The Le Saux Laboratory works towards understanding the molecular events regulating ectopic (abnormal) calcification in soft tissues. For this purpose, we focus on heritable disorders with overlapping calcification phenotypes, specifically pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI). These diseases, through their respective animal models, are pertinent models to learn more about of pathophysiological processes leading to abnormal mineralization in a variety of common disorders such as atherosclerosis, diabetes and kidney diseases

Understanding the molecular basis of ABCC6-related diseases

Physiological mineralization is a multifactorial metabolic process normally restricted to the bones and teeth. The intra- and extracellular mechanisms regulating mineralization rest upon a tightly regulated balance between calcification inhibitors and promoters. In normal circumstances, calcium and inorganic phosphate (Pi) concentrations are near saturation in most soft tissues, which necessitates strong calcification inhibition systems.

Pathological calcification of soft tissues is commonly seen in aging, diabetes, hypercholesterolemia, renal failure, and connective tissue diseases. Abnormal mineralization also occurs in rare genetic disorders such as Pseudoxanthoma elasticum (PXE). Together with Generalized Arterial Calcification of Infancy (GACI) and Calcification of Joints and Arteries (CALJA), these diseases form a spectrum of related disorders that have provided novel molecular insight into the mechanisms involved in the calcification of connective tissues. These three heritable diseases are characterized by abnormal calcification affecting dermal, ocular and cardiovascular tissues, causing substantial morbidity and mortality.

PXE, GACI and CALJA are caused by mutations in ABCC6, ENPP1 and CD73 (NT5E) genes respectively, which contribute to the same molecular pathway (Fig.1). ABCC6 is a membrane transporter mediating the cellular efflux of nucleotides – notably ATP – that are sequentially converted at the cell surface by ectonucleotidases ENPP1 and CD73 (NT5E) into PPi and adenosine, two physiological inhibitors of calcification. While we have shown that reduced plasma PPi plays a prominent role in the calcification phenotype of PXE/GACI, there are still important gaps in the mechanistic understanding of these pathologies that are critical barriers to developing comprehensive diagnostic and therapeutic strategies that could be applied to more common diseases that manifest abnormal calcification.

 

---

Critical barriers.

The central role of ABCC6 in PXE, GACI and DCC is now well established in humans [1] and animal models [2, 3]. However, many aspects of the pathophysiology of ABCC6 dysfunction are still unexplained. ABCC6 mutations cause a broad spectrum of manifestations beyond calcification, including vascular malformation (Rete mirabile, carotid hypoplasia) [4], dyslipidemia and atherosclerosis in humans and mice [5], ischemic stroke [6], inflammation [7], increased infarct size and apoptosis [8].

---

The current goals of the Le Saux laboratory are to provide some answers to the following:

1) The liver expression of ABCC6 is necessary but not sufficient for calcification inhibition[9, 10]. The identification and contribution of peripheral tissues to calcification regulation remains unresolved.

2) Plasma PPi levels fail to explain the wide range of calcification severity in humans [11] and mice [12] and the search for modifier genes has not produced significant results [13-15]. The role of low-grade inflammation in the progression of PXE is not clear [7].

3) Our recent report [16] suggests that ABCC6 has a strong systemic influence on extracellular purinergic metabolism but the cellular and molecular processes in affected tissues have yet to be defined (Fig. 2)

4) The role of ABCC6 in infarct size and cardiac functions is not understood.

5) How does ABCC6 dysfunction influences dyslipidemia and atherosclerosis?

6) At present, only palliative treatments to alleviate some symptoms exist for both PXE and GACI [17-19]. Extensive studies on the mechanism behind calcification has resulted in several novel approaches to treat PXE and GACI. The therapeutic solutions tested in animals and humans include strategies focusing on:

a) The correction/replacement/inhibition of dysfunctional genes/proteins involved in the calcification pathway [20, 21]
b) Supplementation therapies with exogenous compounds

A summary of tested interventions is shown on Figure 3.

