Search Results (67)

Gla-rich protein (GRP), also known as UCMA, is a vitamin K-dependent protein that has emerged as an important regulator of pathological calcification and inflammation. Vascular calcification is a major complication of chronic kidney disease and cardiovascular disorders and is now recognized as an active and tightly regulated process rather than a passive accumulation of minerals. Increasing evidence indicates that GRP plays a protective role in mineral homeostasis through its strong calcium-binding capacity and its dependence on vitamin K-mediated gamma carboxylation. This work represents a comprehensive narrative review aimed at summarizing and critically discussing the current scientific knowledge on GRP. Available experimental and clinical data are analyzed with respect to gene expression, molecular regulation, vitamin K dependency, and underlying mechanisms of action. Particular emphasis is placed on the dual function of GRP in inhibiting ectopic calcification and modulating inflammatory responses. The evidence linking altered GRP levels or changes in its carboxylation status with chronic kidney disease, vascular calcification, calcific aortic valve disease, osteoarthritis, and tumor-associated microcalcifications is systematically examined. Current findings collectively support the concept that GRP is a multifunctional protein operating at the interface of mineral metabolism, inflammation, and tissue remodeling. Despite promising experimental data, important knowledge gaps remain, including the absence of standardized assays capable of distinguishing different GRP forms and the lack of longitudinal clinical studies evaluating its predictive value. This manuscript highlights the potential of GRP as a biomarker of disturbed mineral homeostasis and cardiovascular risk, while emphasizing the need for further research to clarify its precise biological functions and clinical relevance. Full article

The 1st Joint Ectopic Calcification Meeting (JECM) was held in Nancy, France on 24–26 September 2025. In response to the growing need for unified scientific dialogue on soft tissue ectopic calcification, the Joint Ectopic Calcification Meeting (JECM) brought together the communities of INTEC, ISSEC, BBC, iSCCa, and the PXE Budapest meeting. This initiative emerged from concerns over fragmentation in the field, with multiple smaller meetings diluting collaborative potential. By consolidating efforts, JECM aims to foster interdisciplinary exchange, highlight cutting-edge research, and build a flagship event for the ectopic calcification community. With over 100 participants, the inaugural meeting in Nancy marks a promising step toward a more integrated and dynamic future for the field. The abstracts of this year’s meeting oral and poster presentations are collected in this conference paper, with permission from the corresponding authors. Full article

Background/Objectives: Cells derived from direct chemical reprogramming into osteoblasts represent a promising source for bone regeneration, but the efficiency needs improvement. Here, we systematically evaluated whether the natural compound psoralen (Psr) could enhance this process and explored its therapeutic potential and mechanism of action. Methods: Mouse embryonic fibroblasts (MEFs) were treated with a cocktail of forskolin and phenamil (FP), supplemented with Psr. In vitro differentiation was assessed by alkaline phosphatase and Alizarin Red S staining, reverse transcription quantitative PCR, immunofluorescence and Western blot. The bone-regenerative potential of the derived chemically induced osteoblast-like cells (ciOBs) was evaluated in critical-sized calvarial defects, femoral cortical defects and a subcutaneous ectopic implantation model, using micro-computed tomography and histology. Mechanistic insights of Psr were gained by analyzing the adenylyl cyclase 9 (ADCY9)/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP response element-binding protein (CREB) axis using inhibitor SQ22536. Results: Psr acted synergistically with the FP cocktail to drive efficient osteogenic reprogramming of MEFs. At an optimal concentration of 25 μM, Psr enabled the most robust induction of early osteogenic markers and generation of mature, mineralizing ciOBs in vitro. In vivo, FP + Psr-induced ciOBs repaired critical-sized calvarial and femoral cortical defects and generated substantial, vascularized bone tissue in ectopic sites. Mechanistically, Psr co-treatment potently activated the ADCY9/cAMP/PKA/CREB pathway, and pharmacological inhibition of this pathway completely abolished the pro-osteogenic effects of Psr. Conclusions: Psr acts as a potent synergistic enhancer of direct chemical reprogramming, generating functional osteoblast-like cells with robust bone-regenerative capacity via activation of the ADCY9/cAMP/PKA/CREB pathway. Full article

