Yvonne Nitschke

Similarities in the mechanisms of vascular calcification and the processes of bone and cartilage mineralization have come to light in recent years. Although formerly thought to be an inactive process of hydroxyapatite crystal precipitation, presently, vascular calcification is considered a regulated type of tissue mineralization. Moreover, different pathways of tissue mineralization are discussed. Pathological types of calcification are correlated with aging, metabolic disorders, chronic low-grade inflammation, and with genetic and acquired dysregulation of inorganic pyrophosphate (PPi) metabolism. This chapter focuses on recent developments in understanding the mechanisms of vascular calcification with special emphasis on the particular calcification pathway and the impact of deficient inhibition of calcification.

In recent years, mechanisms of arterial calcifications are beginning to be elucidated. Arterial calcification is now considered as an actively regulated process resembling osteogenesis within the arterial wall orchestrated by a number of systemic or constitutively expressed mediators. Genetic studies of rare monogenic human disorders and studies of naturally occurring or mutant mouse models have identified specific inductors and inhibitors of arterial calcification, which can be classified according to the networks they participate in. These networks include ATP and pyrophosphate metabolism, phosphate homeostasis and vitamin D receptor signaling. Furthermore, intracellular signaling molecules, including SMAD6 and a number of systemic circulatory inhibitors of arterial calcification, including fetuin, tumor necrosis factor receptor superfamily member 11b, matrix GLA protein, adiponectin and family with sequence similarity 20 member A have been identified by human and mouse genetics. ...

Generalized arterial calcification of infancy (GACI) is a rare condition characterized by arterial calcification within the internal elastic lamina associated with intimal proliferation, leading to stenosis of great and medium-sized vessels. This disease, caused by mutations in multiple exons of ENPP1, frequently results in death in infancy. Nowadays, the most promising therapeutic compounds for this rare disease are bisphosphonates. We describe a case of GACI associated with the novel mutation c.653A>T (p.D218V) in ENPP1 on both alleles. The male infant was delivered prematurely and developed heart failure, severe hypertension, and diffuse calcifications of all arterial districts. He was treated with etidronate (18 mg/kg/day); however, the clinical condition did not improve, and a resolution of calcifications was not observed. The infant died within the 6th month of life of ischemic heart failure. We conclude that even if the diagnosis of GACI is established early and bisphosphonate treatment is started early, the prognosis can be very poor.

Generalized arterial calcification of infancy (GACI, MIM#208000) is a rare autosomal recessive disorder characterized by extensive calcifications in the media of large- and medium-sized muscular arteries. Most affected children die in early infancy because of cardiac failure. GACI is linked to mutations in the ENPP1 gene, which encodes for an enzyme that generates inorganic pyrophosphate (PP(i)), a potent inhibitor of hydroxyapatite crystal formation. Treatment with bisphosphonates, which are synthetic PP(i) analogues, has been proposed as a means of reducing arterial calcifications in GACI patients, but no formalized treatment approach exists. We report on the long-term survival of a severe case of GACI linked to a novel homozygous missense mutation c.583T/C in the ENPP1 gene, diagnosed prenatally, and treated with bisphosphonates. Intravenous disodium pamidronate (three infusions at days 8, 15, and 18 of 0.25, 0.50, and 0.50 mg/kg, respectively) was changed to oral disodium etidronate (starting dose of 20 mg/kg daily, 50 mg die) at 3 weeks of age. Although the etidronate dose was maintained at 50 mg daily in our patient (corresponding to a progressive decrease from 20 to 5 mg/kg daily), the progressive resolution of arterial calcifications seen by 3 months of age was maintained until 2 years of age. Throughout the 2-year follow-up, our patient developed mild hypophosphatemia, due to renal phosphate wasting, without clinical, biochemical, or radiological sign of rickets. High-dose bisphosphonate therapy may not be necessary for an extended period of time in children with GACI.

Spontaneous pathologic arterial calcifications in childhood can occur in generalized arterial calcification of infancy (GACI) or in pseudoxanthoma elasticum (PXE). GACI is associated with biallelic mutations in ENPP1 in the majority of cases, whereas mutations in ABCC6 ...

Singleton-Merten Syndrome (SMS; MIM 182250) is a rare autosomal-dominant disorder characterized by heart calcifications causing premature death with psoriasis, widened phalanges medullary cavities, focal osteoporosis, delayed tooth eruption, short roots, rapid periodontisis and early loss of teeth through complete root resorption. Objective: To determine the SMS disease causing gene. Methods: 1)Perform whole exome sequencing on four SMS patients/three families; 2)determine structural signficance of mutation; 3)characterize the identified gene expression in affected tissues (qRT-PCR and immunohistochemsitry-IHC); and 4) analyze Affymetrix GeneChip expression profiles of SMS versus control dental cell populations. Results: A missense IFIH1mutation segregated with SMS was identified not present in the Single Nucleotide Polymorphism or the 1000 Genomes databases. IFIH1, located on 2q24.2 with 17 exons, encodes melanoma differentiation associated protein 5 (MDA5) a member of the RIG-I-li...

