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// Qiaoli Li 1 , Joshua Kingman 1 , John P. Sundberg 2 , Michael A. Levine 3 , Jouni Uitto 1 1 Department of Dermatology and Cutaneous Biology, The Sidney Kimmel Medical College at Thomas Jefferson University, Philadelphia, PA, USA 2 The Jackson Laboratory, Bar Harbor, ME, USA 3 Division of Endocrinology and Diabetes, Children’s Hospital of Philadelphia and University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Correspondence to: Qiaoli Li, email: Qiaoli.Li@jefferson.edu Keywords: pseudoxanthoma elasticum, ectopic mineralization, etidronate treatment, bisphosphonates, mouse model Received: March 03, 2016 Accepted: June 09, 2016 Published: July 20, 2016 ABSTRACT 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.

// Joshua Kingman 1 , Jouni Uitto 1 and Qiaoli Li 1 1 Department of Dermatology and Cutaneous Biology, The Sidney Kimmel Medical College, and the PXE International Center of Excellence in Research and Clinical Care, Thomas Jefferson University, Philadelphia, PA, USA Correspondence to: Qiaoli Li, email: // Keywords : mouse model, ectopic mineralization, maternal diet, magnesium, generalized arterial calcification of infancy, Pathology Section Received : October 28, 2016 Accepted : March 16, 2017 Published : March 29, 2017 Abstract 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 Enpp1 asj mutant 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.

// Sarah Y. Siu 1 , Nathaniel A. Dyment 2 , David W. Rowe 2 , John P. Sundberg 3 , Jouni Uitto 1,4 and Qiaoli Li 1,4 1 Department of Dermatology and Cutaneous Biology, The Sidney Kimmel Medical College and The PXE International Center of Excellence in Research and Clinical Care, Thomas Jefferson University, Philadelphia, PA, USA 2 Center for Regenerative Medicine and Skeletal Development, University of Connecticut Health Center, Farmington, CT, USA 3 The Jackson Laboratory, Bar Harbor, ME, USA 4 Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, PA, USA Correspondence to: Qiaoli Li, email: // Keywords : ectopic mineralization; generalized arterial calcification of infancy; mouse models; cryohistology; Pathology Section Received : October 07, 2016 Accepted : November 02, 2016 Published : November 14, 2016 Abstract Generalized arterial calcification of infancy (GACI) is an autosomal recessive disorder characterized by early onset of extensive mineralization of the cardiovascular system. The classical forms of GACI are caused by mutations in the ENPP1 gene, encoding a membrane-bound pyrophosphatase/phosphodiesterase that hydrolyzes ATP to AMP and inorganic pyrophosphate. The asj-2J mouse harboring a spontaneous mutation in the Enpp1 gene has been characterized as a model for GACI. These mutant mice develop ectopic mineralization in skin and vascular connective tissues as well as in cartilage and collagen-rich tendons and ligaments. This study examined in detail the temporal ectopic mineralization phenotype of connective tissues in this mouse model, utilizing a novel cryo-histological method that does not require decalcification of bones. The wild type, heterozygous, and homozygous mice were administered fluorescent mineralization labels at 4 weeks (calcein), 10 weeks (alizarin complexone), and 11 weeks of age (demeclocycline). Twenty-four hours later, outer ears, muzzle skin, trachea, aorta, shoulders, and vertebrae were collected from these mice and examined for progression of mineralization. The results revealed differential timeline for disease initiation and progression in various tissues of this mouse model. It also highlights the advantages of cryo-histological fluorescent imaging technique to study mineral deposition in mouse models of ectopic mineralization disorders.

// Jieyu Zhang 1,2 , Nathaniel A. Dyment 3 , David W. Rowe 3 , Sarah Y. Siu 1 , John P. Sundberg 4 , Jouni Uitto 1 and Qiaoli Li 1 1 Department of Dermatology and Cutaneous Biology, The Sidney Kimmel Medical College at Thomas Jefferson University, Philadelphia, PA, USA 2 Department of Dermatology, The Fourth Military Medical University, Xijing Hospital, Xi’an, China 3 Center for Regenerative Medicine and Skeletal Development, University of Connecticut Health Center, Farmington, CT, USA 4 The Jackson Laboratory, Bar Harbor, ME, USA Correspondence to: Qiaoli Li, email: // Keywords : ectopic mineralization, tendon and ligament calcification, chondrocalcinosis, generalized arterial calcification of infancy, mouse models, Pathology Section Received : October 22, 2015 Accepted : January 31, 2016 Published : February 17, 2016 Abstract Generalized arterial calcification of infancy (GACI), an autosomal recessive disorder caused by mutations in the ENPP1 gene, manifests with extensive mineralization of the cardiovascular system. A spontaneous asj-2J mutant mouse has been characterized as a model for GACI. Previous studies focused on phenotypic characterization of skin and vascular tissues. This study further examined the ectopic mineralization phenotype of cartilage, collagen-rich tendons and ligaments in this mouse model. The mice were placed on either control diet or the “acceleration diet” for up to 12 weeks of age. Soft connective tissues, such as ear (elastic cartilage) and trachea (hyaline cartilage), were processed for standard histology. Assessment of ectopic mineralization in articular cartilage and fibrocartilage as well as tendons and ligaments which are attached to long bones were performed using a novel cryo-histological method without decalcification. These analyses demonstrated ectopic mineralization in cartilages as well as tendons and ligaments in the homozygous asj-2J mice at 12 weeks of age, with the presence of immature osteophytes displaying alkaline phosphatase and tartrate-resistant acid phosphatase activities as early as at 6 weeks of age. Alkaline phosphatase activity was significantly increased in asj-2J mouse serum as compared to wild type mice, indicating increased bone formation rate in these mice. Together, these data highlight the key role of ENPP1 in regulating calcification of both soft and skeletal tissues.