Extra-osseous calcification refers to the pathological deposition of calcium salts in soft tissues. Its most recognized forms affect the cardiovascular system, leading to vascular and heart valve calcifications. This process is active and regulated, involving the phenotype transition of resident cells into osteo/chondrogenic lineage. Chronic kidney disease (CKD) patients frequently suffer from vascular and other soft tissue calcification. OsteoSense dyes are fluorescent imaging agents developed to visualize calcium deposits during bone formation. In addition to its application in bone physiology, it has been used to detect vascular smooth muscle cell calcification in vitro and to evaluate calcification ex vivo. Here, we investigated CKD-associated soft tissue calcification by applying OsteoSense in vivo. CKD was induced by a diet containing adenine and elevated phosphate. OsteoSense (80 nmol/kg body weight) was injected intravenously through the retro-orbital venous sinus 18 h before the measurement on an IVIS Spectrum In Vivo Imaging System. OsteoSense staining detected calcium deposition in the aorta, kidney, heart, lung, and liver in CKD mice. On the other hand, no calcification occurred in the brain, eye, or spleen. OsteoSense positivity in the calcified soft tissues in CKD mice was associated with increased mRNA levels of osteo/chondrogenic transcription factors. Our findings demonstrate that OsteoSense is a sensitive and effective tool for detecting soft tissue calcification in vivo, and may be particularly valuable for studies of CKD-related ectopic calcification. Full article

The 3rd Annual Meeting of the International Network on Ectopic Calcification (INTEC) was held in Faro, Portugal on 12–13 September 2024. This hybrid meeting brought together researchers and clinicians focused on the molecular, (patho)physiological, and clinical aspects of ectopic calcification in hereditary and acquired conditions, as well as in aging. The findings presented in this year’s meeting emphasised the complexity of the field, offering new insights into both mechanistic pathways and translational hurdles. The abstracts of this year’s meeting are collected in this conference paper, with permission from the corresponding authors. Full article

Heterotopic ossification (HO) refers to the pathological formation of bone in soft tissues, typically following trauma, surgical procedures, or as a result of genetic disorders. Notably, injuries to the central nervous system significantly increase the risk of HO, a condition referred to as neurogenic HO (NHO). This review outlines the cellular and molecular mechanisms driving HO, focusing on the inflammatory response, progenitor cell reprogramming, and current treatment strategies. HO is primarily fuelled by a prolonged and dysregulated inflammatory response, characterized by sustained expression of osteoinductive cytokines secreted by M1 macrophages. These cytokines promote the aberrant differentiation of fibro-adipogenic progenitor cells (FAPs) into osteoblasts, leading to ectopic mineralization. Additional factors such as hypoxia, BMP signalling, and mechanotransduction pathways further contribute to extracellular matrix (ECM) remodelling and osteogenic reprogramming of FAPs. In the context of NHO, neuroendocrine mediators enhance ectopic bone formation by influencing both local inflammation and progenitor cell fate decisions. Current treatment options such as nonsteroidal anti-inflammatory drugs (NSAIDs), radiation therapy, and surgical excision offer limited efficacy and are associated with significant risks. Novel therapeutic strategies targeting inflammation, neuropeptide signalling, and calcium metabolism may offer more effective approaches to preventing or mitigating HO progression. Full article

ABCC6, a key regulator in ectopic calcification, plays a crucial role in mineralization through the modulation of extracellular purinergic pathways and production of inorganic pyrophosphate (PPi), which inhibits calcification. Inherited deficiencies in ABCC6 lead to pseudoxanthoma elasticum (PXE) and related conditions, characterized by calcification in various tissues, particularly affecting the skin, eyes, and cardiovascular system. Although PXE does not directly impact the nervous system, secondary neurological issues arise from cerebrovascular complications, increasing the risk of strokes linked to arterial blockages resembling atherosclerosis. This review investigates the connection between ABCC6 mutations and cerebral small vessel disease (SVD), expanding the understanding of PXE and related phenotypes. Mutations in ABCC6, identified as causing PXE, contribute to systemic metabolic dysfunction, with significant implications for cerebrovascular health. An association between ABCC6 mutations and cerebral SVD has been suggested in various studies, particularly in populations with distinct genetic backgrounds. Emerging evidence indicates that pathogenic mutations increase the risk of ischemic strokes, with both homozygous and heterozygous carriers showing susceptibility. Mechanistically, ABCC6 deficiency is implicated in dyslipidemia and atherosclerosis, further exacerbating cerebrovascular risks. Increased arterial pulsatility, linked to carotid siphon calcification, may also contribute to microvascular damage and subsequent brain injury. Understanding these mechanisms is vital for developing targeted diagnostic and therapeutic strategies for managing cerebrovascular risks in PXE patients. This review emphasizes the need for comprehensive genetic screening and the consideration of traditional vascular risk factors in patient management, highlighting the complex interplay between genetic mutations and environmental influences affecting cerebrovascular health. Future research should focus on longitudinal studies to elucidate the causal pathways linking arterial calcification, pulsatility, and brain damage in PXE. Full article

