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.

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.

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.

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.

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.

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.

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.

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.

Activation of BMP4/SMAD pathway by HIF-1α in hypoxic environment promotes osteogenic differentiation of BMSCs and leads to ectopic bone formation.

OBJECTIVE: Heterotopic ossification (HO), also known as ossifying myositis, is a condition that produces abnormal bone and cartilage tissue in the soft tissues. Hypoxia inducible factor lα (HIF-lα) regulates the expression of various genes, which is closely related to the promotion of bone formation, and Drosophila mothers against decapentaplegic protein (SMAD) mediates the signal transduction in the Bone morphogenetic protein (BMP) signaling pathway, which affects the function of osteoblasts and osteoclasts, and thus plays a key role in the regulation of bone remodeling. We aimed to investigate the mechanism by which HIF-1α induces osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in a hypoxic environment. METHODS: A cellular hypoxia model was constructed to verify the expression of HIF-1α, while alizarin red staining was performed to observe the osteogenic differentiation ability of bone marrow mesenchymal stem cells (BMSCs). Alizarin red staining was used to analyze the late mineralization ability of the cells. Western blot analysis was performed to analyze the expression levels of osteogenesis-related factors OCN, OPN proteins as well as the pathway proteins BMP4, p-Smad1/5/8, and Smad1. We also constructed a rat model of ectopic bone formation, observed ectopic ossification by X-ray, and verified the success of the rat model by ELISA of HIF-1α. HE staining was used to observe the matrix and trabecular structure of bone, and Masson staining was used to observe the collagen and trabecular structure of bone. Immunohistochemistry analyzed the expression of OCN and OPN in ectopic bone tissues, and WB analyzed the expression of pathway proteins BMP4, p-Smad1/5/8 and Smad1 in ectopic bone tissues to verify the signaling pathway of ectopic bone formation. RESULTS: Our results indicate that hypoxic environment upregulates HIF-1a expression and activates BMP4/SMAD signaling pathway. This led to an increase in ALP content and enhanced expression of the osteogenesis-related factors OCN and OPN, resulting in enhanced osteogenic differentiation of BMSCs. The results of our in vivo experiments showed that rats inoculated with BMSCs overexpressing HIF-1α showed bony structures in tendon tissues, enhanced expression of the bone signaling pathways BMP4 and p-Smad1/5/8, and enhanced expression levels of the osteogenic-related factors OCN and OPN, resulting in the formation of ectopic bone. CONCLUSIONS: These data further suggest a novel mechanistic view that hypoxic bone marrow BMSCs activate the BMP4/SMAD pathway by up-regulating the expression level of HIF-1α, thereby promoting the secretion of osteogenic factors leading to ectopic bone formation.

Malvidin attenuates trauma-induced heterotopic ossification of tendon in rats by targeting Rheb for degradation via the ubiquitin-proteasome pathway.

The pathogenesis of trauma-induced heterotopic ossification (HO) in the tendon remains unclear, posing a challenging hurdle in treatment. Recognizing inflammation as the root cause of HO, anti-inflammatory agents hold promise for its management. Malvidin (MA), possessing anti-inflammatory properties, emerges as a potential agent to impede HO progression. This study aimed to investigate the effect of MA in treating trauma-induced HO and unravel its underlying mechanisms. Herein, the effectiveness of MA in preventing HO formation was assessed through local injection in a rat model. The potential mechanism underlying MA's treatment was investigated in the tendon-resident progenitor cells of tendon-derived stem cells (TDSCs), exploring its pathway in HO formation. The findings demonstrated that MA effectively hindered the osteogenic differentiation of TDSCs by inhibiting the mTORC1 signalling pathway, consequently impeding the progression of trauma-induced HO of Achilles tendon in rats. Specifically, MA facilitated the degradation of Rheb through the K48-linked ubiquitination-proteasome pathway by modulating USP4 and intercepted the interaction between Rheb and the mTORC1 complex, thus inhibiting the mTORC1 signalling pathway. Hence, MA presents itself as a promising candidate for treating trauma-induced HO in the Achilles tendon, acting by targeting Rheb for degradation through the ubiquitin-proteasome pathway.

