Joshua Kingman

Dual Effects of Bisphosphonates on Ectopic Skin and Vascular Soft Tissue Mineralization versus Bone Microarchitecture in a Mouse Model of Generalized Arterial Calcification of Infancy

Generalized arterial calcification of infancy (GACI) is an intractable ectopic mineralization disorder caused by mutations in the ENPP1 gene resulting in reduced plasma inorganic pyrophosphate levels. We previously characterized the Enpp1asj mutant mouse as a model of GACI, and we have now explored the potential efficacy of bisphosphonates, non-hydrolyzable PPi analogs, in preventing ectopic mineralization in these mice. These mice were maintained on either basic diet (control) or diets containing etidronate or alendronate in three different concentrations (experimental). Considering low bioavailability of bisphosphonates when administered orally, subsequent studies tested the mice with subcutaneous injections of etidronate. The treatments were initiated at 4 weeks of age, and the degree of mineralization was assessed at 12 weeks of age by quantitation of calcium deposits in the muzzle skin containing dermal sheath of vibrissae and in aorta. We found that bisphosphonate treatments significantly reduced mineralization in skin and aorta. These changes in treated mice were accompanied with restoration of their bone microarchitecture, determined bymicrocomputed tomography. The inhibitory capacity of bisphosphonates, with mechanistic implications, was confirmed in a cell-based mineralization assay in vitro. Collectively, these results suggest that bisphosphonate treatment may be beneficial by a dual effect for preventing ectopic soft tissue mineralization while correcting decreased bone mineralization in GACI caused by ENPP1 mutations.

Analysis of CARD14 Polymorphisms in Pityriasis Rubra Pilaris: Activation of NF-κB.

SNP rs Variant Minor allele frequency cDNA Codon Amino acid rs9895931 c.27C>C/T TCC>TCT p.S9S T: 0.0% rs114688446 c.599G>G/A AGC>AAC p.S200N T: 0.8% rs4889990 c.633G>A GAG>GAA p.E211E A: 37.9% rs28674001 c.676-6G>A A: 37.4% rs142246283 c.683T>T/G CTA>CGA p.L228R no data rs61751629 c.1264G>A GAG>AAG p.E422K A: 2.1% rs11658460 c.1323C>T GAC>GAT p.D441D T: 10.2% rs34367357 c.1753G>A GTC>ATC p.V585I A: 5.2% rs2066964 c.1641G>C AGG>AGC p.R547S C: 65.1% rs117918077 c.2044C>C/T CGG>TGG p.R682W T: 1.2% rs11653893 c.2399-4A>G G: 40.4% no data c.2406C>C/A AGC>AGA p.S802R no data rs11652075 c.2458C>T CGG>TGG p.R820W T: 39.9% rs61757652 c.2481C>T CCC>CCT p.P827P T: 6.2% rs139789664 c.2495C>T, CTC>CTT, p.L832L T: 0.5%

Mineral content of the maternal diet influences ectopic mineralization in offspring of Abcc6−/− mice

Ectopic mineralization disorders inflicting the connective tissues display a spectrum of severity, some developing in utero and being diagnosed by prenatal ultrasound. This study was designed to test the hypothesis that the mineral content of maternal diet can influence the mineralization in the offspring. Pregnant Abcc6−/− mice, on 2 different strain backgrounds, were maintained either on normal rodent diet or on “acceleration diet,” rich in phosphate and low in magnesium, which has been previously shown to enhance the mineralization processes. The offspring were examined for mineralization by histopathology of various tissues and quantitated by chemical assay of calcium. The ectopic mineralization in the dermal sheath of vibrissae, a progressive biomarker of the overall mineralization, was readily detectable at the age of 4 weeks in the pups whose mothers were on the acceleration diet, while no evidence of mineralization was noted in those on normal diet. The mineralization of the vibrissae progressively increased when examined at 12 weeks of age. There was a significant reduction in urinary calcium and significant increase in urinary phosphorus concentrations both at 4 and 12 weeks of age in mice on the acceleration diet as compared to those on control diet. The results demonstrate that the mineral content of the maternal diet can influence ectopic mineralization in the offspring of mice genetically predisposed to ectopic mineralization (Abcc6−/−). These observations have implications for dietary management of pregnancies in which the fetus is diagnosed by prenatal ultrasound to have an ectopic mineralization disorder.

