Involvement of the Wnt/ß-catenin signalling pathway in heterotopic ossification and ossification-related diseases.

Heterotopic ossification (HO) is a pathological condition characterized by the formation of bone within soft tissues. The development of HO is a result of abnormal activation of the bone formation programs, where multiple signalling pathways, including Wnt/ß-catenin, BMP and hedgehog signalling, are involved. The Wnt/ß-catenin signalling pathway, a conserved pathway essential for various fundamental activities, has been found to play a significant role in pathological bone formation processes. It regulates angiogenesis, chondrocyte hypertrophy and osteoblast differentiation during the development of HO. More importantly, the crosstalk between Wnt signalling and other factors including BMP, Hedgehog signalling, YAP may contribute in a HO-favourable manner. Moreover, several miRNAs may also be involved in HO formation via the regulation of Wnt signalling. This review aims to summarize the role of Wnt/ß-catenin signalling in the pathogenesis of HO, its interactions with related molecules, and potential preventive and therapeutic measures targeting Wnt/ß-catenin signalling.

Molecular Developmental Biology of Fibrodysplasia Ossificans Progressiva: Measuring the Giant by Its Toe.

When a genetic disease is characterized by the abnormal activation of normal molecular pathways and cellular events, it is illuminating to critically examine the places and times of these activities both in health and disease. Therefore, because heterotopic ossification (HO) in fibrodysplasia ossificans progressiva (FOP) is by far the disease's most prominent symptom, attention is also directed toward the pathways and processes of bone formation during skeletal development. FOP is recognizable by effects of the causative mutation on skeletal development even before HO manifests, specifically in the malformation of the great toes. This signature skeletal phenotype is the most highly penetrant, but is only one among several skeletal abnormalities associated with FOP. Patients may present clinically with joint malformation and ankylosis, particularly in the cervical spine and costovertebral joints, as well as characteristic facial features and a litany of less common, non-skeletal symptoms, all stemming from missense mutations in the ACVR1 gene. In the same way that studying the genetic cause of HO advanced our understanding of HO initiation and progression, insight into the roles of ACVR1 signaling during tissue development, particularly in the musculoskeletal system, can be gained from examining altered skeletal development in individuals with FOP. This review will detail what is known about the molecular mechanisms of developmental phenotypes in FOP and the early role of ACVR1 in skeletal patterning and growth, as well as highlight how better understanding these processes may serve to advance patient care, assessments of patient outcomes, and the fields of bone and joint biology.

Primary cilia mediate skeletogenic BMP and Hedgehog signaling in heterotopic ossification.

Heterotopic ossification (HO), defined as the formation of extraskeletal bone in muscle and soft tissues, is a diverse pathological process caused by either genetic mutations or inciting trauma. Fibrodysplasia ossificans progressiva (FOP) is a genetic form of HO caused by mutations in the bone morphogenetic protein (BMP) type I receptor gene activin A receptor type 1 (ACVR1). These mutations make ACVR1 hypersensitive to BMP and responsive to activin A. Hedgehog (Hh) signaling also contributes to HO development. However, the exact pathophysiology of how skeletogenic cells contribute to endochondral ossification in FOP remains unknown. Here, we showed that the wild-type or FOP-mutant ACVR1 localized in the cilia of stem cells from human exfoliated deciduous teeth with key FOP signaling components, including activin A receptor type 2A/2B, SMAD family member 1/5, and FK506-binding protein 12kD. Cilia suppression by deletion of intraflagellar transport 88 or ADP ribosylation factor like GTPase 3 effectively inhibited pathological BMP and Hh signaling, subdued aberrant chondro-osteogenic differentiation in primary mouse or human FOP cells, and diminished in vivo extraskeletal ossification in Acvr1Q207D, Sox2-Cre; Acvr1R206H/+ FOP mice and in burn tenotomy-treated wild-type mice. Our results provide a rationale for early and localized suppression of cilia in affected tissues after injury as a therapeutic strategy against either genetic or acquired HO.

Elemental and Structural Characterization of Heterotopic Ossification during Achilles Tendon Healing Provides New Insights on the Formation Process.

