Bone morphogenetic protein receptor 2 in patients with idiopathic portal hypertension

In idiopathic portal hypertension (IPH) typical vascular lesions are present in the branches of the portal vein or in the perisinusoidal area of the liver. Similar histological alterations have been reported in the pulmonary vasculature of patients with idiopathic pulmonary artery hypertension (IPAH). As IPAH is associated with mutations of the bone morphogenetic protein receptor 2 (BMPR2) gene, the aim of this study was to investigate whether this association might also be found in patients with IPH. Twenty-three samples belonging to 21 unrelated caucasian patients with IPH followed in the hepatic haemodynamic laboratory of the Hospital Clinic in Barcelona were included in the study. All patients were studied for the entire open reading frame and splice site of the BMPR2 gene by direct sequencing and multiple ligation probe amplification (MLPA) in order to detect large deletions/duplications. None of the 23 patients had pulmonary artery hypertension. Four patients presented one single nucleotide polymorphism (SNP) in intron 5, four patients had a SNP in exon 12 and a SNP in exon 1 was found in two cases. Two patients had both intron 5 and exon 12 polymorphisms. All SNPs were previously described. Except for these three SNPs, neither mutations nor rearrangements have been identified in the BMPR2 gene in this population. We did not detect mutations or rearrangements in the coding region of the BMPR2 gene in our patients with IPH. These findings suggest that, in contrast to IPAH, mutations in BMPR2 are not involved in the pathogenesis of IPH.

Bone morphogenetic protein 2 incorporated into biomimetic coatings retains its biological activity

We have previously shown that proteins can be incorporated into the latticework of calcium phosphate layers when biomimetically coprecipitated with the inorganic components, upon the surfaces of titanium-alloy implants. In the present study, we wished to ascertain whether recombinant human bone morphogenetic protein 2 (rhBMP-2) thus incorporated retained its bioactivity as an osteoinductive agent. Titanium alloy implants were coated biomimetically with a layer of calcium phosphate in the presence of different concentrations of rhBMP-2 (0.1-10 microg/mL). rhBMP-2 was successfully incorporated into the crystal latticework, as revealed by protein blot staining. rhBMP-2 was taken up by the calcium phosphate coatings in a dose-dependent manner, as determined by ELISA. Rat bone marrow stromal cells were grown directly on these coatings for 8 days. Their osteogenicity was then assessed quantitatively by monitoring alkaline phosphatase activity. This parameter increased as a function of rhBMP-2 concentrations within the coating medium. rhBMP-2 incorporated into calcium phosphate coatings was more potent in stimulating the alkaline phosphatase activity of the adhering cell layer than was the freely suspended drug in stimulating that of cell layers grown on a plastic substratum. This system may be of osteoinductive value in orthopedic and dental implant surgery.

Recombinant Human Bone Morphogenetic Protein 9 (rhBMP9) Induced Osteoblastic Behaviour on a Collagen Membrane Compared With rhBMP2

BACKGROUND Bone morphogenetic protein 9 (BMP9) has previously been characterized as one of the most osteogenic growth factors of the BMP-family, however, up until now, these experiments have only been demonstrated using adenovirus-transfection experiments (gene therapy). With the recent development of recombinant human (rh)BMP9, the aim of the present study was to investigate its osteopromotive potential versus rhBMP2 when loaded onto a collagen membrane. METHODS ST2 stromal bone marrow cells were seeded onto 1)control; 2)rhBMP2-low(10ng/ml); 3)rhBMP2-high(100ng/ml); 4)rhBMP9-low(10ng/ml); and 5)rhBMP9-high(100ng/ml) porcine collagen membranes. Groups were then compared for cell adhesion at 8 hours, cell proliferation at 1, 3 and 5 days real-time PCR at 3 and 14 days for genes encoding Runx2, alkaline phosphatase(ALP) and bone sialoprotein(BSP) at 3 and 14 days and alizarin red staining at 14 days. RESULTS While rhBMP2 and rhBMP9 demonstrated little effects on cell attachment and proliferation, pronounced increases were observed on osteoblast differentiation. It was found that all groups significantly induced ALP mRNA levels at 3 days and BSP levels at 14 days, however rhBMP9-high demonstrated significantly higher values when compared to all other groups for ALP levels (5-fold increase at 3 days and 2-fold increase at 14 days). Alizarin red staining further revealed that both concentrations of rhBMP9 induced up to 3-fold more staining when compared to rhBMP2. CONCLUSION These results indicate that the combination of collagen membranes with rhBMP9 significantly induced significantly higher ALP mRNA expression and alizarin red staining when compared to rhBMP2. These findings suggest that rhBMP9 may be a suitable growth factor for future regenerative procedures in bone biology.

