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.

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.

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.

Immunologic Aspects in Fibrodysplasia Ossificans Progressiva.

BACKGROUND: Inflammation is a major driver of heterotopic ossification (HO), a condition of abnormal bone growth in a site that is not normally mineralized. PURPOSE OF REVIEW: This review will examine recent findings on the roles of inflammation and the immune system in fibrodysplasia ossificans progressiva (FOP). FOP is a genetic condition of aggressive and progressive HO formation. We also examine how inflammation may be a valuable target for the treatment of HO. Rationale/Recent findings: Multiple lines of evidence indicate a key role for the immune system in driving FOP pathogenesis. Critical cell types include macrophages, mast cells, and adaptive immune cells, working through hypoxia signaling pathways, stem cell differentiation signaling pathways, vascular regulatory pathways, and inflammatory cytokines. In addition, recent clinical reports suggest a potential role for immune modulators in the management of FOP. FUTURE PERSPECTIVES: The central role of inflammatory mediators in HO suggests that the immune system may be a common target for blocking HO in both FOP and non-genetic forms of HO. Future research focusing on the identification of novel inflammatory targets will help support the testing of potential therapies for FOP and other related conditions.

iMSC-mediated delivery of ACVR2B-Fc fusion protein reduces heterotopic ossification in a mouse model of fibrodysplasia ossificans progressiva.

BACKGROUND: Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease caused by a gain-of-function mutation in ACVR1, which is a bone morphogenetic protein (BMP) type I receptor. Moreover, it causes progressive heterotopic ossification (HO) in connective tissues. Using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) and mouse models, we elucidated the underlying mechanisms of FOP pathogenesis and identified a candidate drug for FOP. METHODS: In the current study, healthy mesenchymal stem/stromal cells derived from iPSCs (iMSCs) expressing ACVR2B-Fc (iMSCACVR2B-Fc), which is a neutralizing receptobody, were constructed. Furthermore, patient-derived iMSCs and FOP mouse model (ACVR1R206H, female) were used to confirm the inhibitory function of ACVR2B-Fc fusion protein secreted by iMSCACVR2B-Fc on BMP signaling pathways and HO development, respectively. RESULTS: We found that secreted ACVR2B-Fc attenuated BMP signaling initiated by Activin-A and BMP-9 in both iMSCs and FOP-iMSCs in vitro. Transplantation of ACVR2B-Fc-expressing iMSCs reduced primary HO in a transgenic mouse model of FOP. Notably, a local injection of ACVR2B-Fc-expressing iMSCs and not an intraperitoneal injection improved the treadmill performance, suggesting compound effects of ACVR2B-Fc and iMSCs. CONCLUSIONS: These results offer a new perspective for treating FOP through stem cell therapy.

Medulloblastoma in a child with osteoma cutis - a rare association due to loss of GNAS expression.

OBJECTIVES: Inactivating GNAS mutations result in varied phenotypes depending on parental origin. Maternally inherited mutations typically lead to hormone resistance and Albright's hereditary osteodystrophy (AHO), characterised by short stature, round facies, brachydactyly and subcutaneous ossifications. Paternal inheritance presents with features of AHO or ectopic ossification without hormone resistance. This report describes the case of a child with osteoma cutis and medulloblastoma. The objective of this report is to highlight the emerging association between inactivating germline GNAS mutations and medulloblastoma, aiming to shed light on its implications for tumor biology and promote future development of targeted surveillance strategies to improve outcomes in paediatric patients with these mutations. CASE PRESENTATION: A 12-month-old boy presented with multiple plaque-like skin lesions. Biopsy confirmed osteoma cutis, prompting genetic testing which confirmed a heterozygous inactivating GNAS mutation. At 2.5 years of age, he developed neurological symptoms and was diagnosed with a desmoplastic nodular medulloblastoma, SHH molecular group, confirmed by MRI and histology. Further analysis indicated a biallelic loss of GNAS in the tumor. CONCLUSIONS: This case provides important insights into the role of GNAS as a tumor suppressor and the emerging association between inactivating GNAS variants and the development of medulloblastoma. The case underscores the importance of careful neurological assessment and ongoing vigilance in children with known inactivating GNAS variants or associated phenotypes. Further work to establish genotype-phenotype correlations is needed to inform optimal management of these patients.

