Search

Introduction The differentiation of dental pulp cells (DPCs) plays an important role in the repair of dental pulp injury. Bone morphogenetic protein 9 (BMP9) is one of the most effective BMPs to induce the differentiation of stem cells. However, the role of BMP9 in promoting the odontogenic differentiation of DPCs and dentinogenesis is worth knowing. Methods Fluorescence in situ hybridization and immunohistochemistry staining were performed to detect the BMP9 expression in human dental pulp. BMP9 was overexpressed in human DPCs (hDPCs), and the mineralization of hDPCs was tested by alkaline phosphatase staining and alizarin red staining. The expression of odontogenic differentiation-related genes was examined by quantitative real-time polymerase chain reaction and western blotting. The subcutaneous transplantation experiment was performed to test the odonto-induction ability of BMP9 in vivo. The rat direct pulp-capping experiment was performed to test the function of BMP9 in promoting dentin formation. Results BMP9 showed an increased expression in odontoblast layer at both the mRNA and protein levels. BMP9 enhanced the mineralization and induced the expression of odontogenic differentiation-related genes in hDPCs. More mineralized nodules, and increased expression of dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP1) were detected in the beta-tricalcium phosphate scaffold/cells composites of BMP9 group compared with the control group. Meanwhile, there was thicker reparative dentin formation in the BMP9 group in the rat pulp exposure experiment. Conclusions BMP9 participates in the process of DPC differentiation and promotes DPC mineralization and dentinogenesis. BMP9 might be a potential therapeutic target in the repair of dental pulp injury.

Venous malformation (VM) is a type of vascular morphogenic defect in humans with an incidence of 1%. Although gene mutation is considered as the most common cause of VM, the pathogenesis of those without gene mutation remains to be elucidated. Here, we aimed to explore the relation of bone morphogenetic protein 9 (BMP9) and development of VM. At first, we found serum and tissue BMP9 expression in VM patients was significantly lower than that in healthy subjects, detected via enzyme-linked immunosorbent assay. Next, with wound healing assay, transwell assay and tube formation assay, we discovered BMP9 could inhibit migration and enhance tube formation activity of human umbilical vein endothelial cells (HUVECs) via receptor activin receptor-like kinase 1 (ALK1). Besides, BMP9 improved the expression of structural proteins alpha-smooth muscle actin (α-SMA) and Desmin in human umbilical vein smooth muscle cells (HUVSMCs) via activation of the SMAD1/5-ID1 pathway, determined by RNA-based next-generation sequencing, qPCR, immunofluorescence and western blotting. Intriguingly, this effect could be blocked by receptor ALK1 inhibitor, SMAD1/5 inhibitor and siRNAs targeting ID1, verifying the BMP9/ALK1/SMAD1/5/ID1/α-SMA pathway. Meanwhile, knocking out BMP9 in C57BL/6 mice embryo led to α-SMA scarcity in walls of lung and mesenteric vessels, as well as walls of small trachea. BMP9-/- zebrafish also exhibited abnormal vascular maturity, indicating a critical role of BMP9 in vascular maturity and remodeling. Finally, a VM mice model revealed that BMP9 might have therapeutic effect in VM progression. Our study discovered that BMP9 might inhibit the occurrence of VM by strengthening the vessel wall and maintaining endothelium quiescence. These findings provide promising evidences of new therapeutic targets that might be used for the management of VM.

Bone morphogenetic protein 9 (BMP9) is an effective osteogenic factor and a promising candidate for bone tissue engineering. The osteoblastic potential of BMP9 needs to be further increased to overcome its shortcomings. However, the details of how BMP9 triggers osteogenic differentiation in mesenchymal stem cells (MSCs) are unclear. In this study, we used real-time PCR, western blot, histochemical staining, mouse ectopic bone formation model, immunofluorescence, immunoprecipitation, and chromatin immunoprecipitation to investigate the role of pyruvate dehydrogenase kinase 4 (PDK4) in BMP9-induced osteogenic differentiation of C3H10T1/2 cells, as well as the underlying mechanism. We found that PDK4 was upregulated by BMP9 in C3H10T1/2 cells. BMP9-induced osteogenic markers and bone mass were increased by PDK4 overexpression, but decreased by PDK4 silencing. β-catenin protein level was increased by BMP9, which was enhanced by PDK overexpression and decreased by PDK4 silencing. BMP9-induced osteogenic markers were reduced by PDK4 silencing, which was almost reversed by β-catenin overexpression. PDK4 increased the BMP9-induced osteogenic markers, which was almost eliminated by β-catenin silencing. Sclerostin was mildly decreased by BMP9 or PDK4, and significantly decreased by combined BMP9 and PDK4. In contrast, sclerostin increased significantly when BMP9 was combined with PDK4 silencing. BMP9-induced p-SMAD1/5/9 was increased by PDK4 overexpression, but was reduced by PDK4 silencing. PDK4 interacts with p-SMAD1/5/9 and regulates the sclerostin promoter. These findings suggest that PDK4 can increase the osteogenic potential of BMP9 by enhancing Wnt/β-catenin signaling via the downregulation of sclerostin. PDK4 may be an effective target to strengthen BMP9-induced osteogenesis.

BACKGROUND: The peptide apelin is expressed in human healthy livers and is implicated in the development of hepatic fibrosis and cirrhosis. Mutations in the bone morphogenetic protein receptor type II (BMPR-II) result in reduced plasma levels of apelin in patients with heritable pulmonary arterial hypertension. Ligands for BMPR-II include bone morphogenetic protein 9 (BMP9), highly expressed in liver, and BMP10, expressed in heart and to a lesser extent liver. However, it is not known whether reductions in BMP9 and/or BMP10, with associated reduction in BMPR-II signalling, correlate with altered levels of apelin in patients with liver fibrosis and cirrhosis. METHODS: Plasma from patients with liver fibrosis (n = 14), cirrhosis (n = 56), and healthy controls (n = 25) was solid-phase extracted using a method optimised for recovery of apelin, which was measured by ELISA. RESULTS: Plasma apelin was significantly reduced in liver fibrosis (8.3 ± 1.2 pg/ml) and cirrhosis (6.5 ± 0.6 pg/ml) patients compared with controls (15.4 ± 2.0 pg/ml). There was no obvious relationship between apelin and BMP 9 or BMP10 previously measured in these patients. Within the cirrhotic group, there was no significant correlation between apelin levels and disease severity scores, age, sex, or treatment with β-blockers. CONCLUSIONS: Apelin was significantly reduced in plasma of patients with both early (fibrosis) and late-stage (cirrhosis) liver disease. Fibrosis is more easily reversible and may represent a potential target for new therapeutic interventions. However, it remains unclear whether apelin signalling is detrimental in liver disease or is beneficial and therefore, whether an apelin antagonist or agonist have clinical use.

