Ectopic Calcification Research Articles

Ectopic Calcification in Congenital Heart Surgery: A Material-Centric Review.

The modern congenital heart surgeon has an array of materials available for cardiovascular repair. With advancements in the surgical outcomes for pediatric cardiac defects, choice of material has become increasingly dependent on late-term complications associated with each material. Calcification is a leading long-term complication and is increasing in prevalence with materials lasting longer in patients. Material calcification can impair functionality, lead to subsequent complications, and require additional interventions. A comprehensive literature review was conducted to investigate ectopic calcification of commonly used materials for congenital heart defect repair. Mechanisms of ectopic calcification among commonly used materials were investigated. Ectopic calcification is initiated by material-specific immunological reactions. Recent efforts have focused on developing new materials that are not prone to calcification. ePTFE was widely used in cardiovascular applications but still has reported instances of calcification in various situations, such as long-term use. Tissue engineering techniques have shown reduced calcification in reports. Calcification can occur in all conventional materials we reviewed and, in some cases, has led to life-threatening complications. Favorable outcomes have been reported with tissue-engineered materials, with the expectation of continued positive results in future reports. With an array of synthetic and biological materials now displaying acceptable surgical and short-term outcomes, there is a pressing need to review the long-term viability of these materials, especially considering improved patient survival to adulthood. Furthermore, developing new materials to mitigate calcification remains a promising avenue of research in this field.

Epigenetic modulation of vascular calcification: Looking for comprehending the role of sirt1 and histone acetylation in VSMC phenotypic transition

In light of the complex origins of ectopic vascular calcification and its significant health implications, this study offers a comprehensive exploration of the molecular dynamics governing vascular smooth muscle cells (VSMCs). Focusing on epigenetic modulation, we investigate the transition from a contractile to a calcifying phenotype in VSMCs, with an emphasis on understanding the role of SIRT1. For this purpose, a single batch of human aortic SMCs, used at a specified passage number to maintain consistency, was subjected to calcium and phosphate overload for up to 72 h. Our findings, validated through RT q-PCR, Western blot, immunofluorescence, and DNA methylation analyses, reveal a complex interplay between acetyltransferases and deacetylases during this phenotypic transition. We highlight HAT1A's critical role in histone acetylation regulation and the involvement of HDACs, as evidenced by subcellular localization studies. Moreover, we demonstrate the modulation of SIRT1 expression, a class III deacetylase, during VSMC calcification, underscoring the influence of DNA methylation in this process. Importantly, the study addresses previously unexplored aspects of the dynamic protein expression patterns observed, providing insight into the counterintuitive expressions of key proteins such as Runx2 and osterix. This research underscores the significant impact of epigenetic mechanisms, particularly the modulation of SIRT1, in the transition from a contractile to a calcifying phenotype in VSMCs, offering potential avenues for further exploration in the context of vascular calcification.

Role of vitamin K2 and vitamin K cycle enzymes in aortic valvular interstitial cell calcification

Abstract Introduction Calcific aortic valve disease (CAVD) is the most common valve disease. Currently, there is no medical treatment available for CAVD. Vitamin K antagonists promote CAVD while population-based studies suggest that dietary intake of vitamin K reduces the incidence of CAVD in adults. However, randomized trials have so far not proven a clear benefit of vitamin K supplementation on the progression of AVS. Consequently, there is a need for a deeper understanding of the role of vitamin K in valvular calcification. This study aims to further investigate mechanisms of action of vitamin K during valvular calcification. Methods and Results Aortic valve samples were obtained from patients undergoing surgical aortic valve replacement for CAVD and valvular interstitial cells (VIC) were isolated by collagenase II digestion (Fig. 1). VIC expressed typical markers like Vimentin and α-SMA and were cultured in a control medium (CM) or further calcified in a pro-calcifying medium (PCM) containing sodium dihydrogen phosphate and ascorbic acid. PCM led to upregulation of Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP) expression, and ALP-activity after 7 and 21 days of culture. Moreover, alizarin red staining visualized significant calcium mineral deposition after 21 days of culture (Fig. 2). The addition of the most potent vitamin K2 derivative (Menaquinone-7, MK-7) improved cell viability under both control and calcifying conditions (Fig. 3). Moreover, MK-7 protected VIC from ferroptotic cell death (Fig. 4) and increased expression of the vitamin K-dependent Matrix-Gla-protein (MGP), a major inhibitor of ectopic calcification (Fig. 5). To fulfill its function as an activator of vitamin K-dependent proteins like MGP, vitamin K needs to be reduced to vitamin K hydroquinone by the vitamin K epoxide reductase enzymes VKORC1 and VKORC1L1. In VIC, using our calcification protocol, treatment with PCM led to the downregulation of VKORC1, but not of VKORC1L1, an enzyme with known anti-oxidative properties (Fig. 6). We next performed siRNA-mediated knockdown of VKORC1 and VKORC1L1 in VIC and observed calcification after 7 and 21 days of culture by measuring expression of ALP, RUNX2, quantifying ALP activity, and staining for calcium minerals (alizarin red). Intriguingly, VKORC1L1-knockdown sharply increased the expression of ALP. To further elucidate these findings, we will perform overexpression experiments to analyze, if VKORC1L1 can alleviate PCM-induced calcification Conclusion: Vitamin K2 inhibits in vitro calcification of valvular interstitial cells and upregulates the calcification inhibitor Matrix-Gla-Protein. Our results suggest the involvement of the vitamin K cycle enzyme VKORC1L1, which is known to exhibit anti-oxidative and cytoprotective effects. Further studies are warranted to shed light on the regulation of the enzymes to improve our understanding.

