Oral supplementation of inorganic pyrophosphate in pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE; OMIM 264800) is a rare heritable multisystem disorder, characterized by ectopic mineralization affecting elastic fibres in the skin, eyes and the cardiovascular system. Skin findings often lead to early diagnosis of PXE, but currently, no specific treatment exists to counteract the progression of symptoms. PXE belongs to a group of Mendelian calcification disorders linked to pyrophosphate metabolism, which also includes generalized arterial calcification of infancy (GACI) and arterial calcification due to CD73 deficiency (ACDC). Inactivating mutations in ABCC6, ENPP1 and NT5E are the genetic cause of these diseases, respectively, and all of them result in reduced inorganic pyrophosphate (PPi ) concentration in the circulation. Although PPi is a strong inhibitor of ectopic calcification, oral supplementation therapy was initially not considered because of its low bioavailability. Our earlier work however demonstrated that orally administered pyrophosphate inhibits ectopic calcification in the animal models of PXE and GACI, and that orally given Na4 P2 O7 is absorbed in humans. Here, we report that gelatin-encapsulated Na2 H2 P2 O7  has similar absorption properties in healthy volunteers and people affected by PXE. The sodium-free K2 H2 P2 O7 form resulted in similar uptake in healthy volunteers and inhibited calcification in Abcc6-/- mice as effectively as its sodium counterpart. Novel pyrophosphate compounds showing higher bioavailability in mice were also identified. Our results provide an important step towards testing oral PPi in clinical trials in PXE, or potentially any condition accompanied by ectopic calcification including diabetes, chronic kidney disease or ageing.

Inhibition of Tissue-Nonspecific Alkaline Phosphatase Attenuates Ectopic Mineralization in the Abcc6-/- Mouse Model of PXE but Not in the Enpp1 Mutant Mouse Models of GACI.

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, is caused by mutations in the ABCC6 gene encoding a putative efflux transporter ABCC6. It was recently shown that the absence of ABCC6-mediated adenosine triphosphate release from the liver and, consequently, reduced inorganic pyrophosphate levels underlie the pathogenesis of PXE. Given that tissue-nonspecific alkaline phosphatase (TNAP), encoded by ALPL, is the enzyme responsible for degrading inorganic pyrophosphate, we hypothesized that reducing TNAP levels either by genetic or pharmacological means would lead to amelioration of the ectopic mineralization phenotype in the Abcc6-/- mouse model of PXE. Thus, we bred Abcc6-/- mice to heterozygous Alpl+/- mice that display approximately 50% plasma TNAP activity. The Abcc6-/-Alpl+/- double-mutant mice showed 52% reduction of mineralization in the muzzle skin compared with the Abcc6-/-Alpl+/+ mice. Subsequently, oral administration of SBI-425, a small molecule inhibitor of TNAP, resulted in 61% reduction of plasma TNAP activity and 58% reduction of mineralization in the muzzle skin of Abcc6-/- mice. By contrast, SBI-425 treatment of Enpp1 mutant mice, another model of ectopic mineralization associated with reduced inorganic pyrophosphate, failed to reduce muzzle skin mineralization. These results suggest that inhibition of TNAP might provide a promising treatment strategy for PXE, a currently intractable disease.

Pseudoxanthoma Elasticum as a Paradigm of Heritable Ectopic Mineralization Disorders: Pathomechanisms and Treatment Development.

Ectopic mineralization is a global problem and leading cause of morbidity and mortality. The pathomechanisms of ectopic mineralization are poorly understood. Recent studies on heritable ectopic mineralization disorders with defined gene defects have been helpful in elucidation of the mechanisms of ectopic mineralization in general. The prototype of such disorders is pseudoxanthoma elasticum (PXE), a late-onset, slowly progressing disorder with multisystem clinical manifestations. Other conditions include generalized arterial calcification of infancy (GACI), characterized by severe, early-onset mineralization of the cardiovascular system, often with early postnatal demise. In addition, arterial calcification due to CD73 deficiency (ACDC) occurs late in life, mostly affecting arteries in the lower extremities in elderly individuals. These three conditions, PXE, GACI, and ACDC, caused by mutations in ABCC6, ENPP1, and NT5E, respectively, are characterized by reduced levels of inorganic pyrophosphate (PPi) in plasma. Because PPi is a powerful antimineralization factor, it has been postulated that reduced PPi is a major determinant for ectopic mineralization in these conditions. These and related observations on complementary mechanisms of ectopic mineralization have resulted in development of potential treatment modalities for PXE, including administration of bisphosphonates, stable PPi analogs with antimineralization activity. It is conceivable that efficient treatments may soon become available for heritable ectopic mineralization disorders with application to common calcification disorders.

