Targeting heterotopic ossification by inhibiting activin receptor­like kinase 2 function (Review).

Heterotopic ossification (HO) refers to the appearance of osteoblasts in soft tissues under pathological conditions, such as trauma or infection. HO arises in an unpredictable way without any recognizable initiation. Activin receptor­like kinase­2 (ALK2) is a type I cell surface receptor for bone morphogenetic proteins (BMPs). The dysregulation of ALK2 signaling is associated with a variety of diseases, including cancer and HO. At present, the prevention and treatment of HO in the clinic predominantly includes nonsteroidal anti­inflammatory drugs (NSAIDs), bisphosphonates and other drug treatments, low­dose local radiation therapy and surgical resection, rehabilitation treatment and physical therapy. However, most of these therapies have adverse effects. These methods do not prevent the occurrence of HO. The pathogenesis of HO is not being specifically targeted; the current treatment strategies target the symptoms, not the disease. These treatments also cannot solve the fundamental problem of the occurrence of HO. Therefore, scholars have been working to develop targeted therapies based on the pathogenesis of HO. The present review focuses on advances in the understanding of the underlying mechanisms of HO, and possible options for the prevention and treatment of HO. In addition, the role of ALK2 in the process of HO is introduced and the progress made towards the targeted inhibition of ALK2 is discussed. The present study aims to offer a platform for further research on possible targets for the prevention and treatment of HO.

Inhibition of phosphatidylinositol 3-kinase α (PI3Kα) prevents heterotopic ossification.

Heterotopic ossification (HO) is the pathological formation of ectopic endochondral bone within soft tissues. HO occurs following mechanical trauma, burns, or congenitally in patients suffering from fibrodysplasia ossificans progressiva (FOP). FOP patients carry a conserved mutation in ACVR1 that becomes neomorphic for activin A responses. Here, we demonstrate the efficacy of BYL719, a PI3Kα inhibitor, in preventing HO in mice. We found that PI3Kα inhibitors reduce SMAD, AKT, and mTOR/S6K activities. Inhibition of PI3Kα also impairs skeletogenic responsiveness to BMPs and the acquired response to activin A of the Acvr1R206H allele. Further, the efficacy of PI3Kα inhibitors was evaluated in transgenic mice expressing Acvr1Q207D . Mice treated daily or intermittently with BYL719 did not show ectopic bone or cartilage formation. Furthermore, the intermittent treatment with BYL719 was not associated with any substantial side effects. Therefore, this work provides evidence supporting PI3Kα inhibition as a therapeutic strategy for HO.

A novel gene (FAM20B encoding glycosaminoglycan xylosylkinase) for neonatal short limb dysplasia resembling Desbuquois dysplasia.

Desbuquois dysplasia (DBQD) is an autosomal recessive heterogeneous disorder characterized by joint laxity and skeletal changes, including a distinctive monkey-wrench appearance of the femora, advanced carpal ossification, and abnormal patterning of the preaxial digits. Two genes for DBQD (CANT1 encoding calcium-activated nucleotidase-1 and XYLT1 encoding xylosyltransferase-1) have been reported. We propose a novel gene for neonatal short limb dysplasia resembling DBQD, based on the phenotype and genotype of two affected siblings. The affected boy and girl died in early infancy and shortly after birth, respectively. The clinical hallmarks included mid-face hypoplasia, thoracic hypoplasia with respiratory failure, very short stature (approximately -7 SD of birth length) with mesomelic shortening of the limbs, and multiple dislocations of the large joints. Radiological examinations showed prominent lesser trochanter, flared metaphyses of the long bones, and joint dislocations. The affected boy had preaxial digital hypoplasia, and the affected girl showed overlapping and syndactyly of the preaxial digits. Molecular analyses of the girl showed compound heterozygous variants in FAM20B (NM_014864: c.174_178delTACCT p.T59Afs*19/c.1038delG p.N347Mfs*4). FAM20B encodes glycosaminoglycan xylosylkinase, which acts downstream of xylosyltransferase-1. Given the fact that FAM20B deficiency causes skeletal phenotypes in mice and zebrafish, these variants are highly probable to be pathogenic.