We currently study two therapeutic solutions in each of these two strategies to ameliorate pathologic calcification with support from pharmaceutical partners. Using preclinical animal models, we examine the repurposing of an FDA-approved drug (Phenylbutyrate or 4-PBA) and the use of new formulation of pyrophosphate (PPi) for therapy of PXE and GACI patients.

[1] O. Le Saux, L. Martin, Z. Aherrahrou, G. Leftheriotis, A. Varadi, C.N. Brampton, The molecular and physiological roles of ABCC6: more than meets the eye, Frontiers in genetics 3 (2012) 289.
[2] Q. Li, H. Guo, D.W. Chou, A. Berndt, J.P. Sundberg, J. Uitto, Mouse models for pseudoxanthoma elasticum: genetic and dietary modulation of the ectopic mineralization phenotypes, PLoS One 9(2) (2014) e89268.
[3] Q. Li, J. Kingman, K. van de Wetering, S. Tannouri, J.P. Sundberg, J. Uitto, Abcc6 knockout rat model highlights the role of liver in PPi homeostasis in pseudoxanthoma elasticum, J Invest Dermatol 137(5) (2017) 1025-1032.
[4] M. Vasseur, B. Carsin-Nicol, J.M. Ebran, S. Willoteaux, L. Martin, G. Leftheriotis, P.X.E.C.C.g. Angers, Carotid rete mirabile and pseudoxanthoma elasticum: an accidental association?, Eur J Vasc Endovasc Surg 42(3) (2011) 292-4.
[5] C. Brampton, V. Pomozi, L.H. Chen, A. Apana, S. McCurdy, J. Zoll, W.A. Boisvert, G. Lambert, D. Henrion, S. Blanchard, S. Kuo, G. Leftheriotis, L. Martin, O. Le Saux, ABCC6 deficiency promotes dyslipidemia and atherosclerosis, Sci Rep 11(1) (2021) 3881.
[6] A.M. Pavlovic, J. Zidverc-Trajkovic, M.M. Milovic, D.M. Pavlovic, Z. Jovanovic, M. Mijajlovic, M. Petrovic, V.S. Kostic, N. Sternic, Cerebral small vessel disease in pseudoxanthoma elasticum: three cases, Can J Neurol Sci 32(1) (2005) 115-8.
[7] P.J. Mention, F. Lacoeuille, G. Leftheriotis, L. Martin, L. Omarjee, 18F-Flurodeoxyglucose and 18F-Sodium Fluoride Positron Emission Tomography/Computed Tomography Imaging of Arterial and Cutaneous Alterations in Pseudoxanthoma Elasticum, Circ Cardiovasc Imaging 11(1) (2018) e007060.
[8] I.N. Mungrue, P. Zhao, Y. Yao, H. Meng, C. Rau, J.V. Havel, T.G. Gorgels, A.A. Bergen, W.R. MacLellan, T.A. Drake, K.I. Bostrom, A.J. Lusis, Abcc6 deficiency causes increased infarct size and apoptosis in a mouse cardiac ischemia-reperfusion model, Arterioscler Thromb Vasc Biol 31(12) (2011) 2806-12.
[9] C. Brampton, Z. Aherrahrou, L.H. Chen, L. Martin, A.A. Bergen, T.G. Gorgels, J. Erdmann, H. Schunkert, Z. Szabo, A. Varadi, O. Le Saux, The level of hepatic ABCC6 expression determines the severity of calcification after cardiac injury, Am J Pathol 184(1) (2014) 159-70.
[10] S.G. Ziegler, C.R. Ferreira, E.G. MacFarlane, R.C. Riddle, R.E. Tomlinson, E.Y. Chew, L. Martin, C.T. Ma, E. Sergienko, A.B. Pinkerton, J.L. Millan, W.A. Gahl, H.C. Dietz, Ectopic calcification in pseudoxanthoma elasticum responds to inhibition of tissue-nonspecific alkaline phosphatase, Sci Transl Med 9(393) (2017).