Background BMP9, originating from the liver, and BMP10 are circulating BMPs that preserve vascular endothelial integrity. We assessed BMP9, BMP10 and soluble endoglin (sEng) levels and their relationships to liver disease severity and associated pulmonary vascular syndromes in a cohort of well-characterised liver disease patients. Methods Plasma samples from patients with liver disease (n = 83) and non-disease controls (n = 21) were assayed for BMP9, BMP10 and sEng. Levels were also assessed in a separate cohort of controls (n = 27) and PoPH patients (n = 8). Expression of mRNA and immunohistochemical staining was undertaken in liver biopsy specimens. Plasma BMP activity was assessed using an endothelial cell bioassay. Findings Plasma BMP9 and BMP10 levels were normal in patients with compensated cirrhosis or fibrosis without cirrhosis, but markedly reduced in patients with decompensated cirrhosis, including those with hepatopulmonary syndrome (HPS) or portopulmonary hypertension (PoPH). Liver biopsy specimens revealed reduced mRNA expression and immunostaining for these ligands. Patient plasma samples with reduced BMP9 and BMP10 levels exhibited low BMP activity that was restored with exogenous BMP9. Endoglin mRNA expression was increased in cirrhotic livers and elevated circulating sEng levels in PoPH and HPS patients suggested increased endothelial sEng shedding in these syndromes. Interpretation Plasma BMP9 and BMP10 levels are reduced in decompensated cirrhosis, leading to reduced circulating BMP activity on the vascular endothelium. The pulmonary complications of cirrhosis, PoPH and HPS, are associated with markedly reduced BMP9 and BMP10 and increased sEng levels, suggesting that supplementation with exogenous ligands might be a therapeutic approach for PoPH and HPS.

Mesenchymal stem cells (MSCs) are multipotent progenitors that can differentiate into multiple lineages including osteoblastic lineage. Osteogenic differentiation of MSCs is a cascade that recapitulates most, if not all, of the molecular events occurring during embryonic skeletal development, which is regulated by numerous signaling pathways including bone morphogenetic proteins (BMPs). Through a comprehensive analysis of the osteogenic activity, we previously demonstrated that BMP9 is the most potent BMP for inducing bone formation from MSCs both in vitro and in vivo. However, as one of the least studied BMPs, the essential mediators of BMP9-induced osteogenic signaling remain elusive. Here we show that BMP9-induced osteogenic signaling in MSCs requires intact Notch signaling. While the expression of Notch receptors and ligands are readily detectable in MSCs, Notch inhibitor and dominant-negative Notch1 effectively inhibit BMP9-induced osteogenic differentiation in vitro and ectopic bone formation in vivo. Genetic disruption of Notch pathway severely impairs BMP9-induced osteogenic differentiation and ectopic bone formation from MSCs. Furthermore, while BMP9-induced expression of early-responsive genes is not affected by defective Notch signaling, BMP9 upregulates the expression of Notch receptors and ligands at the intermediate stage of osteogenic differentiation. Taken together, these results demonstrate that Notch signaling may play an essential role in coordinating BMP9-induced osteogenic differentiation of MSCs., Bone morphogenetic protein (BMP) 9 is a potent inducer of osteogenic differentiation from mesenchymal stem cells, but the mediators of BMP9-induced osteogenesis remain elusive. Here, the authors show that inhibition of Notch1 signaling effectively diminishes BMP9-induced osteogenesis. Genetic disruption of Notch pathway severely impairs BMP9-induced bone formation. Thus, these results demonstrate that Notch signaling may play an essential role in coordinating osteogenic differentiation.

Backgrounds Adipose derived stem cells (ADSCs) could undergo osteogenesis via focal adhesion kinase (FAK) and bone morphogenetic protein (BMP) 9 signals, both of which could affect Wnt-β-catenin signal, a signal pathway closely related to ADSCs osteogenesis. It’s still enigma whether FAK and BMP-9 contribute to osteogenesis. Here, we examined the effect of FAK on BMP9-inducedosteogenic differentiation, unveiled the possible molecular mechanism underling this process. Methods In the present study, ADSCs were isolated and purified, and cells of passage 3 underwent virus mediated transfection to prepare ADSCs with stable FAK shRNA expression. Cell viability and migration were detected by MTT and transwell assay, respectively. Expression of osteogenic gene, phosphorylation of FAK and GSK were detected by western blot. Osteogenic potential was evaluated by activity of alkaline phosphatase (ALP) and calcium deposition by ALP staining and Alizarin Red S staining. Results BMP-9 administration promoted ADSCs osteogenesis. Knocking down FAK attenuated this process, inhibited osteogenic proteins expression through Wnt-β-catenin signal. BMP-9 also triggered ADSCs proliferation and migration, and shFAK antagonized such effects too. Although Wnt signal is affected by FAK shRNA, Smad signal remains intact in ADSCs with shFAK. Conclusion FAK and BMP-9 could cross talk on Wnt signal pathway and promote ADSCs osteogenesis. FAK could participate in BMP-9 induced ADSCs osteogenesis via Wnt signal pathway other than Smads signals (see in graph).

The current aging of the world population will increase the number of people suffering from brain degenerative diseases such as Alzheimer's disease (AD). There are evidence showing that the use of growth factors such as BMP-9 could restored cognitive function as it acts on many AD hallmarks at the same time. However, BMP-9 is a big protein expensive to produce that can hardly access the central nervous system. We have therefore developed a small peptide, SpBMP-9, derived from the knuckle epitope of BMP-9 and showed its therapeutic potential in a previous study. Since it is known that the native protein, BMP-9, can act in synergy with other growth factors in the context of AD, here we study the potential synergistic effect of various combinations of SpBMP-9 with bFGF, EGF, IGF-2 or NGF on the cholinergic differentiation of human neuroblastoma cells SH-SY5Y. We found that, in opposition to IGF-2 or EGF, the combination of SpBMP-9 with bFGF or NGF can stimulate to a greater extent the neurite outgrowth and neuronal differentiation toward the cholinergic phenotype as shown by expression and localization of the neuronal markers NSE and VAchT and the staining of intracellular calcium. Those results strongly suggest that SpBMP-9 plus NGF or bFGF are promising therapeutic combinations against AD that required further attention.

