Ectopic mineralization refers to the deposition of mineralized complexes in the extracellular matrix of soft tissues. Calcific aortic valve disease, vascular calcification, gallstones, kidney stones, and abnormal mineralization in arthritis are common examples of ectopic mineralization. They are debilitating diseases and exhibit excess mortality, disability, and morbidity, which impose on patients with limited social or financial resources. Recent recognition that inflammation plays an important role in ectopic mineralization has attracted the attention of scientists from different research fields. In the present review, we summarize the origin of inflammation in ectopic mineralization and different channels whereby inflammation drives the initiation and progression of ectopic mineralization. The current knowledge of inflammatory milieu in pathological mineralization is reviewed, including how immune cells, pro-inflammatory mediators, and osteogenic signaling pathways induce the osteogenic transition of connective tissue cells, providing nucleating sites and assembly of aberrant minerals. Advances in the understanding of the underlying mechanisms involved in inflammatory-mediated ectopic mineralization enable novel strategies to be developed that may lead to the resolution of these enervating conditions.

In previous studies, we have demonstrated that stress response-induced high glucocorticoid levels could be the underlying cause of traumatic heterotopic ossification (HO), and we have developed a glucocorticoid-induced ectopic mineralization (EM) mouse model by systemic administration of a high dose of dexamethasone (DEX) to animals with muscle injury induced by cardiotoxin injection. In this model, dystrophic calcification (DC) developed into HO in a cell autonomous manner. However, it is not clear how DC is formed after DEX treatment. Therefore, in this study, we aimed to explore how glucocorticoids initiate muscle EM at a cellular and molecular level. We showed that DEX treatment inhibited inflammatory cell infiltration into injured muscle but inflammatory cytokine production in the muscle was significantly increased, suggesting that other non-inflammatory muscle cell types may regulate the inflammatory response and the muscle repair process. Accompanying this phenotype, transforming growth factor β1 (TGF-β1) expression in fibro-adipogenic progenitors (FAPs) was greatly down-regulated. Since TGF-β1 is a strong immune suppressor and FAP\'s regulatory role has a large impact on muscle repair, we hypothesized that down-regulation of TGF-β1 in FAPs after DEX treatment resulted in this hyperinflammatory state and subsequent failed muscle repair and EM formation. To test our hypothesis, we utilized a transgenic mouse model to specifically knock out Tgfb1 gene in PDGFRα positive FAPs to investigate if the transgenic mice could recapitulate the phenotype that was induced by DEX treatment. Our results showed that the transgenic mice completely phenocopied this hyperinflammatory state and spontaneously developed EM following muscle injury. On the contrary, therapeutics that enhanced TGF-β1 signaling in FAPs inhibited the inflammatory response and attenuated muscle EM. In summary, these results indicate that FAPs-derived TGF-β1 is a key molecule in regulating muscle inflammatory response and subsequent EM, and that glucocorticoids exert their effect via down-regulating TGF-β1 in FAPs.
Heterotopic ossification (HO) is abnormal bone formation in soft tissue. Glucocorticoids, which have strong anti-inflammatory properties, have usually been used as HO therapeutics. However, our findings suggest that glucocorticoids can also promote HO formation. In this study, we tried to explain the underlying reason for these seemingly contradictory observations. We showed that glucocorticoids, in addition to exerting an anti-inflammatory effect on inflammatory cells, can also target another type of muscle cell to exert a pro-inflammatory effect. These cells are called fibro-adipogenic progenitors (FAPs), and we demonstrated that FAPs played a master regulatory role in the muscle inflammatory response by modulating the expression of transforming growth factor β1 (TGF-β1), a well-known immune suppressor. In summary, our findings highlighted the importance of FAP TGF-β1 levels in affecting the progression and regression of muscle HO, and provided new treatment options for HO based on their ability to elevate TGF-β1 levels in FAPs.

Raine syndrome (RNS) is a rare autosomal recessive osteosclerotic dysplasia. RNS is caused by loss-of-function disease-causative variants of the FAM20C gene that encodes a kinase that phosphorylates most of the secreted proteins found in the body fluids and extracellular matrix. The most common RNS clinical features are generalized osteosclerosis, facial dysmorphism, intracerebral calcifications and respiratory defects. In non-lethal RNS forms, oral traits include a well-studied hypoplastic amelogenesis imperfecta (AI) and a much less characterized gingival phenotype. We used immunomorphological, biochemical, and siRNA approaches to analyze gingival tissues and primary cultures of gingival fibroblasts of two unrelated, previously reported RNS patients. We showed that fibrosis, pathological gingival calcifications and increased expression of various profibrotic and pro-osteogenic proteins such as POSTN, SPARC and VIM were common findings. Proteomic analysis of differentially expressed proteins demonstrated that proteins involved in extracellular matrix (ECM) regulation and related to the TGFβ/SMAD signaling pathway were increased. Functional analyses confirmed the upregulation of TGFβ/SMAD signaling and subsequently uncovered the involvement of two closely related transcription cofactors important in fibrogenesis, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Knocking down of FAM20C confirmed the TGFβ-YAP/TAZ interplay indicating that a profibrotic loop enabled gingival fibrosis in RNS patients. In summary, our in vivo and in vitro data provide a detailed description of the RNS gingival phenotype. They show that gingival fibrosis and calcifications are associated with, and most likely caused by excessed ECM production and disorganization. They furthermore uncover the contribution of increased TGFβ-YAP/TAZ signaling in the pathogenesis of the gingival fibrosis.