Singleton-Merten syndrome (SMS) is an infrequently described autosomal-dominant disorder characterized by early and extreme aortic and valvular calcification, dental anomalies (early-onset periodontitis and root resorption), osteopenia, and acro-osteolysis. To determine the molecular etiology of this disease, we performed whole-exome sequencing and targeted Sanger sequencing. We identified a common missense mutation, c.2465G>A (p.Arg822Gln), in interferon induced with helicase C domain 1 (IFIH1, encoding melanoma differentiation-associated protein 5 [MDA5]) in four SMS subjects from two families and a simplex case. IFIH1 has been linked to a number of autoimmune disorders, including Aicardi-Goutières syndrome. Immunohistochemistry demonstrated the localization of MDA5 in all affected target tissues. In vitro functional analysis revealed that the IFIH1 c.2465G>A mutation enhanced MDA5 function in interferon beta induction. Interferon signature genes were upregulated in SMS individuals' blood and dental cells. Our data identify a gain-of-function IFIH1 mutation as causing SMS and leading to early arterial calcification and dental inflammation and resorption.

BackgroundCartilage calcification is common in osteoarthritis (OA) and correlates with the severity of cartilage breakdown. Nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) contributes to maintain an optimal balance of pyrophosphate (PPi) and phosphate (Pi) in the extracellular matrix, thereby, together with the phosphate regulating molecules progressive ankylosis protein (ANK) and tissue nonspecific alkaline phosphatase (TNAP), preventing the generation of calcium crystals. Previous data have demonstrated regulation of NPP1 expression by inflammatory mediators and linked it to pathological cartilage calcification.MethodsCartilage explants were obtained from 120 consecuitve patients with OA undergoing joint replacement. Experimental OA was induced in mice by resection of the anterior cruciate ligament. Cartilage calcification was evaluated in human explants by digital contact radiography and in mice by fluorid positron emission tomography (PET)-scanning and μCT. The expression of NPP1, ANK and TNAP was performed by quantitative real time RT-PCR in human cartilage samples and by immunohistochemistry in experimental murine OA. Von Kossa-safranin-O staining was used to measure cartilage and meniscal calcification in mice carrying a homozygous loss of function mutation of enpp1. The severity of OA was quantified using the Mankin Score. Cartilage remodelling was investigated using immunohistological stainings for collagen type I, collagen type X, osteopontin and TRAP.ResultsIn OA patients, the expression of NPP1, but not ANK and TNAP, inversely correlated with cartilage calcification (p<0.05). Such inverse correlation was confirmed in vivo in experimental murine OA. Using Fluoride PET, the authors showed enhanced calcification activity in joints as well as in cartilage of non-weight bearing areas, including ear cartilage, in NPP1−/− mice, thereby suggesting that mechanical stress is not required for induction of calcification. NPP1−/− mice developed typical OA changes as evaluated by histological analysis and in vivo imaging. Intriguingly, calcification processes were associated with increased expression of the hypertrophic marker collagen X and the bone marker collagen I. Finally, the authors detected osteoclasts at the interface between bone and cartilage.ConclusionNPP1 is an important player in OA-associated cartilage calcification. Pathologic calcification in the NPP1−/− mice leads to cartilage remodelling into bone, thereby resembling aspects of hypertrophic maturation. Taken together, the data suggest that OA is characterised by the reactivation of molecular signalling cascades associated with endochondral ossification.

To analyse the function of nucleotide pyrophosphatase phosphodiesterase (NPP1), a member of the pyrophosphate pathway, in osteoarthritis (OA). mRNA expression of NPP1, ANK ankylosing protein and tissue non-specific alkaline phosphatase was assessed by quantitative PCR. NPP1 protein levels were analysed in mouse and human cartilage samples. Bone metabolism was analysed by F18-positron emission tomography-scanning and µCT in ttw/ttw mice. Ttw/ttw mice are mice carrying a loss-of-function mutation in NPP1. Calcification of articular cartilage was assessed using von Kossa staining and OA severity using the Mankin score. Cartilage remodelling was investigated by type X collagen immunohistochemistry. Expression of NPP1, but not the other members of this pathway, inversely correlated with cartilage calcification and OA severity in mouse and humans. Proinflammatory cytokines downregulated the expression of NPP1, demonstrating an influence of inflammation on matrix calcification. Ttw/ttw mutant mice, carrying a loss-of-function mutation in NPP1, exhibit increased bone formation process in joints compared with wild types. Ttw/ttw mice also developed spontaneous OA-like changes, evaluated by histological analysis and in vivo imaging. Ectopic calcifications were associated with increased expression of collagen X in the cartilage. The authors conclude that OA is characterised by the reactivation of molecular signalling cascades involving proinflammatory cytokines, thereby regulating the pyrophosphate pathway which consequently leads to cartilage ossification, at least in part resembling endochondral ossification.