Qiujie Jiang

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

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.

Pseudoxanthoma elasticum : clinical phenotypes, molecular genetics and putative pathomechanisms

Abstract:  Pseudoxanthoma elasticum (PXE), a prototype of heritable multisystem disorders, is characterised by pathologic mineralisation of connective tissues, with primary clinical manifestations in the skin, eyes and the cardiovascular system. The causative gene was initially identified as ABCC6 which encodes an ABC transporter protein (ABCC6) expressed primarily in the liver and the kidneys. The critical role of ABCC6 in ectopic mineralisation has been confirmed by the development of Abcc6−/− knock‐out mice which recapitulate the features of connective tissue mineralisation characteristic of PXE. Over 300 distinct loss‐of‐function mutations representative of over 1000 mutant alleles in ABCC6 have been identified by streamlined mutation detection strategies in this autosomal recessive disease. More recently, missense mutations in the GGCX gene, either in compound heterozygous state or digenic with a recurrent ABCC6 nonsense mutation (p.R1141X), have been identified in patients with PXE‐like cutaneous findings and vitamin K‐dependent coagulation factor deficiency. GGCX encodes a carboxylase which catalyses γ‐glutamyl carboxylation of coagulation factors as well as of matrix gla protein (MGP) which in fully carboxylated form serves as a systemic inhibitor of pathologic mineralisation. Collectively, these observations suggest the hypothesis that a consequence of loss‐of‐function mutations in the ABCC6 gene is the reduced vitamin K‐dependent γ‐glutamyl carboxylation of MGP, with subsequent connective tissue mineralisation. Further progress in understanding the detailed pathomechanisms of PXE should provide novel strategies to counteract, and perhaps cure, this complex heritable disorder at the genome–environment interface.

Pseudoxanthoma Elasticum: Molecular Genetics and Putative Pathomechanisms

Pseudoxanthoma elasticum (PXE), a prototypic heritable disorder with ectopic mineralization, manifests with characteristic skin findings, ocular involvement and cardiovascular problems, with considerable morbidity and mortality. The classic forms of PXE are due to loss-of-function mutations in the ABCC6 gene, which encodes ABCC6, a transmembrane efflux transporter expressed primarily in the liver. Several lines of evidence suggest that PXE is a primary metabolic disorder, which in the absence of ABCC6 transporter activity, displays reduced plasma anti-mineralization capacity due to reduced fetuin-A and matrix gla-protein (MGP) levels. MGP requires to be activated by gamma-glutamyl carboxylation, a vitamin K-dependent reaction, to serve in an anti-mineralization role in the peripheral connective tissue cells. Although the molecules transported from the hepatocytes to circulation by ABCC6 in vivo remain unidentified, it has been hypothesized that a critical vitamin K derivative, such as reduced vitamin K conjugated with glutathione, is secreted to circulation physiologically, but not in the absence of ABCC6 transporter activity. As a result, activation of MGP by gamma-glutamyl carboxylase is diminished, allowing slow yet progressive mineralization of connective tissues characteristic of PXE. Understanding of the pathomechanistic details of PXE provides a basis for the development of targeted molecular therapies for this currently intractable disease.

Elevated Dietary Magnesium Prevents Connective Tissue Mineralization in a Mouse Model of Pseudoxanthoma Elasticum (Abcc6

Pseudoxanthoma elasticum (PXE) is an autosomal recessive multisystem disorder characterized by ectopic connective tissue mineralization, with clinical manifestations primarily in the skin, eyes, and cardiovascular system. There is considerable, both intra- and interfamilial, variability in the spectrum of phenotypic presentation. Previous studies have suggested that mineral content of the diet may modify the severity of the clinical phenotype in PXE. In this study, we utilized a targeted mutant mouse (Abcc6(-/-)) as a model system for PXE. We examined the effects of changes in dietary phosphate and magnesium on the mineralization process using calcification of the connective tissue capsule surrounding the vibrissae as an early phenotypic biomarker. Mice placed on custom-designed diets either high or low in phosphate did not show changes in mineralization, which was similar to that noted in Abcc6(-/-) mice on control diet. However, mice placed on diet enriched in magnesium (fivefold) showed no evidence of connective tissue mineralization in this mouse model of PXE. The inhibitory capacity of magnesium was confirmed in a cell-based mineralization assay system in vitro. Collectively, our observations suggest that assessment of dietary magnesium in patients with PXE may be warranted.

