Saracatinib is an efficacious clinical candidate for fibrodysplasia ossificans progressiva.

Currently, no effective therapies exist for fibrodysplasia ossificans progressiva (FOP), a rare congenital syndrome in which heterotopic bone is formed in soft tissues owing to dysregulated activity of the bone morphogenetic protein (BMP) receptor kinase ALK2 (also known as ACVR1). From a screen of known biologically active compounds, we identified saracatinib as a potent ALK2 kinase inhibitor. In enzymatic and cell-based assays, saracatinib preferentially inhibited ALK2, compared with other receptors of the BMP/TGF-ß signaling pathway, and induced dorsalization in zebrafish embryos consistent with BMP antagonism. We further tested the efficacy of saracatinib using an inducible ACVR1Q207D-transgenic mouse line, which provides a model of heterotopic ossification (HO), as well as an inducible ACVR1R206H-knockin mouse, which serves as a genetically and physiologically faithful FOP model. In both models, saracatinib was well tolerated and potently inhibited the development of HO, even when administered transiently following soft tissue injury. Together, these data suggest that saracatinib is an efficacious clinical candidate for repositioning in FOP treatment, offering an accelerated path to clinical proof-of-efficacy studies and potentially significant benefits to individuals with this devastating condition.

Combined use of leptin and mechanical stress has osteogenic effects on ossification of the posterior longitudinal ligament.

PURPOSE: To evaluate the effects of leptin/leptin receptor (LepR) combined with mechanical stress on the development of ossification of the posterior longitudinal ligament (OPLL), which is a disease characterized by ectopic bone formation of the posterior longitudinal ligament (PLL) and can lead to radiculopathy and myelopathy. METHODS: Six human samples of the PLL were analyzed for the expression of leptin and LepR by RT-PCR and western blotting. PLL cells were stimulated with leptin and mechanical stress delivered via a Flexcell tension system, and osteogenic differentiation was evaluated by RT-PCR and western blotting analysis of osteogenic marker expression as well as by alkaline phosphatase (ALP) staining and alizarin red S staining. Activation of mitogen-activated protein kinase (MAPK), Janus kinase (JAK) 2-signal transducer, activator of transcription (STAT) 3 and phosphatidylinositol 3-kinase (PI3K)-Akt was evaluated by western blotting. RESULTS: Samples from the OPLL group had higher LepR mRNA and protein levels and lower leptin levels than those from healthy controls. Exposure to leptin and Flexcell increased the number of ALP-positive cells and calcium nodules in a dose-dependent manner; this effect was accompanied by upregulation of the osteogenic markers osteocalcin, runt-related transcription factor 2 (RUNX2) and osteopontin. Extracellular signal-regulated kinase, P38 MAPK, JAK2, STAT3, PI3K and Akt signaling, was also activated by the combined effects of leptin and mechanical stress. CONCLUSIONS: Leptin and LepR are differentially expressed in OPLL tissues, and the combined use of leptin/LepR and mechanical stress promotes osteogenic differentiation of PLL cells via MAPK, JAK2-STAT3 and PI3K/Akt signaling. These slides can be retrieved under Electronic Supplementary Material.

Elemene, the essential oil of Curcuma wenyujin, inhibits osteogenic differentiation in ankylosing spondylitis.

OBJECTIVE: To explore the effects of Elemene, the essential oil of Curcuma wenyujin, on Bone morphogenetic protein/drosophila mothers against decapentaplegic proteins (BMP/SMADs) signal pathway in ankylosing spondylitis (AS) fibroblasts. METHODS: Hip joint capsules were obtained from AS patients (n=10) receiving total hip replacement. Healthy hip joint capsules from patients with hip fracture (n=10) receiving surgery were included as a control. Primary fibroblast cell lines were established from these tissue samples. Fibroblasts were incubated with Elemene for 48 hours. The protein expression was detected by Western blot. The mRNA expression was detected by real-time fluorescent quantitative polymerase chain reaction (PCR). RESULTS: The results showed that the expression of proteins including SMAD1, pSMAD1, SMAD4 and Runt-related transcription factor 2 (RUNX2), and mRNA of RUNX2, which were over-expressed in AS fibroblasts were decreased in the AS fibroblasts cultured in medium with Elemene. CONCLUSIONS: Ele could have a hand in anti-osteogenic differentiation of AS fibroblasts by inhibiting the BMP/SMADs signal pathway and subsequently blocking expression of ossification marker genes RUNX2 that initiate the osteogenic differentiation.

Mead acid (20:3n-9) and n-3 polyunsaturated fatty acids are not associated with risk of posterior longitudinal ligament ossification: results of a case-control study.

