Role of osteoclasts in heterotopic ossification enhanced by fibrodysplasia ossificans progressiva-related activin-like kinase 2 mutation in mice

Fibrodysplasia ossificans progressiva (FOP) is a disorder of skeletal malformations and progressive heterotopic ossification. The constitutively activating mutation (R206H) of the bone morphogenetic protein type 1 receptor, activin-like kinase 2 (ALK2), is responsible for the pathogenesis of FOP. Although transfection of the causal mutation of FOP into myoblasts enhances osteoclast formation by transforming growth factor-β (TGF-β), the role of osteoclasts in heterotopic ossification is unknown. We therefore examined the effects of alendronate, SB431542 and SB203580 on heterotopic ossification induced by the causal mutation of FOP. Total bone mineral content as well as numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated and alkaline phosphatase (ALP)-positive cells in heterotopic bone were significantly higher in muscle tissues implanted with ALK2 (R206H)-transfected mouse myoblastic C2C12 cells than in the tissues implanted with empty vector-transfected cells in nude mice. Alendronate, an aminobisphosphonate, did not affect total mineral content or numbers of TRAP-positive multinucleated and ALP-positive cells in heterotopic bone, which were enhanced by the implantation of ALK2 (R206H)-transfected C2C12 cells, although it significantly decreased serum levels of cross-linked C-telopeptide of type I collagen, a bone resorption index. Moreover, neither SB431542, an inhibitor of TGF-β receptor type I kinase, nor SB203580, an inhibitor of p38 mitogen-activated protein kinase, affected the increase in heterotopic ossification due to the implantation of ALK2 (R206H)-transfected C2C12 cells. In conclusion, the present study indicates that osteoclast inhibition does not affect heterotopic ossification enhanced by FOP-related mutation.     

An Acvr1 R206H knock-in mouse has fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP; MIM #135100) is a debilitating genetic disorder of dysregulated cellular differentiation characterized by malformation of the great toes during embryonic skeletal development and by progressive heterotopic endochondral ossification postnatally. Patients with these classic clinical features of FOP have the identical heterozygous single nucleotide substitution (c.617G > A; R206H) in the gene encoding ACVR1/ALK2, a bone morphogenetic protein (BMP) type I receptor. Gene targeting was used to develop an Acvr1 knock‐in model for FOP (Acvr1^R206H/+). Radiographic analysis of Acvr1^R206H/+ chimeric mice revealed that this mutation induced malformed first digits in the hind limbs and postnatal extraskeletal bone formation, recapitulating the human disease. Histological analysis of murine lesions showed inflammatory infiltration and apoptosis of skeletal muscle followed by robust formation of heterotopic bone through an endochondral pathway, identical to that seen in patients. Progenitor cells of a Tie2⁺ lineage participated in each stage of endochondral osteogenesis. We further determined that both wild‐type (WT) and mutant cells are present within the ectopic bone tissue, an unexpected finding that indicates that although the mutation is necessary to induce the bone formation process, the mutation is not required for progenitor cell contribution to bone and cartilage. This unique knock‐in mouse model provides novel insight into the genetic regulation of heterotopic ossification and establishes the first direct in vivo evidence that the R206H mutation in ACVR1 causes FOP. © 2012 American Society for Bone and Mineral Research.     

Dysregulated BMP Signaling and Enhanced Osteogenic Differentiation of Connective Tissue Progenitor Cells From Patients With Fibrodysplasia Ossificans Progressiva (FOP)