Plasma PPi Deficiency Is the Major, but Not the Exclusive, Cause of Ectopic Mineralization in an Abcc6–/– Mouse Model of PXE

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, is caused in most cases by inactivating mutations in the ABCC6 gene. It was recently discovered that absence of ABCC6-mediated adenosine triphosphate release from the liver and consequently reduced plasma inorganic pyrophosphate (PPi) levels underlie PXE. This study examined whether reduced levels of circulating PPi, an antimineralization factor, is the sole mechanism of PXE. The Abcc6-/- and Enpp1asj mice were crossed with transgenic mice expressing human ENPP1, an ectonucleotidase that generates PPi from adenosine triphosphate. We generated Abcc6-/- and Enpp1asj mice, either wild-type or hemizygous for human ENPP1. Plasma levels of PPi and the degree of ectopic mineralization were determined. Overexpression of human ENPP1 in Enpp1asj mice normalized plasma PPi levels to that of wild-type mice and, consequently, completely prevented ectopic mineralization. These changes were accompanied by restoration of their bone microarchitecture. In contrast, although significantly reduced mineralization was noted in Abcc6-/- mice expressing human ENPP1, small mineralization foci were still evident despite increased plasma PPi levels. These results suggest that PPi is the major mediator of ectopic mineralization in PXE, but there might be an alternative, as yet unknown mechanism, independent of PPi, by which ABCC6 prevents ectopic mineralization under physiologic conditions.

Abcc6 Knockout Rat Model Highlights the Role of Liver in PPi Homeostasis in Pseudoxanthoma Elasticum

Pseudoxanthoma elasticum, a heritable ectopic mineralization disorder, is caused by mutations in the ABCC6 gene primarily expressed in the liver and the kidneys. The fundamental question on pathogenesis of pseudoxanthoma elasticum, whether lack of ABCC6 expression in liver or kidney is the primary site of molecular pathology in peripheral tissues, has not been addressed. We generated a series of Abcc6-/- rats as models of pseudoxanthoma elasticum depicting ectopic mineralization in the skin, eyes, and the arterial blood vessels. Plasma inorganic pyrophosphate (PPi) level was reduced (<30%) in the Abcc6-/- rats leading to a lowered PPi/inorganic phosphate plasma ratio. In situ liver and kidney perfusions were performed to determine the relative contribution of these organs to PPi levels in circulation. PPi levels in the perfusates both in the liver and kidney of Abcc6-/- rats were significantly reduced, but the PPi levels in the liver perfusates of wild-type rats were 10-fold higher than that in the kidney perfusates. These observations suggest a critical role of hepatic ABCC6 in contributing to plasma PPi levels, identifying liver as a target of molecular correction to counteract ectopic mineralization in pseudoxanthoma elasticum.

Etidronate prevents, but does not reverse, ectopic mineralization in a mouse model of pseudoxanthoma elasticum (Abcc6-/-).

Pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI) are heritable disorders manifesting with ectopic tissue mineralization. Most cases of PXE and some cases of GACI are caused by mutations in the ABCC6 gene, resulting in reduced plasma pyrophosphate (PPi) levels. There is no effective treatment for these disorders. It has been suggested that administration of bisphosphonates, stable and non-hydrolyzable PPi analogs, could counteract ectopic mineralization in these disorders. In this study we tested the potential efficacy of etidronate, a first generation bisphosphonate, on ectopic mineralization in the muzzle skin of Abcc6−/− mice, a model of PXE. The Abcc6−/− mice received subcutaneous injections of etidronate, 0.283 and 3.40 mg/kg per injection (0.01× and 0.12×), twice a week, in both prevention and reversal studies. Ectopic mineralization in the dermal sheath of vibrissae in muzzle skin was determined by histopathologic analysis and by direct chemical assay for calcium content. Subcutaneous injection of etidronate prevented ectopic mineralization but did not reverse existing mineralization. The effect of etidronate was accompanied by alterations in the trabecular bone microarchitecture, determined by micro-computed tomography. The results suggest that etidronate may offer a potential treatment modality for PXE and GACI caused by ABCC6 mutations. Etidronate therapy should be initiated in PXE patients as soon as the diagnosis is made, with careful monitoring of potential side effects.Pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI) are heritable disorders manifesting with ectopic tissue mineralization. Most cases of PXE and some cases of GACI are caused by mutations in the ABCC6 gene, resulting in reduced plasma pyrophosphate (PPi) levels. There is no effective treatment for these disorders. It has been suggested that administration of bisphosphonates, stable and non-hydrolyzable PPi analogs, could counteract ectopic mineralization in these disorders. In this study we tested the potential efficacy of etidronate, a first generation bisphosphonate, on ectopic mineralization in the muzzle skin of Abcc6-/- mice, a model of PXE. The Abcc6-/- mice received subcutaneous injections of etidronate, 0.283 and 3.40 mg/kg per injection (0.01× and 0.12×), twice a week, in both prevention and reversal studies. Ectopic mineralization in the dermal sheath of vibrissae in muzzle skin was determined by histopathologic analysis and by direct chemical assay for calcium content. Subcutaneous injection of etidronate prevented ectopic mineralization but did not reverse existing mineralization. The effect of etidronate was accompanied by alterations in the trabecular bone microarchitecture, determined by micro-computed tomography. The results suggest that etidronate may offer a potential treatment modality for PXE and GACI caused by ABCC6 mutations. Etidronate therapy should be initiated in PXE patients as soon as the diagnosis is made, with careful monitoring of potential side effects.

Elevated dietary magnesium during pregnancy and postnatal life prevents ectopic mineralization in Enpp1 asj 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.