pyrophosphate and Vascular-Calcification

Conventionally, ATP is considered to be the principal energy source in cells. However, over the last few years, a novel role for ATP as a potent extracellular signaling molecule and the principal source of extracellular pyrophosphate, the main endogenous inhibitor of vascular calcification, has emerged. A large body of evidence suggests that two principal mechanisms are involved in the initiation and progression of ectopic calcification: high phosphate concentration and pyrophosphate deficiency. Pathologic calcification of cardiovascular structures, or vascular calcification, is a feature of several genetic diseases and a common complication of chronic kidney disease, diabetes, and aging. Previous studies have shown that the loss of function of several enzymes and transporters involved in extracellular ATP/pyrophosphate metabolism is associated with vascular calcification. Therefore, pyrophosphate homeostasis should be further studied to facilitate the design of novel therapeutic approaches for ectopic calcification of cardiovascular structures, including strategies to increase pyrophosphate concentrations by targeting the ATP/pyrophosphate metabolism cycle.

Topics: Adenosine Triphosphate; Diphosphates; Homeostasis; Humans; Vascular Calcification

Pathologic soft tissue calcification can occur in both genetic and acquired clinical conditions, causing significant morbidity and mortality. Although the pathomechanisms of pathologic calcification are poorly understood, major progress has been made in recent years in defining the underlying genetic defects in Mendelian disorders of ectopic calcification. This review presents an overview of the pathophysiology of five monogenic disorders of pathologic calcification: pseudoxanthoma elasticum, generalized arterial calcification of infancy, arterial calcification due to deficiency of CD73, ankylosis, and progeria. These hereditary disorders, caused by mutations in genes encoding ATP binding cassette subfamily C member 6, ectonucleotide pyrophosphatase/phosphodiesterase 1, CD73, progressive ankylosis protein, and lamin A/C proteins, respectively, are inorganic pyrophosphate (PPi) deficiency syndromes with reduced circulating levels of PPi, the principal physiologic inhibitor of calcium hydroxyapatite deposition in soft connective tissues. In addition to genetic diseases, PPi deficiency has been encountered in acquired clinical conditions accompanied by pathologic calcification. Because specific and effective treatments are lacking for pathologic calcification, the unifying finding of PPi deficiency suggests that PPi-targeted therapies may be beneficial to counteract pathologic soft tissue calcification in both genetic and acquired diseases.

Topics: Ankylosis; Calcinosis; Choristoma; Diphosphates; Humans; Pseudoxanthoma Elasticum; Syndrome; Vascular Calcification

Cardiovascular complications due to accelerated arterial stiffening and atherosclerosis are the leading cause of morbimortality in Western society. Both pathologies are frequently associated with vascular calcification. Pathologic calcification of cardiovascular structures, or vascular calcification, is associated with several diseases (for example, genetic diseases, diabetes, and chronic kidney disease) and is a common consequence of aging. Calcium phosphate deposition, mainly in the form of hydroxyapatite, is the hallmark of vascular calcification and can occur in the medial layer of arteries (medial calcification), in the atheroma plaque (intimal calcification), and cardiac valves (heart valve calcification). Although various mechanisms have been proposed for the pathogenesis of vascular calcification, our understanding of the pathogenesis of calcification is far from complete. However, in recent years, some risk factors have been identified, including high serum phosphorus concentration (hyperphosphatemia) and defective synthesis of pyrophosphate (pyrophosphate deficiency). The balance between phosphate and pyrophosphate, strictly controlled by several genes, plays a key role in vascular calcification. This review summarizes the current knowledge concerning phosphate and pyrophosphate homeostasis, focusing on the role of extracellular pyrophosphate metabolism in aortic smooth muscle cells and macrophages.

Topics: Diphosphates; Humans; Phosphates; Vascular Calcification

In the past two decades, there has been great progress in identifying the molecular basis and pathomechanistic details in pseudoxanthoma elasticum (PXE), a heritable multisystem ectopic mineralization disorder. Although the identification of pathogenic variants in ABCC6 has been critical for understanding the disease process, genetic modifiers have been disclosed that explain the phenotypic heterogeneity of PXE. Adding to the genetic complexity of PXE are PXE-like phenotypes caused by pathogenic variants in other ectopic mineralization-associated genes. This review summarizes the current knowledge of the genetics and candidate modifier genes in PXE, a multifactorial disease at the genome-environment interface.

Topics: Animals; Diphosphates; Genetic Association Studies; Humans; Mice; Multidrug Resistance-Associated Proteins; Oxidative Stress; Phosphoric Diester Hydrolases; Pseudoxanthoma Elasticum; Pyrophosphatases; Vascular Calcification

Pathological (ectopic) mineralization of soft tissues occurs during aging, in several common conditions such as diabetes, hypercholesterolemia, and renal failure and in certain genetic disorders. Pseudoxanthoma elasticum (PXE), a multi-organ disease affecting dermal, ocular, and cardiovascular tissues, is a model for ectopic mineralization disorders. ABCC6 dysfunction is the primary cause of PXE, but also some cases of generalized arterial calcification of infancy (GACI). ABCC6 deficiency in mice underlies an inducible dystrophic cardiac calcification phenotype (DCC). These calcification diseases are part of a spectrum of mineralization disorders that also includes Calcification of Joints and Arteries (CALJA). Since the identification of ABCC6 as the "PXE gene" and the development of several animal models (mice, rat, and zebrafish), there has been significant progress in our understanding of the molecular genetics, the clinical phenotypes, and pathogenesis of these diseases, which share similarities with more common conditions with abnormal calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into inorganic pyrophosphate (PPi) and adenosine by the ectonucleotidases NPP1 and CD73 (NT5E). PPi is a potent endogenous inhibitor of calcification, whereas adenosine indirectly contributes to calcification inhibition by suppressing the synthesis of tissue non-specific alkaline phosphatase (TNAP). At present, therapies only exist to alleviate symptoms for both PXE and GACI; however, extensive studies have resulted in several novel approaches to treating PXE and GACI. This review seeks to summarize the role of ABCC6 in ectopic calcification in PXE and other calcification disorders, and discuss therapeutic strategies targeting various proteins in the pathway (ABCC6, NPP1, and TNAP) and direct inhibition of calcification via supplementation by various compounds.