The study of FOP, a disabling genetic disorder of progressive heterotopic ossification, is hampered by the lack of readily available connective tissue progenitor cells. We isolated such cells from discarded primary teeth of patients with FOP and controls and discovered dysregulation of BMP signaling and rapid osteoblast differentiation in FOP cells compared with control cells. Introduction: Fibrodysplasia ossificans progressiva (FOP), the most disabling condition of progressive heterotopic ossification in humans, is caused by a recurrent heterozygous missense mutation in activin receptor IA (ACVR1), a bone morphogenetic protein (BMP) type I receptor, in all classically affected individuals. A comprehensive understanding of FOP has been limited, in part, by a lack of readily available connective tissue progenitor cells in which to study the molecular pathology of this disorder. Materials and Methods: We derived connective tissue progenitor cells from discarded primary teeth (SHED cells) of patients with FOP and controls and examined BMP signaling and osteogenic differentiation in these cells. Results: SHED cells transmitted BMP signals through both the SMAD and p38 mitogen‐activated protein kinase (MAPK) pathways and responded to BMP4 treatment by inducing BMP responsive genes. FOP cells showed ligand‐independent BMP signaling and ligand‐dependent hyper‐responsiveness to BMP stimulation. Furthermore, FOP cells showed more rapid differentiation to an osteogenic phenotype than control cells. Conclusions: This is the first study of BMP signaling and osteogenic differentiation in connective tissue progenitor cells from patients with FOP. Our data strongly support both basal and ligand‐stimulated dysregulation of BMP signaling consistent with in silico studies of the mutant ACVR1 receptor in this condition. This study substantially extends our understanding of dysregulated BMP signaling in a progenitor cell population relevant to the pathogenesis of this catastrophic disorder of progressive ectopic ossification.     

Palovarotene Inhibits Heterotopic Ossification and Maintains Limb Mobility and Growth in Mice With the Human ACVR1R206H Fibrodysplasia Ossificans Progressiva (FOP) Mutation

Fibrodysplasia ossificans progressiva (FOP), a rare and as yet untreatable genetic disorder of progressive extraskeletal ossification, is the most disabling form of heterotopic ossification (HO) in humans and causes skeletal deformities, movement impairment, and premature death. Most FOP patients carry an activating mutation in a bone morphogenetic protein (BMP) type I receptor gene, ACVR1^R206H, that promotes ectopic chondrogenesis and osteogenesis and, in turn, HO. We showed previously that the retinoic acid receptor γ (RARγ) agonist palovarotene effectively inhibited HO in injury‐induced and genetic mouse models of the disease. Here we report that the drug additionally prevents spontaneous HO, using a novel conditional‐on knock‐in mouse line carrying the human ACVR1^R206H mutation for classic FOP. In addition, palovarotene restored long bone growth, maintained growth plate function, and protected growing mutant neonates when given to lactating mothers. Importantly, palovarotene maintained joint, limb, and body motion, providing clear evidence for its encompassing therapeutic potential as a treatment for FOP. © 2016 American Society for Bone and Mineral Research.     

Elevated BMP and Mechanical Signaling Through YAP1/RhoA Poises FOP Mesenchymal Progenitors for Osteogenesis

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by the formation of extraskeletal bone, or heterotopic ossification (HO), in soft connective tissues such as skeletal muscle. All familial and sporadic cases with a classic clinical presentation of FOP carry a gain-of-function mutation (R206H; c.617 G > A) in ACVR1, a cell surface receptor that mediates bone morphogenetic protein (BMP) signaling. The BMP signaling pathway is recognized for its chondro/osteogenic-induction potential, and HO in FOP patients forms ectopic but qualitatively normal endochondral bone tissue through misdirected cell fate decisions by tissue-resident mesenchymal stem cells. In addition to biochemical ligand-receptor signaling, mechanical cues from the physical environment are transduced to activate intracellular signaling, a process known as mechanotransduction, and can influence cell fates. Utilizing an established mesenchymal stem cell model of mouse embryonic fibroblasts (MEFs) from the Acvr1^R206H/+ mouse model that mimics the human disease, we demonstrated that activation of the mechanotransductive effectors Rho/ROCK and YAP1 are increased in Acvr1^R206H/+ cells. We show that on softer substrates, a condition associated with low mechanical signaling, the morphology of Acvr1^R206H/+ cells is similar to the morphology of control Acvr1^+/+ cells on stiffer substrates, a condition that activates mechanotransduction. We further determined that Acvr1^R206H/+ cells are poised for osteogenic differentiation, expressing increased levels of chondro/osteogenic markers compared with Acvr1^+/+ cells. We also identified increased YAP1 nuclear localization in Acvr1^R206H/+ cells, which can be rescued by either BMP inhibition or Rho antagonism. Our results establish RhoA and YAP1 signaling as modulators of mechanotransduction in FOP and suggest that aberrant mechanical signals, combined with and as a result of the increased BMP pathway signaling through mutant ACVR1, lead to misinterpretation of the cellular microenvironment and a heightened sensitivity to mechanical stimuli that promotes commitment of Acvr1^R206H/+ progenitor cells to chondro/osteogenic lineages.     

