Therapeutics Development for Pseudoxanthoma Elasticum and Related Ectopic Mineralization Disorders: Update 2020

Pseudoxanthoma elasticum (PXE), the prototype of heritable ectopic mineralization disorders, manifests with deposition of calcium hydroxyapatite crystals in the skin, eyes and arterial blood vessels. This autosomal recessive disorder, due to mutations in ABCC6, is usually diagnosed around the second decade of life. In the spectrum of heritable ectopic mineralization disorders are also generalized arterial calcification of infancy (GACI), with extremely severe arterial calcification diagnosed by prenatal ultrasound or perinatally, and arterial calcification due to CD73 deficiency (ACDC) manifesting with arterial and juxta-articular mineralization in the elderly; the latter disorders are caused by mutations in ENPP1 and NT5E, respectively. The unifying pathomechanistic feature in these three conditions is reduced plasma levels of inorganic pyrophosphate (PPi), a powerful endogenous inhibitor of ectopic mineralization. Several on-going attempts to develop treatments for these conditions, either with the goal to normalize PPi plasma levels or by means of preventing calcium hydroxyapatite deposition independent of PPi, are in advanced preclinical levels or in early clinical trials. This overview summarizes the prospects of treatment development for ectopic mineralization disorders, with PXE, GACI and ACDC as the target diseases, from the 2020 vantage point.

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ABCC6, Pyrophosphate and Ectopic Calcification: Therapeutic Solutions

International Journal of Molecular Sciences ◽

10.3390/ijms22094555 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4555

Author(s):

Briana K. Shimada ◽

Viola Pomozi ◽

Janna Zoll ◽

Sheree Kuo ◽

Ludovic Martin ◽

...

Keyword(s):

Soft Tissues ◽

Genetic Disorders ◽

Pseudoxanthoma Elasticum ◽

Arterial Calcification ◽

Ectopic Calcification ◽

Direct Inhibition ◽

Inorganic Pyrophosphate ◽

Cardiovascular Tissues ◽

Ectopic Mineralization

Pathological (ectopic) mineralization of soft tissues occurs during aging, in several common conditions such as diabetes, hypercholesterolemia, and renal failure and in certain genetic disorders. Pseudoxanthoma elasticum (PXE), a multi-organ disease affecting dermal, ocular, and cardiovascular tissues, is a model for ectopic mineralization disorders. ABCC6 dysfunction is the primary cause of PXE, but also some cases of generalized arterial calcification of infancy (GACI). ABCC6 deficiency in mice underlies an inducible dystrophic cardiac calcification phenotype (DCC). These calcification diseases are part of a spectrum of mineralization disorders that also includes Calcification of Joints and Arteries (CALJA). Since the identification of ABCC6 as the “PXE gene” and the development of several animal models (mice, rat, and zebrafish), there has been significant progress in our understanding of the molecular genetics, the clinical phenotypes, and pathogenesis of these diseases, which share similarities with more common conditions with abnormal calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into inorganic pyrophosphate (PPi) and adenosine by the ectonucleotidases NPP1 and CD73 (NT5E). PPi is a potent endogenous inhibitor of calcification, whereas adenosine indirectly contributes to calcification inhibition by suppressing the synthesis of tissue non-specific alkaline phosphatase (TNAP). At present, therapies only exist to alleviate symptoms for both PXE and GACI; however, extensive studies have resulted in several novel approaches to treating PXE and GACI. This review seeks to summarize the role of ABCC6 in ectopic calcification in PXE and other calcification disorders, and discuss therapeutic strategies targeting various proteins in the pathway (ABCC6, NPP1, and TNAP) and direct inhibition of calcification via supplementation by various compounds.

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Pseudoxanthoma elasticum with prominent arterial calcifications evoking CD73 deficiency

Vascular Medicine ◽

10.1177/1358863x19853360 ◽

2019 ◽

Vol 24 (5) ◽

pp. 461-464 ◽

Cited By ~ 4

Author(s):

Magali Devriese ◽

Anne Legrand ◽

Marie-Cécile Courtois ◽

Xavier Jeunemaitre ◽

Juliette Albuisson

Keyword(s):