Ossification of the posterior longitudinal ligament (OPLL) involves the replacement of ligamentous tissue with ectopic bone. Although genetics and heritability appear to be involved in the development of OPLL, its pathogenesis remains to be elucidated. Given previous findings that 5,8,11-eicosatrienoic acid [20:3n-9, Mead acid (MA)] has depressive effects on osteoblastic activity and anti-angiogenic effects, and that n-3 polyunsaturated fatty acids (PUFAs) have a preventive effect on heterotopic ossification, we hypothesized that both fatty acids would be involved in OPLL development. To examine the biological significance of these and other fatty acids in OPLL, we conducted this case-control study involving 106 patients with cervical OPLL and 109 age matched controls. Fatty acid composition was determined from plasma samples by gas chromatography. Associations between fatty acid levels and incident OPLL were evaluated by logistic regression. Contrary to our expectations, we found no significant differences between patients and controls in the levels of MA or n-3 PUFAs (e.g., eicosapentaenoic acid and docosahexaenoic acid). Logistic regression analysis did not reveal any associations with OPLL risk for MA or n-3 PUFAs. In conclusion, no potential role was found for MA or n-3 PUFAs in ectopic bone formation in the spinal canal.

Clinically applicable antianginal agents suppress osteoblastic transformation of myogenic cells and heterotopic ossifications in mice.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by progressive heterotopic ossification. FOP is caused by a gain-of-function mutation in ACVR1 encoding the bone morphogenetic protein type II receptor, ACVR1/ALK2. The mutant receptor causes upregulation of a transcriptional factor, Id1. No therapy is available to prevent the progressive heterotopic ossification in FOP. In an effort to search for clinically applicable drugs for FOP, we screened 1,040 FDA-approved drugs for suppression of the Id1 promoter activated by the mutant ACVR1/ALK2 in C2C12 cells. We found that that two antianginal agents, fendiline hydrochloride and perhexiline maleate, suppressed the Id1 promoter in a dose-dependent manner. The drugs also suppressed the expression of native Id1 mRNA and alkaline phosphatase in a dose-dependent manner. Perhexiline but not fendiline downregulated phosphorylation of Smad 1/5/8 driven by bone morphogenetic protein (BMP)-2. We implanted crude BMPs in muscles of ddY mice and fed them fendiline or perhexiline for 30 days. Mice taking perhexiline showed a 38.0 % reduction in the volume of heterotopic ossification compared to controls, whereas mice taking fendiline showed a slight reduction of heterotopic ossification. Fendiline, perhexiline, and their possible derivatives are potentially applicable to clinical practice to prevent devastating heterotopic ossification in FOP.

Changes in basic metabolic elements associated with the degeneration and ossification of ligamenta flava.

OBJECTIVE: To determine the association between levels of basic metabolic elements and degeneration and ossification of the ligamentum flavum (LF). SUBJECTS: Fourteen consecutive patients with degenerative lumbar stenosis, 11 with ossification of the thoracic ligamenta flava, and 11 control subjects. METHODS: The basic elements of calcium (Ca), phosphorus (P), magnesium (Mg), zinc (Zn), copper (Cu), manganese (Mn), molybdenum (Mo), and fluoride (F) in the specimens were measured using atomic absorption spectrometry, the phosphomolybdic blue method, and a fluoride-selected electrode. RESULTS: Ca content and the ratio of Ca/Mg in the LF specimens increased significantly in the sequence of control, degeneration, and ossification groups. Compared with values for the control group, the Zn, Mn, and Mo contents in the ossification and degeneration groups were significantly lower (P < 0.01); in contrast, Cu content was significantly higher (P < 0.01). As to F, its content in the specimens of the ossification group was much higher than those in the degeneration and control groups (P < 0.01); the F content in the ligamenta flava and sera from patients with fluorosis was also significantly higher than in those from patients without fluorosis (P < 0.01). Compared with the control group, there were no differences in the F content in serum from patients without fluorosis; however, the F content in ligamenta flava specimens from patients without fluorosis was significantly higher (P < 0.01). CONCLUSIONS: There are trends in the contents of basic metabolic elements in the degeneration and ossification of ligamenta flava. These basic metabolic elements may play an important role in this process.

Heterotopic ossification: review of histologic findings and tissue distribution in a 10-year experience.