Axonal Energy Crisis and Calcium Phosphate Dysregulation as Pathogenesis of Optic Disc Drusen.

Soft tissue ectopic calcification is due to abnormal accumulation of calcium and phosphate outside the bone. It is the result of the failure of a complex, active, and highly regulated process - much of which is still not well understood. Some of our understanding of ectopic calcification come from studies on diseases such as atherosclerosis, aortic valve disease and kidney stone disease. In the eye, the most common causes of visual defects due to ectopic calcification include optic disc drusen (ODD) and age-related macular degeneration (AMD). In ODD, ectopic calcification occurs only in the most anterior, unmyelinated portion of optic nerve - the sole output of the eye and particularly susceptible to hypoxic and metabolic stress. In this article, we review the effects of hypoxia on mitochondrial function and calcium regulation and delineate the key processes likely involved in ectopic calcification. We propose a working hypothesis for ODD centering on the role of hypoxia-induced calcium and phosphate overload, mitochondrial dysfunction and osteogenic differentiation, leading to hydroxyapatite deposition and biomineralization.

Matrix vesicles from osteoblasts promote atherosclerotic calcification

Atherosclerotic calcification often coincides with osteoporosis, suggesting a potential interplay between bone and vascular mineralization. Osteoblast-derived matrix vesicles (Ost-MVs), pivotal in bone mineralization, have emerged as potential contributors to ectopic vascular calcification. However, the precise role of Ost-MVs in vascular calcification and the underlying mechanisms remain elusive. In this study, we observed a concomitant increase in atherosclerotic calcification and bone loss, accompanied by elevated release of Ost-MVs into circulation. We demonstrate that circulating Ost-MVs target plaque lesions in the setting of atherosclerosis. Mechanistically, vascular injury facilitates transendothelial transport of Ost-MVs, collagen І remodeling promotes Ost-MVs aggregation, and vascular smooth muscle cell (VSMC) phenotypic switching enhances MV uptake. These pathological changes during atherosclerosis collectively contribute to Ost-MVs recruitment into the vasculature. Furthermore, Ost-MVs and VSMC-derived matrix vesicles (VSMC-MVs) exacerbate calcification via the Ras-Raf-ERK pathway. Our findings unveil a novel Ost-MVs-mediated mechanism participating in vascular calcification and enriching our understanding of bone-vascular crosstalk.