Heterotopic ossification (HO) in tendons can lead to increased pain and poor tendon function. Although it is believed to share some characteristics with bone, the structural and elemental compositions of HO deposits have not been fully elucidated. This study utilizes a multimodal and multiscale approach for structural and elemental characterization of HO deposits in healing rat Achilles tendons at 3, 6, 12, 16, and 20 weeks post transection. The microscale tomography and scanning electron microscopy results indicate increased mineral density and Ca/P ratio in the maturing HO deposits (12 and 20 weeks), when compared to the early time points (3 weeks). Visually, the mature HO deposits present microstructures similar to calcaneal bone. Through synchrotron-based X-ray scattering and fluorescence, the hydroxyapatite (HA) crystallites are shorter along the c-axis and become larger in the ab-plane with increasing healing time, while the HA crystal thickness remains within the reference values for bone. At the mineralization boundary, the overlap between high levels of calcium and prominent crystallite formation was outlined by the presence of zinc and iron. In the mature HO deposits, the calcium content was highest, and zinc was more present internally, which could be indicative of HO deposit remodeling. This study emphasizes the structural and elemental similarities between the calcaneal bone and HO deposits.

Endogenous hydrogen sulfide accelerated trauma-induced heterotopic ossification through the Ca2+/ERK pathway-enhanced aberrant osteogenic activity.

Unveiling of the mechanism involved in the occurrence and development of trauma-induced heterotopic ossification (tHO) is highly demanding due to current ineffective clinical treatment for it. Previous studies proposed that hydrogen sulfide (H2S) was vital for fate determination of stem cells, suggesting a potential role in the regulation of tHO development. In the current study, We found that expression of metabolic enzyme within sulfur conversion pathway was enhanced after tendon injury, leading to H2S accumulation within the tHO region. Increased production of endogenous H2S was shown to promote aberrant osteogenic activity of tendon-derived stem cells (TDSCs), which accelerated tHO formation. The inhibition of metabolic enzyme of H2S production or directly absorption of H2S could abolished osteogenic induction of TDSCs and the formation of tHO. Mechanistically, through RNA sequencing combined with rescue experiments, we demonstrated that activation of Ca2+/ERK pathway was the downstream molecular event of H2S-induced osteogenic commitment of TDSCs and tHO. For treatment strategy exploration, zine oxide nanoparticles (ZnO) as an effective H2S elimination material was validated to ideally halt the tHO formation in this study. Furthermore, in terms of chirality of nanoparticles, D-ZnO or L-ZnO nanoparticles showed superiority over R-ZnO nanoparticles in both clearing of H2S and inhibition of tHO. Our study not only revealed the mechanism of tHO through the endogenous gas signaling event from a new perspective, but also presented a applicable platform for elimination of the inordinate gas production, thus aiding the development of clinical treatment for tHO.

Disturbed glycolipid metabolism activates CXCL13-CXCR5 axis in senescent TSCs to promote heterotopic ossification.

Heterotopic ossification (HO) occurs as a common complication after injury, while its risk factor and mechanism remain unclear, which restricts the development of pharmacological treatment. Clinical research suggests that diabetes mellitus (DM) patients are prone to developing HO in the tendon, but solid evidence and mechanical research are still needed. Here, we combined the clinical samples and the DM mice model to identify that disordered glycolipid metabolism aggravates the senescence of tendon-derived stem cells (TSCs) and promotes osteogenic differentiation. Then, combining the RNA-seq results of the aging tendon, we detected the abnormally activated autocrine CXCL13-CXCR5 axis in TSCs cultured in a high fat, high glucose (HFHG) environment and also in the aged tendon. Genetic inhibition of CXCL13 successfully alleviated HO formation in DM mice, providing a potential therapeutic target for suppressing HO formation in DM patients after trauma or surgery.

AMPK induces PIAS3 mediated SUMOylation of E3 ubiquitin ligase Smurf1 impairing osteogenic differentiation and traumatic heterotopic ossification.

AMP-activated protein kinase (AMPK) is a typical sensor of intracellular energy metabolism. Our previous study revealed the role of activated AMPK in the suppression of osteogenic differentiation and traumatic heterotopic ossification, but the underlying mechanism remains poorly understood. The E3 ubiquitin ligase Smurf1 is a crucial regulator of osteogenic differentiation and bone formation. We report here that Smurf1 is primarily SUMOylated at a C-terminal lysine residue (K324), which enhances its activity, facilitating ALK2 proteolysis and subsequent bone morphogenetic protein (BMP) signaling pathway inhibition. Furthermore, SUMOylation of the SUMO E3 ligase PIAS3 and Smurf1 SUMOylation was suppressed during the osteogenic differentiation and traumatic heterotopic ossification. More importantly, we found that AMPK activation enhances the SUMOylation of Smurf1, which is mediated by PIAS3 and increases the association between PIAS3 and AMPK. Overall, our study revealed that Smurf1 can be SUMOylated by PIAS3, Furthermore, Smurf1 SUMOylation mediates osteogenic differentiation and traumatic heterotopic ossification through suppression of the BMP signaling pathway. This study revealed that promotion of Smurf1 SUMOylation by AMPK activation may be implicated in traumatic heterotopic ossification treatment.