Vascular Calcification ◽

Pseudoxanthoma Elasticum ◽

Arterial Calcification ◽

Elastic Fibers ◽

Connective Tissues ◽

Tibial Arteries ◽

Ectopic Mineralization ◽

Abcc6 Gene ◽

Next Generation Sequencing Ngs ◽

Eye Lesions

Pseudoxanthoma elasticum (PXE) is a rare disorder characterized by skin, eye, and cardiovascular lesions due to ectopic mineralization and fragmentation of elastic fibers of connective tissues. We present an atypical case of PXE with diffuse vascular calcification and negligible skin and eye lesions. The patient was a 37-year-old man suffering from severe bilateral arterial calcifications in superficial femoral and posterior tibial arteries. Eye fundoscopy and skin examination were first considered normal. This phenotype suggested first the diagnosis of Arterial Calcification due to Deficiency of CD73 (ACDC) characterized by mutations in NT5E gene. However, we found two variants in ABCC6 gene, and no variant in NT5E. Skin reexamination revealed few lateral skin papules confined to the scalp. Phenotypic overlap was described in vascular calcification disorders, between GACI and PXE phenotypes, and we discuss here expansion of this overlap, including ACDC phenotype. Identification of these expanding and overlapping phenotypes was enabled by genetic screening of the corresponding genes, in a systematic approach. We propose to create a calcification next generation sequencing (NGS) panel with NT5E, GGCX, ENPP1, and ABCC6 genes to improve the molecular diagnosis of vascular calcification.

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Dysregulation of gene expression in ABCC6 knockdown HepG2 cells

Cellular & Molecular Biology Letters ◽

10.2478/s11658-014-0208-2 ◽

2014 ◽

Vol 19 (4) ◽

Cited By ~ 17

Author(s):

Rocchina Miglionico ◽

Maria Armentano ◽

Monica Carmosino ◽

Antonella Salvia ◽

Flavia Cuviello ◽

...

Keyword(s):

Hepg2 Cells ◽

Autosomal Recessive ◽

Pseudoxanthoma Elasticum ◽

Arterial Calcification ◽

Elastic Fibers ◽

Ectopic Calcification ◽

Ectopic Mineralization ◽

Abcc6 Gene ◽

Basolateral Plasma Membrane

AbstractABCC6 protein is an ATP-dependent transporter that is mainly found in the basolateral plasma membrane of hepatocytes. ABCC6 deficiency is the primary cause of several forms of ectopic mineralization syndrome. Mutations in the human ABCC6 gene cause pseudoxanthoma elasticum (PXE), an autosomal recessive disease characterized by ectopic calcification of the elastic fibers in dermal, ocular and vascular tissues. Mutations in the mouse ABCC6 gene were also associated with dystrophic cardiac calcification. Reduced levels of ABCC6 protein were found in a β-thalassemic mouse model. Moreover, some cases of generalized arterial calcification in infancy are due to ABCC6 mutations. In order to study the role of ABCC6 in the pathogenesis of ectopic mineralization, the expressions of genes involved in this process were evaluated in HepG2 cells upon stable knockdown of ABCC6 by small hairpin RNA (shRNA) technology. ABCC6 knockdown in HepG2 cells causes a significant upregulation of the genes promoting mineralization, such as TNAP, and a parallel downregulation of genes with anti-mineralization activity, such as NT5E, Fetuin A and Osteopontin. Although the absence of ABCC6 has been already associated with ectopic mineralization syndromes, this study is the first to show a direct relationship between reduced ABCC6 levels and the expression of pro-mineralization genes in hepatocytes.

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Targeted Ablation of the Abcc6 Gene Results in Ectopic Mineralization of Connective Tissues

Molecular and Cellular Biology ◽

10.1128/mcb.25.18.8299-8310.2005 ◽

2005 ◽

Vol 25 (18) ◽

pp. 8299-8310 ◽

Cited By ~ 141

Author(s):

John F. Klement ◽

Yasushi Matsuzaki ◽

Qiu-Jie Jiang ◽

Joseph Terlizzi ◽

Hae Young Choi ◽

...

Keyword(s):

Complex Disease ◽

Soft Tissues ◽

Subcutaneous Tissue ◽

Pseudoxanthoma Elasticum ◽

Connective Tissues ◽

Alizarin Red ◽

Arterial Blood ◽

Ectopic Mineralization ◽

Abcc6 Gene ◽

Total Body

ABSTRACT Pseudoxanthoma elasticum (PXE), characterized by connective tissue mineralization of the skin, eyes, and cardiovascular system, is caused by mutations in the ABCC6 gene. ABCC6 encodes multidrug resistance-associated protein 6 (MRP6), which is expressed primarily in the liver and kidneys. Mechanisms producing ectopic mineralization as a result of these mutations remain unclear. To elucidate this complex disease, a transgenic mouse was generated by targeted ablation of the mouse Abcc6 gene. Abcc6 null mice were negative for Mrp6 expression in the liver, and complete necropsies revealed profound mineralization of several tissues, including skin, arterial blood vessels, and retina, while heterozygous animals were indistinguishable from the wild-type mice. Particularly striking was the mineralization of vibrissae, as confirmed by von Kossa and alizarin red stains. Electron microscopy revealed mineralization affecting both elastic structures and collagen fibers. Mineralization of vibrissae was noted as early as 5 weeks of age and was progressive with age in Abcc6 −/− mice but was not observed in Abcc6 +/− or Abcc6 +/+ mice up to 2 years of age. A total body computerized tomography scan of Abcc6 −/− mice revealed mineralization in skin and subcutaneous tissue as well as in the kidneys. These data demonstrate aberrant mineralization of soft tissues in PXE-affected organs, and, consequently, these mice recapitulate features of this complex disease.