Novel treatment for PXE: Recombinant ENPP1 enzyme therapy

Pseudoxanthoma elasticum (PXE) is a genetic multisystem ectopic calcification disorder caused by inactivating mutations in the ABCC6 gene encoding ABCC6, a hepatic efflux transporter. ABCC6-mediated ATP secretion by the liver is the main source of plasma inorganic pyrophosphate (PPi), a potent endogenous calcification inhibitor. The deficiency of plasma PPi underpins PXE. Recent studies demonstrated that INZ-701, a recombinant human ENPP1, the principal PPi-generating enzyme, restored plasma PPi levels and prevented ectopic calcification in the muzzle skin of an Abcc6-/- mouse model of PXE. This study examined the pharmacokinetics, pharmacodynamics, and potency of a new ENPP1-Fc isoform, BL-1118, in Abcc6-/- mice. When Abcc6-/- mice received a single subcutaneous injection of BL-1118 at 0.25, 0.5, or 1 mg/kg, they had dose-dependent elevations in plasma ENPP1 enzyme activity and PPi levels, with an enzyme half-life of approximately 100 hours. When Abcc6-/- mice were injected weekly from 5 to 15 weeks of age, BL-1118 dose-dependently increased steady-state plasma ENPP1 activity and PPi levels and significantly reduced ectopic calcification in the muzzle skin and kidneys. These results suggest that BL-1118 is a promising enzyme therapy for PXE, a disease currently lacking preventive or therapeutic options.

Role of CD34 in calcification of human aortic valve interstitial cells from patients with aortic valve stenosis

Various osteogenic factors are involved in ectopic human aortic valve calcification; however, the key cell species involved in calcification remains unclear. In a previous study, we reported that mesenchymal stem (CD73, 90, 105) and endothelial (VEGFR2) cell markers are positive in almost all human aortic valve interstitial cells (HAVICs) obtained from a patient with calcified aortic valve stenosis (CAVS). Further, CD34-negative HAVICs are highly sensitive to calcification stimulations. Here, we aimed to pathophysiologically clarify the role of CD34 in HAVICs obtained from individual patients with severe CAVS. A DNA microarray between CD34-positive and CD34-negative HAVICs, separated by fluorescence-activated cell sorting, indicated that tenascin X (TNX) mRNA expression significantly decreased in CD34-negative cells. Furthermore, the inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1β significantly downregulated CD34 expression in HAVICs. TGF-β, a key cytokine of endothelial-mesenchymal transition, did not affect HAVIC calcification. CD34 overexpression strongly inhibited TNF-α- and IL-1β-induced calcification and maintained TNX mRNA expression. These results suggest one possibility that CD34 is an inhibitory regulator of valve calcification. Furthermore, TNF-α- and IL-1β-induced CD34 downregulation in HAVICs contributes to HAVIC calcification by downregulating TNX protein expression.

Biology of bone mineralization and ectopic calcifications: the same actors for different plays

Bone has several crucial functions. It is essential for locomotion and allows our body to stand erect against gravity. A mismatch between the mechanical stresses applied to it and its mechanical resistance leads to fractures. Bone also has numerous endocrine functions. It acts as a reservoir for minerals such as calcium and phosphorus, making it the target of calciotropic hormones that mobilize these minerals, particularly calcium, according to the body's needs. Additionally, bone secretes hormones, notably fibroblast growth factor 23 (FGF23), which regulates urinary excretion of phosphate and the bioavailability of active vitamin D. Bone mineralization is the process that facilitates the organized deposition of minerals in the bone matrix, providing rigidity and appropriate mechanical resistance. This process is compromised in genetically related bone mineralization disorders, such as those causing hypophosphatemia or hypophosphatasia. Conversely, calcification can be pathological, affecting soft tissues like the blood vessels, as seen in generalized arterial calcification of infancy (GACI) or arterial calcification due to CD73 deficiency (ACDC). The aim of this article is to first present the composition and structure of the mineralized bone matrix, to review the current understanding of the molecular mechanisms of mineralization, and finally to discuss the conditions associated with ectopic calcification and the underlying mechanisms.

Autosomal recessive hypophosphatemic rickets type 2 due to ENPP1 deficiency (ARHR2)

Autosomal recessive hypophosphatemic rickets type 2 (ARHR2; MIM #613312) is a very rare disorder caused by biallelic loss-of-function mutations in the ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) gene. ENPP1 deficiency encompasses a spectrum of phenotypes that includes, in addition to ARHR2, generalized arterial calcification of infancy (GACI), ossification of the posterior longitudinal ligament (OPLL), and pseudoxanthoma elasticum. ARHR2 can be found in GACI survivors, but it may also be the first manifestation of ENPP1 deficiency. Although the precise mechanisms are not fully elucidated, patients with GACI and ARHR2 have elevated serum FGF23 levels, leading to renal phosphate wasting and hypophosphatemia. As a result, the clinical and radiological phenotype of ARHR2 patients is very similar to that of patients affected with other forms of hypophosphatemic rickets, such as X-linked hypophosphatemia. Patients show signs of rickets (abnormal mineralization of growth plates in children) and osteomalacia (abnormal bone mineralization in children and adults) of varying severity. Clinical manifestations specific to ENPP1 loss-of-function mutations and common to GACI, such as ectopic calcifications (valvular, arterial, or periarticular), deafness, OPLL, and PXE, may also be found. Genetic confirmation of the disease is important so as to ensure that patients receive the appropriate treatment or have the opportunity to participate in clinical trials to evaluate the safety and efficacy of novel and promising recombinant enzyme therapies.