Bone morphogenetic proteins (BMPs) are expressed in different cell types of the human ovarian follicle and play important roles in the regulation of ovarian function. BMP-9, also known as growth differentiation factor-2 (GDF-2), belongs to the transforming growth factor-beta (TGF-β) superfamily. BMP-9 is mainly synthesized in the liver and secreted into the blood which allows it to regulate various physiological and pathological functions. To date, the expression of BMP-9 in the human ovary and its function in human granulosa cells remains unknown. In the present study, we detect the protein expression of BMP-9 in the human follicular fluid. Using the primary culture of human granulosa-lutein (hGL) cells obtained from patients undergoing in vitro fertilization as a cell model, we show that treatment with BMP-9 downregulates steroidogenic acute regulatory protein (StAR) expression and suppresses progesterone (P4) production. The expression levels of the P450 side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD) are not affected by BMP-9 treatment. Mechanistically, treatment of hGL cells with BMP-9 activates both SMAD1/5/8 and SMAD2/3 signaling pathways. Blocking the activations of SMAD1/5/8 and SMAD2/3 by pharmacological inhibitors or knockdown of SMAD4 attenuates the inhibitory effects of BMP-9 on StAR expression and P4 production. This study reveals a novel function of BMP-9 in the regulation of ovarian steroidogenesis.

OTULIN coordinates with LUBAC to edit linear polyubiquitin chains in embryonic development, autoimmunity, and inflammatory diseases. However, the mechanism by which angiogenesis, especially that of endothelial cells (ECs), is regulated by linear ubiquitination remains unclear. Here, we reveal that constitutive or EC-specific deletion of Otulin resulted in arteriovenous malformations and embryonic lethality. LUBAC conjugates linear ubiquitin chains onto Activin receptor-like kinase 1 (ALK1), which is responsible for angiogenesis defects, inhibiting ALK1 enzyme activity and Smad1/5 activation. Conversely, OTULIN deubiquitinates ALK1 to promote Smad1/5 activation. Consistently, embryonic survival of Otulin-deficient mice was prolonged by BMP9 pretreatment or EC-specific ALK1Q200D (constitutively active) knockin. Moreover, mutant ALK1 from type 2 hereditary hemorrhagic telangiectasia (HHT2) patients exhibited excessive linear ubiquitination and increased HOIP binding. As such, a HOIP inhibitor restricted the excessive angiogenesis of ECs derived from ALK1G309S-expressing HHT2 patients. These results show that OTULIN and LUBAC govern ALK1 activity to balance EC angiogenesis.

Our previous study confirmed that bone morphogenetic protein 9 (BMP9) participated in the development of nonalcoholic steatohepatitis (NASH) by affecting macrophage polarization. The focus of this study was to further confirm the role of macrophages in BMP9-mediated NASH and to analyze the underlying mechanism. In vivo, mice that were administered adeno-associated viral (AAV) vectors containing a null transgene (AAV-null) or the BMP9 transgene (AAV-BMP9) were divided into methionine- and choline-deficient (MCD) and control diet (CD) groups, and they were administered either control liposomes or clodronate liposomes via tail vein injection, the latter to deplete macrophages. The mice were sacrificed after 4 weeks of MCD diet feeding. In vitro, RAW264.7 cells were pretreated with or without BAY11-7085 (an NF-κB inhibitor) and stimulated with recombinant human BMP9 (rh-BMP9). To explore the underlying mechanism of action of BMP9, primary human monocyte-derived macrophages were additionally investigated and immunohistochemistry, biochemical assays, qRT-PCR, and Western blotting were used. The characteristics of NASH-related inflammation were assessed by hepatic histological analysis. Serum AST and ALT and hepatic triglyceride were examined by biochemical assays. We found that the expression of M1 macrophage genes (including CD86, IL1β, IL6, MCP-1 and TNFα) and the number of M1 macrophages (iNOS+ macrophages) in the liver were significantly elevated after BMP9 overexpression and BMP9 directly upregulated TLR4 expression in MCD-induced NASH. These effects were eliminated by macrophage depletion. In vitro, we discovered that BMP9 enhanced the nuclear translocation of NF-κB to induce macrophage M1 polarization in RAW264.7 cells and it promoted LPS-mediated activation of the NF-κB pathway in primary human macrophages. Taken together, this study demonstrates that BMP9 promotes NASH development by directly acting on macrophages.

Recent studies indicated a lower osteogenic differentiation potential of adipose tissue-derived stromal cells (ASCs) compared to bone marrow derived mesenchymal stromal cells. The aim of this study was to evaluate the effects of potent combinations of highly osteogenic bone morphogenetic proteins (BMPs) in order to enhance the osteogenic differentiation potential of ASCs. Human ASCs were cultured for 10 days in the presence of osteogenic medium consisting of dexamethasone, ß-glycerophosphate and ascorbat-2-phosphate (OM) supplemented with BMP-2, BMP-6, BMP-9+IGF-2 and BMP-2,-6,-9 (day 1+2: 50 ng/ml, days 3-6: 100 ng/ml, days 7-10: 200 ng/ml). The formation of the osteoblast phenotype was evaluated by quantification of osteoblast-related marker genes using real-time polymerase chain reaction (RT-PCR). Matrix mineralization was assessed by Alizarin Red S staining. Statistical analysis was carried out using the one-way analysis of variance (ANOVA) followed by the Scheffe's post hoc procedure. Osteogenic medium (OM) significantly increased the expression of alkaline phosphatase (ALP) and osteocalcin (p 0.05) and led to a stable matrix mineralization. Under the influence of BMP-9+IGF-2 and BMP-2,-6,-9 the ALP expression further increased compared to ASCs cultured with OM only (p 0.01). However, multiple osteogenic markers showed no change or decreased under the influence of OM and BMP combinations (p 0.05). The current results indicate a restricted osteogenic differentiation potential of ASCs and suggest careful reconsideration of their use in bone tissue engineering applications.