Role of altered signal transduction in heterotopic ossification and fibrodysplasia ossificans progressiva

Heterotopic ossification is a pathologic condition in which bone tissue is formed outside of the skeleton, within soft tissues of the body. The extraskeletal bone that forms in these disorders is normal; the cellular mechanisms that direct cell fate decisions are dysregulated. Patients with fibrodysplasia ossificans progressiva (FOP), a rare human genetic disorder of extensive and progressive heterotopic ossification, have malformations of normal skeletal elements, identifying the causative gene mutation and its relevant signaling pathways as key regulators of skeletal development and of cell fate decisions by adult stem cells. The discovery that mildly activating mutations in ACVR1/ALK2, a bone morphogenetic protein (BMP) type I receptor, is the cause of FOP has provided opportunities to identify previously unknown functions for this receptor and for BMP signaling and to develop new diagnostic and therapeutic strategies for FOP and other more common forms of heterotopic ossification, as well as tissue engineering applications.     

ACVR1-Fc suppresses BMP signaling and chondro-osseous differentiation in an in vitro model of Fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare and devastating genetic disease of heterotopic endochondral ossification (HEO), and currently no effective therapies are available for this disease. A recurrent causative heterozygous mutation (c.617 G > A; R206H) for FOP was identified in activin receptor type IA (ACVR1), a bone morphogenetic protein (BMP) type I receptor. This mutation aberrantly activates the BMP-Smad1/5/8 signaling pathway and leads to HEO in FOP patients. Here we report development of a soluble recombinant ACVR1-Fc fusion protein by combining the extracellular domain of human wild type ACVR1 and the Fc portion of human immunoglobulin gamma 1 (IgG1). The ACVR1-Fc fusion protein significantly down-regulated the dysregulated BMP signaling caused by the FOP ACVR1 mutation and effectively suppressed chondro-osseous differentiation in a previously described cellular FOP model, human umbilical vein endothelial cells (HUVECs) that were infected with adenovirus-ACVR1^R206H (HUVEC^R206H). This ACVR1-Fc fusion protein holds great promise for prevention and treatment of HEO in FOP and related diseases.     

Possible involvement of bone morphogenetic protein 2 in heterotopic ossification in metastatic lesion from urothelial carcinoma of bladder

Heterotopic bone formation caused by urothelial carcinoma is rare. The precise mechanism of heterotopic ossification is still unknown. We report a case of urothelial carcinoma with heterotopic bone formation in a metastatic site and investigate the expression of bone morphogenetic protein 2 (BMP-2) and the BMP receptor (BMPR)-Ib using immunohistochemistry. Positive staining of BMP-2 was observed in the cytoplasm of tumor cells in both bladder and psoas lesions. In addition, positive staining of BMPR-Ib was seen in osteoblast-like cells adjacent to bone formation in the psoas metastasis. The heterotopic ossification may result from the metaplasia of pluripotent stem cells into osteoblast cells induced by BMP-2 in a paracrine fashion.     

Cellular Hypoxia Promotes Heterotopic Ossification by Amplifying BMP Signaling

Hypoxia and inflammation are implicated in the episodic induction of heterotopic endochondral ossification (HEO); however, the molecular mechanisms are unknown. HIF‐1α integrates the cellular response to both hypoxia and inflammation and is a prime candidate for regulating HEO. We investigated the role of hypoxia and HIF‐1α in fibrodysplasia ossificans progressiva (FOP), the most catastrophic form of HEO in humans. We found that HIF‐1α increases the intensity and duration of canonical bone morphogenetic protein (BMP) signaling through Rabaptin 5 (RABEP1)‐mediated retention of Activin A receptor, type I (ACVR1), a BMP receptor, in the endosomal compartment of hypoxic connective tissue progenitor cells from patients with FOP. We further show that early inflammatory FOP lesions in humans and in a mouse model are markedly hypoxic, and inhibition of HIF‐1α by genetic or pharmacologic means restores canonical BMP signaling to normoxic levels in human FOP cells and profoundly reduces HEO in a constitutively active Acvr1^Q207D/+ mouse model of FOP. Thus, an inflammation and cellular oxygen‐sensing mechanism that modulates intracellular retention of a mutant BMP receptor determines, in part, its pathologic activity in FOP. Our study provides critical insight into a previously unrecognized role of HIF‐1α in the hypoxic amplification of BMP signaling and in the episodic induction of HEO in FOP and further identifies HIF‐1α as a therapeutic target for FOP and perhaps nongenetic forms of HEO. © 2016 American Society for Bone and Mineral Research.     