Background/Objectives: Ectopic calcification is a pathological condition characterized by the mineralization of soft tissues due to the deposition of calcium phosphate crystals. Hexasodium fytate (CSL525, previously known as SNF472) is a crystallization inhibitor being developed for the treatment of ectopic calcification-related disorders. Our aim was to investigate CSL525 for the treatment of soft-tissue calcification disorders in animal models of pseudoxanthoma elasticum and calcinosis cutis. Methods: In a first study, abcc6^-/- zebrafish larvae were exposed to 1 mM CSL525 for 7 days or kept under the same conditions without CSL525, and spinal mineralization was quantified. In a second study, abcc6^-/- mice were administered subcutaneously with CSL525 at 15 mg/kg thrice weekly for eight weeks. Vehicle-treated WT (C57BL/6J) and abcc6^-/- mice served as controls, and muzzle skin calcification was quantified. In a third study, calcinosis cutis was induced in rats through subcutaneous administration of 0.15 mg FeCl₃ at two sites in the thorax. Rats were administered either subcutaneous CSL525 (60 mg/kg) or vehicle (0.9% NaCl), and calcium content was measured in the skin. Results: CSL525 significantly reduced the calcified area (~40%) in abcc6a^-/- zebrafish larvae. The abcc6^-/- mice receiving CSL525 showed a 57% inhibition of muzzle calcification compared to vehicle-treated abcc6^-/- mice. CSL525 inhibited skin calcification development by 60% in the calcinosis cutis rat model. Conclusions: CSL525 may prove beneficial not only in preventing the progression of cardiovascular calcification but also in treating other ectopic calcification conditions, including skin calcification associated with genetic disorders such as PXE. Full article

Thalassemia, once associated with limited survival, now sees extended life expectancy due to treatment advancements, but new complications such as pseudoxanthoma elasticum (PXE)-like syndrome are emerging. In fact, thalassemia patients develop PXE-like features more frequently than the general population. These features include skin lesions, ocular changes, and vascular issues like arterial calcifications, all linked to oxidative damage from iron overload. PXE-like syndrome in thalassemia mimics inherited PXE but is acquired. The underlying cause is thought to be oxidative stress due to iron overload, which induces free radicals and damages elastic tissues. Unlike inherited PXE, this form does not involve mutations in the ABCC6 gene, suggesting different pathogenic mechanisms, including abnormal fibroblast metabolism and oxidative processes. The vascular calcification seen in this syndrome often follows elastic fiber degeneration, with proteoglycans and glycoproteins acting as nucleation sites for mineralization. The condition can lead to severe cardiovascular and gastrointestinal complications. Studies have shown a significant incidence of PXE-like skin lesions in thalassemia patients, with some dying from cardiovascular complications. Research on ABCC6, a transporter protein involved in ectopic mineralization, has highlighted its role in various conditions, including PXE, beta-thalassemia, and generalized arterial calcification of infancy. ABCC6 mutations or reduced expression led to ectopic mineralization, affecting cardiovascular, ocular, and dermal tissues. The exact molecular mechanisms linking ABCC6 deficiency to ectopic mineralization remain unclear, though it is known to influence calcification-modulating proteins. This review focuses on the role of ABCC6 in the pathogenesis of calcifications, especially intracranial vascular calcifications in PXE and beta-thalassemia. Full article