Heterotopic ossification (HO) within tissues involved by a pathologic process is a well-recognized phenomenon. It is most frequently observed in atherosclerotic plaques, in soft tissue around joints, and in the central nervous system. Less frequently, carcinomas and some benign neoplasms will undergo heterotopic ossification. We performed a retrospective review of our experience with HO over a 10-year period to determine the frequency and tissue site distribution of heterotopic ossification. A computerized review of surgical pathology records of approximately 126,000 reports revealed 85 cases in which heterotopic ossification, ectopic bone or metaplastic bone was specifically mentioned in the surgical pathology diagnosis. Twenty-two cases were neoplasms of non-osseous tissues, and 63 cases were non-neoplastic lesions. Immunohistochemical staining for bone morphogenic proteins (BMP) 1, 4, and 6 was performed. Fourteen cases showed staining for BMP-1, 22 cases showed staining for BMP-4, and five cases showed weak staining for BMP-6. HO is a relatively infrequent finding and is more commonly seen in degenerative and reparative conditions than in neoplasms.

Low dose fibroblast growth factor-2 (FGF-2) enhances bone morphogenetic protein-2 (BMP-2)-induced ectopic bone formation in mice.

To examine how fibroblast growth factor-2 (FGF-2) affects the BMP signaling pathway during bone morphogenetic protein-2 (BMP-2)-induced ectopic bone formation, we implanted type I collagen disks containing constant amounts of BMP-2 (5 micrograms) and varying amounts of FGF-2 onto the back muscles of adult male mice. We then performed histological analyses and histomorphometry, and measured bone mineral density and radiopaque area on the discs 1, 2, and 3 weeks after implantation. We also determined the expression profiles of several genes involved in bone formation and the BMP signaling pathway in the muscle that had been adjacent to the implanted disc and in muscle-derived primary culture cells that had similarly been treated with a constant concentration of BMP-2 and a varying concentration of FGF-2. In the presence of a constant amount of BMP-2, we confirmed that low doses of FGF-2 increased ectopic bone formation in vivo and high doses inhibited bone formation. Northern and/or Western blots of recovered muscle from the in vivo experiment and treated muscle-derived primary culture cells from the in vitro experiment revealed that low doses of FGF-2, but not high doses, increased the expression BMP receptor (BMPR)-1B, phosphorylated Smad1, Noggin, and Osteocalcin. Our results indicate that low-dose FGF-2 may facilitate BMP-2-induced ectopic bone formation by altering the expression of BMPRs on the surface of bone forming progenitor cells. They also indicate that the inhibitory effect of high-dose FGF-2 is not mediated via increased expression of the BMP inhibitor Noggin.

Heterotopic ossification in rectal cancer: Rare finding with a novel proposed mechanism.

The rare finding of heterotopic ossification in a case of primary rectal adenocarcinoma is described along with a review of the literature. Immunohistochemistry for a bone morphogenic protein (BMP-2) and fibroblast growth factor (FGF-2), both of which induce and stimulate bone formation, was performed and revealed overexpression of BMP-2 by the tumor cells, elucidating a possible mechanism which up to now had been based merely on speculation.

Radiographic and morphologic studies of multiple miliary osteomas of cadaver skin.

OBJECTIVES: The purpose of this study was to examine the frequency, chemical composition, and radiographic and morphologic features of multiple miliary osteomas of the facial skin. STUDY DESIGN: Facial skin was obtained from 33 cadavers. After contact radiographs were taken, osteomas were examined by photo and scanning electron microscopy. Radiographic microanalysis of inorganic elements of the osteomas was also performed. In addition, clinical dental radiographic examinations were performed on 158 living subjects. RESULTS: Radiographic examination revealed milia-like osteomas in the facial skin of all of the cadavers. Nodules of 0.5 to 2.0 mm in diameter were scattered symmetrically in the skin of the cheek, mandibular angle, and forehead. Each nodule consisted of a concentric, multilamellated, osteoid cortex and an adipose medulla. Microanalysis suggested the presence of hydroxyapatite similar in composition to that of normal cortical bone. The condition was detected in 28% (44/158) of the living subjects by clinical dental radiographic examination. CONCLUSION: Multiple miliary osteoma is a very common condition and may not be related to specific diseases.

X-ray microanalysis of ossified auricles in Addison's disease.

The element content of ossified auricles in two patients with Addison's disease was determined by X-ray microanalysis. The results showed a similar element content of the ossified auricles of the Addison's patients and control bone specimens. The calcium and phosphorus content of the petrified auricles was, however, slightly decreased compared with the iliac bone biopsies of the patients. The sulfur content of the ossified auricles was higher than that of their bones, but markedly lower than control auricular cartilage. Iron, sodium, and chloride contents were similar in the ossified auricles and in the normal auricular cartilage, whereas only trace amounts of these elements were detected in the bone. The auricles of the patients with Addison's disease possessed relatively high aluminium content compared with both control bone and cartilage. The element content of the bone biopsies from Addison patients was comparable to control bone. The presence of trace amounts of aluminium in the petrified auricles of Addison's patients is probably attributed to a long period of aluminium hydroxide consumption.