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

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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The Role of Vitamin K and Its Related Compounds in Mendelian and Acquired Ectopic Mineralization Disorders

International Journal of Molecular Sciences ◽

10.3390/ijms20092142 ◽

2019 ◽

Vol 20 (9) ◽

pp. 2142

Author(s):

Lukas Nollet ◽

Matthias Van Gils ◽

Shana Verschuere ◽

Olivier Vanakker

Keyword(s):

Vitamin K ◽

Current Knowledge ◽

Pseudoxanthoma Elasticum ◽

Ectopic Calcification ◽

Multifactorial Diseases ◽

Multiple Organs ◽

Keutel Syndrome ◽

Ectopic Mineralization ◽

Related Compounds

Ectopic mineralization disorders comprise a broad spectrum of inherited or acquired diseases characterized by aberrant deposition of calcium crystals in multiple organs, such as the skin, eyes, kidneys, and blood vessels. Although the precise mechanisms leading to ectopic calcification are still incompletely known to date, various molecular targets leading to a disturbed balance between pro- and anti-mineralizing pathways have been identified in recent years. Vitamin K and its related compounds, mainly those post-translationally activated by vitamin K-dependent carboxylation, may play an important role in the pathogenesis of ectopic mineralization as has been demonstrated in studies on rare Mendelian diseases, but also on highly prevalent disorders, like vascular calcification. This narrative review compiles and summarizes the current knowledge regarding the role of vitamin K, its metabolism, and associated compounds in the pathophysiology of both monogenic ectopic mineralization disorders, like pseudoxanthoma elasticum or Keutel syndrome, as well as acquired multifactorial diseases, like chronic kidney disease. Clinical and molecular aspects of the various disorders are discussed according to the state-of-the-art, followed by a comprehensive literature review regarding the role of vitamin K in molecular pathophysiology and as a therapeutic target in both human and animal models of ectopic mineralization disorders.

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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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Vitamin K-dependent carboxylation of matrix Gla-protein: a crucial switch to control ectopic mineralization

Trends in Molecular Medicine ◽

10.1016/j.molmed.2012.12.008 ◽

2013 ◽

Vol 19 (4) ◽

pp. 217-226 ◽

Cited By ~ 154

Author(s):

Leon J. Schurgers ◽

Jouni Uitto ◽

Chris P. Reutelingsperger

Keyword(s):

Vitamin K ◽

Protein A ◽

Matrix Gla Protein ◽

Ectopic Mineralization

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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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Administration of vitamin K does not counteract the ectopic mineralization of connective tissues inAbcc6-/-mice, a model for pseudoxanthoma elasticum

Cell Cycle ◽

10.4161/cc.10.4.14862 ◽

2011 ◽

Vol 10 (4) ◽

pp. 701-707 ◽

Cited By ~ 32

Author(s):

Qiujie Jiang ◽

Qiaoli Li ◽

Alix E. Grand-Pierre ◽

Leon J. Schurgers ◽

Jouni Uitto

Keyword(s):

Vitamin K ◽

Pseudoxanthoma Elasticum ◽

Connective Tissues ◽

Ectopic Mineralization

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Relationship Between Mitochondrial Structure and Bioenergetics in Pseudoxanthoma elasticum Dermal Fibroblasts

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.610266 ◽

2020 ◽

Vol 8 ◽

Author(s):

Francesco Demetrio Lofaro ◽

Federica Boraldi ◽

Maria Garcia-Fernandez ◽

Lara Estrella ◽

Pedro Valdivielso ◽

...

Keyword(s):