Characterization of a shorter recombinant polypeptide chain of bone morphogenetic protein 2 on osteoblast behaviour

BACKGROUND Recombinant bone morphogenetic protein two (rhBMP2) has been utilised for a variety of clinical applications in orthopaedic surgery and dental procedures. Despite its widespread use, concerns have been raised regarding its short half-life and transient bioactivity in vivo. Recent investigation aimed at developing rhBMP2 synthesized from a shorter polypeptide chain (108 amino acids) has been undertaken. METHODS The osteopromotive properties of BMP2 were investigated on cell behaviour. Five concentrations of rhBMP2_108 including 10, 50, 100, 200 and 500 ng/ml were compared to a commercially available rhBMP2 (100 ng/ml). Each of the working concentrations of rhBMP2_108 were investigated on MC3T3-E1 osteoblasts for their ability to induce osteoblast recruitment, proliferation and differentiation as assessed by alkaline phosphatase (ALP) staining, alizarin red staining, and real-time PCR for genes encoding ALP, osteocalcin (OCN), collagen-1 (COL-1) and Runx2. RESULTS The results demonstrate that all concentrations of rhBMP2_108 significantly improved cell recruitment and proliferation of osteoblasts at 5 days post seeding. Furthermore, rhBMP2_108 had the most pronounced effects on osteoblast differentiation. It was found that rhBMP2_108 had over a four fold significant increase in ALP activity at seven and 14 days post-seeding and the concentrations ranging from 50 to 200 ng/ml demonstrated the most pronounced effects. Analysis of real-time PCR for genes encoding ALP, OCN, COL-1 and Runx2 further confirmed dose-dependant increases at 14 days post-seeding. Furthermore, alizarin red staining demonstrated a concentration dependant increase in staining at 14 days. CONCLUSION The results from the present study demonstrate that this shorter polypeptide chain of rhBMP2_108 is equally as bioactive as commercially available rhBMP2 for the recruitment of progenitor cells by facilitating their differentiation towards the osteoblast lineage. Future in vivo study are necessary to investigate its bioactivity.

Roles of Bone Morphogenetic Protein Type I Receptors and Smad Proteins in Osteoblast and Chondroblast Differentiation

The biological effects of type I serine/threonine kinase receptors and Smad proteins were examined using an adenovirus-based vector system. Constitutively active forms of bone morphogenetic protein (BMP) type I receptors (BMPR-IA and BMPR-IB; BMPR-I group) and those of activin receptor–like kinase (ALK)-1 and ALK-2 (ALK-1 group) induced alkaline phosphatase activity in C2C12 cells. Receptor-regulated Smads (R-Smads) that act in the BMP pathways, such as Smad1 and Smad5, also induced the alkaline phosphatase activity in C2C12 cells. BMP-6 dramatically enhanced alkaline phosphatase activity induced by Smad1 or Smad5, probably because of the nuclear translocation of R-Smads triggered by the ligand. Inhibitory Smads, i.e., Smad6 and Smad7, repressed the alkaline phosphatase activity induced by BMP-6 or the type I receptors. Chondrogenic differentiation of ATDC5 cells was induced by the receptors of the BMPR-I group but not by those of the ALK-1 group. However, kinase-inactive forms of the receptors of the ALK-1 and BMPR-I groups blocked chondrogenic differentiation. Although R-Smads failed to induce cartilage nodule formation, inhibitory Smads blocked it. Osteoblast differentiation induced by BMPs is thus mediated mainly via the Smad-signaling pathway, whereas chondrogenic differentiation may be transmitted by Smad-dependent and independent pathways.