Upregulated synthesis of extracellular matrix (ECM) proteins by myofibroblasts is a common phenomenon in the development of fibrosis. Although the role of TGF-β in fibrosis development has been extensively studied, the involvement of other members of this superfamily of cytokines, the bone morphogenetic proteins (BMPs) in organ fibrosis has given contradictory results. BMP9 is the main ligand for activin receptor-like kinase-1 (ALK1) TGF-β1 type I receptor and its effect on fibrosis development is unknown. Our purpose was to study the effect of BMP9 in ECM protein synthesis in fibroblasts, as well as the involved receptors and signaling pathways. In cultured mice fibroblasts, BMP9 induces an increase in collagen, fibronectin and connective tissue growth factor expression, associated with Smad1/5/8, Smad2/3 and Erk1/2 activation. ALK5 inhibition with SB431542 or ALK1/2/3/6 with dorsomorphin-1, inhibition of Smad3 activation with SIS3, and inhibition of the MAPK/Erk1/2 with U0126, demonstrates the involvement of these pathways in BMP9-induced ECM synthesis in MEFs. Whereas BMP9 induced Smad1/5/8 phosphorylation through ALK1, it also induces Smad2/3 phosphorylation through ALK5 but only in the presence of ALK1. Summarizing, this is the first study that accurately identifies BMP9 as a profibrotic factor in fibroblasts that promotes ECM protein expression through ALK1 and ALK5 receptors.

Transforming growth factor-β (TGF-β) family ligands are pleiotropic cytokines. Their physiological activities are not determined by a simple coupling of stimulus and response, but depend critically on context, i.e. the interplay of receptors, ligands, and regulators that form the TGF-β signal transduction system of a cell or tissue. How these different components combine to regulate signaling activities remains poorly understood. Here, we describe a ligand-mediated mechanism of signaling regulation. Based on the observation that the type II TGF-β family receptors ActRIIA, ActRIIB, and BMPRII interact with a large group of overlapping ligands at overlapping epitopes, we hypothesized high affinity ligands compete with low affinity ligands for receptor binding and signaling. We show activin A and other high affinity ligands directly inhibited signaling by the low affinity ligands BMP-2, BMP-7, and BMP-9. We demonstrate activin A functions as a competitive inhibitor that blocks the ligand binding epitope on type II receptors. We propose binding competition and signaling antagonism are integral functions of the TGF-β signal transduction system. These functions could help explain how activin A modulates physiological signaling during extraordinary cellular responses, such as injury and wound healing, and how activin A could elicit disease phenotypes such as cancer-related muscle wasting and fibrosis.

Methotrexate is the most common first-line chemotherapy for low-risk gestational trophoblastic neoplasia (GTN). Uterine artery pulsatility index (UAPI) is an ultrasound marker for tumor vascularity that has been associated with an increased risk of methotrexate resistance. The combination of circulating angiogenic factor levels with UAPI data may improve the capacity of this model to predict chemoresistance.This was a single-center cohort study of women newly diagnosed between January 2008 and June 2012 with low-risk GTN during postmolar surveillance and treated with single-agent methotrexate at Charing Cross Hospital, a UK national center for treatment of gestational trophoblastic disease. Two hundred seventeen women underwent an ultrasound for UAPI measurement prior to initiation of chemotherapy. To examine serologic markers of methotrexate resistance among this cohort, we performed a case-control study using archived serum from 76 patients who could be matched based on prognostic risk score. Serum samples were examined by immunoassay to measure 8 different angiogenic factors (VEGF-A, FGF-basic, PLGF-1, PDGF-BB, EGF, ANGPT2, BMP-9, and ENG). Receiver-operator characteristic area under the curve (AUC) values were calculated for the ability of each analyte to correctly classify patients as methotrexate sensitive (MTX-S) or resistant (MTX-R).Total human chorionic gonadotropin levels were similar between the MTX-S and MTX-R groups. UAPI values were significantly higher in MTX-S (median 1.30 [interquartile range {IQR} = 0.80-1.90]) compared to MTX-R patients (median 0.875 [IQR = 0.60-1.30]; P0.0001) with AUC 0.68 (95% confidence interval 0.61-0.76; P0.0001). In univariate analysis, only BMP-9 concentrations were significantly different between groups, lower among MTX-S (median of 225 ng/L, IQR = 170-287) compared to MTX-R patients (median 280 ng/L [IQR = 200-339]; P= 0.03). Combining UAPI with BMP-9 concentration improved prediction for chemoresistance with AUC 0.77 (95% confidence interval 0.66-0.88; P0.0001).Circulating levels of BMP-9 are elevated in newly diagnosed women with low-risk GTN destined to fail primary methotrexate therapy. A combined test using serum BMP-9 plus UAPI might improve prediction of MTX-R in low-risk GTN.

Highlights • Apelin has been implicated in hepatic fibrosis and cirrhosis suggesting that plasma levels of this peptide might be a biomarker of liver disease. • Previous studies of fibrosis and cirrhosis patients reported variable plasma concentrations of apelin ranging from 200 pg/ml to 10,000 pg/ml. • Using solid-phase extraction, we report decreased plasma levels of apelin in fibrosis and cirrhosis patients compared to healthy volunteers. • Decreased circulating apelin may be a biomarker of liver fibrosis that occurs in early-stage liver disease. • Fibrosis, unlike cirrhosis, is easily reversible and may represent a target for new therapeutic interventions., Background The peptide apelin is expressed in human healthy livers and is implicated in the development of hepatic fibrosis and cirrhosis. Mutations in the bone morphogenetic protein receptor type II (BMPR-II) result in reduced plasma levels of apelin in patients with heritable pulmonary arterial hypertension. Ligands for BMPR-II include bone morphogenetic protein 9 (BMP9), highly expressed in liver, and BMP10, expressed in heart and to a lesser extent liver. However, it is not known whether reductions in BMP9 and/or BMP10, with associated reduction in BMPR-II signalling, correlate with altered levels of apelin in patients with liver fibrosis and cirrhosis. Methods Plasma from patients with liver fibrosis (n = 14), cirrhosis (n = 56), and healthy controls (n = 25) was solid-phase extracted using a method optimised for recovery of apelin, which was measured by ELISA. Results Plasma apelin was significantly reduced in liver fibrosis (8.3 ± 1.2 pg/ml) and cirrhosis (6.5 ± 0.6 pg/ml) patients compared with controls (15.4 ± 2.0 pg/ml). There was no obvious relationship between apelin and BMP 9 or BMP10 previously measured in these patients. Within the cirrhotic group, there was no significant correlation between apelin levels and disease severity scores, age, sex, or treatment with β-blockers. Conclusions Apelin was significantly reduced in plasma of patients with both early (fibrosis) and late-stage (cirrhosis) liver disease. Fibrosis is more easily reversible and may represent a potential target for new therapeutic interventions. However, it remains unclear whether apelin signalling is detrimental in liver disease or is beneficial and therefore, whether an apelin antagonist or agonist have clinical use.