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A Reference Range for Plasma Levels of Inorganic Pyrophosphate in Children Using the ATP Sulfurylase Method

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/clinem/dgab615 ◽

2021 ◽

Author(s):

Eva Bernhard ◽

Yvonne Nitschke ◽

Gus Khursigara ◽

Yves Sabbagh ◽

Yongbao Wang ◽

...

Keyword(s):

Plasma Levels ◽

Reference Range ◽

Pediatric Population ◽

Hypophosphatemic Rickets ◽

Arterial Calcification ◽

Analytical Sensitivity ◽

Atp Sulfurylase ◽

Inherited Disorders ◽

Inorganic Pyrophosphate ◽

Standard Range

Abstract Purpose Generalized arterial calcification of infancy, pseudoxanthoma elasticum, autosomal recessive hypophosphatemic rickets type 2, and hypophosphatasia are rare inherited disorders associated with altered plasma levels of inorganic pyrophosphate (PPi). In this study, we aimed to establish a reference range for plasma PPi in the pediatric population, which would be essential to support its use as a biomarker in children with mineralization disorders. Methods Plasma samples were collected from 200 children aged 1 day to 18 years who underwent blood testing for medical conditions not affecting plasma PPi levels. PPi was measured in proband plasma utilizing a validated adenosine triphosphate (ATP) sulfurylase method. Results The analytical sensitivity of the ATP sulfurylase assay consisted of 0.15 to 10 µM PPi. Inter- and intra-assay coefficients of variability on identical samples were below 10%. The standard range of PPi in the blood plasma of children and adolescents aged 0 to 18 years was calculated as 2.36 to 4.44 µM, with a median of 3.17 µM, with no difference between male and female probands. PPi plasma levels did not differ significantly in different pediatric age groups. Main Conclusions Our results yielded no noteworthy discrepancy to the reported standard range of plasma PPi in adults (2-5 µM). We propose the described ATP sulfurylase method as a diagnostic tool to measure PPi levels in plasma as a biomarker in the pediatric population.

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Nonlinear optical microscopy is a novel tool for the analysis of cutaneous alterations in pseudoxanthoma elasticum

Lasers in Medical Science ◽

10.1007/s10103-020-03027-w ◽

2020 ◽

Vol 35 (8) ◽

pp. 1821-1830 ◽

Cited By ~ 1

Author(s):

Norbert Kiss ◽

Luca Fésűs ◽

Szabolcs Bozsányi ◽

Flóra Szeri ◽

Matthias Van Gils ◽

...

Keyword(s):

Early Diagnosis ◽

Nonlinear Optical ◽

Second Harmonic ◽

Histopathological Examination ◽

Pseudoxanthoma Elasticum ◽

Calcium Deposition ◽

Inorganic Pyrophosphate ◽

Ectopic Mineralization ◽

New Variant ◽

Cardiovascular Assessment

Abstract Pseudoxanthoma elasticum (PXE, OMIM 264800) is a rare autosomal recessive disorder with ectopic mineralization and fragmentation of elastin fibers. It is caused by mutations of the ABCC6 gene that leads to decreased serum levels of inorganic pyrophosphate (PPi) anti-mineralization factor. The occurrence of severe complications among PXE patients highlights the importance of early diagnosis so that prompt multidisciplinary care can be provided to patients. We aimed to examine dermal connective tissue with nonlinear optical (NLO) techniques, as collagen emits second-harmonic generation (SHG) signal, while elastin can be excited by two-photon excitation fluorescence (TPF). We performed molecular genetic analysis, ophthalmological and cardiovascular assessment, plasma PPi measurement, conventional histopathological examination, and ex vivo SHG and TPF imaging in five patients with PXE and five age- and gender-matched healthy controls. Pathological mutations including one new variant were found in the ABCC6 gene in all PXE patients and their plasma PPi level was significantly lower compared with controls. Degradation and mineralization of elastin fibers and extensive calcium deposition in the mid-dermis was visualized and quantified together with the alterations of the collagen structure in PXE. Our data suggests that NLO provides high-resolution imaging of the specific histopathological features of PXE-affected skin. In vivo NLO may be a promising tool in the assessment of PXE, promoting early diagnosis and follow-up.