Background/Objectives: Delays in bone healing and complications of remodeling constitute a major medical problem—particularly in older adults and patients with comorbidities. Current therapeutic approaches are based on strategies that promote bone regeneration. We recently identified a disaccharide compound (DP2) that enhances in vitro mineralization in human osteoblast cells via the early activation of Runx2 and the induction of osteoblast differentiation. Methods: First, a calcium quantification assay was performed to assess mineralization in MC3T3-E1 cells. Next, microcomputed tomography and histological analyses were used to examine in vivo bone repair in a rat 5 mm cranial defect model following the implantation of DP2 coupled to a micro/macroporous biphasic CaP ceramic (MBCP⁺) or collagen scaffold. Results: Here, we demonstrated that DP2 induced osteogenic differentiation and significantly elevated calcium matrix deposition in the murine preosteoblast cell line MC3T3-E1. We found that treatment with DP2 coupled to MBCP⁺ repaired the calvarial defect on post-implantation day 91. It significantly increased bone mineral density starting on day 29 post-treatment. In addition, DP2 did not induce ectopic bone formation. Conclusions: Taken as a whole, these results show that DP2 is a promising candidate treatment for delayed bone healing. Full article

The Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1) ectoenzyme regulates vascular intimal proliferation and mineralization of bone and soft tissues. ENPP1 variants cause Generalized Arterial Calcification of Infancy (GACI), a rare genetic disorder characterized by ectopic calcification, intimal proliferation, and stenosis of large- and medium-sized arteries. ENPP1 hydrolyzes extracellular ATP to pyrophosphate (PP_i) and AMP. AMP is the precursor of adenosine, which has been implicated in the control of neointimal formation. Herein, we demonstrate that an ENPP1-Fc recombinant therapeutic inhibits proliferation of vascular smooth muscle cells (VSMCs) in vitro and in vivo. Addition of ENPP1 and ATP to cultured VSMCs generated AMP, which was metabolized to adenosine. It also significantly decreased cell proliferation. AMP or adenosine alone inhibited VSMC growth. Inhibition of ecto-5′-nucleotidase CD73 decreased adenosine accumulation and suppressed the anti-proliferative effects of ENPP1/ATP. Addition of AMP increased cAMP synthesis and phosphorylation of VASP at Ser157. This AMP-mediated cAMP increase was abrogated by CD73 inhibitors or by A_2aR and A_2bR antagonists. Ligation of the carotid artery promoted neointimal hyperplasia in wild-type mice, which was exacerbated in ENPP1-deficient ttw/ttw mice. Prophylactic or therapeutic treatments with ENPP1 significantly reduced intimal hyperplasia not only in ttw/ttw but also in wild-type mice. These findings provide the first insight into the mechanism of the anti-proliferative effect of ENPP1 and broaden its potential therapeutic applications beyond enzyme replacement therapy. Full article

Putatively, tooth agenesis was attributed to the initiation failure of tooth germs, though little is known about the histological and molecular alterations. To address if constitutively active FGF signaling is associated with tooth agenesis, we activated Fgf8 in dental mesenchyme with Osr-cre knock-in allele in mice (Osr2-cre^KI; Rosa26R-Fgf8) and found incisor agenesis and molar microdontia. The cell survival assay showed tremendous apoptosis in both the Osr2-cre^KI; Rosa26R-Fgf8 incisor epithelium and mesenchyme, which initiated incisor regression from cap stage. In situ hybridization displayed vanished Shh transcription, and immunostaining exhibited reduced Runx2 expression and enlarged mesenchymal Lef1 domain in Osr2-cre^KI; Rosa26R-Fgf8 incisors, both of which were suggested to enhance apoptosis. In contrast, Osr2-cre^KI; Rosa26R-Fgf8 molar germs displayed mildly suppressed Shh transcription, and the increased expression of Ectodin, Runx2 and Lef1. Although mildly smaller than WT controls prenatally, the Osr2-cre^KI; Rosa26R-Fgf8 molar germs produced a miniature tooth with impaired mineralization after a 6-week sub-renal culture. Intriguingly, the implanted Osr2-cre^KI; Rosa26R-Fgf8 molar germs exhibited delayed odontoblast differentiation and accelerated ameloblast maturation. Collectively, the ectopically activated Fgf8 in dental mesenchyme caused incisor agenesis by triggering incisor regression and postnatal molar microdontia. Our findings reported tooth agenesis resulting from the regression from the early bell stage and implicated a correlation between tooth agenesis and microdontia. Full article