Fibrosis is a common endpoint for chronic organic diseases. Many signaling pathways and cytokines are involved in the development of fibrosis. Recently, bone morphogenetic protein 9 (BMP9), a member of transforming growth factor-beta (TGF-β) superfamily, has been considered as a pivotal regulator in tissue fibrosis, and exerts its diverse effects on the activation of fibroblasts and the development of fibrotic diseases. BMP9 exhibits its functional roles largely through binding to type I BMP receptor activin receptor-like kinase 1 (ALK1), and then directly activates small mother against decapentaplegic (Smad) signaling which promotes or limits the expressions of pro-fibrotic genes. Accumulating studies have demonstrated that the aberrant BMP9/ALK1/Smad signaling pathway affects the fibrogenesis of various organs, but the exact role of BMP9 in fibrosis remains elusive and debatable. In the present review, reports from in vitro, in vivo and clinical studies regarding the functions and underlying mechanisms of the BMP9 signaling in tissue fibrosis, are widely summarized and discussed. In addition, the major components of the BMP9 signaling pathway and the potential interventions targeting this pathway are also described. This review will enrich our understanding of the BMP9 signaling pathway in organ fibrosis, and provide a novel clue for the potential interventions of organ fibrosis.

Inflammation is a common situation during bone healing and is recognized to inhibit osteogenic differentiation and bone formation. However, the effect of inflammation on BMP-9-induced osteoblastic differentiation remains unclear. In the present study, we found that an inflammatory environment triggered by lipopolysaccharide (LPS) in vitro suppressed BMP-9-induced osteogenic differentiation. In addition, LPS decreased BMP-9-induced phosphorylation of Smad1/5/8 and showed obvious inhibitory effects on BMP-9-induced Smad signaling. We then confirmed that LPS and BMP-9 can activate p38MAPK and ERK1/2 signaling, and that LPS stimulation reduces BMP-9-induced Runx2 expression through the activation of p38MAPK and ERK1/2 signaling. Finally, we determined that blockade of MAPK signaling by specific inhibitors reverses the inhibitory effect of LPS on BMP-9-induced osteogenic differentiation, and that MAPK acts as a mediator of the negative regulatory role of LPS in BMP-9-induced activation of Smad signaling. Based on these results, we conclude that the LPS-mediated inflammatory environment inhibits BMP-9-induced osteogenic differentiation, via crosstalk between BMP/MAPK and Smad signaling. The elucidation of these mechanisms may hasten the development of new strategies and improve the osteoinductive efficacy of BMP-9 in the clinic, resulting in enhanced osteoblastic differentiation and bone formation.

A variety of different growth factors, most notably bone morphogenetic proteins (BMPs), have been shown to stimulate the osteogenic differentiation of mesenchymal stromal cells (MSCs) in vitro. Yet, due to the lack of comparative studies it remains unclear which protocol is the most effective in the induction of osteogenesis in MSC cultures. The aim of this study was to compare the most potent growth factors in regard to their osteoinductive potential. Human MSCs were cultured for 10 days in the presence of BMP-2, BMP-6, BMP-9 + IGF-2 and BMP-2, -6, -9 (day 1 + 2: 50 ng/ml; days 3-6: 100 ng/ml; days 7-10: 200 ng/ml). The formation of the osteoblast phenotype was assessed by quantification of osteoblast-related marker genes using reverse transcription polymerase chain reaction (RT-PCR) and alkaline phosphatase (ALP) staining. Matrix mineralization was assessed by alizarin red S and von Kossa staining. Statistical analysis was carried out using the one-way analysis of variance (ANOVA) followed by Scheffe's post hoc procedure. Among the tested growth factors the combination of BMP-2 + BMP-6 + BMP-9 most effectively induced the upregulation of collagen type I, collagen type V, osteocalcin, alkaline phosphatase, RUNX2, BMP-2, osteonectin and DLX5 (p0.01) and resulted in a consistent matrix mineralization. The findings suggest the combined addition of BMP-2, BMP-6 and BMP-9 to the osteoinductive culture medium containing dexamethasone, β-glycerophosphate and ascorbate-2-phosphate produces more potent osteoblast differentiation of human MSCs in vitro.

Type I collagen is one of the most widely used materials for drug delivery in tissue repair. It is the reference carrier for delivering growth factors like bone morphogenetic proteins (BMPs such as BMP-2 and BMP-7) for bone repair. Since BMPs are expensive to produce, we have developed a peptide derived from BMP-9 (pBMP-9) that is 300 times less expensive than the entire protein while still promoting osteogenic differentiation. We have now evaluated the effects of the collagen concentration and the initial pBMP-9 load on peptide release. We then developed a model of pBMP-9 release kinetics by finite differences using a system based on Fick's second law in which the interactions between the peptide and collagen fibers are assumed to follow Langmuir adsorption kinetics. The Langmuir isotherms suggest that the structure of the collagen gel influences the strength of its electrostatic interaction with the peptide, since increasing the collagen concentration decreased the affinity of pBMP-9 for the collagen. The resulting model of the mechanism accurately reflects the experimental data and the parameters estimated indicate that the diffusivities with the different collagen concentrations are similar, whereas the mass transfer coefficient increases with the collagen concentration. The results also indicate that perfect sink conditions cannot be assumed and suggest the presence of an optimal collagen concentration. Finally, we have correlated our conclusions with the differences in collagen fiber organization observed by transmission electron microscopy.