Relaxin Augments BMP‐2–Induced Osteoblast Differentiation and Bone Formation

Relaxin (Rln), a polypeptide hormone of the insulin superfamily, is an ovarian peptide hormone that is involved in a diverse range of physiological and pathological reactions. In this study, we investigated the effect of Rln on bone morphogenetic protein 2 (BMP‐2)‐induced osteoblast differentiation and bone formation. Expression of Rln receptors was examined in the primary mouse bone marrow stem cells (BMSCs) and mouse embryonic fibroblast cell line C3H/10T1/2 cells by RT‐PCR and Western blot during BMP‐2–induced osteoblast differentiation. The effect of Rln on osteoblast differentiation and mineralization was evaluated by measuring the alkaline phosphatase activity, osteocalcin production, and Alizarin red S staining. For the in vivo evaluation, BMP‐2 and/or Rln were administered with type I collagen into the back of mice, and after 3 weeks, bone formation was analyzed by micro–computed tomography (µCT). Western blot was performed to determine the effect of Rln on osteoblast differentiation‐related signaling pathway. Expression of Rxfp 1 in BMSCs and C3H/10T1/2 cells was significantly increased by BMP‐2. In vitro, Rln augmented BMP‐2–induced alkaline phosphatase expression, osteocalcin production, and matrix mineralization in BMSCs and C3H/10T1/2 cells. In addition, in vivo administration of Rln enhanced BMP‐2–induced bone formation in a dose‐dependent manner. Interestingly, Rln synergistically increased and sustained BMP‐2–induced Smad, p38, and transforming growth factor‐β activated kinase (TAK) 1 phosphorylation. BMP‐2–induced Runx 2 expression and activity were also significantly augmented by Rln. These results show that Rln enhanced synergistically BMP‐2–induced osteoblast differentiation and bone formation through its receptor, Rxfp 1, by augmenting and sustaining BMP‐2–induced Smad and p38 phosphorylation, which upregulate Runx 2 expression and activity. These results suggest that Rln might be useful for therapeutic application in destructive bone diseases. © 2014 American Society for Bone and Mineral Research.     

Dysregulation of the BMP-p38 MAPK signaling pathway in cells from patients with fibrodysplasia ossificans progressiva (FOP).

FOP is a disabling disorder in which skeletal muscle is progressively replaced with bone. Lymphocytes, our model system for examining BMP signaling, cannot signal through the canonical Smad pathway unless exogenous Smad1 is supplied, providing a unique cell type in which the BMP-p38 MAPK pathway can be examined. FOP lymphocytes exhibit defects in the BMP-p38 MAPK pathway, suggesting that altered BMP signaling underlies ectopic bone formation in this disease. INTRODUCTION: Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder characterized by progressive heterotopic ossification of connective tissues. Whereas the primary genetic defect in this condition is unknown, BMP4 mRNA and protein and BMP receptor type IA (BMPRIA) protein are overexpressed in cultured lymphocytes from FOP patients, supporting that altered BMP signaling is involved in this disease. In this study, we examined downstream signaling targets to study the BMP-Smad and BMP-p38 mitogen-activated protein kinase (MAPK) pathways in FOP. MATERIALS AND METHODS: Protein phosphorylation was assayed by immunoblots, and p38 MAPK activity was measured by kinase assays. To examine BMP target genes, the mRNA expression of ID1, ID3, and MSX2 was determined by quantitative real-time PCR. Statistical analysis was performed using Student's t-test or ANOVA. RESULTS: FOP lymphocytes exhibited increased levels of p38 phosphorylation and p38 MAPK activity in response to BMP4 stimulation. Furthermore, in response to BMP4, FOP cells overexpressed the downstream signaling targets ID1 by 5-fold and ID3 by 3-fold compared with controls. ID1 and ID3 mRNA induction was specifically blocked with a p38 MAPK inhibitor, but not extracellular signal-related kinase (ERK) or c-Jun N-terminal kinase (JNK) inhibitors. MSX2, a known Smad pathway target gene, is not upregulated in control or FOP cells in response to BMP, suggesting that lymphocytes do not use this limb of the BMP pathway. However, introduction of Smad1 into lymphocytes made the cells competent to regulate MSX2 mRNA after BMP4 treatment. CONCLUSIONS: Lymphocytes are a cell system that signals primarily through the BMP-p38 MAPK pathway rather than the BMP-Smad pathway in response to BMP4. The p38 MAPK pathway is dysregulated in FOP lymphocytes, which may play a role in the pathogenesis of FOP.     