Mutation in bone morphogenetic protein receptor-IB is associated with increased ovulation rate in Booroola Mérino ewes

Ewes from the Booroola strain of Australian Mérino sheep are characterized by high ovulation rate and litter size. This phenotype is due to the action of the FecBB allele of a major gene named FecB, as determined by statistical analysis of phenotypic data. By genetic analysis of 31 informative half-sib families from heterozygous sires, we showed that the FecB locus is situated in the region of ovine chromosome 6 corresponding to the human chromosome 4q22–23 that contains the bone morphogenetic protein receptor IB (BMPR-IB) gene encoding a member of the transforming growth factor-β (TGF-β) receptor family. A nonconservative substitution (Q249R) in the BMPR-IB coding sequence was found to be associated fully with the hyperprolificacy phenotype of Booroola ewes. In vitro, ovarian granulosa cells from FecBB/FecBB ewes were less responsive than granulosa cells from FecB+/FecB+ ewes to the inhibitory effect on steroidogenesis of GDF-5 and BMP-4, natural ligands of BMPR-IB. It is suggested that in FecBB/FecBB ewes, BMPR-IB would be inactivated partially, leading to an advanced differentiation of granulosa cells and an advanced maturation of ovulatory follicles.

Competition between noggin and bone morphogenetic protein 4 activities may regulate dorsalization during Xenopus development.

Bone morphogenetic protein 4 (BMP-4) induces ventral mesoderm but represses dorsal mesoderm formation in Xenopus embryos. We show that BMP-4 inhibits two signaling pathways regulating dorsal mesoderm formation, the induction of dorsal mesoderm (Spemann organizer) and the dorsalization of ventral mesoderm. Ectopic expression of BMP-4 RNA reduces goosecoid and forkhead-1 transcription in whole embryos and in activin-treated animal cap explants. Embryos and animal caps overexpressing BMP-4 transcribe high levels of genes expressed in ventral mesoderm (Xbra, Xwnt-8, Xpo, Mix.1, XMyoD). The Spemann organizer is ventralized in these embryos; abnormally high levels of Xwnt-8 mRNA and low levels of goosecoid mRNA are detected in the organizer. In addition, the organizer loses the ability to dorsalize neighboring ventral marginal zone to muscle. Overexpression of BMP-4 in ventral mesoderm inhibits its response to dorsalization signals. Ventral marginal zone explants ectopically expressing BMP-4 form less muscle when treated with soluble noggin protein or when juxtaposed to a normal Spemann organizer in comparison to control explants. Endogenous BMP-4 transcripts are downregulated in ventral marginal zone explants dorsalized by noggin, in contrast to untreated explants. Thus, while BMP-4 inhibits noggin protein activity, noggin downregulates BMP-4 expression by dorsalizing ventral marginal zone to muscle. Noggin and BMP-4 activities may control the lateral extent of dorsalization within the marginal zone. Competition between these two molecules may determine the final degree of muscle formation in the marginal zone, thus defining the border between dorsolateral and ventral mesoderm.

Elevated hepatocyte growth factor levels in osteoarthritis osteoblasts contribute to their altered response to bone morphogenetic protein-2 and reduced mineralization capacity

International audience

The influence of Bone Morphogenic Protein-2 on the consolidation phase in a distraction osteogenesis model.