Topics: 5'-Nucleotidase; Animals; ATP-Binding Cassette Transporters; Calcification, Physiologic; Calcinosis; Diphosphates; GPI-Linked Proteins; Humans; Joint Diseases; Mice; Multidrug Resistance-Associated Proteins; Phosphoric Diester Hydrolases; Pseudoxanthoma Elasticum; Pyrophosphatases; Rats; Vascular Calcification; Vascular Diseases

This review summarizes current understanding of generalized arterial calcification of infancy (GACI), emphasizing pathophysiology, clinical presentation, and approaches and controversies in management.. Identification of causative ENPP1 mutations revealed that GACI arises from deficiencies in inorganic pyrophosphate (leading to calcifications) and adenosine monophosphate (leading to intimal proliferation). Identification of genotypic and phenotypic overlap with pseudoxanthoma elasticum and autosomal recessive hypophosphatemic rickets further advanced understanding of GACI as a complex, multisystemic disease. Clinical data is limited to small, retrospective samples; it is therefore unknown whether commonly used medications, such as bisphosphonates and hypophosphatemia treatment, are therapeutic or potentially harmful. ENPP1-Fc replacement represents a promising approach warranting further study. Knowledge gaps in natural history place clinicians at high risk of assigning causality to interventions that are correlated with changes in clinical status. There is thus a critical need for improved natural history studies to develop and test targeted therapies.

Topics: Adenosine Monophosphate; Bone Density Conservation Agents; Calcinosis; Cardiovascular Agents; Chelating Agents; Diphosphates; Diphosphonates; Familial Hypophosphatemic Rickets; Genotype; Hearing Loss; Humans; Multidrug Resistance-Associated Proteins; Phenotype; Phosphoric Diester Hydrolases; Pseudoxanthoma Elasticum; Pyrophosphatases; Thiosulfates; Tooth Diseases; Vascular Calcification; Vitamin D

Pseudoxanthoma elasticum is a rare disease mainly due to

Topics: 5'-Nucleotidase; Diphosphates; GPI-Linked Proteins; Humans; Kidney Calculi; Multidrug Resistance-Associated Proteins; Mutation; Pseudoxanthoma Elasticum; Rare Diseases; Urolithiasis; Vascular Calcification

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.

Topics: 5'-Nucleotidase; Diphosphates; Diphosphonates; GPI-Linked Proteins; Humans; Multidrug Resistance-Associated Proteins; Phosphoric Diester Hydrolases; Pseudoxanthoma Elasticum; Pyrophosphatases; Vascular Calcification

In recent years, the mechanisms and clinical significance of vascular calcification have been increasingly investigated. For over a century, however, pathologists have recognized that vascular calcification is a form of heterotopic ossification. In this review, we aim to describe the pathology and molecular processes of vascular ossification, to characterize its clinical significance and treatment options, and to elucidate areas that require further investigation. The molecular mechanisms of vascular ossification involve the activation of regulators including bone morphogenic proteins and chondrogenic transcription factors and the loss of mineralization inhibitors like fetuin-A and pyrophosphate. Although few studies have examined the gross pathology of vascular ossification, the presence of these molecular regulators and evidence of microfractures and cartilage have been demonstrated on heart valves and atherosclerotic plaques. These changes are often triggered by common inflammatory and metabolic disorders like diabetes, hyperlipidemia, and chronic kidney disease. The increasing prevalence of these diseases warrants further research into the clinical significance of vascular ossification and future treatment options.

Topics: 5'-Nucleotidase; alpha-2-HS-Glycoprotein; Animals; Diphosphates; GPI-Linked Proteins; Humans; Ossification, Heterotopic; RANK Ligand; Vascular Calcification

Alkaline phosphatases (APs) remove the phosphate (dephosphorylation) needed in multiple metabolic processes (from many molecules such as proteins, nucleotides, or pyrophosphate). Therefore, APs are important for bone mineralization but paradoxically they can also be deleterious for other processes, such as vascular calcification and the increasingly known cross-talk between bone and vessels. A proper balance between beneficial and harmful activities is further complicated in the context of chronic kidney disease (CKD). In this narrative review, we will briefly update the complexity of the enzyme, including its different isoforms such as the bone-specific alkaline phosphatase or the most recently discovered B1x. We will also analyze the correlations and potential discrepancies with parathyroid hormone and bone turnover and, most importantly, the valuable recent associations of AP's with cardiovascular disease and/or vascular calcification, and survival. Finally, a basic knowledge of the synthetic and degradation pathways of APs promises to open new therapeutic strategies for the treatment of the CKD-Mineral and Bone Disorder (CKD-MBD) in the near future, as well as for other processes such as sepsis, acute kidney injury, inflammation, endothelial dysfunction, metabolic syndrome or, in diabetes, cardiovascular complications. However, no studies have been done using APs as a primary therapeutic target for clinical outcomes, and therefore, AP's levels cannot yet be used alone as an isolated primary target in the treatment of CKD-MBD. Nonetheless, its diagnostic and prognostic potential should be underlined.

Topics: Alkaline Phosphatase; Animals; Bone Remodeling; Chronic Kidney Disease-Mineral and Bone Disorder; Diphosphates; Humans; Inflammation; Isoenzymes; Parathyroid Glands; Parathyroid Hormone; Phosphates; Phosphorus; Proportional Hazards Models; Treatment Outcome; Vascular Calcification

Pathologic cardiovascular calcification is associated with a number of conditions and is a common complication of chronic kidney disease. Because ambient calcium and phosphate levels together with properties of the vascular matrix favor calcification even under normal conditions, endogenous inhibitors such as pyrophosphate play a key role in prevention. Genetic diseases and animal models have elucidated the metabolism of extracellular pyrophosphate and demonstrated the importance of pyrophosphate deficiency in vascular calcification. Therapies based on pyrophosphate metabolism have been effective in animal models, including renal failure, and hold promise as future therapies to prevent vascular calcification.

Topics: Animals; Blood Vessels; Calcium; Diphosphates; Down-Regulation; Genetic Predisposition to Disease; Humans; Renal Insufficiency, Chronic; Risk Factors; Vascular Calcification

Pseudoxanthoma elasticum is a prototype of heritable ectopic mineralization disorders, with phenotypic overlap with generalized arterial calcification of infancy and arterial calcification due to CD73 deficiency. Recent observations have suggested that the reduced inorganic pyrophosphate/phosphate ratio is the cause of soft connective tissue mineralization in these disorders. PXE International, a patient advocacy organization, supports research in part by sponsoring biennial research symposia on these disorders; the latest meeting was held in September 2016 at Thomas Jefferson University, Philadelphia. This report summarizes the progress in pseudoxanthoma elasticum and other ectopic mineralization disorders, as presented in the symposium, with focus on translational aspects of precision medicine toward improved diagnostics and treatment development for these currently intractable disorders.