An mTOR Signaling Modulator Suppressed Heterotopic Ossification of Fibrodysplasia Ossificans Progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare and intractable disorder characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor gain-of-function mutations in ACVR1 (FOP-ACVR1), a type I receptor for bone morphogenetic proteins. Despite numerous studies, no drugs have been approved for FOP. Here, we developed a high-throughput screening (HTS) system focused on the constitutive activation of FOP-ACVR1 by utilizing a chondrogenic ATDC5 cell line that stably expresses FOP-ACVR1. After HTS of 5,000 small-molecule compounds, we identified two hit compounds that are effective at suppressing the enhanced chondrogenesis of FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) and suppressed the heterotopic ossification (HO) of multiple model mice, including FOP-ACVR1 transgenic mice and HO model mice utilizing FOP-iPSCs. Furthermore, we revealed that one of the hit compounds is an mTOR signaling modulator that indirectly inhibits mTOR signaling. Our results demonstrate that these hit compounds could contribute to future drug repositioning and the mechanistic analysis of mTOR signaling.

Leptin accelerates the pathogenesis of heterotopic ossification in rat tendon tissues via mTORC1 signaling.

Leptin, an adipocyte-derived cytokine associated with bone metabolism, is believed to play a critical role in the pathogenesis of heterotopic ossification (HO). The effect and underlying action mechanism of leptin were investigated on osteogenic differentiation of tendon-derived stem cells (TDSCs) in vitro and the HO formation in rat tendons. Isolated rat TDSCs were treated with various concentrations of leptin in the presence or absence of mTORC1 signaling specific inhibitor rapamycin in vitro. A rat model with Achilles tenotomy was employed to evaluate the effect of leptin on HO formation together with or without rapamycin treatment. In vitro studies with TDSCs showed that leptin increased the expression of osteogenic biomarkers (alkaline phosphatase, runt-related transcription factor 2, osterix, osteocalcin) and enhanced mineralization of TDSCs via activating the mTORC1 signal pathway (as indicated by phosphorylation of p70 ribosomal S6 kinase 1 and p70 ribosomal S6). However, mTORC1 signaling blockade with rapamycin treatment suppressed leptin-induced osteogenic differentiation and mineralization. In vivo studies showed that leptin promoted HO formation in the Achilles tendon after tenotomy, and rapamycin treatment blocked leptin-induced HO formation. In conclusion, leptin can promote TDSC osteogenic differentiation and heterotopic bone formation via mTORC1 signaling in both vitro and vivo model, which provides a new potential therapeutic target for HO prevention.

Mutations in Biosynthetic Enzymes for the Protein Linker Region of Chondroitin/Dermatan/Heparan Sulfate Cause Skeletal and Skin Dysplasias.

Glycosaminoglycans, including chondroitin, dermatan, and heparan sulfate, have various roles in a wide range of biological events such as cell signaling, cell proliferation, tissue morphogenesis, and interactions with various growth factors. Their polysaccharides covalently attach to the serine residues on specific core proteins through the common linker region tetrasaccharide, -xylose-galactose-galactose-glucuronic acid, which is produced through the stepwise addition of respective monosaccharides by four distinct glycosyltransferases. Mutations in the human genes encoding the glycosyltransferases responsible for the biosynthesis of the linker region tetrasaccharide cause a number of genetic disorders, called glycosaminoglycan linkeropathies, including Desbuquois dysplasia type 2, spondyloepimetaphyseal dysplasia, Ehlers-Danlos syndrome, and Larsen syndrome. This review focused on recent studies on genetic diseases caused by defects in the biosynthesis of the common linker region tetrasaccharide.

Treatment of heterotopic ossification through remote ATP hydrolysis.

Heterotopic ossification (HO) is the pathologic development of ectopic bone in soft tissues because of a local or systemic inflammatory insult, such as burn injury or trauma. In HO, mesenchymal stem cells (MSCs) are inappropriately activated to undergo osteogenic differentiation. Through the correlation of in vitro assays and in vivo studies (dorsal scald burn with Achilles tenotomy), we have shown that burn injury enhances the osteogenic potential of MSCs and causes ectopic endochondral heterotopic bone formation and functional contractures through bone morphogenetic protein-mediated canonical SMAD signaling. We further demonstrated a prevention strategy for HO through adenosine triphosphate (ATP) hydrolysis at the burn site using apyrase. Burn site apyrase treatment decreased ATP, increased adenosine 3',5'-monophosphate, and decreased phosphorylation of SMAD1/5/8 in MSCs in vitro. This ATP hydrolysis also decreased HO formation and mitigated functional impairment in vivo. Similarly, selective inhibition of SMAD1/5/8 phosphorylation with LDN-193189 decreased HO formation and increased range of motion at the injury site in our burn model in vivo. Our results suggest that burn injury-exacerbated HO formation can be treated through therapeutics that target burn site ATP hydrolysis and modulation of SMAD1/5/8 phosphorylation.