Lysyl oxidase (LOX)-mediated extracellular matrix crosslinking modulates calcification in atherosclerosis and aortic valve disease; however, this enzyme also induces oxidative stress. We addressed the contribution of LOX-dependent oxidative stress to cardiovascular calcification. LOX is upregulated in human-calcified atherosclerotic lesions and atheromas from atherosclerosis-challenged LOX transgenic mice (TgLOX^VSMC) and colocalized with a marker of oxidative stress (8-oxo-deoxyguanosine) in vascular smooth muscle cells (VSMCs). Similarly, in calcific aortic valves, high LOX expression was detected in valvular interstitial cells (VICs) positive for 8-oxo-deoxyguanosine, while LOX and LOXL2 expression correlated with osteogenic markers (SPP1 and RUNX2) and NOX2. In human VICs, mito-TEMPO and TEMPOL attenuated the increase in superoxide anion levels and the mineralization induced by osteogenic media (OM). Likewise, in OM-exposed VICs, β-aminopropionitrile (a LOX inhibitor) ameliorated both oxidative stress and calcification. Gain- and loss-of-function approaches in VICs demonstrated that while LOX silencing negatively modulates oxidative stress and calcification induced by OM, lentiviral LOX overexpression exacerbated oxidative stress and VIC calcification, effects that were prevented by mito-TEMPO, TEMPOL, and β-aminopropionitrile. Our data indicate that LOX-induced oxidative stress participates in the procalcifying effects of LOX activity in ectopic cardiovascular calcification, and highlight the multifaceted role played by LOX isoenzymes in cardiovascular diseases. Full article

There is no mouse model of patellar tendinopathy. This study aimed to establish a mouse inflammatory and degenerative patellar tendon injury model, which will facilitate research on patellar tendinopathy using advanced molecular tools including transgenic models. Collagenase at different doses (low dose (LD), medium dose (MD), high dose (HD)) or saline was injected over the mouse patellar tendon. At weeks 1, 2, 4, and 8 post-injection, the tendons were harvested for histology and further examined by micro-computed tomography (microCT) imaging at week 8. The optimal dose group and the saline group were further evaluated by immunohistochemical staining, gait pattern, and biomechanical properties. The histopathological score increased dose-dependently post-collagenase injection. Ectopic mineralization was observed and increased with collagenase dose. The LD group was selected for further analysis. The expression of IL-10, TNF-α, and MMP-1 significantly increased post-injection. The changes of limb idleness index (ΔLII) compared to preinjury state were significantly higher, while the ultimate load, stiffness, ultimate stress, and maximum Young’s modulus were significantly lower in the LD group compared to the saline group. A mouse inflammatory degenerative model of patellar tendon injury resembling tendinopathy was established as indicated by the dose-dependent increase in tendon histopathology, ectopic calcification, decrease in biomechanical properties, and pain-associated gait changes. Full article

In pulpitis, dentinal restorative processes are considerably associated with undifferentiated mesenchymal cells in the pulp. This study aimed to investigate strategies to improve the odonto/osteogenic differentiation of dental pulp stem cells (DPSCs) in an inflammatory environment. After pretreatment of DPSCs with 20 ng/mL tumor necrosis factor-induced protein-6 (TSG-6), DPSCs were cultured in an inflammation-inducing solution. Real-time polymerase chain reaction and Western blotting were performed to measure the expression levels of nuclear factor kappa B (NF-κB) and odonto/osteogenic differentiation markers, respectively. Cell Counting Kit-8 and 5-ethynyl-2′-deoxyuridine assays were used to assess cell proliferation and activity. Subcutaneous ectopic osteogenesis and mandibular bone cultures were performed to assess the effects of TSG-6 in vivo. The expression levels of odonto/osteogenic markers were higher in TSG-6-pre-treated DPSCs than nontreated DPSCs, whereas NF-κB-related proteins were lower after the induction of inflammation. An anti-CD44 antibody counteracted the rescue effect of TSG-6 on DPSC activity and mineralization in an inflammatory environment. Exogenous administration of TSG-6 enhanced the anti-inflammatory properties of DPSCs and partially restored their mineralization function by inhibiting NF-κB signaling. The mechanism of action of TSG-6 was attributed to its interaction with CD44. These findings reveal novel mechanisms by which DPSCs counter inflammation and provide a basis for the treatment of pulpitis. Full article