Topics: 5'-Nucleotidase; Alkaline Phosphatase; Animals; Biopsy, Needle; Clinical Trials as Topic; Congresses as Topic; Diphosphates; Disease Models, Animal; Etidronic Acid; Genetic Predisposition to Disease; GPI-Linked Proteins; Humans; Immunohistochemistry; Internationality; Mice; Mutation; Phosphoric Diester Hydrolases; Pseudoxanthoma Elasticum; Pyrophosphatases; Rare Diseases; Vascular Calcification

We give an update on the etiology and potential treatment options of rare inherited monogenic disorders associated with arterial calcification and calcific cardiac valve disease.. Genetic studies of rare inherited syndromes have identified key regulators of ectopic calcification. Based on the pathogenic principles causing the diseases, these can be classified into three groups: (1) disorders of an increased extracellular inorganic phosphate/inorganic pyrophosphate ratio (generalized arterial calcification of infancy, pseudoxanthoma elasticum, arterial calcification and distal joint calcification, progeria, idiopathic basal ganglia calcification, and hyperphosphatemic familial tumoral calcinosis; (2) interferonopathies (Singleton-Merten syndrome); and (3) others, including Keutel syndrome and Gaucher disease type IIIC. Although some of the identified causative mechanisms are not easy to target for treatment, it has become clear that a disturbed serum phosphate/pyrophosphate ratio is a major force triggering arterial and cardiac valve calcification. Further studies will focus on targeting the phosphate/pyrophosphate ratio to effectively prevent and treat these calcific disease phenotypes.

Topics: Abnormalities, Multiple; Aortic Diseases; Basal Ganglia Diseases; Calcinosis; Cartilage Diseases; Dental Enamel Hypoplasia; Diphosphates; Enzyme Replacement Therapy; Gaucher Disease; Hand Deformities, Congenital; Humans; Hyperostosis, Cortical, Congenital; Hyperphosphatemia; Interferons; Metacarpus; Muscular Diseases; Odontodysplasia; Osteoporosis; Phosphates; Progeria; Pseudoxanthoma Elasticum; Pulmonary Valve Stenosis; Vascular Calcification

Heritable ectopic mineralization disorders represent a phenotypically diverse group of conditions characterized by deposition of calcium phosphate complexes in soft connective tissues. The prototype of such conditions is pseudoxanthoma elasticum, and related conditions with overlapping clinical features include generalized arterial calcification of infancy and arterial calcification due to CD73 deficiency. Molecular genetic investigations have revealed mutations in the genes physiologically involved in generation of inorganic pyrophosphate and inorganic phosphate, and the findings suggest a unifying pathomechanism relating to reduced inorganic pyrophosphate/inorganic phosphate ratio. This hypothesis is based on the notion that inorganic pyrophosphate serves as a powerful inhibitor of mineralization, whereas inorganic phosphate is a promineralization factor, and an appropriate inorganic pyrophosphate/inorganic phosphate ratio is critical for prevention of ectopic mineralization under homeostatic conditions.

Topics: 5'-Nucleotidase; Animals; Biomedical Research; Diphosphates; Forecasting; Genetic Predisposition to Disease; GPI-Linked Proteins; Humans; Mice; Mice, Knockout; Molecular Biology; Mutation; Phenotype; Pseudoxanthoma Elasticum; Rare Diseases; Vascular Calcification

Vascular calcification is an independent risk factor for the development of cardiovascular disease and is classified into two types based on the site of calcification : intimal atherosclerotic calcification and Mönckeberg's medial calcification. Matrix vesicles released from macrophages and vascular smooth muscle cells (VSMC) during apoptosis play a pivotal role in formation of fine granular calcification, while osteogenic differentiation of VSMC contributes to progression of advanced calcification. Recent noninvasive imaging studies of atherosclerotic calcification provide robust evidence that inflammation precedes active calcification, leading to establish the inflammation-dependent calcification paradigm. On the other hand, elastin degradation by increased elastolytic activities and disturbance of regulatory systems of extracellular pyrophosphate metabolism play an important role in development of Mönckeberg's medial calcification.

Topics: Animals; Apoptosis; Calcinosis; Cardiovascular Diseases; Cellular Senescence; Diphosphates; Elastin; Extracellular Matrix; Humans; Macrophages; Mice; Muscle, Smooth, Vascular; Osteogenesis; Risk Factors; Vascular Calcification

Carbonic anhydrases are a group of isoenzymes that catalyze the reversible conversion of carbon dioxide into bicarbonate. They participate in a constellation of physiological processes in humans, including respiration, bone metabolism, and the formation of body fluids, including urine, bile, pancreatic juice, gastric secretion, saliva, aqueous humor, cerebrospinal fluid, and sweat. In addition, carbonic anhydrase may provide carbon dioxide/bicarbonate to carboxylation reactions that incorporate carbon dioxide to substrates. Several isoforms of carbonic anhydrase have been identified in humans, but their precise physiological role and the consequences of their dysfunction are mostly unknown. Carbonic anhydrase isoenzymes are involved in calcification processes in a number of biological systems, including the formation of calcareous spicules from sponges, the formation of shell in some animals, and the precipitation of calcium salts induced by several microorganisms, particularly urease-producing bacteria. In human tissues, carbonic anhydrase is implicated in calcification processes either directly by facilitating calcium carbonate deposition which in turn serves to facilitate calcium phosphate mineralization, or indirectly via its action upon γ-glutamyl-carboxylase, a carboxylase that enables the biological activation of proteins involved in calcification, such as matrix Gla protein, bone Gla protein, and Gla-rich protein. Carbonic anhydrase is implicated in calcification of human tissues, including bone and soft-tissue calcification in rheumatological disorders such as ankylosing spondylitis and dermatomyositis. Carbonic anhydrase may be also involved in bile and kidney stone formation and carcinoma-associated microcalcifications. The aim of this review is to evaluate the possible association between carbonic anhydrase isoenzymes and vascular calcification in humans.

Topics: Animals; Blood Vessels; Calcium-Binding Proteins; Carbon-Carbon Ligases; Carbonic Anhydrases; Diphosphates; Extracellular Matrix Proteins; Humans; Isoenzymes; Matrix Gla Protein; Osteocalcin; Signal Transduction; Vascular 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. Based on the in vivo evidence of their functional relevance, these proteins will serve as excellent targets for the prevention and treatment of arterial calcification. In this review we discuss the functional role of the identified modulators of arterial calcification and describe the networks they belong to.