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.

The HIF-1α and mTOR Pathways Amplify Heterotopic Ossification.

Fibrodysplasia ossificans progressiva (FOP; MIM# 135100) is an ultra-rare congenital disorder caused by gain-of-function point mutations in the Activin receptor A type I (ACVR1, also known as ALK2) gene. FOP is characterized by episodic heterotopic ossification (HO) in skeletal muscles, tendons, ligaments, or other soft tissues that progressively causes irreversible loss of mobility. FOP mutations cause mild ligand-independent constitutive activation as well as ligand-dependent bone morphogenetic protein (BMP) pathway hypersensitivity of mutant ACVR1. BMP signaling is also a key pathway for mediating acquired HO. However, HO is a highly complex biological process involving multiple interacting signaling pathways. Among them, the hypoxia-inducible factor (HIF) and mechanistic target of rapamycin (mTOR) pathways are intimately involved in both genetic and acquired HO formation. HIF-1α inhibition or mTOR inhibition reduces HO formation in mouse models of FOP or acquired HO in part by de-amplifying the BMP pathway signaling. Here, we review the recent progress on the mechanisms of the HIF-1α and mTOR pathways in the amplification of HO lesions and discuss the future directions and strategies to translate the targeting of HIF-1α and the mTOR pathways into clinical interventions for FOP and other forms of HO.

Sex as a Critical Variable in Basic and Pre-Clinical Studies of Fibrodysplasia Ossificans Progressiva.

Heterotopic ossification (HO) is most dramatically manifested in the rare and severely debilitating disease, fibrodysplasia ossificans progressiva (FOP), in which heterotopic bone progressively accumulates in skeletal muscles and associated soft tissues. The great majority of FOP cases are caused by a single amino acid substitution in the type 1 bone morphogenetic protein (BMP) receptor ACVR1, a mutation that imparts responsiveness to activin A. Although it is well-established that biological sex is a critical variable in a range of physiological and disease processes, the impact of sex on HO in animal models of FOP has not been explored. We show that female FOP mice exhibit both significantly greater and more variable HO responses after muscle injury. Additionally, the incidence of spontaneous HO was significantly greater in female mice. This sex dimorphism is not dependent on gonadally derived sex hormones, and reciprocal cell transplantations indicate that apparent differences in osteogenic activity are intrinsic to the sex of the transplanted cells. By circumventing the absolute requirement for activin A using an agonist of mutant ACVR1, we show that the female-specific response to muscle injury or BMP2 implantation is dependent on activin A. These data identify sex as a critical variable in basic and pre-clinical studies of FOP.

Oxidative phosphorylation is a pivotal therapeutic target of fibrodysplasia ossificans progressiva.

Heterotopic ossification (HO) is a non-physiological bone formation where soft tissue progenitor cells differentiate into chondrogenic cells. In fibrodysplasia ossificans progressiva (FOP), a rare genetic disease characterized by progressive and systemic HO, the Activin A/mutated ACVR1/mTORC1 cascade induces HO in progenitors in muscle tissues. The relevant biological processes aberrantly regulated by activated mTORC1 remain unclear, however. RNA-sequencing analyses revealed the enrichment of genes involved in oxidative phosphorylation (OXPHOS) during Activin A-induced chondrogenesis of mesenchymal stem cells derived from FOP patient-specific induced pluripotent stem cells. Functional analyses showed a metabolic transition from glycolysis to OXPHOS during chondrogenesis, along with increased mitochondrial biogenesis. mTORC1 inhibition by rapamycin suppressed OXPHOS, whereas OXPHOS inhibitor IACS-010759 inhibited cartilage matrix formation in vitro, indicating that OXPHOS is principally involved in mTORC1-induced chondrogenesis. Furthermore, IACS-010759 inhibited the muscle injury-induced enrichment of fibro/adipogenic progenitor genes and HO in transgenic mice carrying the mutated human ACVR1. These data indicated that OXPHOS is a critical downstream mediator of mTORC1 signaling in chondrogenesis and therefore is a potential FOP therapeutic target.