Matrix metalloproteinase-9 is a diagnostic marker of heterotopic ossification in a murine model.

Heterotopic ossification (HO) is a serious disorder that occurs when there is aberrant bone morphogenic protein (BMP) signaling in soft tissues. Currently, there are no methods to detect HO before mineralization occurs. Yet once mineralization occurs, there are no effective treatments, short of surgery, to reverse HO. Herein, we used in vivo molecular imaging and confirmatory ex vivo tissue analyses of an established murine animal model of BMP-induced HO to show that matrix metalloproteinase-9 (MMP-9) can be detected as an early-stage biomarker before mineralization. Ex vivo analyses show that active MMP-9 protein is significantly elevated within tissues undergoing HO as early as 48 h after BMP induction, with its expression co-localizing to nerves and vessels. In vivo molecular imaging with a dual-labeled near-infrared fluorescence and micro-positron emission tomography (µPET) agent specific to MMP-2/-9 expression paralleled the ex vivo observations and reflected the site of HO formation as detected from microcomputed tomography 7 days later. The results suggest that the MMP-9 is a biomarker of the early extracellular matrix (ECM) re-organization and could be used as an in vivo diagnostic with confirmatory ex vivo tissue analysis for detecting HO or conversely for monitoring the success of tissue-engineered bone implants that employ ECM biology for engraftment.

In vitro analyses of the dysregulated R206H ALK2 kinase-FKBP12 interaction associated with heterotopic ossification in FOP.

A single recurrent mutation in the regulatory subdomain of a bone morphogenetic protein type I receptor kinase has been linked to heterotopic ossification in classic fibrodysplasia ossificans progressiva (FOP). As a result of a substitution at 1 residue by only 1 other side chain (Arg206His) in just 1 of the 4 type I BMP receptors (ALK2/ACVR1), soft connective tissues progressively metamorphose through an endochondral process into cartilage that is replaced by bone. The substitution of arginine for histidine, also a basic residue yet with the singular property of ionization/protonation over the physiological pH range, led to the hypothesis of an aberrant, pH-sensitive switch mechanism for the ligand-independent activation of BMP signaling through the mutant receptor kinase in patients presenting with classic FOP. To test a potential aspect of the putative pH-dependent mechanism, i.e. loss of autoinhibition of the kinase mediated by the inhibitory protein FKBP12, in vitrointeraction analyses with purified wild-type and R206H ALK2 kinase and FKBP12 proteins were performed. Interactions between the kinases and inhibitory proteins were analyzed qualitatively and quantitatively by native gel electrophoresis and HPLC size exclusion chromatography and with an optical biosensor (Octet; ForteBio). Binding of inhibitory protein by the R206H mutant was diminished 3-fold relative to the wild type kinase at a physiological pH, yet below this value (<~7.5) pronounced nonspecific interactions, particularly with the mutant, prevented comparative evaluations. In conclusion, substitution with histidine leads to partial loss of inhibition of the mutant type I receptor through diminished binding of FKBP12, which may act as a gradient reader in morphogenetic contexts.

Stimulation of ectopic bone formation in response to BMP-2 by Rho kinase inhibitor: a pilot study.

The small GTPase Rho and Rho-associated protein kinase (Rho kinase, ROCK) signal participates in a variety of biological functions including vascular contraction, tumor invasion, and penile erection. Evidence also suggests Rho-ROCK is involved in signaling for mesenchymal cellular differentiation. However, whether it is involved in osteoblastic differentiation is unknown. We therefore asked whether Rho-ROCK signaling participates in recombinant human bone morphogenetic protein (rhBMP-2)-induced osteogenesis both in vitro and in vivo. Continuous delivery of a specific ROCK inhibitor (Y-27632) enhanced ectopic bone formation induced by rhBMP-2 impregnated into an atelocollagen carrier in mice without affecting systemic bone metabolism. Treatment with Y-27632 also enhanced the osteoblastic differentiation of cultured murine neonatal calvarial cells. These effects were associated with increased expression of BMP-4 gene. Expression of a dominant negative mutant of ROCK in ST2 cells promoted osteoblastic differentiation, while a constitutively active mutant of ROCK attenuated osteoblastic differentiation and the ROCK inhibitor reversed this phenotype. Thus, ROCK inhibits osteogenesis, and a ROCK inhibitor in combination with the local delivery of rhBMP/collagen composite may be clinically applicable for stimulating bone formation.

