca10(PO4)6(OH)2), but the crystal formation happens via a number of short-lived intermediates such as octacalcium phosphate and amorphous calcium phosphate [1]. The intra- and extracellular mechanisms regulating mineralization rest upon a tightly regulated balance between calcification inhibitors and promoters. Under normal circumstances, calcium and inorganic phosphate (Pi) concentrations are near saturation in most soft tissues, which necessitates strong calcification inhibition systems [2].

**Citation:** Shimada, B.K.; Pomozi, V.; Zoll, J.; Kuo, S.; Martin, L.; Le Saux, O. ABCC6, Pyrophosphate and Ectopic Calcification: Therapeutic Solutions. *Int. J. Mol. Sci.* **2021**, *22*, 4555. https:// doi.org/10.3390/ijms22094555

Academic Editor: Thomas Falguières

Received: 22 March 2021 Accepted: 21 April 2021 Published: 27 April 2021

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Ectopic calcification can lead to clinical symptoms when it occurs in cardiovascular tissues and has been the object of intense research focus. Many host, environmental, and genetic factors contributing to this calcification have been identified [3], but there are still gaps in our understanding [4]. In recent years, the identification of mutations in the ATPbinding cassette (ABC) transporter ABCC6 [5–7] and the characterization of its function [8] has provided new molecular insight into the regulation of ectopic calcification inhibition in relation to pyrophosphate (PPi). ABCC6 mediates the cellular efflux of nucleotides, notably ATP, which is rapidly converted into PPi and adenosine at the cellular surface by the ectonucleotidases NPP1 (encoded by *ENPP1*) and CD73 (encoded by *NT5E*) [8–11]. Both PPi and indirectly adenosine are major inhibitors of calcification. ABCC6 deficiencies underlie the calcification disorders PXE (MIM#264800) and a cardiac calcification phenotype (DCC) described in mice [12–14]. GACI (MIM#208000) is primarily linked to NPP1, the key enzyme that generates PPi [15]. However, some cases of GACI (MIM#614473) are caused by *ABCC6* mutations, while some PXE patients carry disease-causing variants only in *ENPP1* [16,17]. CALJA (MIM#211800) is due to mutations in CD73 (*NT5E*) [10]. PXE, together with GACI and CALJA form a spectrum of diseases with overlapping calcification processes but with distinct clinical features. If PXE, GACI, and DCC result from a deficit in PPi production [18,19], CALJA is caused by increased PPi degradation [10,11] (Figure 1). The explanation of these phenotypes places ABCC6 as an upstream modulator of an extracellular purinergic pathway that, among other things, inhibits mineralization by regulating the Pi/PPi ratio in connective tissues (Figure 1). Although it has been recently claimed that ABCC6 acts downstream of NPP1 [20], this hypothesis is highly controversial in the field [21] and contradicts most studies [8,9,11,18]. This pathway (Figure 1) and the molecular cascade leading to PPi generation and TNAP inhibition offers many opportunities for therapeutic interventions in the case of PXE and also GACI.

**Figure 1.** The ABCC6 pathway influences calcification and extracellular purinergic metabolism. ABCC6 facilitates the cellular efflux of ATP from liver and other tissues/cells, which is quickly converted to pyrophosphate (PPi), a potent inhibitor of mineralization. Decreased plasma PPi levels cause calcification in PXE and GACI. CD73 activity leads to adenosine production, which affects many biological activities including the inhibition of TNAP synthesis. TNAP degrades PPi into inorganic phosphate (Pi), an activator of calcification, which leads to vascular calcification in CAL JA patients.

At present, only palliative treatments to alleviate some symptoms exist for both PXE and GACI [22–24]. Extensive studies on the mechanism behind calcification has resulted in several novel approaches to treating PXE and GACI. The therapeutic solutions envisioned and tested in animals include strategies focusing on two distinct aspects of the ABCC6 pathway: (1) the correction/replacement/inhibition of dysfunctional genes/proteins involved in the calcification pathway [9,18], and (2) supplementation therapies with exogenous compounds. Drugs targeting ABCC6, NPP1 and TNAP aim at correcting parts of the ABCC6 pathway, whereas exogenous compounds such as magnesium, vitamin K, bisphosphonates (PPi analogs), PPi and recently phytic acid are intended to directly inhibit calcification.

In this article, we provide an overview of ABCC6 as a key regulator of ectopic calcification in PXE (and GACI). We will discuss possible treatment options that have been explored in recent years and speculate as to what future treatments might be. Because ABCC6 and NPP1 are functionally related proteins interchangeably causing PXE and

GACI [17], we address herein some aspects of GACI and NPP1, which can be relevant for therapeutic interventions.