Topics: Adenosine Triphosphate; Animals; Diphosphates; Humans; Mice; Receptors, Calcitriol; Signal Transduction; Vascular Calcification

Calcium supplements have been associated with increased cardiovascular risk, but the mechanism is unknown. We investigated the effects of calcium supplements on the propensity of serum to calcify, based on the transition time of primary to secondary calciprotein particles (T50). Changes in serum calcium were related to changes in T50.. Calcium supplements have been associated with increased cardiovascular risk; however, it is unknown whether this is related to an increase in vascular calcification.. We investigated the acute and 3-month effects of calcium supplements on the propensity of serum to calcify, based on the transition time of primary to secondary calciprotein particles (T50), and on three possible regulators of calcification: fetuin-A, pyrophosphate and fibroblast growth factor-23 (FGF23). We randomized 41 postmenopausal women to 1 g/day of calcium as carbonate, or to a placebo containing no calcium. Measurements were performed at baseline and then 4 and 8 h after their first dose, and after 3 months of supplementation. Fetuin-A, pyrophosphate and FGF23 were measured in the first 10 participants allocated to calcium carbonate and placebo who completed the study.. T50 declined in both groups, the changes tending to be greater in the calcium group. Pyrophosphate declined from baseline in the placebo group at 4 h and was different from the calcium group at this time point (p = 0.04). There were no other significant between-groups differences. The changes in serum total calcium from baseline were significantly related to changes in T50 at 4 h (r = -0.32, p = 0.05) and 8 h (r = -0.39, p = 0.01), to fetuin-A at 3 months (r = 0.57, p = 0.01) and to pyrophosphate at 4 h (r = 0.61, p = 0.02).. These correlative findings suggest that serum calcium concentrations modulate the propensity of serum to calcify (T50), and possibly produce counter-regulatory changes in pyrophosphate and fetuin-A. This provides a possible mechanism by which calcium supplements might influence vascular calcification.

Topics: Aged; alpha-2-HS-Glycoprotein; Biomarkers; Bone Density Conservation Agents; Calcium; Calcium Carbonate; Calcium Citrate; Dietary Supplements; Diphosphates; Drug Administration Schedule; Female; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Humans; Middle Aged; Vascular Calcification

Ectopic calcification is characterized by inappropriate deposition of calcium mineral in nonskeletal connective tissues and can cause significant morbidity and mortality, particularly when it affects the cardiovascular system. Identification of the metabolic and genetic determinants of ectopic calcification could help distinguish individuals at the greatest risk of developing these pathological calcifications and could guide development of medical interventions. Inorganic pyrophosphate (PP

Topics: Bone and Bones; Calcium, Dietary; Diphosphates; Humans; Minerals; Vascular Calcification

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 (PP

Topics: Animals; Dietary Supplements; Diphosphates; Humans; Mice; Mutation; Phosphoric Diester Hydrolases; Pseudoxanthoma Elasticum; Pyrophosphatases; Vascular Calcification

(1) Background: Tissue non-specific alkaline phosphatase (TNAP) is suspected to induce atherosclerosis plaque calcification. TNAP, during physiological mineralization, hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PP

Topics: Adenosine Triphosphate; Alkaline Phosphatase; Animals; Aorta; Ascorbic Acid; Atherosclerosis; Cell Transdifferentiation; Chondrocytes; Diphosphates; Glycerophosphates; Humans; Magnetic Resonance Spectroscopy; Mice; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphates; Vascular Calcification

Trauma-induced calcification is the pathological consequence of complex injuries which often affect the central nervous system and other parts of the body simultaneously. We demonstrated by an animal model recapitulating the calcification of the above condition that adrenaline transmits the stress signal of brain injury to the calcifying tissues. We have also found that although the level of plasma pyrophosphate, the endogenous inhibitor of calcification, was normal in calcifying animals, it could not counteract the acute calcification. However, externally added pyrophosphate inhibited calcification even when it was administered after the complex injuries. Our finding suggests a potentially powerful clinical intervention of calcification triggered by polytrauma injuries which has no effective treatment.

Topics: Adrenergic Antagonists; Animals; Brain Injuries, Traumatic; Cardiotoxins; Diphosphates; Disease Models, Animal; Epinephrine; Female; Gene Expression Regulation; Mice, Inbred C57BL; Muscle, Skeletal; Ossification, Heterotopic; Receptors, Adrenergic; Vascular Calcification; X-Ray Microtomography

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

Topics: Adenosine Triphosphate; Alkaline Phosphatase; Animals; Diphosphates; Disease Models, Animal; Drug Evaluation, Preclinical; Female; Humans; Liver; Male; Mice; Mice, Knockout; Multidrug Resistance-Associated Proteins; Mutation; Niacinamide; Phosphoric Diester Hydrolases; Pseudoxanthoma Elasticum; Pyrophosphatases; Skin; Sulfonamides; Vascular Calcification

Objective- Hydroxyapatite deposition on the medial layer of the aortic walls is the hallmark of vascular calcification and the most common complication in aging individuals and in patients with diabetes mellitus and those undergoing hemodialysis. Extracellular pyrophosphate is a potent physicochemical inhibitor of hydroxyapatite crystal formation. This study analyzed changes in extracellular pyrophosphate metabolism during the phosphate-induced calcification process. Approach and Results- Phosphate-induced calcification of ex vivo-cultured aortic rings resulted in calcium accumulation after 7 days. This accumulation was enhanced when aortic walls were devitalized. BMP2 (bone morphogenic protein 2) expression was associated with calcium accumulation in cultured aortic rings, as well as in cultured vascular smooth muscle cells (VSMCs) and in calcitriol-induced calcification in rats. Hydroxyapatite dose dependently induced BMP2 overexpression in VSMCs. Moreover, TNAP (tissue nonspecific alkaline phosphatase) mRNA levels and activity were found to be downregulated in early phases and upregulated in later phases of calcification in all 3 models studied. eNPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) increased from early to later phases of calcification, whereas eNTPD1 (ectonucleoside triphosphate diphosphohydrolase 1) was downregulated during later phases. Synthesis of pyrophosphate in VSMCs increased significantly over time, in all 3 models studied. Because the rate of pyrophosphate hydrolysis was 10× slower than the rate of pyrophosphate synthesis, pyrophosphate synthesis is determined mainly by the ratio of eNPP1 to eNTPD1 activity. Hydroxyapatite also induces increments both in TNAP and eNPP1/eNTPD1 ratio in VSMCs. Conclusions- Pyrophosphate synthesis increases in VSMCs during phosphate-induced calcification because of compensatory regulation of extracellular pyrophosphate metabolism.