RAF1-rearranged Spindle Cell Mesenchymal Tumor With Calcification and Heterotopic Ossification: A Case Report and Review of Literature.

We report an exceptional case of a spindle cell mesenchymal tumor with S100 and CD34 co-reactivity, which harbored a SLMAP::RAF1 fusion. To the best of our knowledge, this is the second case of a spindle cell mesenchymal tumor with S100 and CD34 co-reactivity with this specific fusion. Remarkable is the presence of calcification and heterotopic ossification in the center of our lesion, a feature that, to our knowledge, has not been described yet in RAF1-rearranged spindle cell mesenchymal tumors.

A fibrodysplasia ossificans progressiva patient with a rare missense mutation in ACVR1 detected on 18F-FDG PET/CT.

Fibrodysplasia ossificans progressiva (FOP) is an exceedingly rare human genetic disorder characterized by the progressive and incapacitating formation of ectopic bone outside the skeleton. We report a case of FOP patient with mutations within the ACVR1 gene (c.982G>A; p.G328R). 18F-FDG positron emission tomography/computed tomography (PET/CT) was carried out for disease assessment. Previous studies have shown increased FDG uptake in regions of heterotopic ossification (HO) in FOP. However, in our study, the PET/CT features demonstrate that active ossificans exhibit increased 18F-FDG uptake, whereas end-stage ossifications do not. Collectively, 18F-FDG PET/CT emerges as a prospective approach to evaluate medication efficacy in the early stages, directing early intervention and pharmacological management of FOP before ossifications formation.

Navigating the Complex Landscape of Fibrodysplasia Ossificans Progressiva: From Current Paradigms to Therapeutic Frontiers.

Fibrodysplasia ossificans progressiva (FOP) is an enigmatic, ultra-rare genetic disorder characterized by progressive heterotopic ossification, wherein soft connective tissues undergo pathological transformation into bone structures. This incapacitating process severely limits patient mobility and poses formidable challenges for therapeutic intervention. Predominantly caused by missense mutations in the ACVR1 gene, this disorder has hitherto defied comprehensive mechanistic understanding and effective treatment paradigms. This write-up offers a comprehensive overview of the contemporary understanding of FOP's complex pathobiology, underscored by advances in molecular genetics and proteomic studies. We delve into targeted therapy, spanning genetic therapeutics, enzymatic and transcriptional modulation, stem cell therapies, and innovative immunotherapies. We also highlight the intricate complexities surrounding clinical trial design for ultra-rare disorders like FOP, addressing fundamental statistical limitations, ethical conundrums, and methodological advancements essential for the success of interventional studies. We advocate for the adoption of a multi-disciplinary approach that converges bench-to-bedside research, clinical expertise, and ethical considerations to tackle the challenges of ultra-rare diseases like FOP and comparable ultra-rare diseases. In essence, this manuscript serves a dual purpose: as a definitive scientific resource for ongoing and future FOP research and a call to action for innovative solutions to address methodological and ethical challenges that impede progress in the broader field of medical research into ultra-rare conditions.

GNAS locus: bone related diseases and mouse models.

GNASis a complex locus characterized by multiple transcripts and an imprinting effect. It orchestrates a variety of physiological processes via numerous signaling pathways. Human diseases associated with the GNAS gene encompass fibrous dysplasia (FD), Albright's Hereditary Osteodystrophy (AHO), parathyroid hormone(PTH) resistance, and Progressive Osseous Heteroplasia (POH), among others. To facilitate the study of the GNAS locus and its associated diseases, researchers have developed a range of mouse models. In this review, we will systematically explore the GNAS locus, its related signaling pathways, the bone diseases associated with it, and the mouse models pertinent to these bone diseases.

PTGR1 is involved in cell proliferation in thoracic ossification of the ligamentum flavum.