BMP-9, whose expression is highest in liver cells, has been demonstrated to regulate expression of enzymes involved in glucose homeostasis. However, the underlying mechanism of this effect has yet to be elucidated. We observed that MB109, a recombinant BMP-9 derivative, enhanced brown adipogenesis of human adipose tissue derived stem cells. With this observation of the cell culture system, we hypothesized that MB109 may be able to improve glucose metabolism by regulating expression of brown adipogenic genes. Systemic intraperitoneal injection of MB109 (200 μg/kg/wk) suppressed weight gaining of high fat diet-induced obese mice by reducing sizes of white adipocytes and decreased 16 h fasting blood glucose levels without changing food consumption or apparent behavioral performances. MB109 induced expression of brown adipogenic genes in the subcutaneous but not in the visceral fat tissues from the mice fed with high fat diet. In addition, systematic injection of MB109 enhanced fatty acid synthase expression in the liver of obese mice, which may help attenuate an obesity-associated increase of blood glucose levels. Our results demonstrate a role of BMP-9 in brown adipogenesis and suppressing pathophysiology of high fat diet-induced obesity, presumably through the activin receptor like kinase 1 signaling pathway.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease; its pathogenesis remains unclear. Fibroblast-like synoviocytes (FLSs) play a vital role in the pathogenesis of RA. BMP9, a member of the bone morphogenetic protein (BMP) family, has been reported to play a critical role in both normal physiological processes and the pathology of various diseases. In this study, we explored the function and underlying mechanisms of BMP9 in the proliferation and migration of RA FLSs. We found that BMP9 expression was significantly downregulated in the synovial tissues of RA patients, compared with those of OA patients; BMP9 expression was also low in adjuvant-induced arthritis (AA) samples. Additionally, inhibition of BMP9 expression by BMP9 siRNA increased the proliferation of AA FLSs, and the expression of c-Myc, Cyclin D1, MMP-2, and MMP-9, but not TIMP-1, in AA FLSs. However, AA FLSs transfected with the overexpression vector PEX-3-BMP9 showed reduced proliferation and expression of c-Myc, Cyclin D1, MMP-2, and MMP-9, but not TIMP-1. Further studies indicate that BMP9 may induce the activation of the PI3K/AKT signaling pathway. Thus, these data indicate that BMP9 may play a critical role in the proliferation and migration of FLSs through the activation of the AKT signaling pathway.

Dedifferentiated fat (DFAT) cells, which are isolated from mature adipocytes using the ceiling culture method, exhibit similar characteristics to mesenchymal stem cells, and possess adipogenic, osteogenic, chondrogenic, and myogenic potentials. Bone morphogenetic protein (BMP)-2 and -9, members of the transforming growth factor-β superfamily, exhibit the most potent osteogenic activity of this growth factor family. However, the effects of BMP-2 and BMP-9 on the osteogenic differentiation of DFAT remain unknown. Here, we examined the effects of BMP-2 and BMP-9 on osteoblastic differentiation of rat DFAT (rDFAT) cells in the presence or absence of FK506, an immunosuppressive agent. Co-stimulation with BMP-9 and FK506 induced gene expression of runx2, osterix, and bone sialoprotein, and ALP activity compared with BMP-9 alone, BMP-2 alone and BMP-2+FK506 in rDFAT cells. Furthermore, it caused mineralization of cultures and phosphorylation of smad1/5/8, compared with BMP-9 alone. The ALP activity induced by BMP-9+FK506 was not influenced by addition of noggin, a BMP antagonist. Our data suggest that the combination of BMP-9 and FK506 potently induces osteoblastic differentiation of rDFAT cells.

Biomaterials functionalized by adhesive peptides improve the cell-substratum interaction. However, their influence on the response of cells to growth factors is still poorly understood. We have shown that bone morphogenetic protein (BMP) 2 activates the Smad pathway only in murine MC3T3-E1 preosteoblasts attached to polycaprolactone (PCL) film functionalized by RGD peptides derived from bone sialoprotein (pRGD). We have now analysed the way recombinant human BMP-2 and/or BMP-9 (0.38 nM) influence the signal transduction and differentiation of MC3T3-E1 preosteoblasts attached to PCL-pRGD. While kinetics of MAPK activation were similar in cells treated by BMP-2 and BMP-9, different kinetics of Smad activation and β-catenin stabilization were observed. BMP-2 induced Smad1/5/8 phosphorylation within 0.5 and BMP-9 within 4 h, while the β-catenin was lower at 2 h only in cells treated with BMP-9. However, both BMPs induced the translocation of phosphorylated Smad1/5/8 to the nucleus at 4 h and increased Dlx5, osterix and osteocalcin transcripts as well as alkaline phosphatase activity at 72 h. A BMP-2/BMP-9 combination that maintained the β-catenin amount constant but reduced that of phosphorylated Smad within 4 h had quite similar effect than BMP-2 alone. It is therefore important to determine how biomimetic materials influence the response of cells to BMPs.

Activin receptor-like kinase 1 (ALK1), an endothelial cell-specific type I receptor of the TGF-β superfamily, is an important regulator of normal blood vessel development as well as pathological tumor angiogenesis. As such, ALK1 is an important therapeutic target. Thus, several ALK1-directed agents are currently in clinical trials as anti-angiogenic cancer therapeutics. Given the biological and clinical importance of the ALK1 signaling pathway, we sought to elucidate the biophysical and structural basis underlying ALK1 signaling. The TGF-β family ligands BMP9 and BMP10 as well as the three type II TGF-β family receptors ActRIIA, ActRIIB, and BMPRII have been implicated in ALK1 signaling. Here, we provide a kinetic and thermodynamic analysis of BMP9 and BMP10 interactions with ALK1 and type II receptors. Our data show that BMP9 displays a significant discrimination in type II receptor binding, whereas BMP10 does not. We also report the crystal structure of a fully assembled ternary complex of BMP9 with the extracellular domains of ALK1 and ActRIIB. The structure reveals that the high specificity of ALK1 for BMP9/10 is determined by a novel orientation of ALK1 with respect to BMP9, which leads to a unique set of receptor-ligand interactions. In addition, the structure explains how BMP9 discriminates between low and high affinity type II receptors. Taken together, our findings provide structural and mechanistic insights into ALK1 signaling that could serve as a basis for novel anti-angiogenic therapies.