Topics: Alkaline Phosphatase; Animals; Antigens, CD; Aorta; Apyrase; Bone Morphogenetic Protein 2; Cell Proliferation; Cells, Cultured; Diphosphates; Down-Regulation; Durapatite; Extracellular Space; Gene Expression; Hydrolysis; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphates; Phosphoric Diester Hydrolases; Pyrophosphatases; Rats, Sprague-Dawley; RNA, Messenger; Up-Regulation; Vascular Calcification

Generalized arterial calcification of infancy (GACI) is a rare, life-threatening disorder caused by loss-of-function mutations in the gene encoding ectonucleotide pyrophosphatase phosphodiesterase 1 (

Topics: Animals; Blood Pressure; Cardiovascular System; Diphosphates; Disease Models, Animal; Enzyme Replacement Therapy; Humans; Mice; Mice, Inbred BALB C; Organ Specificity; Phosphoric Diester Hydrolases; Pyrophosphatases; Vascular Calcification

Phosphorus is an essential nutrient involved in many pathobiological processes. Less than 1% of phosphorus is found in extracellular fluids as inorganic phosphate ion (Pi) in solution. High serum Pi level promotes ectopic calcification in many tissues, including blood vessels. Here, we studied the effect of elevated Pi concentration on macrophage polarization and calcification. Macrophages, present in virtually all tissues, play key roles in health and disease and display remarkable plasticity, being able to change their physiology in response to environmental cues.. High-throughput transcriptomic analysis and functional studies demonstrated that Pi induces unpolarized macrophages to adopt a phenotype closely resembling that of alternatively-activated M2 macrophages, as revealed by arginine hydrolysis and energetic and antioxidant profiles. Pi-induced macrophages showed an anti-calcifying action mediated by increased availability of extracellular ATP and pyrophosphate.. We conclude that the ability of Pi-activated macrophages to prevent calcium-phosphate deposition is a compensatory mechanism protecting tissues from hyperphosphatemia-induced pathologic calcification.

Topics: Adenosine Triphosphate; Animals; Antioxidants; Calcium; Diphosphates; Flow Cytometry; Macrophages; Male; Mice; Mice, Inbred C57BL; Phosphates; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Vascular Calcification

Various disorders including pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI), which are caused by inactivating mutations in

Topics: Administration, Oral; Adult; Aged; Animals; ATP-Binding Cassette Transporters; Calcium; Connective Tissue; Diphosphates; Disease Models, Animal; Female; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Multidrug Resistance-Associated Proteins; Myocardium; Phosphoric Diester Hydrolases; Pregnancy; Pseudoxanthoma Elasticum; Pyrophosphatases; Vascular Calcification; Young Adult

Topics: Computed Tomography Angiography; Coronary Angiography; Diphosphates; Humans; Phosphates; Vascular Calcification

Calcium-phosphate deposition (CPD) in atherosclerotic lesions, which begins in middle age and increases with aging, is a major independent predictor of future cardiovascular disease morbi-mortality. Remodeling of atherosclerotic vessels during aging is regulated in part by intimal macrophages, which can polarize to phenotypically distinct populations with distinct functions. This study tested the hypothesis that classically activated macrophages (M1φs) and alternatively activated macrophages (M2φs) differently affect vascular smooth muscle cell (VSMC) calcification and investigated the underlying mechanisms. We analyzed mouse VSMC-macrophage cocultures using a transwell system. Coculture of VSMCs with M2φs significantly reduced CPD, but coculture with M1φs had no effect. The anticalcific effect of M2φs was associated with elevated amounts of extracellular ATP and pyrophosphate (PPi), two potent inhibitors of CPD, and was lost upon forced hydrolysis of these metabolites. In M2φs and VSMC-M2φs cocultures, analysis of the ectoenzymes that regulate extracellular ATP/PPi metabolism revealed increased mRNA expression and activity of ectoenzyme nucleotide pyrophosphatase/phosphodiesterase-1, which synthesizes PPi from ATP, without changes in tissue-nonspecific alkaline phosphatase, which hydrolyzes PPi In conclusion, increased accumulation of extracellular ATP and PPi by alternatively activated mouse M2φs inhibits CPD. These results reveal novel mechanisms underlying macrophage-dependent control of intimal calcification.

Topics: Adenosine Triphosphate; Animals; Cells, Cultured; Diphosphates; Extracellular Fluid; Macrophage Activation; Male; Mice; Mice, Inbred C57BL; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Vascular Calcification

Vascular calcification significantly contributes to mortality in chronic kidney disease (CKD) patients. Sevelamer and pyrophosphate (PPi) have proven to be effective in preventing vascular calcification, the former by controlling intestinal phosphate absorption, the latter by directly interfering with the hydroxyapatite crystal formation. Since most patients present with established vascular calcification, it is important to evaluate whether these compounds may also halt or reverse the progression of preexisting vascular calcification. CKD and vascular calcification were induced in male Wistar rats by a 0.75 % adenine low protein diet for 4 weeks. Treatment with PPi (30 or 120 µmol/kg/day), sevelamer carbonate (1500 mg/kg/day) or vehicle was started at the time point at which vascular calcification was present and continued for 3 weeks. Hyperphosphatemia and vascular calcification developed prior to treatment. A significant progression of aortic calcification in vehicle-treated rats with CKD was observed over the final 3-week period. Sevelamer treatment significantly reduced further progression of aortic calcification as compared to the vehicle control. No such an effect was seen for either PPi dose. Sevelamer but not PPi treatment resulted in an increase in both osteoblast and osteoid perimeter. Our study shows that sevelamer was able to reduce the progression of moderate to severe preexisting aortic calcification in a CKD rat model. Higher doses of PPi may be required to induce a similar reduction of severe established arterial calcification in this CKD model.

Topics: Animals; Aorta; Chelating Agents; Diphosphates; Durapatite; Male; Rats; Rats, Wistar; Renal Insufficiency, Chronic; Sevelamer; Vascular Calcification

Ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (E-NPP1), encoded by ENPP1, is a plasma membrane protein that generates inorganic pyrophosphate (PP

Topics: Animals; Cell Membrane; Chlorocebus aethiops; COS Cells; Diphosphates; Down-Regulation; HEK293 Cells; Humans; Mutagenesis, Site-Directed; Mutation, Missense; Phosphoric Diester Hydrolases; Pyrophosphatases; Vascular Calcification