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Faculty Opinions recommendation of ABCC6 prevents ectopic mineralization seen in pseudoxanthoma elasticum by inducing cellular nucleotide release.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718188211.793523492 ◽

2016 ◽

Author(s):

Bruno Stieger

Keyword(s):

Pseudoxanthoma Elasticum ◽

Ectopic Mineralization ◽

Nucleotide Release

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Disruption of Abcc6 Transporter in Zebrafish Causes Ocular Calcification and Cardiac Fibrosis

International Journal of Molecular Sciences ◽

10.3390/ijms22010278 ◽

2020 ◽

Vol 22 (1) ◽

pp. 278

Author(s):

Jianjian Sun ◽

Peilu She ◽

Xu Liu ◽

Bangjun Gao ◽

Daqin Jin ◽

...

Keyword(s):

Vitamin K ◽

Cardiac Fibrosis ◽

Clinical Manifestations ◽

Pseudoxanthoma Elasticum ◽

Matrix Gla Protein ◽

Ectopic Calcification ◽

Transcription Activator ◽

Multisystem Disorder ◽

Ectopic Mineralization ◽

Extensive Calcification

Pseudoxanthoma elasticum (PXE), caused by ABCC6/MRP6 mutation, is a heritable multisystem disorder in humans. The progressive clinical manifestations of PXE are accompanied by ectopic mineralization in various connective tissues. However, the pathomechanisms underlying the PXE multisystem disorder remains obscure, and effective treatment is currently available. In this study, we generated zebrafish abcc6a mutants using the transcription activator-like effector nuclease (TALEN) technique. In young adult zebrafish, abcc6a is expressed in the eyes, heart, intestine, and other tissues. abcc6a mutants exhibit extensive calcification in the ocular sclera and Bruch’s membrane, recapitulating part of the PXE manifestations. Mutations in abcc6a upregulate extracellular matrix (ECM) genes, leading to fibrotic heart with reduced cardiomyocyte number. We found that abcc6a mutation reduced levels of both vitamin K and pyrophosphate (PPi) in the serum and diverse tissues. Vitamin K administration increased the gamma-glutamyl carboxylated form of matrix gla protein (cMGP), alleviating ectopic calcification and fibrosis in vertebrae, eyes, and hearts. Our findings contribute to a comprehensive understanding of PXE pathophysiology from zebrafish models.

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Structural and Functional Characterization of the ABCC6 Transporter in Hepatic Cells: Role on PXE, Cancer Therapy and Drug Resistance

International Journal of Molecular Sciences ◽

10.3390/ijms22062858 ◽

2021 ◽

Vol 22 (6) ◽

pp. 2858

Author(s):

Faustino Bisaccia ◽

Prashant Koshal ◽

Vittorio Abruzzese ◽

Maria Antonietta Castiglione Morelli ◽

Angela Ostuni

Keyword(s):

Cancer Therapy ◽

Functional Characterization ◽

Physiological Role ◽

Pseudoxanthoma Elasticum ◽

Connective Tissues ◽

Functional Studies ◽

Extracellular Milieu ◽

Hepatic Cells ◽

Ectopic Mineralization ◽

Anti Cancer

Pseudoxanthoma elasticum (PXE) is a complex autosomal recessive disease caused by mutations of ABCC6 transporter and characterized by ectopic mineralization of soft connective tissues. Compared to the other ABC transporters, very few studies are available to explain the structural components and working of a full ABCC6 transporter, which may provide some idea about its physiological role in humans. Some studies suggest that mutations of ABCC6 in the liver lead to a decrease in some circulating factor and indicate that PXE is a metabolic disease. It has been reported that ABCC6 mediates the efflux of ATP, which is hydrolyzed in PPi and AMP; in the extracellular milieu, PPi gives potent anti-mineralization effect, whereas AMP is hydrolyzed to Pi and adenosine which affects some cellular properties by modulating the purinergic pathway. Structural and functional studies have demonstrated that silencing or inhibition of ABCC6 with probenecid changed the expression of several genes and proteins such as NT5E and TNAP, as well as Lamin, and CDK1, which are involved in cell motility and cell cycle. Furthermore, a change in cytoskeleton rearrangement and decreased motility of HepG2 cells makes ABCC6 a potential target for anti-cancer therapy. Collectively, these findings suggested that ABCC6 transporter performs functions that modify both the external and internal compartments of the cells.

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