The role of endothelial‐mesenchymal transition in heterotopic ossification

Heterotopic ossification (HO) is a process by which bone forms in soft tissues, in response to injury, inflammation, or genetic disease. This usually occurs by initial cartilage formation, followed by endochondral ossification. A rare disease called fibrodysplasia ossificans progressiva (FOP) allows this mechanism to be induced by a combination of genetic mutation and acute inflammatory responses. FOP patients experience progressive HO throughout their lifetime and form an ectopic skeleton. Recent studies on FOP have suggested that heterotopic cartilage and bone is of endothelial origin. Vascular endothelial cells differentiate into skeletal cells through a mesenchymal stem cell intermediate that is generated by endothelial‐mesenchymal transition (EndMT). Local inflammatory signals and/or other changes in the tissue microenvironment mediate the differentiation of endothelial‐derived mesenchymal stem cells into chondrocytes and osteoblasts to induce HO. We discuss the current evidence for the endothelial contribution to heterotopic bone formation. © 2012 American Society for Bone and Mineral Research.     

Skeletal metamorphosis in fibrodysplasia ossificans progressiva (FOP)

Metamorphosis, the transformation of one normal tissue or organ system into another, is a biological process rarely studied in higher vertebrates or mammals, but exemplified pathologically by the extremely disabling autosomal dominant disorder fibrodysplasia ossificans progressiva (FOP). The recurrent single nucleotide missense mutation in the gene encoding activin receptor IA/activin-like kinase-2 (ACVR1/ALK2), a bone morphogenetic protein type I receptor that causes skeletal metamorphosis in all classically affected individuals worldwide, is the first identified human metamorphogene. Physiological studies of this metamorphogene are beginning to provide deep insight into a highly conserved signaling pathway that regulates tissue stability following morphogenesis, and that when damaged at a highly specific locus (c.617G > A; R206H), and triggered by an inflammatory stimulus permits the renegade metamorphosis of normal functioning connective tissue into a highly ramified skeleton of heterotopic bone. A comprehensive understanding of the process of skeletal metamorphosis, as revealed by the rare condition FOP, will lead to the development of more effective treatments for FOP and, possibly, for more common disorders of skeletal metamorphosis.     

Biphasic effects of transforming growth factor β on bone morphogenetic protein–induced osteoblast differentiation

Bone morphogenetic proteins (BMPs) exert an important role in skeletal development, adult bone homeostasis, and fracture healing and have demonstrated clinical utility for bone regeneration. However, BMPs fall short as regenerative agents because high doses need to be used to obtain therapeutic effects. Determining the molecular mechanisms controlling BMP‐induced bone formation may lead to the development of more effective BMP‐based therapies. To identify kinases mediating BMP‐induced osteoblast differentiation, we performed an siRNA screen to find kinases modulating BMP‐6‐induced alkaline phosphatase (ALP) activity. Surprisingly, although transforming growth factor β (TGF‐β) generally is considered to antagonize BMP‐induced osteoblast differentiation, C2C12 cells transfected with siRNAs targeting TGF‐β receptors displayed reduced BMP‐6‐induced ALP activity. Furthermore, pharmacologic inhibitors blocking the TGF‐β type I receptor impaired BMP‐induced ALP activity in KS483 and C2C12 cells and mineralization of KS483 cells. Consistently, costimulation with BMPs and TGF‐β further increased expression of osteoblast‐specific genes, ALP activity, and mineralization of KS483 cells and primary mesenchymal stem cells compared with BMPs alone. The stimulatory and inhibitory effects of TGF‐β were found to depend on timing and duration of the costimulation. TGF‐β inhibited BMP‐induced activation of a BMP‐Smad‐dependent luciferase reporter, suggesting that the stimulatory effect of TGF‐β is not due to increased BMP‐Smad activity. TGF‐β also inhibited the BMP‐induced expression of the BMP antagonist noggin and prolonged BMP activity. In conclusion, TGF‐β, besides acting as an inhibitor, also can, by dampening the noggin‐mediated negative‐feedback loop, enhance BMP‐induced osteoblast differentiation, which might be beneficial in fracture healing. © 2011 American Society for Bone and Mineral Research.     