From inflammation to bone formation: the intricate role of neutrophils in skeletal muscle injury and traumatic heterotopic ossification.

Neutrophils are emerging as an important player in skeletal muscle injury and repair. Neutrophils accumulate in injured tissue, thus releasing inflammatory factors, proteases and neutrophil extracellular traps (NETs) to clear muscle debris and pathogens when skeletal muscle is damaged. During the process of muscle repair, neutrophils can promote self-renewal and angiogenesis in satellite cells. When neutrophils are abnormally overactivated, neutrophils cause collagen deposition, functional impairment of satellite cells, and damage to the skeletal muscle vascular endothelium. Heterotopic ossification (HO) refers to abnormal bone formation in soft tissue. Skeletal muscle injury is one of the main causes of traumatic HO (tHO). Neutrophils play a pivotal role in activating BMPs and TGF-ß signals, thus promoting the differentiation of mesenchymal stem cells and progenitor cells into osteoblasts or osteoclasts to facilitate HO. Furthermore, NETs are specifically localized at the site of HO, thereby accelerating the formation of HO. Additionally, the overactivation of neutrophils contributes to the disruption of immune homeostasis to trigger HO. An understanding of the diverse roles of neutrophils will not only provide more information on the pathogenesis of skeletal muscle injury for repair and HO but also provides a foundation for the development of more efficacious treatment modalities for HO.

Exploring the Link Between Autophagy-Lysosomal Dysfunction and Early Heterotopic Ossification in Tendons.

Heterotopic ossification (HO), the pathological formation of bone within soft tissues such as tendon and muscle, is a notable complication resulting from severe injury. While soft tissue injury is necessary for HO development, the specific molecular pathology responsible for trauma-induced HO remains a mystery. The previous study detected abnormal autophagy function in the early stages of tendon HO. Nevertheless, it remains to be determined whether autophagy governs the process of HO generation. Here, trauma-induced tendon HO model is used to investigate the relationship between autophagy and tendon calcification. In the early stages of tenotomy, it is observed that autophagic flux is significantly impaired and that blocking autophagic flux promoted the development of more rampant calcification. Moreover, Gt(ROSA)26sor transgenic mouse model experiments disclosed lysosomal acid dysfunction as chief reason behind impaired autophagic flux. Stimulating V-ATPase activity reinstated both lysosomal acid functioning and autophagic flux, thereby reversing tendon HO. This present study demonstrates that autophagy-lysosomal dysfunction triggers HO in the stages of tendon injury, with potential therapeutic targeting implications for HO.

An ALK2 inhibitor, BLU-782, prevents heterotopic ossification in a mouse model of fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease driven by gain-of-function variants in activin receptor-like kinase 2 (ALK2), the most common variant being ALK2R206H. In FOP, ALK2 variants display increased and dysregulated signaling through the bone morphogenetic protein (BMP) pathway resulting in progressive and permanent replacement of skeletal muscle and connective tissues with heterotopic bone, ultimately leading to severe debilitation and premature death. Here, we describe the discovery of BLU-782 (IPN60130), a small-molecule ALK2R206H inhibitor developed for the treatment of FOP. A small-molecule library was screened in a biochemical ALK2 binding assay to identify potent ALK2 binding compounds. Iterative rounds of structure-guided drug design were used to optimize compounds for ALK2R206H binding, ALK2 selectivity, and other desirable pharmacokinetic properties. BLU-782 preferentially bound to ALK2R206H with high affinity, inhibiting signaling from ALK2R206H and other rare FOP variants in cells in vitro without affecting signaling of closely related homologs ALK1, ALK3, and ALK6. In vivo efficacy of BLU-782 was demonstrated using a conditional knock-in ALK2R206H mouse model, where prophylactic oral dosing reduced edema and prevented cartilage and heterotopic ossification (HO) in both muscle and bone injury models. BLU-782 treatment preserved the normal muscle-healing response in ALK2R206H mice. Delayed dosing revealed a short 2-day window after injury when BLU-782 treatment prevented HO in ALK2R206H mice, but dosing delays of 4 days or longer abrogated HO prevention. Together, these data suggest that BLU-782 may be a candidate for prevention of HO in FOP.