Mutations in the GGCX and ABCC6 Genes in a Family with Pseudoxanthoma Elasticum-Like Phenotypes

A characteristic feature of classic pseudoxanthoma elasticum (PXE), an autosomal recessive disorder caused by mutations in the ABCC6 gene, is aberrant mineralization of connective tissues, particularly the elastic fibers. Here, we report a family with PXE-like cutaneous features in association with multiple coagulation factor deficiency, an autosomal recessive disorder associated with GGCX mutations. The proband and her sister, both with severe skin findings with extensive mineralization, were compound heterozygotes for missense mutations in the GGCX gene, which were shown to result in reduced gamma-glutamyl carboxylase activity and in undercarboxylation of matrix gla protein. The probands mother and aunt, also manifesting with PXE-like skin changes, were heterozygous carriers of a missense mutation (p.V255M) in GGCX and a null mutation (p.R1141X) in the ABCC6 gene, suggesting digenic nature of their skin findings. Thus, reduced gamma-glutamyl carboxylase activity in individuals either compound heterozygous for a missense mutation in GGCX or with haploinsufficiency in GGCX in combination with heterozygosity for ABCC6 gene expression results in aberrant mineralization of skin leading to PXE-like phenotype. These findings expand the molecular basis of PXE-like phenotypes, and suggest a role for multiple genetic factors in pathologic tissue mineralization in general.

Administration of vitamin K does not counteract the ectopic mineralization of connective tissues in Abcc6 (-/-) mice, a model for pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE) is a heritable multisystem disorder manifesting with ectopic calcification of peripheral connective tissues, caused by mutations in the ABCC6 gene. Alterations in vitamin K metabolism have been suggested to contribute to the pathomechanisms of the mineralization process. In this study we administered vitamin K or its glutathione conjugate (K3-GSH) into Abcc6-/- mice which recapitulate features of PXE. Oral administration of vitamin K2 in dosages, which vastly exceed the amounts in control diet or the recommended amounts for humans, did not alter the ectopic mineralization in Abcc6-/- mice. Similarly, intravenous administration of K3-GSH did not alter the degree of mineralization. Testing of vitamin K2, K3 and K3-GSH in an in vitro calcification system provided no evidence of mineralization inhibition. Collectively, our data suggest that vitamin K deficiency in the peripheral tissues is not a simple explanation for development of mineral deposits in PXE.

Restricting dietary magnesium accelerates ectopic connective tissue mineralization in a mouse model of pseudoxanthoma elasticum (Abcc6

Ectopic mineralization, linked to a number of diseases, is a major cause of morbidity and mortality in humans. Pseudoxanthoma elasticum (PXE) is a heritable multisystem disorder characterized by calcium phosphate deposition in various tissues. The mineral content of diet has been suggested to modify the disease severity in PXE. The aim of this study is to explore the role of diet with reduced magnesium in modifying tissue mineralization in a mouse model of PXE. Abcc6−/− mice were placed on either standard rodent diet (control) or an experimental diet low in magnesium at weaning (4 weeks) and examined for mineralization in the skin and internal organs at the ages of 1.5, 2 or 6 months by computerized morphometric analysis of histopathological sections and by chemical assay of calcium and phosphate. Abcc6−/− mice on experimental diet demonstrated an accelerated, early‐onset mineralization of connective tissues, as compared to control mice. Wild‐type or heterozygous mice on experimental diet did not show evidence of mineralization up to 6 months of age. All mice on experimental diet showed decreased urinary calcium, increased urinary phosphate and elevated parathyroid serum levels. However, no difference in bone density at 6 months of age was noted. Our findings indicate that the mineral content, particularly magnesium, can modify the extent and the onset of mineralization in Abcc6−/− mice and suggest that dietary magnesium levels may contribute to the phenotypic variability of PXE. The control of mineralization by dietary magnesium may have broader implications in general population in the context of vascular mineralization.