Elevated dietary magnesium during pregnancy and postnatal life prevents ectopic mineralization in Enpp1asj mice, a model for generalized arterial calcification of infancy.

Generalized arterial calcification of infancy (GACI) is an autosomal recessive disorder caused by mutations in the ENPP1 gene. It is characterized by mineralization of the arterial blood vessels, often diagnosed prenatally, and associated with death in early childhood. There is no effective treatment for this devastating disorder. We previously characterized the Enpp1asjmutant mouse as a model of GACI, and we have now explored the effect of elevated dietary magnesium (five-fold) in pregnant mothers and continuing for the first 14 weeks of postnatal life. The mothers were kept on either control diet or experimental diet supplemented with magnesium. Upon weaning at 4 weeks of age the pups were placed either on control diet or high magnesium diet. The degree of mineralization was assessed at 14 weeks of age by histopathology and a chemical calcium assay in muzzle skin, kidney and aorta. Mice placed on high magnesium diet showed little, if any, evidence of mineralization when their corresponding mothers were also placed on diet enriched with magnesium during pregnancy and nursing. The reduced ectopic mineralization in these mice was accompanied by increased calcium and magnesium content in the urine, suggesting that magnesium competes calcium-phosphate binding thereby preventing the mineral deposition. These results have implications for dietary management of pregnancies in which the fetus is suspected of having GACI. Moreover, augmenting a diet with high magnesium may be beneficial for other ectopic mineralization diseases, including nephrocalcinosis.

Next-generation sequencing for mutation detection in heritable skin diseases: the paradigm of pseudoxanthoma elasticum.

Next-generation sequencing applied either to the entire genome or to a subset, such as a whole exome, has revolutionized the search for pathogenic mutations in heritable diseases, including genodermatoses. In this issue, Hosen et al. applied whole-exome sequencing to identify potential pathogenic mutations in four candidate genes associated with pseudoxanthoma elasticum, the prototype of ectopic mineralization disorders. The study highlights the advantages of this approach over traditional Sanger sequencing, including expedience and cost, but it also illustrates some of the challenges encountered in implementing this rapidly evolving technology.

The effects of bisphosphonates on ectopic soft tissue mineralization caused by mutations in the ABCC6 gene.

Pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI) are heritable ectopic mineralization disorders. Most cases of PXE and many cases of GACI harbor mutations in the ABCC6 gene. There is no effective treatment for these disorders. We explored the potential efficacy of bisphosphonates to prevent ectopic calcification caused by ABCC6 mutations by feeding Abcc6(-/-) mice with diet containing etidronate disodium (ETD) or alendronate sodium trihydrate (AST) in quantities corresponding to 1x, 5x, or 12x of the doses used to treat osteoporosis in humans. The mice were placed on diet at 4 weeks of age, and the degree of mineralization was assessed at 12 weeks by quantitation of the calcium deposits in the dermal sheath of vibrissae, a progressive biomarker of the mineralization, by computerized morphometry of histopathologic sections and by direct chemical assay of calcium. We found that ETD, but not AST, at the 12x dosage, significantly reduced mineralization, suggesting that selected bisphosphonates may be helpful for prevention of mineral deposits in PXE and GACI caused by mutations in the ABCC6 gene, when combined with careful monitoring of efficacy and potential side-effects.

Genetic modulation of nephrocalcinosis in mouse models of ectopic mineralization: the Abcc6(tm1Jfk) and Enpp1(asj) mutant mice.

Ectopic mineralization of renal tissues in nephrocalcinosis is a complex, multifactorial process. The purpose of this study was to examine the role of genetic modulation and the role of diet in nephrocalcinosis using two established mouse models of ectopic mineralization, Abcc6(tm1Jfk) and Enpp1(asj) mice, which serve as models for pseudoxanthoma elasticum and generalized arterial calcification of infancy, two heritable disorders, respectively. These mutant mice, when on standard rodent diet, develop nephrocalcinosis only at a very late age. In contrast, when placed on an 'acceleration diet' composed of increased phosphate and reduced magnesium content, they showed extensive mineralization of the kidneys affecting primarily the medullary tubules as well as arcuate and renal arteries, as examined by histopathology and quantitated by chemical assay for calcium. Mineralization could also be detected noninvasively by micro computed tomography. Whereas the heterozygous mice did not develop nephrocalcinosis, compound heterozygous mice carrying both mutant alleles, Abcc6(tm1Jfk/+) and Enpp1(+/asj), developed ectopic mineralization similar to that noted in homozygous mice for either gene, indicating that deletion of one Abcc6 allele along with Enpp1 haploinsufficiency resulted in renal mineralization. Thus, synergistic genetic defects in the complex mineralization/antimineralization network can profoundly modulate the degree of ectopic mineralization in nephrocalcinosis.