Endoglin (CD105), a transmembrane protein of the transforming growth factor β superfamily, plays a crucial role in angiogenesis. Mutations in endoglin result in the vascular defect known as hereditary hemorrhagic telangiectasia (HHT1). The soluble form of endoglin was suggested to contribute to the pathogenesis of preeclampsia. To obtain further insight into its function, we cloned, expressed, purified, and characterized the extracellular domain (ECD) of mouse and human endoglin fused to an immunoglobulin Fc domain. We found that mouse and human endoglin ECD-Fc bound directly, specifically, and with high affinity to bone morphogenetic proteins 9 and 10 (BMP9 and BMP10) in surface plasmon resonance (Biacore) and cell-based assays. We performed a function mapping analysis of the different domains of endoglin by examining their contributions to the selectivity and biological activity of the protein. The BMP9/BMP10 binding site was localized to the orphan domain of human endoglin composed of the amino acid sequence 26-359. We established that endoglin and type II receptors bind to overlapping sites on BMP9. In the in vivo chick chorioallantoic membrane assay, the mouse and the truncated human endoglin ECD-Fc both significantly reduced VEGF-induced vessel formation. Finally, murine endoglin ECD-Fc acted as an anti-angiogenic factor that decreased blood vessel sprouting in VEGF/FGF-induced angiogenesis in in vivo angioreactors and reduced the tumor burden in the colon-26 mouse tumor model. Together our findings indicate an important role of soluble endoglin ECD in the regulation of angiogenesis and highlight efficacy of endoglin-Fc as a potential anti-angiogenesis therapeutic agent.

Nonunion fractures present a challenge to orthopedics with no optimal solution. In-vivo DNA electroporation is a gene-delivery technique that can potentially accelerate regenerative processes. We hypothesized that in vivo electroporation of an osteogenic gene in a nonunion radius bone defect site would induce fracture repair. Nonunion fracture was created in the radii of C3H/HeN mice, into which a collagen sponge was placed. To allow for recruitment of host progenitor cells (HPCs) into the implanted sponge, the mice were housed for 10 days before electroporation. Mice were electroporated with either bone morphogenetic protein 9 (BMP-9) plasmid, Luciferase plasmid or injected with BMP-9 plasmid but not electroporated. In vivo bioluminescent imaging indicated that gene expression was localized to the defect site. Microcomputed tomography (µCT) and histological analysis of murine radii electroporated with BMP-9 demonstrated bone formation bridging the bone gap, whereas in the control groups the defect remained unbridged. Population of the implanted collagen sponge by HPCs transfected with the injected plasmid following electroporation was noted. Our data indicate that regeneration of nonunion bone defect can be attained by performing in vivo electroporation with an osteogenic gene combined with recruitment of HPCs. This gene therapy approach may pave the way for regeneration of other skeletal tissues.

Mutations in transforming growth factor-beta (TGF-beta) receptor superfamily members underlie conditions characterized by vascular dysplasia. Mutations in endoglin and activin-like kinase receptor 1 (ALK1) cause hereditary hemorrhagic telangiectasia, whereas bone morphogenetic protein type II receptor (BMPR-II) mutations underlie familial pulmonary arterial hypertension. To understand the functional roles of these receptors, we examined their relative contributions to BMP signaling in human pulmonary artery endothelial cells (HPAECs). BMP9 potently and selectively induced Smad1/5 phosphorylation and Id gene expression in HPAECs. Contrary to expectations, BMP9 also stimulated Smad2 activation. Furthermore, BMP9 induced the expression of interleukin 8 and E-selectin. Using small interfering RNA, we demonstrate that the type I receptor, ALK1, is essential for these responses. However, small interfering RNA and inhibitor studies showed no involvement of ALK5 or endoglin. We further demonstrate that, of the candidate type II receptors, BMPR-II predominantly mediated IL-8 and E-selectin induction and mitogenic inhibition by BMP9. Conversely, activin receptor type II (ActR-II) contributed more to BMP9-mediated Smad2 activation. Only abolition of both type II receptors significantly reduced the Smad1/5 and Id responses. Both ALK1 and BMPR-II contributed to growth inhibition of HPAECs, whereas ActR-II was not involved. Taken together, our findings demonstrate the critical role of type II receptors in balancing BMP9 signaling via ALK1 and emphasize the essential role for BMPR-II in a subset of BMP9 responses (interleukin 8, E-selectin, and proliferation). This differential signaling may contribute to the contrasting pathologies of hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension.

Methamphetamine (MA) is a drug of abuse as well as a dopaminergic neurotoxin. We have previously demonstrated that pretreatment with bone morphogenetic protein 7 (BMP7) reduced 6-hydroxydopamine-mediated neurodegeneration in a rodent model of Parkinson’s disease. In this study, we examined the neuroprotective effects of BMP7 against MA-mediated toxicity in dopaminergic neurons. Primary dopaminergic neurons, prepared from rat embryonic ventral mesencephalic tissue, were treated with MA. High doses of MA decreased tyrosine hydroxylase immunoreactivity (THir) while increasing terminal deoxynucleotidyl transferase–mediated dNTP nick end labeling. These toxicities were significantly antagonized by BMP7. Interaction of BMP7 and MA in vivo was first examined in CD1 mice. High doses of MA (10 mg/kg ×4 s.c.) significantly reduced locomotor activity and THir in striatum. I.c.v. administration of BMP7 antagonized these changes. In BMP7 +/− mice, MA suppressed locomotor activity and reduced TH immunoreactivity in nigra reticulata to a greater degree than in wild type BMP7 +/+ mice, suggesting that deficiency in BMP7 expression increases vulnerability to MA insults. Since BMP7 +/− mice also carry a LacZ-expressing reporter allele at the BMP7 locus, the expression of BMP7 was indirectly measured through the enzymatic activity of β-galactosidase (β-gal) in BMP7 +/− mice. High doses of MA significantly suppressed β-gal activity in striatum, suggesting that MA may inhibit BMP7 expression at the terminals of the nigrostriatal pathway. A similar effect was also found in CD1 mice in that high doses of MA suppressed BMP7 mRNA expression in nigra. In conclusion, our data indicate that MA can cause lesioning in the nigrostriatal dopaminergic terminals and that BMP7 is protective against MA-mediated neurotoxicity in central dopaminergic neurons. Published by Elsevier Ltd on behalf of IBRO.