Thoracic ossification of the ligamentum flavum (TOLF) is a heterotopic ossification of spinal ligaments, leading to serious myelopathy. TOLF underlying mechanisms are not well understood. Our iTRAQ analysis have identified ten inflammatory factors related to TOLF, including l. We found that PTGR1 expressions increased in TOLF by RT-PCR and western blot in this study. Both cell proliferation and differentiation are important for the process of bone formation. In our previous study, we demonstrated that TOLF primary cells grew faster than control cells. It was reported that knockdown of PTGR1 inhibited cell proliferation. We hypothesize that PTGR1 may participate in cell proliferation in TOLF. To test this hypothesis, TOLF primary cells were treated for 24h with PTGR1. We observed that PTGR1 increased cell proliferation. The effect of PTGR1 on cell proliferation related genes was examined in TOLF primary cells. Our results showed that PTGR1 was able to activate expressions of c-Myc and CyclinD1. Moreover, blocking JNK pathway by selective JNK inhibitor SP600125 eliminated the positive effect of PTGR1 on c-Myc expression, indicating that PTGR1 activated the expression of c-Myc via JNK pathway. Our new findings suggest that PTGR1 is involved in cell proliferation of TOLF.

Spinal cord injury dysregulates fibro-adipogenic progenitors miRNAs signaling to promote neurogenic heterotopic ossifications.

Neurogenic heterotopic ossifications are intramuscular bone formations developing following central nervous system injury. The pathophysiology is poorly understood and current treatments for this debilitating condition remain unsatisfying. Here we explored the role of miRNAs in a clinically relevant mouse model that combines muscle and spinal cord injury, and in patients' cells. We found an osteo-suppressive miRNAs response in injured muscle that was hindered when the spinal cord injury was associated. In isolated fibro-adipogenic progenitors from damaged muscle (cells at the origin of ossification), spinal cord injury induced a downregulation of osteo-suppressive miRNAs while osteogenic markers were overexpressed. The overexpression of selected miRNAs in patient's fibro-adipogenic progenitors inhibited mineralization and osteo-chondrogenic markers in vitro. Altogether, we highlighted an osteo-suppressive mechanism involving multiple miRNAs in response to muscle injury that prevents osteogenic commitment which is ablated by the neurologic lesion in heterotopic ossification pathogenesis. This provides new research hypotheses for preventive treatments.

[Recommendations for the healthcare of patients with FOP]. / Empfehlungen zur Versorgung von Patient:innen mit FOP.

BACKGROUND: Fibrodysplasia ossificans progressiva (FOP) is a very rare, severe genetic disorder triggered by a gain-of-function mutation in the ACVR1 gene that codes for the type I bone morphogenetic protein (BMP) receptor ACVR1 (activin A receptor-type 1), also known as ALK2 (activin receptor-like kinase-2). It leads to the onset and progression of heterotopic ossification (HO) in soft and connective tissue. HO is often preceded by episodes of soft tissue swelling or flare-ups. Flare-ups, characteristic of FOP, may be induced by trauma, infection, vaccination, or other medications, as well as surgical procedures or may occur spontaneously. As patients age, they develop severe mobility limitations due to progressive HO formation, including immobility, causing a shortened life expectancy. FOP's first characteristic clinical sign is the congenital malformation of one or both big toes with valgus axis deviation, which is present in almost all patients. To confirm the diagnosis, molecular genetic analysis of the ACVR1 gene is possible. AIM OF THE RECOMMENDATIONS: This white paper aims to provide an overview of the necessary prerequisites and conditions for the care of patients with FOP and positively contribute to patients with FOP by improving the overall availability of knowledge. To achieve this, relevant aspects of the care of the very rare disease FOP are presented, from the initial diagnosis to the care in regular care based on the authors' knowledge (German FOP network) and the international FOP Treatment Guidelines. The recommendations presented here are addressed to all actors and decision-makers in the health care system and are also intended to inform patients and the public.

Coding and noncoding RNA profile of human heterotopic ossifications - Risk factors and biomarkers.