Human genetic diseases of phosphate and pyrophosphate metabolism

In humans, physiological bone and tooth mineralization is a complex cell-mediated process. Prerequisites for proper mineralization include sufficient amounts of minerals (calcium and phosphate [Pi]) to initiate the formation and the growth of apatite crystals and adequate amounts of mineralization inhibitors, such as pyrophosphate (PPi), to prevent uncontrolled extraskeletal mineralization.In this review, we provide an overview of the genetics of human disorders of mineralization, focusing on Pi and PPi metabolism and transport diseases, as the Pi/PPi ratio is an important determinant of crystal production in vivo. Variants in genes implicated in the homeostasis of this ratio may lead to a systemic or local increased Pi/PPi ratio, either by increasing the Pi concentration or by decreasing the PPi concentration, resulting in ectopic calcifications; conversely, variants may lead to a decreased Pi/PPi ratio, resulting in defective mineralization.Owing to the implication of common pathways and, occasionally, to some extent of clinical overlap, an accurate diagnosis and understanding of the pathophysiology of these disorders may be challenging. However, precise molecular characterization of these conditions not only facilitates their diagnosis, but also helps to gather evidence regarding the pathophysiology and phenotype–genotype correlation to improve medical care and develop innovative therapeutics.

An Insight in to Cadaveric Ectopic Calcification: A Case Report

Abstract: Pathological Calcification is an anomalous accumulation of calcium salts along with minimal quantities of iron, magnesium and other minerals in bodily tissues except teeth and bone causing hardening of the affected tissues. The location of the calcification and the presence or absence of mineral balance can be used to categorize it. The atypical mineral deposition processes in biological systems can sometimes be referred to as pathological calcification. During the routine dissection in National Institute of Ayurveda, Jaipur it was observed that numerous pathological calcifications were present both on osseous and extra osseous sites which were different from the normal human anatomy. The subject was a 90-year-old male of North Indian origin. Case examples illustrate different types and locations of calcification, shedding light on the anatomical distribution and potential clinical relevance.

A high-calcium environment induced ectopic calcification ofrenal interstitial fibroblasts via TFPI-2-DCHS1-ALP/ENPP1 axis to participate inRandall's plaque formation.

Randall's plaques (RP) serve as anchoring sites for calcium oxalate (CaOx) stones, but the underlying mechanism remains unclear. Renal interstitium with a high-calcium environment is identified as pathogenesis of RP formation where the role of human renal interstitial fibroblasts (hRIFs) was highlighted. Our study aims to elucidate the potential mechanism by which a high-calcium environment drives ectopic calcification of hRIFs to participate in RP formation. Alizarin Red staining demonstrated calcium nodules in hRIFs treated with high-calcium medium. Utilizing transcriptome sequencing, tissue factor pathway inhibitor-2 (TFPI-2) was found to be upregulated in high-calcium-induced hRIFs and RP tissues, and TFPI-2 promoted high-calcium-induced calcification of hRIFs. Subsequently, the downstream regulator of TFPI2 was screened by transcriptome sequencing analysis of hRIFs with TFPI-2 knockdown or overexpressed. Dachsous Cadherin Related 1 (DCHS1) knockdown was identified to suppress the calcification of hRIFs enhanced by TFPI-2. Further investigation revealed that TFPI-2/DCHS1 axis promoted high-calcium-induced calcification of hRIFs via disturbing the balance of ENPP1/ALP activities, but without effect on the canonical osteogenic markers, such as osteopontin (OPN), osteogenic factors runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2). In summary, our study mimicked the high-calcium environment observed in CaOx stone patients with hypercalciuria, and discovered that the high-calcium drove ectopic calcification of hRIFs via a novel TFPI-2-DCHS1-ALP/ENPP1 pathway rather than adaption of osteogenic phenotypes to participate in RP formation.