Hepcidin, the principal regulator of the iron metabolism, is up-regulated in response to inflammatory stimuli, bone morphogenic proteins (BMPs), and iron excess. There are two murine hepcidin genes: hepcidin-1 (Hamp1) and hepcidin-2 (Hamp2). Hamp1 gene responds to both IL-6 and BMPs while Hamp2 responds to neither. We replaced the putative functional regulatory motifs of the Hamp1 promoter with the corresponding putative “non-functional” Hamp2 motifs and vice versa in reporter constructs. Conversion of the Hamp1 STAT site into the Hamp2 site reduced the basal level of reporter expression but did not affect IL-6 and BMP responsiveness; replacing Hamp2 site with the Hamp1 site only resulted in partial responsiveness. These data are in contrast to the role of the STAT site in the human hepcidin promoter which is important in both basal level and IL-6 inducible promoter activity. The murine AP1, E-box and TIEG motifs were found to neither influence the basal level of expression of Hamp1 and HAMP promoters nor play a critical role in the IL-6 and BMP-9 induced response. Our data suggest that the STAT site (nt −148 to −130) is important for the regulation of basal level expression of Hamp1 but there are additional regions that are responsible for the IL-6 and BMP-9 responsiveness within the Hamp1 promoter.

Myogenic differentiation is suppressed in vitro by unknown factors present in fetal bovine serum (FBS). We found that specific inhibitors of bone morphogenetic proteins (BMPs) stimulated myogenic differentiation even in the presence of 20% FBS, which in turn activated specific BMP signaling. Moreover, these specific BMP inhibitors blocked maturation of osteoblastic cells induced by FBS, indicating that BMP-like factor(s) in serum regulate both myogenic and osteoblastic differentiation. The factor identified had an apparent molecular weight (Mw) of over 100 kDa on a Superdex 200 column for molecular sieving HPLC, but an apparent Mw of 33 kDa on SDS–PAGE under non-reducing conditions. Analysis of a purified preparation from FBS (5 L) by liquid chromatography-tandem mass spectrometry revealed the presence of an amino acid sequence conserved between mature human and murine BMP-4. This is the first study to show that BMP-4 is present in FBS as a large complex.

Osteoblast lineage-specific differentiation of mesenchymal stem cells is a well regulated but poorly understood process. Both bone morphogenetic proteins (BMPs) and Wnt signaling are implicated in regulating osteoblast differentiation and bone formation. Here we analyzed the expression profiles of mesenchymal stem cells stimulated with Wnt3A and osteogenic BMPs, and we identified connective tissue growth factor (CTGF) as a potential target of Wnt and BMP signaling. We confirmed the microarray results, and we demonstrated that CTGF was up-regulated at the early stage of BMP-9 and Wnt3A stimulations and that Wnt3A-regulated CTGF expression was beta-catenin-dependent. RNA interference-mediated knockdown of CTGF expression significantly diminished BMP-9-induced, but not Wnt3A-induced, osteogenic differentiation, suggesting that Wnt3A may also regulate osteoblast differentiation in a CTGF-independent fashion. However, constitutive expression of CTGF was shown to inhibit both BMP-9- and Wnt3A-induced osteogenic differentiation. Exogenous expression of CTGF was shown to promote cell migration and recruitment of mesenchymal stem cells. Our findings demonstrate that CTGF is up-regulated by Wnt3A and BMP-9 at the early stage of osteogenic differentiation, which may regulate the proliferation and recruitment of osteoprogenitor cells; however, CTGF is down-regulated as the differentiation potential of committed pre-osteoblasts increases, strongly suggesting that tight regulation of CTGF expression may be essential for normal osteoblast differentiation of mesenchymal stem cells.

Bone morphogenetic proteins (BMPs) belong to the TGF-beta superfamily and play an important role in development and in many cellular processes. We have found that BMP-2, BMP-6, and BMP-9 induce the most potent osteogenic differentiation of mesenchymal stem cells. Expression profiling analysis has revealed that the Inhibitors of DNA binding/differentiation (Id)-1, Id-2, and Id-3 are among the most significantly up-regulated genes upon BMP-2, BMP-6, or BMP-9 stimulation. Here, we sought to determine the functional role of these Id proteins in BMP-induced osteoblast differentiation. We demonstrated that the expression of Id-1, Id-2, and Id-3 genes was significantly induced at the early stage of BMP-9 stimulation and returned to basal levels at 3 days after stimulation. RNA interference-mediated knockdown of Id expression significantly diminished the BMP-9-induced osteogenic differentiation of mesenchymal progenitor cells. Surprisingly, a constitutive overexpression of these Id genes also inhibited osteoblast differentiation initiated by BMP-9. Furthermore, we demonstrated that BMP-9-regulated Id expression is Smad4-dependent. Overexpression of the three Id genes was shown to promote cell proliferation that was coupled with an inhibition of osteogenic differentiation. Thus, our findings suggest that the Id helix-loop-helix proteins may play an important role in promoting the proliferation of early osteoblast progenitor cells and that Id expression must be down-regulated during the terminal differentiation of committed osteoblasts, suggesting that a balanced regulation of Id expression may be critical to BMP-induced osteoblast lineage-specific differentiation of mesenchymal stem cells.

Different animal strains have different genetic backgrounds that influence their physiological function and pathological process. The differences in genetic background may affect the efficiency of adenoviral infection and target gene expression and further cause different gene therapy results when target genes are delivered with adenoviral vectors. In this study, ectopic bone was not seen in ADCMVBMP4 injection sites, but was formed in ADCMVBMP9 injection sites in all rat strains. The mean volumes of bone induced with ADCMVBMP9 were 0.87 +/- 0.2 cm3 in Wistar, 0.26 +/- 0.1 cm3 in Long-Evans, 0.34 +/- 0.2 cm3 in Sprague-Dawley, 0.44 +/- 0.1 cm3 in ACI, 0.66 +/- 0.2 cm3 in PVG, and 0.58 +/- 0.1 cm3 in Fischer 344 rats. This indicates that ADCMVBMP9 has different bone formation potentials in different immunocompetent rat strains (P = 0.02). The basic levels of CD4+ and CD8+ T cells in blood before viral infection and titers of adenoviral neutralizing antibodies 30 days post-viral infection were significantly different among rat strains (P0.01). The efficiencies of target gene expression delivered with adenovirus were also significantly different in primary muscle cell cultures from different rat strains (P0.01). The different osteogenic potentials of ADCMVBMP9 among rat strains may be, in part, due to the differences in immune factors and target gene expression efficiency in muscle tissue.