Activation of c‐Jun NH2‐terminal kinase 1 increases cellular responsiveness to BMP‐2 and decreases binding of inhibitory Smad6 to the type 1 BMP receptor

Bone morphogenetic protein 2 (BMP‐2) plays a critical role in the differentiation of precursor cells and has been approved for clinical application to induce new bone formation. To date, unexpectedly high doses of recombinant BMP‐2 have been required to induce bone healing in humans. Thus, enhancing cellular responsiveness to BMP‐2 potentially has critically important clinical implications. BMP responsiveness may be modulated in part by cross‐talk with other signaling pathways, including mitogen‐activated protein kinases (MAPKs). c‐Jun NH₂‐terminal kinase (JNK) is a MAPK that has been reported to be required for late‐stage differentiation of preosteoblasts and BMP‐2‐induced differentiation of preosteoblasts and pleuripotent cells. In this study we determined that MC3T3‐E1‐clone 24 cells (MC‐24) can be induced by BMP‐2 to differentiate into mineralizing osteoblast cultures. Using this inducible system, we employed both JNK loss‐of‐function and gain‐of‐function reagents to make three key observations: (1) JNK is required for phosphorylation of Smad1 by BMP‐2 and subsequent activation of Smad1 signaling and osteoblast differentiation, (2) JNK1, but not JNK2, is required for BMP‐2‐induced formation of mineralized nodules, and (3) JNK1 activation decreases binding of inhibitory Smad6 to the type I BMP receptor (BMPR‐I) and reciprocally increases binding of Smad1, both observations that would increase responsiveness to BMP‐2. Understanding this and other pathways that lead to increased cellular responsiveness to BMPs could greatly aid more cost‐effective and safe clinical delivery of these important molecules. © 2011 American Society for Bone and Mineral Research.     

Clearance of Senescent Cells From Injured Muscle Abrogates Heterotopic Ossification in Mouse Models of Fibrodysplasia Ossificans Progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease caused by mutations in activin A receptor type I/activin-like kinase 2 (ACVR1/ALK2), a bone morphogenetic protein (BMP) type I receptor, resulting in the formation of extraskeletal or heterotopic ossification (HO) and other features consistent with premature aging. During the first decade of life, episodic bouts of inflammatory swellings (flare-ups) occur, which are typically triggered by soft tissue trauma. Through an endochondral process, these exacerbations ultimately result in skeletal muscles, tendons, ligaments, fascia, and aponeuroses transforming into ectopic bone, rendering movement impossible. We have previously shown that soft tissue injury causes early FOP lesions characterized by cellular hypoxia, cellular damage, and local inflammation. Here we show that muscle injury in FOP also results in senescent cell accumulation, and that senescence promotes tissue reprogramming toward a chondrogenic fate in FOP muscle but not wild-type (WT) muscle. Using a combination of senolytic drugs we show that senescent cell clearance and reduction in the senescence associated secretory phenotype (SASP) ameliorate HO in mouse models of FOP. We conclude that injury-induced senescent cell burden and the SASP contribute to FOP lesion formation and that tissue reprogramming in FOP is mediated by cellular senescence, altering myogenic cell fate toward a chondrogenic cell fate. Furthermore, pharmacological removal of senescent cells abrogates tissue reprogramming and HO formation. Here we provide proof-of-principle evidence for senolytic drugs as a future therapeutic strategy in FOP. © 2021 American Society for Bone and Mineral Research (ASBMR).