We asked whether locally applied recombinant-Bone Morphogenic Protein-2 (rh-BMP-2) with an absorbable Type I collagen sponge (ACS) carrier could enhance the consolidation phase in a callotasis model. We performed unilateral transverse osteotomy of the tibia in 21 immature male rabbits. After a latency period of 7 days, a 3-weeks distraction was begun at a rate of 0.5mm/12h. At the end of the distraction period (Day 28) animals were randomly divided into three groups and underwent a second surgical procedure: 6 rabbits in Group I (Control group; the callus was exposed and nothing was added), 6 rabbits in Group II (ACS group; receiving the absorbable collagen sponge soaked with saline) and 9 rabbits in Group III (rh-BMP-2/ACS group; receiving the ACS soaked with 100μg/kg of rh-BMP-2, Inductos(®), Medtronic). Starting at Day 28 we assessed quantitative and qualitative radiographic parameters as well as densitometric parameters every two weeks (Days 28, 42, 56, 70 and 84). Animals were sacrificed after 8 weeks of consolidation (Day 84). Qualitative radiographic evaluation revealed hypertrophic calluses in the Group III animals. The rh-BMP-2/ACS also influenced the development of the cortex of the calluses as shown by the modified radiographic patterns in Group III when compared to Groups I and II. Densitometric analysis revealed the bone mineral content (BMC) was significantly higher in the rh-BMP-2/ACS treated animals (Group III).

Resveratrol improves bone repair by modulation of bone morphogenetic proteins and osteopontin gene expression in rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bovine Bone Matrix Action Associated With Morphogenetic Protein in Bone Defects in Rats Submitted to Alcoholism

Extended excessive alcohol use causes changes in bone tissue, thus affecting osteogenesis. The objective of this study was to evaluate if demineralized bone matrix (Gen-ox (R)) associated with bone morphogenetic protein (Gen-pro (R)) changes bone neoformation in rats submitted to experimental alcoholism. Forty male rats (Rattus norvegicus) were separated into 2 groups of 20 animals each: Group E1, which received ethyl alcohol at 25% and had the surgical cavity filled in only with blood clot; and Group E2. which received ethyl alcohol at 25% and had the surgical cavity filled in with demineralized bovine cortical bone associated with bone morphogenetic protein. The animals were submitted to a three-week period of gradual adaptation to alcohol, and then continued receiving alcohol at 25% for 90 days, when the surgical cavity was made. After the surgery, the animals continued consuming alcohol until reaching the sacrifice periods of 10, 20, 40, and 60 days, when the tibias were removed for histological processing. Results showed that surgical cavity repair and bone marrow reorganization occurred faster in Group E1 than in Group E2. At the end of the experiment, it was observed that animals in Group E2 had thick bony trabeculae surrounding the implanted material particles and a small area of connective tissue in the surface region. In conclusion, the implanted material did not accelerate bone neoformation, rather it served as a structure for osteogenesis.

Bone Deposition, Bone Resorption, and Osteosarcoma

Bone deposition and bone resorption are ongoing dynamic processes, constituting bone remodeling. Some bone tumors, such as osteosarcoma (OS), stimulate focal bone deposition. OS is the most common primary bone tumor in children and young adults. A complex network of genes regulates bone remodeling and alterations in its expression levels can influence the genesis and progression of bone diseases, including OS. We hypothesized that the expression profiles of bone remodeling regulator genes would be correlated with OS biology and clinical features. We used real-time PCR to evaluate the mRNA levels of the tartrate-resistant acid phosphatase (ACP5), colony stimulating factor-1 (CSF1R), bone morphogenetic protein 7 (BMP7), collagen, type XI, alpha 2 (COL11A2), and protein tyrosine phosphatases zeta 1 (PTPRZ1) genes, in 30 OS tumor samples and correlated with clinical and histological data. All genes analyzed, except CSF1R, were differentially expressed when compared with normal bone expression profiles. In our results, OS patients with high levels of COL11A2 mRNA showed worse overall (p = 0.041) and event free survival (p = 0.037). Also, a trend for better overall survival was observed in patients with samples showing higher expression of BMP7 (p =0.067). COL11A2 overexpression and BMP7 underexpression could collaborate to OS tumor growth, through its central role in bone remodeling process. (C) 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1142-1148, 2010