Mutant Enpp1asj mice as a model for generalized arterial calcification of infancy.

Generalized arterial calcification of infancy (GACI), an autosomal recessive disorder, is characterized by early mineralization of blood vessels, often diagnosed by prenatal ultrasound and usually resulting in demise during the first year of life. It is caused in most cases by mutations in the ENPP1 gene, encoding an enzyme that hydrolyzes ATP to AMP and inorganic pyrophosphate, the latter being a powerful anti-mineralization factor. Recently, a novel mouse phenotype was recognized as a result of ENU mutagenesis - those mice developed stiffening of the joints, hence the mutant mouse was named 'ages with stiffened joints' (asj). These mice harbor a missense mutation, p.V246D, in the Enpp1 gene. Here we demonstrate that the mutant ENPP1 protein is largely absent in the liver of asj mice, and the lack of enzymatic activity results in reduced inorganic pyrophosphate (PPi) levels in the plasma, accompanied by extensive mineralization of a number of tissues, including arterial blood vessels. The progress of mineralization is highly dependent on the mineral composition of the diet, with significant shortening of the lifespan on a diet enriched in phosphorus and low in magnesium. These results suggest that the asj mouse can serve as an animal model for GACI.

Warfarin accelerates ectopic mineralization in Abcc6(-/-) mice: clinical relevance to pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE) is a multisystem ectopic mineralization disorder caused by mutations in the ABCC6 gene. Warfarin, a commonly used anticoagulant, is associated with increased mineralization of the arterial blood vessels and cardiac valves. We hypothesized that warfarin may accelerate ectopic tissue mineralization in PXE, with clinical consequences. To test this hypothesis, we developed a model in which Abcc6(-/-) mice, which recapitulate features of PXE, were fed a diet supplemented with warfarin and vitamin K1. Warfarin action was confirmed by significantly increased serum levels of oxidized vitamin K. For mice placed on a warfarin-containing diet, quantitative chemical and morphometric analyses revealed massive accumulation of mineral deposits in a number of tissues. Mice fed a warfarin-containing diet were also shown to have abundant uncarboxylated form of matrix Gla protein, which allowed progressive tissue mineralization to ensue. To explore the clinical relevance of these findings, 1747 patients with PXE from the approximately 4000 patients in the PXE International database were surveyed about the use of warfarin. Of the 539 respondents, 2.6% reported past or present use of warfarin. Based on the prevalence of PXE (approximately 1:50,000), thousands of patients with PXE worldwide may be at risk for worsening of PXE as a result of warfarin therapy.

Magnesium reduces carotid intima-media thickness in a mouse model of pseudoxanthoma elasticum: a novel treatment biomarker.

Pseudoxanthoma elasticum (PXE), which demonstrates progressive build-up of calcium phosphate and proteoglycan deposits in skin, eye, and arteries, has been associated with myocardial infarctions, stroke, and blindness. In a mouse model of PXE, a magnesium-enriched diet prevents mineralization of the vibrissae capsule, an early biomarker for PXE. However, biomarkers for therapeutic responses in PXE have not been identified in humans. Because PXE patients have an increased carotid intima-media thickness (CIMT), a risk factor for cardiovascular disease and stroke, we analyzed the feasibility of CIMT as a treatment endpoint before and after magnesium supplementation in a mouse model of PXE (Abcc6(-/-) ). CIMT was measured in 1-year-old Abcc6(-/-) and Abcc6(+/+) mice fed either standard rodent diet with or without magnesium oxide supplementation for 2 months. Baseline CIMT in Abcc6(-/-) versus Abcc6(+/+) mice was increased (p value = 0.009), whereas CIMT in magnesium-treated versus untreated Abcc6(-/-) mice was reduced (p value = 0.024). CIMT is a novel treatment endpoint in this mouse model and may serve as a predictive biomarker of therapeutic response in PXE patients. In that context, magnesium oxide significantly reduced CIMT in PXE mice, and may be useful for disease prevention in PXE patients.

[Study on the extraction of polyphenol from Scindapsus officinalis with ultrasonic wave technology optimized by central composite design-response surface method].

OBJECTIVE: To optimize ultrasonic extraction technology process conditions of polyphenol from Scindapsus officinalis by the response surface method. METHODS: Based on ethanol concentration, ultrasonic time, the liquid-solid ratio of single factor experiment, the principle of design for 3 star factor 3 level response surface methodology was applied. With FC extraction method for determination of polyphenols, the response surface optimization extraction conditions were studied. RESULTS: The ethanol concentration of 61.14%, ultrasonic wave extracting time of 59.73 min and the ratio of solvent volume of 27.72:1 (Extract 3 times) were selected as the optimum conditions,the extraction yield of polyphenols was 1.352%, with the theoretical 1.361% for the relative error of -0.66%. CONCLUSION: Ultrasonic extraction is a good method for saving time, energy and material,and can be applied to the polyphenols extraction. Central composite design-response surface optimization can get better ecasting results.