Small Charged Molecule-Mediated Fibrillar Mineralization: Implications for Ectopic Calcification.

Numerous small biomolecules exist in the human body and play roles in various biological and pathological processes. Small molecules are believed not to induce intrafibrillar mineralization alone. They are required to work in synergy with noncollagenous proteins (NCPs) and their analogs, e.g. polyelectrolytes, for inducing intrafibrillar mineralization, as the polymer-induced liquid-like precursor (PILP) process has been well-documented. In this study, we demonstrate that small charged molecules alone, such as sodium tripolyphosphate, sodium citrate, and (3-aminopropyl) triethoxysilane, could directly mediate fibrillar mineralization. We propose that small charged molecules might be immobilized in collagen fibrils to form the polyelectrolyte-like collagen complex (PLCC) via hydrogen bonds. The PLCC could attract CaP precursors along with calcium and phosphate ions for inducing mineralization without any polyelectrolyte additives. The small charged molecule-mediated mineralization process was evidenced by Cryo-TEM, AFM, SEM, FTIR, ICP-OES, etc., as the PLCC exhibited both characteristic features of collagen fibrils and polyelectrolyte with increased charges, hydrophilicity, and density. This might hint at one mechanism of pathological biomineralization, especially for understanding the ectopic calcification process.

Long noncoding RNA MALAT1 mediates fibrous topography-driven pathologic calcification through trans-differentiation of myoblasts

Prosthesis-induced pathological calcification is a significant challenge in biomaterial applications and is often associated with various reconstructive medical procedures. It is uncertain whether the fibrous extracellular matrix (ECM) adjacent to biomaterials directly triggers osteogenic trans-differentiation in nearby cells. To investigate this possibility, we engineered a heterogeneous polystyrene fibrous matrix (PSF) designed to mimic the ECM. Our findings revealed that the myoblasts grown on this PSF acquired osteogenic properties, resulting in mineralization both in vitro and in vivo. Transcriptomic analyses indicated a notable upregulation in the expression of the long noncoding RNA metastsis-associated lung adenocarcinoma transcript 1 (Malat1) in the C2C12 myoblasts cultured on PSF. Intriguingly, silencing Malat1 curtailed the PSF-induced mineralization and downregulated the expression of bone morphogenetic proteins (Bmps) and osteogenic markers. Further, we found that PSF prompted the activation of Yap1 signaling and epigenetic modifications in the Malat1 promoter, crucial for the expression of Malat1. These results indicate that the fibrous matrix adjacent to biomaterials can instigate Malat1 upregulation, subsequently driving osteogenic trans-differentiation in myoblasts and ectopic calcification through its transcriptional regulation of osteogenic genes, including Bmps. Our findings point to a novel therapeutic avenue for mitigating prosthesis-induced pathological calcification, heralding new possibilities in the field of biomaterial-based therapies.

ENPP1 enzyme replacement therapy improves ectopic calcification but does not rescue skeletal phenotype in a mouse model for craniometaphyseal dysplasia.

Craniometaphyseal dysplasia (CMD) is a rare genetic bone disorder, characterized by progressive thickening of craniofacial bones and flared metaphyses of long bones. Craniofacial hyperostosis leads to the obstruction of neural foramina and neurological symptoms such as facial palsy, blindness, deafness, or severe headache. Mutations in ANKH (mouse ortholog ANK), a transporter of small molecules such as citrate and ATP, are responsible for autosomal dominant CMD. Knock-in (KI) mice carrying an ANKF377del mutation (AnkKI/KI ) replicate many features of human CMD. Pyrophosphate (PPi) levels in plasma are significantly reduced in AnkKI/KI mice. PPi is a potent inhibitor of mineralization. To examine the extent to which restoration of circulating PPi levels may prevent the development of a CMD-like phenotype, we treated AnkKI/KI mice with the recombinant human ENPP1-Fc protein IMA2a. ENPP1 hydrolyzes ATP into AMP and PPi. Male and female Ank+/+ and AnkKI/KI mice (n ≥ 6/group) were subcutaneously injected with IMA2a or vehicle weekly for 12wk, starting at the age of 1wk. Plasma ENPP1 activity significantly increased in AnkKI/KI mice injected with IMA2a (Vehicle/IMA2a: 28.15 ± 1.65/482.7 ± 331.2 mOD/min; p <.01), which resulted in the successful restoration of plasma PPi levels (Ank+/+ /AnkKI/KI vehicle treatment/AnkKI/KI IMA2a: 0.94 ± 0.5/0.43 ± 0.2/1.29 ± 0.8μM; p <.01). We examined the skeletal phenotype by X-Ray imaging and μCT. IMA2a treatment of AnkKI/KI mice did not significantly correct CMD features such as the abnormal shape of femurs, increased bone mass of mandibles, hyperostotic craniofacial bones, or the narrowed foramen magnum. However, μCT imaging showed ectopic calcification near basioccipital bones at the level of the foramen magnum and on joints of AnkKI/KI mice. Interestingly, IMA2a treatment significantly reduced the volume of calcified nodules at both sites. Our data demonstrate that IMA2a is sufficient to restore plasma PPi levels and reduce ectopic calcification but fails to rescue skeletal abnormalities in AnkKI/KI mice under our treatment conditions.