Generalized arterial calcification of infancy (GACI) is a rare genetic disease characterized by arterial calcifications or stenoses and hypertension. GACI is caused by mutations in the ENPP1 or ABCC6 genes, and it often causes intrauterine or early infancy death. Here, we report a case of rare GACI caused by a homozygous variation in ENPP1, in a Chinese infant initially presenting with hypertension. The proband was an 8-month-old boy with in utero tricuspid valve calcification, presenting with hypertension at birth. Enhanced computed tomography revealed extensive arterial calcification. Genetic testing identified a homozygous variation in ENPP1 (c.783C > G p.Y261X), which led to the diagnosis of GACI. This mutation has been reported in only three Chinese patients, which all initially presented with hypophosphatemic rickets rather than GACI. This case enriches the clinical and genetic spectrum of ENPP1 mutations and reminds us that GACI should be considered in an infant presenting with hypertension and extensive arterial calcification, and that genetic testing should be performed.

BACKGROUND: Pseudoxanthoma elasticum (PXE), a monogenic disorder resulting in calcification affecting the skin, eyes and peripheral arteries, is caused by mutations in the ABCC6 gene, and is associated with low plasma inorganic pyrophosphate (PPi). It is unknown how ABCC6 genotype affects plasma PPi.
METHODS: We studied the association of ABCC6 genotype (192 patients with biallelic pathogenic ABCC6 mutations) and PPi levels, and its association with the severity of arterial and ophthalmological phenotypes. ABCC6 variants were classified as truncating or non-truncating, and three groups of the 192 patients were formed: those with truncating mutations on both chromosomes (n = 121), those with two non-truncating mutations (n = 10), and a group who had one truncating and one non-truncating ABCC6 mutation (n = 61). The hypothesis formulated before this study was that there was a negative association between PPi level and disease severity.
RESULTS: Our findings confirm low PPi in PXE compared with healthy controls (0.53 ± 0.15 vs. 1.13 ± 0.29 µM, p < 0.01). The PPi of patients correlated with increasing age (β: 0.05 µM, 95% CI: 0.03-0.06 per 10 years) and was higher in females (0.55 ± 0.17 vs. 0.51 ± 0.13 µM in males, p = 0.03). However, no association between PPi and PXE phenotypes was found. When adjusted for age and sex, no association between PPi and ABCC6 genotype was found.
CONCLUSIONS: Our data suggest that the relationship between ABCC6 mutations and reduced plasma PPi may not be as direct as previously thought. PPi levels varied widely, even in patients with the same ABCC6 mutations, further suggesting a lack of direct correlation between them, even though the ABCC6 protein-mediated pathway is responsible for ~60% of this metabolite in the circulation. We discuss potential factors that may perturb the expected associations between ABCC6 genotype and PPi and between PPi and disease severity. Our findings support the argument that predictions of pathogenicity made on the basis of mutations (or on the structure of the mutated protein) could be misleading.

Inorganic pyrophosphate (PPi) is a crucial extracellular mineralization regulator. Low plasma PPi concentrations underlie the soft tissue calcification present in several rare hereditary mineralization disorders as well as in more common conditions like chronic kidney disease and diabetes. Even though deregulated plasma PPi homeostasis is known to be linked to multiple human diseases, there is currently no reliable assay for its quantification. We here describe a PPi assay that employs the enzyme ATP sulfurylase to convert PPi into ATP. Generated ATP is subsequently quantified by firefly luciferase-based bioluminescence. An internal ATP standard was used to correct for sample-specific interference by matrix compounds on firefly luciferase activity. The assay was validated and shows excellent precision (< 3.5%) and accuracy (93-106%) of PPi spiked into human plasma samples. We found that of several anticoagulants tested only EDTA effectively blocked conversion of ATP into PPi in plasma after blood collection. Moreover, filtration over a 300,000-Da molecular weight cut-off membrane reduced variability of plasma PPi and removed ATP present in a membrane-enclosed compartment, possibly platelets. Applied to plasma samples of wild-type and Abcc6-/- rats, an animal model with established low circulating levels of PPi, the new assay showed lower variability than the assay that was previously in routine use in our laboratory. In conclusion, we here report a new and robust assay to determine PPi concentrations in plasma, which outperforms currently available assays because of its high sensitivity, precision, and accuracy.