The effects of COX-1 and COX-2 inhibitors on prostaglandin synthesis and the formation of heterotopic bone in a rat model.

INTRODUCTION: Traumatic heterotopic ossification (HO) is a common clinical condition associated with various orthopedic procedures that involve injury to soft tissues near bone. In this study, we tested the hypothesis that the prophylactic effects of NSAID's in the treatment of HO are mediated via inhibition of the COX-2 enzyme. Here we describe a rat model that simulates HO in the human that was used to test the above hypothesis. MATERIALS AND METHODS: Heterotopic ossification was surgically induced in the quadriceps by injury to the muscle and femoral periosteum and transplantation of donor bone marrow cells containing osteoprogenitors into the site of injury. HO was imaged and quantified by micro-CT scanning of femurs removed from sacrificed animals at 6 weeks post-injury, three-dimensional computer reconstructions of the scanned bones and computer-assisted morphometric analysis. Prostaglandin E(2) (PGE(2)) synthesis was quantified using an enzyme immunoassay system. The effects of a nonselective COX inhibitor or specific inhibitors of COX-1 or COX-2 following oral administration on the content of ectopic bone and PGE(2) were also measured. RESULTS: Micro-CT and histological analyses demonstrated that all of the femurs in operated limbs developed HO in the vastus lateralis muscle belly of the quadriceps close to the anterior femur. Only the COX-1,2 nonselective and COX-2 inhibitors significantly decreased HO formation (by about one-third in each case; P < 0.05). PGE(2) synthesis at the site of injury was increased 50- and 100-fold (to 25 ng/g tissue) within 1 and 7 days, respectively, post-injury with the levels declining to near baseline within 2 weeks of surgery. Both the COX-1,2 nonselective and COX-2 inhibitors significantly decreased PGE(2) levels to 25% of control HO levels within 24 h of the first administration, even at low dosages. The COX-1 inhibitor only produced the same effect after 1 week of administration. CONCLUSION: These findings suggest that although inhibitors of COX-2 or COX-1 reduced PGE(2) synthesis, only the COX-2 enzyme plays a role in the mechanism of traumatic HO.

Contribution of matrix vesicles and alkaline phosphatase to ectopic bone formation.

Endochondral calcification involves the participation of matrix vesicles (MVs), but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 +/- 103 nm. While the role of tissue-nonspecific alkaline phosphatase (TNAP) during mineralization involves hydrolysis of inorganic pyrophosphate (PPi), it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP), ATP and PPi. The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNPP, ATP and PPi as substrates. While the enzymatic efficiency (k cat/Km) remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k cat/Km for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PPi, respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment.

Contribution of matrix vesicles and alkaline phosphatase to ectopic bone formation

Endochondral calcification involves the participation of matrix vesicles (MVs), but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 ± 103 nm. While the role of tissue-nonspecific alkaline phosphatase (TNAP) during mineralization involves hydrolysis of inorganic pyrophosphate (PPi), it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP), ATP and PPi. The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNPP, ATP and PPi as substrates. While the enzymatic efficiency (k cat/Km) remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k cat/Km for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PPi, respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment.

The value of serum creatine kinase in early diagnosis of heterotopic ossification.

BACKGROUND: Heterotopic ossification (HO) is a complication of spinal cord injury (SCI) characterized by formation of ectopic bone. Early diagnosis is critical, but available diagnostic methods have drawbacks. Serum creatine kinase may be a marker for the development and severity of HO. PARTICIPANTS: 18 SCI patients with diagnosed HO based on clinical findings and bone scintigraphy. METHODS: Serum creatine kinase levels were taken at the time of diagnosis of HO and during subsequent etidronate therapy. RESULTS: Of the 14 patients with normal creatine kinase values, 13 had no evidence of HO on follow-up radiographic examination. Of the 4 patients with elevated creatine kinase, all developed radiographic signs of HO. CONCLUSION: Elevated serum creatine kinase may be associated with a more aggressive course of HO as well as resistance to etidronate therapy. Further studies are needed to determine whether creatine kinase may serve as a marker for early, active HO.

Stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10) expression in developing human bone: potential roles in skeletal development.