Topics: Animals; Aorta; Aortic Diseases; Diphosphates; Vascular Calcification

We previously reported that oxidized low density lipoprotein (oxLDL) accelerated the calcification in aorta of rats and rat vascular smooth muscle cells (RVSMCs). However, the molecular mechanism underlying the acceleration remains poorly understood. The present study aimed to investigate the role of calpain-1, Ca2+-sensitive intracellular cysteine proteases, in the vascular calcification of rats treated with both high dose of vitamin D2 and high cholesterol diet. The results showed that calpain activity significantly increased in calcified aortic tissue of rats and RVSMCs treated with oxLDL. Specific calpain inhibitor I (CAI, 0.5mg/kg, intraperitoneal) inhibited the vascular calcification in rats with hypercholesterolemia accompanied by the increase in the level of extracellular inorganic pyrophosphate (PPi), the endogenous inhibitor of vascular calcification. In addition, CAI increased the content of adenosine triphosphate (ATP), decreased the activity, mRNA and protein expression of alkaline phosphatase (ALP) and reduced the production of superoxide anion in calcified aortic tissue. CAI also increased the activity of ATP synthase as well as protein expression of ATP5D, δ subunit of ATP synthase. In the in vitro study, suppression of calpain-1 using siRNA assay inhibited the calcium deposition, increased the levels of PPi and ATP, improved the activity of ATP synthase as well as protein expression of ATP5D in RVSMCs treated with oxLDL. Calpain-1 suppression also decreased the activity, mRNA and protein expression of ALP and reduced the mitochondrial ROS (Mito-ROS) production in RVSMCs. However, mito-TEMPO, the mitochondria-targeted ROS scavenger, reduced the calcium deposition, increased the PPi in culture medium, decreased the activity, mRNA and protein expression of ALP in RVSMCs treated with oxLDL. Taken together, the results suggested that calpain-1 activation plays critical role in vascular calcification caused by oxLDL, which might be mediated by PPi metabolism disorder. The results also implied that Mito-ROS might contribute to the PPi metabolism disorder through regulation of the activity and expression of ALP.

Topics: Animals; Aorta; Calcium; Calpain; Cell Line; Diphosphates; Glycoproteins; Hypercholesterolemia; Lipoproteins, LDL; Male; Mitochondria; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Proton-Translocating ATPases; Rats; Rats, Sprague-Dawley; Vascular Calcification

Chronic kidney disease (CKD) is generally associated with disturbances of mineral and bone metabolism. They contribute to the development of vascular calcification (VC), a strong, independent predictor of cardiovascular risk. Pyrophosphate (PPi), an endogenous inhibitor of hydroxyapatite formation, has been shown to slow the progression of VC in uremic animals. Since in patients with CKD treatment is usually initiated for already existing calcifications, we aimed to compare the efficacy of PPi therapy with that of the phosphate binder sevelamer, using a uremic apolipoprotein-E knockout mouse model with advanced VCs. After CKD creation or sham surgery, 12-week-old female mice were randomized to one sham group and four CKD groups (n = 18-19/group). Treatment was initiated 8 weeks after left nephrectomy allowing prior VC development. Uremic groups received either intraperitoneal PPi (high dose, 1.65 mg/kg or low dose, 0.33 mg/kg per day), oral sevelamer (3 % in diet), or placebo treatment for 8 weeks. Both intima and media calcifications worsened with time in placebo-treated CKD mice, based on both quantitative image analysis and biochemical measurements. Progression of calcification between 8 and 16 weeks was entirely halted by PPi treatment, as it was by sevelamer treatment. PPi did not induce consistent bone histomorphometry changes. Finally, the beneficial vascular action of PPi probably involved mechanisms different from that of sevelamer. Further studies are needed to gain more precise insight into underlying mechanisms and to see whether PPi administration may also be useful in patients with CKD and VC.

Topics: Animals; Apolipoproteins E; Diphosphates; Disease Models, Animal; Disease Progression; Infusions, Parenteral; Mice; Mice, Knockout; Renal Insufficiency, Chronic; Uremia; Vascular Calcification

Vascular calcification (VC) is a risk factor for cardiovascular mortality in the setting of chronic kidney disease (CKD). Pyrophosphate (PPi), an endogenous molecule that inhibits hydroxyapatite crystal formation, has been shown to prevent the development of VC in animal models of CKD. However, the possibility of harmful effects of exogenous administration of PPi on bone requires further investigation. To this end, we examined by histomorphometry the bone of CKD mice after intraperitoneal PPi administration. After CKD creation or sham surgery, 10-week-old female apolipoprotein-E knockout (apoE(-/-)) mice were randomized to one non-CKD group or 4 CKD groups (n = 10-35/group) treated with placebo or three distinct doses of PPi, and fed with standard diet. Eight weeks later, the animals were killed. Serum and femurs were sampled. Femurs were processed for bone histomorphometry. Placebo-treated CKD mice had significantly higher values of osteoid volume, osteoid surface and bone formation rate than sham-placebo mice with normal renal function. Slightly higher osteoid values were observed in CKD mice in response to very low PPi dose (OV/BV, O.Th and ObS/BS) and, for one parameter measured, to high PPi dose (O.Th), compared to placebo-treated CKD mice. Treatment with PPi did not modify any other structural parameters. Mineral apposition rates, and other parameters of bone formation and resorption were not significantly different among the treated animal groups or control CKD placebo group. In conclusion, PPi does not appear to be deleterious to bone tissue in apoE(-/-) mice with CKD, although a possible stimulatory PPi effect on osteoid formation may be worth further investigation.

Topics: Animals; Apolipoproteins E; Bone Density; Dialysis Solutions; Diphosphates; Female; Femur; Mice; Mice, Knockout; Peritoneal Dialysis; Renal Insufficiency, Chronic; Vascular Calcification

Plasma levels of pyrophosphate, an endogenous inhibitor of vascular calcification, are reduced in end-stage renal disease and correlate inversely with arterial calcification. However, it is not known whether the low plasma levels are directly pathogenic or are merely a marker of reduced tissue levels. This was tested in an animal model in which aortas were transplanted between normal mice and Enpp1(-/-) mice lacking ectonucleotide pyrophosphatase phosphodiesterase, the enzyme that synthesizes extracellular pyrophosphate. Enpp1(-/-) mice had very low plasma pyrophosphate and developed aortic calcification by 2 months that was greatly accelerated with a high-phosphate diet. Aortas of Enpp1(-/-) mice showed no further calcification after transplantation into wild-type mice fed a high-phosphate diet. Aorta allografts of wild-type mice calcified in Enpp1(-/-) mice but less so than the adjacent recipient Enpp1(-/-) aorta. Donor and recipient aortic calcium contents did not differ in transplants between wild-type and Enpp1(-/-) mice, demonstrating that transplantation per se did not affect calcification. Histology revealed medial calcification with no signs of rejection. Thus, normal levels of extracellular pyrophosphate are sufficient to prevent vascular calcification, and systemic Enpp1 deficiency is sufficient to produce vascular calcification despite normal vascular extracellular pyrophosphate production. This establishes an important role for circulating extracellular pyrophosphate in preventing vascular calcification.