暂无摘要。

Pseudoxanthoma elasticum (PXE, OMIM # 264800) is an autosomal recessive, multisystemic disorder, associated with mutations of the ABCC6 gene. Ectopic mineralization is in the background of the clinical manifestations of the disease. Calcium-salt crystals are deposited primarily in the skin, in the Bruch membrane of the eyes, and in the vascular endothelium. Thus, in addition to the skin lesions, visual impairment and cardiovascular involvement also occur. Clinical symptoms show varying severity and display heterogeneous appearance. The identification of the phenotype and care of the patients require a multidisciplinary perspective based on the collaboration of a dermatologist, ophthalmologist, cardiologist, and clinical geneticist. The aim of our work is to describe the development of symptoms of the disease, in order to facilitate the diagnosis. In addition, we aim to draw attention to the importance of early diagnosis of pseudoxanthoma elasticum, and to present modern diagnostic methods. Considering the development of severe systemic complications, the early diagnosis with the collaboration between related specialists is crucial to provide optimal clinical care and management of the patients.
Összefoglaló. A pseudoxanthoma elasticum (PXE, OMIM # 264800) egy autoszomális recesszív módon öröklődő multiszisztémás érintettséggel járó kórkép, melynek háttérében az ABCC6 gén mutációi állnak. A tünetek kialakulásának oka az ektópiás mineralizáció. Kalcium-só kristályok rakódnak le elsősorban a bőrben, a szem Bruch-membránjában és az erek endotheliumában, így a bőrelváltozások mellett a látás csökkenése és cardiovascularis eltérések is jelentkezhetnek. A klinikai tünetek változó súlyosságúak lehetnek, heterogén megjelenésűek. A betegek fenotípusának azonosítása, valamint gondozása multidiszciplináris feladat, bőrgyógyász, szemész, kardiológus és klinikai genetikus együttműködésén alapul. Célunk, hogy bemutassuk a betegségben előforduló tüneteket, melyek ismerete megkönnyíti a kórkép felismerését, illetve hogy felhívjuk a figyelmet a korai diagnózis fontosságára és ismertessük a korszerű diagnosztikai módszereket. A súlyos szisztémás tünetek kialakulása miatt rendkívüli jelentőséggel bír a társszakmák együttműködése, hogy a korai diagnózis által időben megfelelő gondozásban és terápiában részesülhessenek a betegek. Orv Hetil. 2022; 163(18): 702–711.

Chronic low back pain is a highly prevalent health condition intricately linked to intervertebral disc degeneration. One of the prominent features of disc degeneration that is commonly observed with aging is dystrophic calcification. ATP-binding cassette sub-family C member 6 (ABCC6), a presumed ATP efflux transporter, is a key regulator of systemic levels of the mineralization inhibitor pyrophosphate (PPi). Mutations in ABCC6 result in pseudoxanthoma elasticum (PXE), a progressive human metabolic disorder characterized by mineralization of the skin and elastic tissues. The implications of ABCC6 loss-of-function on pathological mineralization of structures in the spine, however, are unknown. Using the Abcc6 -/- mouse model of PXE, we investigated age-dependent changes in the vertebral bone and intervertebral disc. Abcc6 -/- mice exhibited diminished trabecular bone quality parameters at 7 months, which remained significantly lower than the wild-type mice at 18 months of age. Abcc6 -/- vertebrae showed increased TRAP staining along with decreased TNAP staining, suggesting an enhanced bone resorption as well as decreased bone formation. Surprisingly, however, loss of ABCC6 resulted only in a mild, aging disc phenotype without evidence of dystrophic mineralization. Finally, we tested the utility of oral K3Citrate to treat the vertebral phenotype since it is shown to regulate hydroxyapatite mechanical behavior. The treatment resulted in inhibition of the osteoclastic response and an early improvement in mechanical properties of the bone underscoring the promise of potassium citrate as a therapeutic agent. Our data suggest that although ectopic mineralization is tightly regulated in the disc, loss of ABCC6 compromises vertebral bone quality and dysregulates osteoblast-osteoclast coupling.

Dystrophic calcification is a common histopathologic finding that can be concomitant with a plethora of diseases, ranging from self-limited infections to insidious malignancies. Gamna-Gandy bodies (GGBs) are a form of dystrophic calcification associated with chronic hemolysis and are typically observed in the spleen. In this report, we present the case of a 92-year-old man who presented with a 4-mm blue papule that was biopsied given the concern for a blue nevus. The subsequent histopathologic examination of the biopsy specimen showed a dermal organizing hematoma adjacent to pale-yellow to brown, refractile material within fibrotic collagen consistent with GGBs. Scanning electron microscopy with energy-dispersive x-ray analysis (SEM/EDXA) revealed that the structures were composed of carbon (39%), oxygen (32%), iron (16%), phosphorus (7%), calcium (5%), and sodium (1%). Fourier transform infrared spectroscopy identified amorphous calcium phosphate. GGBs have not been previously described in the skin and have been rarely characterized with SEM/EDXA in other sites.