Heterotopic ossification (HO) means the formation of bone in muscles and soft tissues, such as ligaments or tendons. HO could have a genetic history or develop after a traumatic event, as a result of muscle injury, fractures, burns, surgery, or neurological disorders. Many lines of evidence suggest that the formation of HO is related to the pathological differentiation of stem or progenitor cells present within soft tissues or mobilized from the bone marrow. The cells responsible for the initiation and progression of HO are generally called HO precursor cells. The exact mechanisms behind the development of HO are not fully understood. However, several factors have been identified as potential contributors. For example, local tissue injury and inflammation disturb soft tissue homeostasis. Inflammatory cells release growth factors and cytokines that promote osteogenic or chondrogenic differentiation of HO precursor cells. The bone morphogenetic protein (BMP) is one of the main factors involved in the development of HO. In this study, next-generation sequencing (NGS) and RT-qPCR were performed to analyze the differences in mRNA, miRNA, and lncRNA expression profiles between muscles, control bone samples, and HO samples coming from patients who underwent total hip replacement (THR). As a result, crucial changes in the level of gene expression between HO and healthy tissues were identified. The bioinformatic analysis allowed to describe the processes most severely impacted, as well as genes which level differed the most significantly between HO and control samples. Our analysis showed that the level of transcripts involved in leukocyte migration, differentiation, and activation, as well as markers of chronic inflammatory diseases, that is, miR-148, increased in HO, as compared to muscle. Furthermore, the levels of miR-195 and miR-143, which are involved in angiogenesis, were up-regulated in HO, as compared to bone. Thus, we suggested that inflammation and angiogenesis play an important role in HO formation. Importantly, we noticed that HO is characterized by a higher level of TLR3 expression, compared to muscle and bone. Thus, we suggest that infection may also be a risk factor in HO development. Furthermore, an increased level of transcripts coding proteins involved in osteogenesis and signaling pathways, such as ALPL, SP7, BGLAP, BMP8A, BMP8B, SMPD3 was noticed in HO, as compared to muscles. Interestingly, miR-99b, miR-146, miR-204, and LINC00320 were up-regulated in HO, comparing to muscles and bone. Therefore, we suggested that these molecules could be important biomarkers of HO formation and a potential target for therapies.

The serum levels of activin A and bone morphogenetic protein-4 and -6 in patients with fibrodysplasia ossificans progressiva.

BACKGROUND: Fibrodysplasia ossificans progressiva (FOP) is an ultrarare and disabling genetic disorder of connective tissue characterized by congenital malformation of the great toes, and progressive heterotopic ossification (HO) in soft connective tissues. A gain-of-function mutation of activin A receptor type I (ACVR1) enables ACVR1 to recognize activin A as an agonist with bone morphogenetic protein (BMP) signalling that leads to HO. Previous studies confirmed that activin A stimulates BMP signalling in vitro and drives HO in mouse models of FOP. However, the roles for BMP4 and BMP6 in FOP are supported only by correlative evidence in vitro. Thus, it remains unclear whether the circulating levels of activin A, BMP4 and BMP6 correlate with flare-ups in FOP patients. Hence, we investigated the protein levels of activin A, BMP4 and BMP6 in the serum of FOP patients. RESULTS: We recruited 16 untreated FOP patients and 16 age- and sex- matched healthy control subjects in this study. The 16 FOP patients were retrospectively divided into the flare-up group (n = 8) and remission group (n = 8) depending on whether they had flare-ups or worsening of any joint movement in the last 6 months. The serum activin A, BMP4 and BMP6 levels were detected by enzyme-linked immunosorbent assay. The serum activin A, BMP4 and BMP6 levels were slightly higher in FOP patients (median: 434.05 pg/mL, 459.48 pg/mL and 67.84 pg/mL) versus healthy control subjects (median: 364.14 pg/mL, 450.39 pg/mL and 55.36 pg/mL). However, there were no statistically significant differences between the two groups (p > 0.05 for all items), nor were there significant differences between the flare-up and remission groups of FOP (p > 0.05 for all items). Univariate and multivariate logistic regression analyses showed that age, sex, and serum activin A, BMP4 and BMP6 levels were not related to flare-up in FOP patients. CONCLUSIONS: There were no significant differences in the serum levels of activin A, BMP4 and BMP6 in FOP patients compared with healthy control subjects. Serum activin A, BMP4 and BMP6 proteins might not be the stimulators for FOP flare-up, and may not be biomarkers for FOP diagnosis.