Topics: Animals; Aorta; Aortic Diseases; Calcium; Diphosphates; Disease Models, Animal; Disease Progression; Mice, Inbred C57BL; Mice, Knockout; Phosphoric Diester Hydrolases; Phosphorus, Dietary; Pyrophosphatases; Time Factors; Vascular Calcification

Arterial medial calcification is a major complication in patients with chronic kidney disease and diabetes. It has been hypothesized that a high concentration of inorganic phosphate (Pi) induces calcification in vascular smooth muscle cells (vSMCs). However, the role of transforming growth factor-β (TGF-β)/Smad3 signaling in Pi-induced vascular calcification remains controversial. The aim of this study was to investigate the possible involvement of Smad3 in Pi-induced vascular calcification. We compared the degree of Pi-induced vSMC calcification between vSMCs isolated from wild-type (Smad3(+/+)) and Smad3-deficient (Smad3(-/-)) mice. We found that vSMCs from Smad3(+/+) mice had less calcium (Ca) than those from Smad3(-/-) mice when they were exposed to high concentrations of Pi and Ca (Pi+Ca). The phosphorylation of Smad3 was induced in Smad3(+/+) vSMCs by exposure to Pi+Ca. The concentration of extracellular pyrophosphate (ePPi) was lower in Smad3(-/-) vSMCs than in Smad3(+/+) vSMCs and was significantly increased in Smad3(+/+) vSMCs by treatment with TGF-β1. Also, the addition of a small amount of PPi to culture medium significantly decreased the deposition of Ca in both Smad3(+/+) and Smad3(-/-) vSMCs. Ectonucleotide phosphatase/phosphodiesterase1 (Enpp1) was decreased at the mRNA, protein, and enzymatic activity levels in Smad3(-/-) vSMCs compared with Smad3(+/+) vSMCs. A ChIP assay showed that phosphorylated Smad3 directly binds to the Enpp1 gene. Furthermore, the calcification of aortic segments was attenuated by treatment with TGF-β1 only in Smad3(+/+) mice. Taken together, we conclude that Pi-induced vSMC calcification is suppressed by Smad3 via an increase in ePPi.

Topics: Animals; Blotting, Western; Chromatin Immunoprecipitation; Diphosphates; Male; Mice; Mice, Knockout; Muscle, Smooth, Vascular; Phosphoric Diester Hydrolases; Pyrophosphatases; Real-Time Polymerase Chain Reaction; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta1; Vascular Calcification

Progerin is a mutant form of lamin A responsible for Hutchinson-Gilford progeria syndrome (HGPS), a premature aging disorder characterized by excessive atherosclerosis and vascular calcification that leads to premature death, predominantly of myocardial infarction or stroke. The goal of this study was to investigate mechanisms that cause excessive vascular calcification in HGPS.. We performed expression and functional studies in wild-type mice and knock-in Lmna(G609G/+) mice expressing progerin, which mimic the main clinical manifestations of HGPS. Lmna(G609G/+) mice showed excessive aortic calcification, and primary aortic vascular smooth muscle cells from these progeroid animals had an impaired capacity to inhibit vascular calcification. This defect in progerin-expressing vascular smooth muscle cells is associated with increased expression and activity of tissue-nonspecific alkaline phosphatase and mitochondrial dysfunction, which leads to reduced ATP synthesis. Accordingly, Lmna(G609G/+) vascular smooth muscle cells are defective for the production and extracellular accumulation of pyrophosphate, a major inhibitor of vascular calcification. We also found increased alkaline phosphatase activity and reduced ATP and pyrophosphate levels in plasma of Lmna(G609G/+) mice without changes in phosphorus and calcium. Treatment with pyrophosphate inhibited vascular calcification in progeroid mice.. Excessive vascular calcification in Lmna(G609G) mice is caused by reduced extracellular accumulation of pyrophosphate that results from increased tissue-nonspecific alkaline phosphatase activity and diminished ATP availability caused by mitochondrial dysfunction in vascular smooth muscle cells. Excessive calcification is ameliorated on pyrophosphate treatment. These findings reveal a previously undefined pathogenic process in HGPS that may also contribute to vascular calcification in normal aging, because progerin progressively accumulates in the vascular tissue of individuals without HGPS.

Topics: Adenosine Triphosphate; Alkaline Phosphatase; Animals; Aorta; Cells, Cultured; Diphosphates; Disease Models, Animal; Lamin Type A; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Mitochondria, Muscle; Muscle, Smooth, Vascular; Progeria; Treatment Outcome; Vascular Calcification

Topics: Animals; Diphosphates; Male; Progeria; Vascular Calcification

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.

Topics: Animals; Base Sequence; Calcification, Physiologic; Calcium; Diet; Diphosphates; Disease Models, Animal; DNA Mutational Analysis; Genotyping Techniques; Kaplan-Meier Estimate; Kidney; Mice; Mice, Mutant Strains; Molecular Sequence Data; Phenotype; Phosphoric Diester Hydrolases; Phosphorus; Pyrophosphatases; RNA, Messenger; Spectrometry, X-Ray Emission; Tomography, X-Ray Computed; Vascular Calcification; Vibrissae

Vascular calcification is a major risk factor of cardiovascular mortality, particularly for patients with end-stage renal disease and diabetes. Although chronic inflammation is one of the etiologic factors, the underlying mechanism is not fully understood. To clarify this, we studied how nuclear factor-kappa B (NF-κB) induction, a mediator of inflammation, might promote vascular calcification. Activation of NF-κB by tumor necrosis factor (TNF) promoted inorganic phosphate-induced calcification in human aortic smooth muscle cells. Pyrophosphate (an inhibitor of calcification) efflux to the extracellular matrix was suppressed along with the decreased expression of ankylosis protein homolog (ANKH), a transmembrane protein that controls pyrophosphate efflux of cells. The restoration of ANKH expression in these cells overcame the decreased pyrophosphate efflux and calcification. Tristetraprolin, a downstream product of NF-κB activation, may mediate destabilization of ANKH mRNA as its knockdown by shRNA increased ANKH expression and decreased calcification. Furthermore, a rat chronic renal failure model, with increased serum TNF levels, activated NF-κB and decreased ANKH levels. In contrast, the inhibition of NF-κB maintained ANKH expression and attenuated vascular calcification both in vivo and in vitro. Both human calcified atherosclerotic lesions and arteries from patients with chronic kidney disease had activated NF-κB and decreased ANKH expression. Thus, TNF-activated NF-κB promotes inflammation-accelerated vascular calcification by inhibiting ankylosis protein homolog expression and consequent pyrophosphate secretion.