[11] Y. Nitschke, G. Baujat, U. Botschen, T. Wittkampf, M. du Moulin, J. Stella, M. Le Merrer, G. Guest, K. Lambot, M.F. Tazarourte-Pinturier, N. Chassaing, O. Roche, I. Feenstra, K. Loechner, C. Deshpande, S.J. Garber, R. Chikarmane, B. Steinmann, T. Shahinyan, L. Martorell, J. Davies, W.E. Smith, S.G. Kahler, M. McCulloch, E. Wraige, L. Loidi, W. Hohne, L. Martin, S. Hadj-Rabia, R. Terkeltaub, F. Rutsch, Generalized arterial calcification of infancy and pseudoxanthoma elasticum can be caused by mutations in either ENPP1 or ABCC6, Am J Hum Genet 90(1) (2012) 25-39.
[12] Y. Le Corre, O. Le Saux, F. Froeliger, H. Libouban, G. Kauffenstein, S. Willoteaux, G. Leftheriotis, L. Martin, Quantification of the calcification phenotype of abcc6-deficient mice with microcomputed tomography, Am J Pathol 180(6) (2012) 2208-13.
[13] D. Hendig, C. Knabbe, C. Gotting, New insights into the pathogenesis of pseudoxanthoma elasticum and related soft tissue calcification disorders by identifying genetic interactions and modifiers, Frontiers in genetics 4 (2013) 114.
[14] M.J. Hosen, F. Van Nieuwerburgh, W. Steyaert, D. Deforce, L. Martin, G. Leftheriotis, A. De Paepe, P.J. Coucke, O.M. Vanakker, Efficiency of exome sequencing for the molecular diagnosis of pseudoxanthoma elasticum, J Invest Dermatol 135(4) (2015) 992-998.
[15] O.M. Vanakker, M.J. Hosen, A.D. Paepe, The ABCC6 transporter: what lessons can be learnt from other ATP-binding cassette transporters?, Frontiers in genetics 4 (2013) 203.
[16] G. Kauffenstein, G.G. Yegutkin, S. Khiati, V. Pomozi, O. Le Saux, G. Leftheriotis, G. Lenaers, D. Henrion, L. Martin, Alteration of Extracellular Nucleotide Metabolism in Pseudoxanthoma Elasticum, J Invest Dermatol 138(8) (2018) 1862-1870.
[17] S. Akhtar Ali, C. Ng, J.K. Votava-Smith, L.M. Randolph, P. Pitukcheewanont, Bisphosphonate therapy in an infant with generalized arterial calcification with an ABCC6 mutation, Osteoporos Int 29(11) (2018) 2575-2579.
[18] T. Edouard, G. Chabot, J. Miro, D.C. Buhas, Y. Nitschke, C. Lapierre, F. Rutsch, N. Alos, Efficacy and safety of 2-year etidronate treatment in a child with generalized arterial calcification of infancy, Eur J Pediatr 170(12) (2011) 1585-90.
[19] R.P. Finger, P. Charbel Issa, S. Schmitz-Valckenberg, F.G. Holz, H.N. Scholl, Long-term effectiveness of intravitreal bevacizumab for choroidal neovascularization secondary to angioid streaks in pseudoxanthoma elasticum, Retina 31(7) (2011) 1268-78.
[20] R.S. Jansen, S. Duijst, S. Mahakena, D. Sommer, F. Szeri, A. Varadi, A. Plomp, A.A. Bergen, R.P. Oude Elferink, P. Borst, K. van de Wetering, ABCC6-mediated ATP secretion by the liver is the main source of the mineralization inhibitor inorganic pyrophosphate in the systemic circulation-brief report, Arterioscler Thromb Vasc Biol 34(9) (2014) 1985-9.
[21] V. Pomozi, C. Brampton, K. van de Wetering, J. Zoll, B. Calio, K. Pham, J.B. Owens, J. Marh, S. Moisyadi, A. Varadi, L. Martin, C. Bauer, J. Erdmann, Z. Aherrahrou, O. Le Saux, Pyrophosphate Supplementation Prevents Chronic and Acute Calcification in ABCC6-Deficient Mice, Am J Pathol 187(6) (2017) 1258-1272.