Vegfc-expressing cells form heterotopic bone after musculoskeletal injury.

Heterotopic ossification (HO) is a challenging condition that occurs after musculoskeletal injury and is characterized by the formation of bone in non-skeletal tissues. While the effect of HO on blood vessels is well established, little is known about its impact on lymphatic vessels. Here, we use a mouse model of traumatic HO to investigate the relationship between HO and lymphatic vessels. We show that injury triggers lymphangiogenesis at the injury site, which is associated with elevated vascular endothelial growth factor C (VEGF-C) levels. Through single-cell transcriptomic analyses, we identify mesenchymal progenitor cells and tenocytes as sources of Vegfc. We demonstrate by lineage tracing that Vegfc-expressing cells undergo osteochondral differentiation and contribute to the formation of HO. Last, we show that Vegfc haploinsufficiency results in a nearly 50% reduction in lymphangiogenesis and HO formation. These findings shed light on the complex mechanisms underlying HO formation and its impact on lymphatic vessels.

Cell Senescence in Heterotopic Ossification.

The formation of bone outside the normal skeleton, or heterotopic ossification (HO), occurs through genetic and acquired mechanisms. Fibrodysplasia ossificans progressiva (FOP), the most devastating genetic condition of HO, is due to mutations in the ACVR1/ALK2 gene and is relentlessly progressive. Acquired HO is mostly precipitated by injury or orthopedic surgical procedures but can also be associated with certain conditions related to aging. Cellular senescence is a hallmark of aging and thought to be a tumor-suppressive mechanism with characteristic features such as irreversible growth arrest, apoptosis resistance, and an inflammatory senescence-associated secretory phenotype (SASP). Here, we review possible roles for cellular senescence in HO and how targeting senescent cells may provide new therapeutic approaches to both FOP and acquired forms of HO.

KLF5 promotes the ossification process of ligamentum flavum by transcriptionally activating CX43.

BACKGROUND: Ossification of ligamentum flavum (OLF) is a prevalent degenerative spinal disease, typically causing severe neurological dysfunction. Kruppel-like factor 5 (KLF5) plays an essential role in the regulation of skeletal development. However, the mechanism KLF5 plays in OLF remains unclear, necessitating further investigative studies. METHODS: qRT-PCR, immunofluorescent staining and western blot were used to measure the expression of KLF5. Alkaline Phosphatase (ALP) staining, Alizarin red staining (ARS), and the expression of Runt-related transcription factor 2 (RUNX2), osteopontin (OPN), and osteocalcin (OCN) were used to evaluate the osteogenic differentiation. Luciferase activity assay and ChIP-PCR were performed to investigate the molecular mechanisms. RESULTS: KLF5 was significantly upregulated in OLF fibroblasts in contrast to normal ligamentum flavum (LF) fibroblasts. Silencing KLF5 diminished osteogenic markers and mineralized nodules, while its overexpression had the opposite effect, confirming KLF5's role in promoting ossification. Moreover, KLF5 promotes the ossification of LF by activating the transcription of Connexin 43 (CX43), and overexpressing CX43 could reverse the suppressive impact of KLF5 knockdown on OLF fibroblasts' osteogenesis. CONCLUSION: KLF5 promotes the OLF by transcriptionally activating CX43. This finding contributes significantly to our understanding of OLF and may provide new therapeutic targets.

Roles of Plasminogen Activator Inhibitor-1 in Heterotopic Ossification Induced by Achilles Tenotomy in Thermal Injured Mice.