Stromelysin, a member of the matrix metalloproteinase family, demonstrates wide substrate specificity with the ability to degrade proteoglycan, fibronectin, laminin, casein, and the nonhelical region of collagen. The two forms of stromelysin (SL), types 1 (MMP-3) and 2 (MMP-10), share 82% sequence homology, but exhibit differences in cellular synthesis and inducibility by cytokines and growth factors in vitro. However, the distribution of the two isoforms in bone has not been reported. We investigated the presence of SL-1 and SL-2 in human osteophytic and neonatal rib bone using immunohistochemistry and, combined with a new method of in situ zymography, determined the activity of the immunolocalized stromelysins. Latent SL-1 was strongly expressed in the extracellular matrix in fibrous tissue surrounding areas of endochondral ossification in osteophytes, and adjacent to the periosteum of fetal rib bone. Active SL-1 expression was detected in osteocytes and the matrix surrounding osteocytic lacunae. SL-2 showed intense cell-associated staining at sites of resorption in areas of endochondral ossification and in resorptive cells at the chondro-osseous junction, which correlated with enzyme activity detected by zymography. Within the rib, active SL-2 expression was localized in chondrocytes of the growth plate, whereas only occasional SL-1 signal was evident. Vascular areas showed strong SL-2 staining with some proteolytic activity. SL-2, but not SL-1, was strongly expressed in osteoclasts and most mononuclear cells within the marrow. At sites of bone formation both isoforms were expressed by osteoblasts with SL-1 also present in osteoid. These results demonstrate, for the first time, the differential expression of SL-1 and SL-2 in developing human bone, indicating specific roles for the two isoforms. In situ zymography demonstrates that SL-2 is produced in an active form with associated degradation, whereas SL-1, in a matrix-bound proenzyme form, may act as a reservoir for later activation.

Osteoinduction capability of recombinant human bone morphogenetic protein-2 in intramuscular and subcutaneous sites: an experimental study.

The osteoinduction capability of recombinant human bone morphogenetic protein-2 (rhBMP-2) in the muscle and in the subcutaneous tissue in Wistar rats (n = 20) was evaluated, using atelopeptide type-I collagen as a carrier. The alkaline phosphatase (ALP) activity and calcium (Ca) content were quantitatively analyzed 1, 3, 7 and 21 days after the implantation of 5 micrograms of rhBMP-2. At 3 days, the ALP activity began to increase gradually. The Ca content showed a slow increase until 7 days and was markedly elevated at 21 days. There was no significant difference observed between the intramuscular and subcutaneous sites until 3 days. However, at 7 days, both the ALP activity and Ca content were significantly higher intramuscularly than subcutaneously. Also, at 21 days they were higher in the muscle than in the subcutaneous tissue. These results suggest that the difference in osteoinduction could be related to the partial pressure of oxygen or the blood supply in the intramuscular and subcutaneous sites, and that immature mesenchymal cells in the muscle could more easily differentiate into osteoblasts, leading to osteoinduction. This study clearly demonstrated that even a small amount (5 micrograms) or rhBMP-2 induces new bone in the subcutaneous tissue, which has a lesser blood flow than the muscle.

Computerized quantitative radionuclide assessment of heterotopic ossification in spinal cord injury patients.

We evaluated the progression of heterotopic ossification (HO) in 17 spinal cord injury patients by comparing radiographs, quantitative radionuclide bone scans, and serum alkaline phosphatase levels. Evidence of maturation of HO appeared earlier (3 months to 6 years post injury) in radiographs, whereas, during the same time frame, radioactive nuclide assessment showed continued progression of HO in 10 out of the 17 patients. The evolution of HO appeared to take place over a period ranging between 3 and 80 months. We believe that stabilization of HO may be reasonably defined in terms of uptake ratios of 2.0 or less in patients with initial uptake ratios over 3.0 but below 5.0, and of ratios of 3.0 or less when the initial values are over 5.0.

Serum alkaline phosphatase and inorganic phosphorus values in spinal cord injury patients with heterotopic ossification.

The blood chemistry was studied in 140 spinal cord injury (SCI) patients (acute injury ward), including 18 patients who developed heterotopic ossification (HO). Comparisons between the HO and non-HO groups were made to determine if the alkaline phosphatase (AP), inorganic phosphorus (P), or calcium (Ca) levels were of diagnostic value. The results showed that AP, P, and Ca by themselves were of little help in the diagnosis of HO. However, the combination of elevated AP and P was significant, especially if both were consistently elevated. There were no significant differences between the HO and non-HO groups concerning completeness or level of spinal injury.