Complete list of published work

https://www.ncbi.nlm.nih.gov/myncbi/olivier.le%20saux.1/bibliography/public/

Selected Publications

⦁ Le Saux O, Urban Z, Tschuch C, Csiszar K, Bacchelli B, et al. Mutations in a gene encoding an ABC transporter cause pseudoxanthoma elasticum. Nat Genet. 2000 Jun;25(2):223-7. PMID: 10835642
⦁ Le Saux O, Beck K, Sachsinger C, Silvestri C, Treiber C, et al. A spectrum of ABCC6 mutations is responsible for pseudoxanthoma elasticum. Am J Hum Genet. 2001 Oct;69(4):749-64. PubMed PMID: 11536079
⦁ Iliás A, Urbán Z, Seidl TL, Le Saux O, Sinkó E, et al. Loss of ATP-dependent transport activity in pseudoxanthoma elasticum-associated mutants of human ABCC6 (MRP6). J Biol Chem. 2002 May 10;277(19):16860-7. PMID: 11880368
⦁ Le Saux O, Beck K, Sachsinger C, Treiber C, Göring HH, et al. Evidence for a founder effect for pseudoxanthoma elasticum in the Afrikaner population of South Africa. Hum Genet. 2002 Oct;111(4-5):331-8. PMID: 12384774
⦁ Le Saux O, Bunda S, VanWart CM, Douet V, Got L, et al. Serum factors from pseudoxanthoma elasticum patients alter elastic fiber formation in vitro. J Invest Dermatol. 2006 Jul;126(7):1497-505. PMID: 16543900
⦁ Douet V, VanWart CM, Heller MB, Reinhard S, Le Saux O. HNF4alpha and NF-E2 are key transcriptional regulators of the murine Abcc6 gene expression. Biochim Biophys Acta. 2006 Aug-Sep;1759(8-9):426-36. PubMed PMID: 16997394
⦁ Douet V, Heller MB, Le Saux O. DNA methylation and Sp1 binding determine the tissue-specific transcriptional activity of the mouse Abcc6 promoter. Biochem Biophys Res Commun. 2007 Mar 2;354(1):66-71. PMID: 17214963
⦁ Hamlin N, Beck K, Bacchelli B, Cianciulli P, Pasquali-Ronchetti I, et al. Acquired Pseudoxanthoma elasticum-like syndrome in beta-thalassaemia patients. Br J Haematol. 2003 Sep;122(5):852-4. PMID: 12930400
⦁ Pomozi V., Brampton C., van de Wetering K., Zoll J., Calio B., et al. Pyrophosphate supplementation prevents chronic and acute calcification in ABCC6 deficient mice. Am. J. Pathol. 2017, 187(6):1258-1272. PMID: 28416300
⦁ Dedinszki D., Szeri F., Kozák E., Pomozi V., Tőkési N., et al. Oral administration of Pyrophosphate Inhibits Connective Tissue Calcification. 2017, EMBO Mol. Medicine. 2017 9(11):1463-1470. PMID: 28701330
⦁ Pomozi V., Julian C.B., Zoll J., Pham K., Kuo S., Tőkési N., Martin L., Váradi A. and Le Saux O. Dietary pyrophosphate modulates calcification in a mouse model of pseudoxanthoma elasticum: implication for treatment of patients. J. Invest. Dermatol 2019, 139(5):1082-108. PMID:30468740
⦁ C. Brampton, V. Pomozi, L. Chen, A. Apana, S. McCurdy, J. Zoll, W. A. Boisvert, G. Lambert, D. Henrion, S. Blanchard, S. Kuo, G. Leftheriotis, L. Martin and O. Le Saux. ABCC6 deficiency promotes dyslipemdia and atherosclerosis. 2021. Sci Rep.11(1):3881. PMID: 33594095
⦁ B.K. Shimada, V. Pomozi, J. Zoll, S. Kuo, L. Martin and O. Le Saux. ABCC6, pyrophosphate and ectopic calcification: therapeutic solutions. 2021. Int. J. Mol. Sci. 2021 Apr 27;22(9):4555. PMID: 33925341