Pseudoxanthoma elasticum, the paradigm of heritable ectopic mineralization disorders - can diet help?

Pseudoxanthoma elasticum (PXE) is a heritable multi-system disorder manifesting with characteristic cutaneous lesions, associated with ocular findings and cardiovascular involvement. The skin lesions, yellowish papules which coalesce into plaques of inelastic and leathery skin, demonstrate by histopathologic and ultrastructural examinations ectopic mineralization of dermal connective tissues, primarily the elastic structures. PXE is inherited in an autosomal recessive fashion due to mutations in the ABCC6 gene. Significant insights into the pathogenesis of PXE have been recently obtained from observations on the Abcc6(-/-) knockout mouse which mimics the genetic, histopathologic and ultrastructural features of PXE. This mouse model has provided a platform to test various treatment modalities to counteract the mineralization phenotypes. One of the intriguing findings emanating from these studies is that supplementation of the mouse diet with magnesium, at levels that are ∼5-fold higher than those in control diet, completely inhibits the development of tissue mineralization. These and related observations suggest that changes in the diet might counteract the progression of PXE and improve the quality of life of patients with 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.

Magnesium carbonate-containing phosphate binder prevents connective tissue mineralization in Abcc6(-/-) mice-potential for treatment of pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE) is a heritable disorder characterized by ectopic mineralization of connective tissues primarily in the skin, eyes, and the cardiovascular system. PXE is caused by mutations in the ABCC6 gene. While PXE is associated with considerable morbidity and mortality, there is currently no effective or specific treatment. In this study, we tested oral phosphate binders for treatment of a mouse model of PXE which we have developed by targeted ablation of the corresponding mouse gene (Abcc6(-/-)). This "knock-out" (KO) mouse model recapitulates features of PXE and demonstrates mineralization of a number of tissues, including the connective tissue capsule surrounding vibrissae in the muzzle skin which serves as an early biomarker of the mineralization process. Treatment of these mice with a magnesium carbonate-enriched diet (magnesium concentration being 5-fold higher than in the control diet) completely prevented mineralization of the vibrissae up to 6 months of age, as demonstrated by computerized morphometric analysis of histopathology as well as by calcium and phosphate chemical assays. The magnesium carbonate-enriched diet also prevented the progression of mineralization when the mice were placed on that experimental diet at 3 months of age and followed up to 6 months of age. Treatment with magnesium carbonate was associated with a slight increase in the serum concentration of magnesium, with no effect on serum calcium and phosphorus levels. In contrast, concentration of calcium in the urine was increased over 10-fold while the concentration of phosphorus was markedly decreased, being essentially undetectable after long-term (> 4 month) treatment. No significant changes were noted in the serum parathyroid hormone levels. Computerized axial tomography scan of bones in mice placed on magnesium carbonate-enriched diet showed no differences in the bone density compared to mice on the control diet, and chemical assays showed a small increase in the calcium and phosphate content of the femurs by chemical assay, in comparison to mice on control diet. Similar experiments with another experimental diet supplemented with lanthanum carbonate did not interfere with the mineralization process in Abcc6(-/-) mice. These results suggest that magnesium carbonate may offer a potential treatment modality for PXE, a currently intractable disease, as well as for other conditions characterized by ectopic mineralization of connective tissues.

Pseudoxanthoma elasticum: oxidative stress and antioxidant diet in a mouse model (Abcc6-/-).

Pseudoxanthoma elasticum (PXE), a multisystem disorder characterized by ectopic mineralization of soft connective tissues, is caused by mutations in the ABCC6 gene. The pathomechanistic details of the mineralization process are largely unknown, but oxidative stress has been suggested to play a role. In this study, we tested Abcc6(-/-) mice, which serve as a model of PXE, for markers of the oxidative stress in the liver and serum. The total antioxidant capacity as well as markers of protein oxidation and lipid peroxidation suggested the presence of chronic oxidative stress. Feeding these mice for 5 months with a diet supplemented with antioxidants (vitamins C and E, selenium, and N-acetylcysteine) countered the oxidative stress but did not modify the ectopic mineralization process. These results suggest that the Abcc6(-/-) mice suffer from chronic oxidative stress but this does not contribute to connective tissue mineralization, the hallmark of PXE.

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

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.