Oxidative Phosphorylation ◽

Clinical Manifestations ◽

Pseudoxanthoma Elasticum ◽

Dermal Fibroblasts ◽

Morphological Data ◽

Functional Changes ◽

Ectopic Mineralization ◽

Mitochondrial Functions ◽

Extracellular Matrix Components ◽

Pathologic Calcification

Pseudoxanthoma elasticum (PXE) is a genetic disease considered as a paradigm of ectopic mineralization disorders, being characterized by multisystem clinical manifestations due to progressive calcification of skin, eyes, and the cardiovascular system, resembling an age-related phenotype. Although fibroblasts do not express the pathogenic ABCC6 gene, nevertheless these cells are still under investigation because they regulate connective tissue homeostasis, generating the “arena” where cells and extracellular matrix components can promote pathologic calcification and where activation of pro-osteogenic factors can be associated to pathways involving mitochondrial metabolism. The aim of the present study was to integrate structural and bioenergenetic features to deeply investigate mitochondria from control and from PXE fibroblasts cultured in standard conditions and to explore the role of mitochondria in the development of the PXE fibroblasts’ pathologic phenotype. Proteomic, biochemical, and morphological data provide new evidence that in basal culture conditions (1) the protein profile of PXE mitochondria reveals a number of differentially expressed proteins, suggesting changes in redox balance, oxidative phosphorylation, and calcium homeostasis in addition to modified structure and organization, (2) measure of oxygen consumption indicates that the PXE mitochondria have a low ability to cope with a sudden increased need for ATP via oxidative phosphorylation, (3) mitochondrial membranes are highly polarized in PXE fibroblasts, and this condition contributes to increased reactive oxygen species levels, (4) ultrastructural alterations in PXE mitochondria are associated with functional changes, and (5) PXE fibroblasts exhibit a more abundant, branched, and interconnected mitochondrial network compared to control cells, indicating that fusion prevail over fission events. In summary, the present study demonstrates that mitochondria are modified in PXE fibroblasts. Since mitochondria are key players in the development of the aging process, fibroblasts cultured from aged individuals or aged in vitro are more prone to calcify, and in PXE, calcified tissues remind features of premature aging syndromes; it can be hypothesized that mitochondria represent a common link contributing to the development of ectopic calcification in aging and in diseases. Therefore, ameliorating mitochondrial functions and cell metabolism could open new strategies to positively regulate a number of signaling pathways associated to pathologic calcification.

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Matrix Gla protein in tumoral pathology

Medicine and Pharmacy Reports ◽

10.15386/cjmed-579 ◽

2016 ◽

Vol 89 (3) ◽

pp. 319-321 ◽

Cited By ~ 6

Author(s):

Simona Roxana Gheorghe ◽

Alexandra Mărioara Crăciun

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle Cells ◽

Vitamin K ◽

Matrix Gla Protein ◽

Ectopic Calcification ◽

Clinical Implications ◽

Pathological Angiogenesis ◽

Different Types ◽

Dependent Protein

Matrix Gla protein is a vitamin K-dependent protein secreted by chondrocytes and vascular smooth muscle cells. The presence of matrix Gla protein was reported in arterial and venous walls, lungs, kidney, uterus, heart, tooth cementum and eyes. Several studies identified matrix Gla protein in tumoral pathology.Until recently, it was thought to only have an inhibitory role of physiological and ectopic calcification. New studies demonstrated that it also has a role in physiological and pathological angiogenesis, as well as in tumorigenesis.The aim of this review is to report the latest findings related to the expression and clinical implications of matrix Gla protein in different types of cancer with an emphasis on cerebral tumors.

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Vitamin K for the Treatment of Cardiovascular Disease in End-Stage Renal Disease Patients: Is there Hope?

Current Vascular Pharmacology ◽

10.2174/1570161118666200320111745 ◽

2020 ◽

Vol 19 (1) ◽

pp. 77-90 ◽

Cited By ~ 2

Author(s):

Stefanos Roumeliotis ◽

Athanasios Roumeliotis ◽

Evangelia Dounousi ◽

Theodoros Eleftheriadis ◽

Vassilios Liakopoulos

Keyword(s):

Renal Disease ◽

Vascular Calcification ◽

Vitamin K ◽

End Stage Renal Disease ◽

Matrix Gla Protein ◽

Ectopic Mineralization ◽

Cv Disease ◽

Stage Renal Disease ◽

End Stage ◽

Esrd Patients

In Chronic Kidney Disease, vascular calcification (VC) is highly prevalent even at early stages and is gradually enhanced, along with disease progression to End-Stage Renal Disease (ESRD). The calcification pattern in uremia includes all types of mineralization and contributes to the heavy cardiovascular (CV) burden that is common in these patients. Ectopic mineralization is the result of the imbalance between inhibitors and promoters of vascular calcification, with the latter overwhelming the former. The most powerful, natural inhibitor of calcification is Matrix Gla Protein (MGP), a small vitamin K dependent protein, secreted by chondrocytes and vascular smooth muscle cells. In uremia, MGP was reported as the only molecule able to reverse VC by “sweeping” calcium and hydroxyapatite crystals away from the arterial wall. To become biologically active, this protein needs to undergo carboxylation and phosphorylation, reactions highly dependent on vitamin K status. The inactive form of MGP reflects the deficiency of vitamin K and has been associated with CV events and mortality in ESRD patients. During the past decade, vitamin K status has emerged as a novel risk factor for vascular calcification and CV disease in various populations, including dialysis patients. This review presents evidence regarding the association between vitamin K and CV disease in ESRD patients, which are prone to atherosclerosis and atheromatosis.

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