Overexpression of fetuin-a counteracts ectopic mineralization in a mouse model of pseudoxanthoma elasticum (abcc6(-/-)).

The pathologic hallmark of pseudoxanthoma elasticum (PXE) is ectopic mineralization of soft connective tissues. Recent studies have suggested that PXE is a metabolic disease, and perturbations in a number of circulatory factors have been postulated. One of them is fetuin-A, a 60-kDa glycoprotein synthesized in the liver and secreted into blood. Observations in targeted mutant mice (Ahsg(-/-)) and in cell culture model systems have shown that fetuin-A is a powerful anti-mineralization factor in circulation, and the serum levels of fetuin-A in patients with PXE as well as in a mouse model of PXE (Abcc6(-/-)) have been shown to be reduced by up to 30%. In this study, we tested the hypothesis that overexpression of fetuin-A in Abcc6(-/-) mice counteracts the ectopic mineralization. Delivery of an expression construct containing full-length mouse fetuin-A complementary DNA (cDNA), linked to a His-tag, to the liver of these mice resulted in elevated serum levels of this protein. As a consequence, soft tissue mineralization, which is a characteristic of Abcc6(-/-) mice, was reduced by approximately 70% at 12 weeks of age, but the effect was transient when examined 4 weeks later. The results suggest that normalization of serum fetuin-A, either through gene therapy approaches or by direct protein delivery to the circulation, may offer strategies for treating PXE and perhaps other heritable disorders of soft tissue mineralization.

Ectopic mineralization of connective tissue in Abcc6–/– mice: effects of dietary modifications and a phosphate binder – a preliminary study

Abstract:  Pseudoxanthoma elasticum (PXE), a heritable multisystem disorder, is caused by mutations in the ABCC6 gene. We have developed a murine model for PXE by targeted inactivation of the corresponding mouse gene. A feature of this mouse model is ectopic mineralization of connective tissue capsule surrounding the bulb of vibrissae. This study was designed to investigate the effect of dietary sevelamer hydrochloride (Renagel®), a phosphate binder, and specific mineral modifications on ectopic mineralization of connective tissue in Abcc6–/– mice. Three groups were fed a specific diet: (i) a standard rodent diet, (ii) a standard rodent diet supplemented with sevelamer hydrochloride, and (iii) a custom experimental diet with specific mineral modifications (high phosphorus, low calcium and low magnesium). The degree of mineralization was determined in hematoxylin‐eosin‐stained sections using computerized morphometric analysis and by chemical assays to measure the calcium and phosphorus content of the vibrissae. The results indicated increased mineralization in the Abcc6–/– mice fed a standard diet or a diet with mineral modifications as compared with control mice fed a standard diet. However, feeding Abcc6–/– mice with diet supplemented with sevelamer hydrochloride did not improve mineralization, in comparison to mice fed with normal diet. Collectively, these results suggest that the mineralization process in PXE may be exacerbated by changes in mineral intake. The role of dietary minerals, and phosphorus in particular, as well as that of phosphate binders, in ectopic mineralization of PXE, merits further investigation.

Ectopic mineralization disorders of the extracellular matrix of connective tissue: molecular genetics and pathomechanisms of aberrant calcification.

Ectopic mineralization of connective tissues is a complex process leading to deposition of calcium phosphate complexes in the extracellular matrix, particularly affecting the skin and the arterial blood vessels and common in age-associated disorders. A number of initiating and contributing metabolic and environmental factors are linked to aberrant mineralization in these diseases, making the identification of precise pathomechanistic pathways exceedingly difficult. However, there has been significant recent progress in understanding the ectopic mineralization processes through study of heritable single-gene disorders, which have allowed identification of discrete pathways and contributing factors leading to aberrant connective tissue mineralization. These studies have provided support for the concept of an intricate mineralization/anti-mineralization network present in peripheral connective tissues, providing a perspective to development of pharmacologic approaches to limit the phenotypic consequences of ectopic mineralization. This overview summarizes the current knowledge of ectopic heritable mineralization disorders, with accompanying animal models, focusing on pseudoxanthoma elasticum and generalized arterial calcification of infancy, two autosomal recessive diseases manifesting with extensive connective tissue mineralization in the skin and the cardiovascular system.