Heterotopic ossification (HO) is the process by which ectopic bone forms at an extraskeletal site. Inflammatory conditions induce plasminogen activator inhibitor 1 (PAI-1), an inhibitor of fibrinolysis, which regulates osteogenesis. In the present study, we investigated the roles of PAI-1 in the pathophysiology of HO induced by trauma/burn treatment using PAI-1-deficient mice. PAI-1 deficiency significantly promoted HO and increased the number of alkaline phosphatase (ALP)-positive cells in Achilles tendons after trauma/burn treatment. The mRNA levels of inflammation markers were elevated in Achilles tendons of both wild-type and PAI-1-deficient mice after trauma/burn treatment and PAI-1 mRNA levels were elevated in Achilles tendons of wild-type mice. PAI-1 deficiency significantly up-regulated the expression of Runx2, Osterix, and type 1 collagen in Achilles tendons 9 weeks after trauma/burn treatment in mice. In in vitro experiments, PAI-1 deficiency significantly increased ALP activity and mineralization in mouse osteoblasts. Moreover, PAI-1 deficiency significantly increased ALP activity and up-regulated osteocalcin expression during osteoblastic differentiation from mouse adipose-tissue-derived stem cells, but suppressed the chondrogenic differentiation of these cells. In conclusion, the present study showed that PAI-1 deficiency promoted HO in Achilles tendons after trauma/burn treatment partly by enhancing osteoblast differentiation and ALP activity in mice. Endogenous PAI-1 may play protective roles against HO after injury and inflammation.

Matrix metalloproteinase-9 deficiency confers resilience in fibrodysplasia ossificans progressiva in a man and mice.

Single case studies of extraordinary disease resilience may provide therapeutic insight into conditions for which no definitive treatments exist. An otherwise healthy 35-year-old man (patient-R) with the canonical pathogenic ACVR1R206H variant and the classic congenital great toe malformation of fibrodysplasia ossificans progressiva (FOP) had extreme paucity of post-natal heterotopic ossification (HO) and nearly normal mobility. We hypothesized that patient-R lacked a sufficient post-natal inflammatory trigger for HO. A plasma biomarker survey revealed a reduction in total matrix metalloproteinase-9 (MMP-9) compared to healthy controls and individuals with quiescent FOP. Whole exome sequencing identified compound heterozygous variants in MMP-9 (c.59C > T, p.A20V and c.493G > A, p.D165N). Structural analysis of the D165N variant predicted both decreased MMP-9 secretion and activity that were confirmed by enzyme-linked immunosorbent assay and gelatin zymography. Further, human proinflammatory M1-like macrophages expressing either MMP-9 variant produced significantly less Activin A, an obligate ligand for HO in FOP, compared to wildtype controls. Importantly, MMP-9 inhibition by genetic, biologic, or pharmacologic means in multiple FOP mouse models abrogated trauma-induced HO, sequestered Activin A in the extracellular matrix (ECM), and induced regeneration of injured skeletal muscle. Our data suggest that MMP-9 is a druggable node linking inflammation to HO, orchestrates an existential role in the pathogenesis of FOP, and illustrates that a single patient's clinical phenotype can reveal critical molecular mechanisms of disease that unveil novel treatment strategies.

A healthy 35-year-old man (patient-R) with the classic fibrodysplasia ossificans progressiva (FOP) mutation and the congenital great toe malformation of FOP had extreme lack of heterotopic ossification (HO) and nearly normal mobility. We hypothesized that patient-R lacked a sufficient inflammatory trigger for HO. Blood tests revealed a reduction in the level of an inflammatory protein called matrix metalloproteinase-9 (MMP-9) compared to other individuals with FOP as well as healthy controls. DNA analysis in patient-R identified mutations in MMP-9, one of which predicted decreased activity of MMP-9 which was confirmed by further testing. Inflammatory cells (macrophages) expressing the MMP-9 mutations identified in patient-R produced significantly less Activin A, an obligate stimulus for HO in FOP. In order to determine if MMP-9 deficiency was a cause of HO prevention in FOP, we inhibited MMP-9 activity by genetic, biologic, or pharmacologic means in FOP mouse models and showed that MMP-9 inhibition prevented or dramatically decreased trauma-induced HO in FOP, locked-up Activin A in the extracellular matrix, and induced regeneration of injured skeletal muscle. Our data show that MMP-9 links inflammation to HO and illustrate that one patient's clinical picture can reveal critical molecular mechanisms of disease that unveil new treatment strategies.

Hedgehog Signaling Controls Chondrogenesis and Ectopic Bone Formation via the Yap-Ihh Axis.

Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disorder characterized by abnormal bone formation due to ACVR1 gene mutations. The identification of the molecular mechanisms underlying the ectopic bone formation and expansion in FOP is critical for the effective treatment or prevention of HO. Here we find that Hh signaling activation is required for the aberrant ectopic bone formation in FOP. We show that the expression of Indian hedgehog (Ihh), a Hh ligand, as well as downstream Hh signaling, was increased in ectopic bone lesions in Acvr1R206H; ScxCre mice. Pharmacological treatment with an Ihh-neutralizing monoclonal antibody dramatically reduced chondrogenesis and ectopic bone formation. Moreover, we find that the activation of Yap in the FOP mouse model and the genetic deletion of Yap halted ectopic bone formation and decreased Ihh expression. Our mechanistic studies showed that Yap and Smad1 directly bind to the Ihh promoter and coordinate to induce chondrogenesis by promoting Ihh expression. Therefore, the Yap activation in FOP lesions promoted ectopic bone formation and expansion in both cell-autonomous and non-cell-autonomous manners. These results uncovered the crucial role of the Yap-Ihh axis in FOP pathogenesis, suggesting the inhibition of Ihh or Yap as a potential therapeutic strategy to prevent and reduce HO.

The HIF-1α/PLOD2 axis integrates extracellular matrix organization and cell metabolism leading to aberrant musculoskeletal repair.

While hypoxic signaling has been shown to play a role in many cellular processes, its role in metabolism-linked extracellular matrix (ECM) organization and downstream processes of cell fate after musculoskeletal injury remains to be determined. Heterotopic ossification (HO) is a debilitating condition where abnormal bone formation occurs within extra-skeletal tissues. Hypoxia and hypoxia-inducible factor 1α (HIF-1α) activation have been shown to promote HO. However, the underlying molecular mechanisms by which the HIF-1α pathway in mesenchymal progenitor cells (MPCs) contributes to pathologic bone formation remain to be elucidated. Here, we used a proven mouse injury-induced HO model to investigate the role of HIF-1α on aberrant cell fate. Using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics analyses of the HO site, we found that collagen ECM organization is the most highly up-regulated biological process in MPCs. Zeugopod mesenchymal cell-specific deletion of Hif1α (Hoxa11-CreERT2; Hif1afl/fl) significantly mitigated HO in vivo. ScRNA-seq analysis of these Hoxa11-CreERT2; Hif1afl/fl mice identified the PLOD2/LOX pathway for collagen cross-linking as downstream of the HIF-1α regulation of HO. Importantly, our scRNA-seq data and mechanistic studies further uncovered that glucose metabolism in MPCs is most highly impacted by HIF-1α deletion. From a translational aspect, a pan-LOX inhibitor significantly decreased HO. A newly screened compound revealed that the inhibition of PLOD2 activity in MPCs significantly decreased osteogenic differentiation and glycolytic metabolism. This suggests that the HIF-1α/PLOD2/LOX axis linked to metabolism regulates HO-forming MPC fate. These results suggest that the HIF-1α/PLOD2/LOX pathway represents a promising strategy to mitigate HO formation.

Intersections of Fibrodysplasia Ossificans Progressiva and Traumatic Heterotopic Ossification.

Heterotopic ossification (HO) is a debilitating pathology where ectopic bone develops in areas of soft tissue. HO can develop as a consequence of traumatic insult or as a result of dysregulated osteogenic signaling, as in the case of the orphan disease fibrodysplasia ossificans progressiva (FOP). Traumatic HO (tHO) formation is mediated by the complex interplay of signaling between progenitor, inflammatory, and nerve cells, among others, making it a challenging process to understand. Research into the pathogenesis of genetically mediated HO (gHO) in FOP has established a pathway involving uninhibited activin-like kinase 2 receptor (ALK2) signaling that leads to downstream osteogenesis. Current methods of diagnosis and treatment lag behind pre-mature HO detection and progressive HO accumulation, resulting in irreversible decreases in range of motion and chronic pain for patients. As such, it is necessary to draw on advancements made in the study of tHO and gHO to better diagnose, comprehend, prevent, and treat both.