Topics: Animals; Atherosclerosis; Diphosphates; Disease Models, Animal; Disease Progression; Down-Regulation; Genes, Reporter; HEK293 Cells; Humans; I-kappa B Proteins; Inflammation Mediators; Kidney Failure, Chronic; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NF-kappa B; NF-KappaB Inhibitor alpha; Osteogenesis; Phosphate Transport Proteins; Promoter Regions, Genetic; Rats; Rats, Wistar; RNA Interference; RNA Stability; Signal Transduction; Time Factors; Transfection; Tristetraprolin; Tumor Necrosis Factor-alpha; Vascular Calcification

The high rate of cardiovascular mortality in patients with end-stage renal disease (ESRD) is a significant barrier to improved life expectancy. Unique in this population is the marked development and aggressive worsening of vascular calcification (VC). Pyrophosphate (PPi), an endogenous molecule, appears to naturally inhibit soft tissue calcification, but may be depressed in chronic kidney disease (CKD) and ESRD. Although once thought to be a promising therapeutic, PPi's very short half-life in circulation curtailed earlier studies. We tested the possibility that a slow, continuous entry of PPi into the circulation and prevention of VC might be achieved by daily peritoneal dialysis (PD).. Pharmacokinetic studies were first carried out in rats with renal impairment resulting from a 5/6 nephrectomy. Efficacy studies were then performed in the apolipoprotein E gene knockout mouse model overlaid with CKD. PPi was delivered by means of a permanent peritoneal catheter in a solution simulating PD, but without the timed removal of spent dialysate. von Kossa's staining followed by semiquantitative morphological image processing, with separation of inside (intimal) and outside (presumed medial) lesions, was used to determine aortic root calcification.. In comparison to an intravenous bolus, delivery of PPi in a PD solution resulted in a slower, extended delivery over >4 h. Next, the efficacy studies showed that a 6-day/week PD-simulated administration of PPi resulted in a dose-dependent inhibition of aortic calcification in both intimal and medial lesions. A dose-response effect on total aortic calcification was also documented, with a full inhibition seen at the highest dose. A limited peritoneal catheter-related inflammation was observed, as expected, and included the placebo-treated control groups. This inflammatory response could have masked a lower level PPi-specific adverse effect, but none was observed.. Our findings suggest potential for PPi, administered during PD, to prevent the development of VC and to potentially extend the life of ESRD patients.

Topics: Animals; Apolipoproteins E; Calcium; Dialysis Solutions; Diphosphates; Female; Half-Life; Male; Mice; Mice, Knockout; Peritoneal Dialysis; Rats; Rats, Sprague-Dawley; Renal Insufficiency; Tissue Distribution; Uremia; Vascular Calcification

Extracellular inorganic pyrophosphate (ePP(i)) is an important endogenous inhibitor of vascular calcification, but it is not known whether systemic or local vascular PP(i) metabolism controls calcification. To determine the role of ePP(i) in vascular smooth muscle, we identified the pathways responsible for ePP(i) production and hydrolysis in rat and mouse aortas and manipulated them to demonstrate their role in the calcification of isolated aortas in culture. Rat and mouse aortas contained mRNA for ectonucleotide pyrophosphatase/phosphodiesterases (NPP1-3), the putative PP(i) transporter ANK, and tissue-nonspecific alkaline phosphatase (TNAP). Synthesis of PP(i) from ATP in aortas was blocked by β,γ-methylene-ATP, an inhibitor of NPPs. Aortas from mice lacking NPP1 (Enpp1(-/-)) did not synthesize PP(i) from ATP and exhibited increased calcification in culture. Although ANK-mediated transport of PP(i) could not be demonstrated in aortas, aortas from mutant (ank/ank) mice calcified more in culture than did aortas from normal (ANK/ANK) mice. Hydrolysis of PP(i) was reduced 25% by β,γ-methylene-ATP and 50% by inhibition of TNAP. Hydrolysis of PP(i) was increased in cells overexpressing TNAP or NPP3 but not NPP1 and was not reduced in Enpp1(-/-) aortas. Overexpression of TNAP increased calcification of cultured aortas. The results show that smooth muscle NPP1 and TNAP control vascular calcification through effects on synthesis and hydrolysis of ePP(i), indicating an important inhibitory role of locally produced PP(i). Smooth muscle ANK also affects calcification, but this may not be mediated through transport of PP(i). NPP3 is identified as an additional pyrophosphatase that could influence vascular calcification.

Topics: Adenosine Triphosphate; Adenoviridae; Alkaline Phosphatase; Animals; Arteries; Calcification, Physiologic; Diphosphates; DNA Primers; Extracellular Space; Humans; Mice; Mice, Knockout; Muscle, Smooth, Vascular; Organ Culture Techniques; Phosphoric Diester Hydrolases; Pyrophosphatases; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transfection; Vascular Calcification

Calcium phosphate deposition (CPD) is the hallmark of vascular smooth muscle cell (VSMC) calcification. CPD is a thermodynamically-favored process under physiological conditions. Hydroxyapatite, the most common calcium phosphate in calcified arteries, is passively formed during VSMC calcification, independently on any direct cellular activity. Furthermore, in recent years it has been demonstrated there is an anti-calcifying effect by extracellular pyrophosphate, an endogenous inhibitor of CPD, both in vitro and in vivo, which directly blocks hydroxyapatite formation.. We have used the in vitro calcification model without cellular activity, by treating confluent rat aortic VSMC with paraformaldehyde. Fixed cells were incubated with the indicated media to obtain or inhibit calcification. The calcium content was determined colorimetrically. Calcification was observed after 3 weeks (21 days) using a physiological concentration of calcium (1.8 mmol/L) and phosphate (1 mmol/L). Calcium deposition was directly proportional to the amount of phosphate in the media, with a calcification rate of 3.5, 7.5, and 14.3 µg·cm⁻²·day⁻¹, using 1, 2, and 4 mmol/L of phosphate, respectively. Under physiological conditions, pyrophosphate inhibits CPD with an IC₅₀ of ≍200 nmol/L.. CPD occurs under a physiological concentration of calcium and phosphate, but this deposition is completely inhibited in the presence of a physiological concentration of pyrophosphate (3-5 µmol/L).

Topics: Animals; Calcium; Cells, Cultured; Diphosphates; Durapatite; Models, Biological; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Rats; Time Factors; Vascular Calcification