Supplements in Rare Bone Diseases

AbstractDespite having different aetiologies, different rare bone diseases (RBDs) such as hypophosphatasia (HPP), autosomal dominant hypophosphatemic rickets (ADHR), X-linked hypophosphatemia (XLH) and osteogenesis imperfecta (OI) share common clinical features such as growth disturbances, pathological fractures, pseudo-fractures and chronic musculoskeletal pain. The role of micronutrients including minerals, trace elements and vitamin D in the physiological bone metabolism are well established. A significant share of RBD patients suffer from nutritional deficiencies due to the underlying disease or do not achieve the recommended daily intake (RDI) for micronutrients. The supplementation of micronutrients in RBDs should have the goal of achieving the RDI and promoting bone metabolism without increasing the burden of disease. Specific diets and an increased intake of specific micronutrients could potentially improve some of the disease symptoms, however special caution should be taken to avoid over-supplementation and to avoid adverse effects such as hypercalciuria, ectopic calcifications, GI-upset and nephrocalcinosis in case of calcium over-supplementation.

Converging Mechanisms of Vascular and Cartilaginous Calcification.

Physiological calcification occurs in bones and epiphyseal cartilage as they grow, whereas ectopic calcification occurs in blood vessels, cartilage, and soft tissues. Although it was formerly thought to be a passive and degenerative process associated with aging, ectopic calcification has been identified as an active cell-mediated process resembling osteogenesis, and an increasing number of studies have provided evidence for this paradigm shift. A significant association between vascular calcification and cardiovascular risk has been demonstrated by various studies, which have shown that arterial calcification has predictive value for future coronary events. With respect to cartilaginous calcification, calcium phosphate or hydroxyapatite crystals can form asymptomatic deposits in joints or periarticular tissues, contributing to the pathophysiology of osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, tendinitis, and bursitis. The risk factors and sequence of events that initiate ectopic calcification, as well as the mechanisms that prevent the development of this pathology, are still topics of debate. Consequently, in this review, we focus on the nexus of the mechanisms underlying vascular and cartilaginous calcifications, trying to circumscribe the similarities and disparities between them to provide more clarity in this regard.

Activation of Nuclear Factor Erythroid 2-Related Factor 2 Transcriptionally Upregulates Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 Expression and Inhibits Ectopic Calcification in Mice.

Calcification plays a key role in biological processes, and breakdown of the regulatory mechanism results in a pathological state such as ectopic calcification. We hypothesized that ENPP1, the enzyme that produces the calcification inhibitor pyrophosphate, is transcriptionally regulated by Nrf2, and that Nrf2 activation augments ENPP1 expression to inhibit ectopic calcification. Cell culture experiments were performed using mouse osteoblastic cell line MC3T3-E1. Nrf2 was activated by 5-aminolevulinic acid and sodium ferrous citrate. Nrf2 overexpression was induced by the transient transfection of an Nrf2 expression plasmid. ENPP1 expression was monitored by real-time RT-PCR. Because the promoter region of ENPP1 contains several Nrf2-binding sites, chromatin immunoprecipitation using an anti-Nrf2 antibody followed by real-time PCR (ChIP-qPCR) was performed. The relationship between Nrf2 activation and osteoblastic differentiation was examined by alkaline phosphatase (ALP) and Alizarin red staining. We used mice with a hypomorphic mutation in ENPP1 (ttw mice) to analyze whether Nrf2 activation inhibits ectopic calcification. Nrf2 and Nrf2 overexpression augmented ENPP1 expression and inhibited osteoblastic differentiation, as indicated by ALP expression and calcium deposits. ChIP-qPCR showed that some putative Nrf2-binding sites in the ENPP1 promoter region were bound by Nrf2. Nrf2 activation inhibited ectopic calcification in mice. ENPP1 gene expression was transcriptionally regulated by Nrf2, and Nrf2 activation augmented ENPP1 expression, leading to the attenuation of osteoblastic differentiation and ectopic calcification in vitro and in vivo. Nrf2 activation has a therapeutic potential for preventing ectopic calcification.

Structural and molecular imaging-based characterization of soft tissue and vascular calcification in hyperphosphatemic familial tumoral calcinosis.

Hyperphosphatemic familial tumoral calcinosis (HFTC) is a rare disorder caused by deficient FGF23 signaling and resultant ectopic calcification. Here, we systematically characterized and quantified macro- and micro-calcification in a HFTC cohort using CT and 18F-sodium fluoride PET/CT (18F-NaF PET/CT). Fourier-transform infrared (FTIR) spectroscopy was performed on 4 phenotypically different calcifications from a patient with HFTC, showing the dominant component to be hydroxyapatite. Eleven patients with HFTC were studied with CT and/or 18F-NaF PET/CT. Qualitative review was done to describe the spectrum of imaging findings on both modalities. CT-based measures of volume (eg, total calcific burden and lesion volume) and density (Hounsfield units) were quantified and compared to PET-based measures of mineralization activity (eg, mean standardized uptake values-SUVs). Microcalcification scores were calculated for the vasculature of 6 patients using 18F-NaF PET/CT and visualized on a standardized vascular atlas. Ectopic calcifications were present in 82% of patients, predominantly near joints and the distal extremities. Considerable heterogeneity was observed in total calcific burden per patient (823.0 ± 670.1cm3, n = 9) and lesion volume (282.5 ± 414.8cm3, n = 27). The largest lesions were found at the hips and shoulders. 18F-NaF PET offered the ability to differentiate active vs quiescent calcifications. Calcifications were also noted in multiple anatomic locations, including brain parenchyma (50%). Vascular calcification was seen in the abdominal aorta, carotid, and coronaries in 50%, 73%, and 50%, respectively. 18F-NaF-avid, but CT-negative calcification was seen in a 17-year-old patient, implicating early onset vascular calcification. This first systematic assessment of calcifications in a cohort of patients with HFTC has identified the early onset, prevalence, and extent of calcification. It supports 18F-NaF PET/CT as a clinical tool for distinguishing between active and inactive calcification, informing disease progression, and quantification of ectopic and vascular disease burden.

Lead Acetate-Injected Mice is an Animal Model for Extrapolation of Calcifying Response to Humans Due to Low Involvement of Bone Resorption.

Vascular calcification affects the prognosis of patients with renal failure. Bisphosphonates are regarded as candidate anti-calcifying drugs because of their inhibitory effects on both calcium-phosphate aggregation and bone resorption. However, calcification in well-known rodent models is dependent upon bone resorption accompanied by excessive bone turnover, making it difficult to estimate accurately the anti-calcifying potential of drugs. Therefore, models with low bone resorption are required to extrapolate anti-calcifying effects to humans. Three bisphosphonates (etidronate, alendronate, and FYB-931) were characterised for their inhibitory effects on bone resorption in vivo and calcium-phosphate aggregation estimated by calciprotein particle formation in vitro. Then, their effects were examined using two models inducing ectopic calcification: the site where lead acetate was subcutaneously injected into mice and the transplanted, aorta obtained from a donor rat. The inhibitory effects of bisphosphonates on bone resorption and calcium-phosphate aggregation were alendronate > FYB-931 > etidronate and FYB-931 > alendronate = etidronate, respectively. In the lead acetate-induced model, calcification was most potently suppressed by FYB-931, followed by alendronate and etidronate. In the aorta-transplanted model, only FYB-931 suppressed calcification at a high dose. In both the models, no correlation was observed between calcification and bone resorption marker, tartrate-resistant acid phosphatase (TRACP). Results from the lead acetate-induced model showed that inhibitory potency against calcium-phosphate aggregation contributed to calcification inhibition. The two calcification models, especially the lead acetate-induced model, may be ideal for the extrapolation of calcifying response to humans because of calcium-phosphate aggregation rather than bone resorption as its mechanism.