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Chondroprogenitors (CPCs) have emerged as a promising cellular therapy for cartilage-related pathologies due to their inherent primed chondrogenic potential. Studies report that the addition of growth factors such as parathyroid hormone (PTH) and Bone Morphogenic Protein (BMP) enhance the chondroinducive potential in chondrocytes and mesenchymal stem cells. This study evaluated if supplementation of the standard culture medium for cell expansion with 1-34 PTH and BMP-9 would enhance the chondrogenic potential of CPCs and reduce their hypertrophic tendency.Human chondrocytes were isolated from patients undergoing total knee replacement for osteoarthritis (n = 3). Following fibronectin adhesion assay, passage 1 CPCs were divided and further expanded under three culture conditions (a) control, i.e., cells continued under standard culture conditions, (b) 1-34 PTH group, additional intermittent 6 h exposure with 1-34 PTH and (c) BMP-9 group, additional BMP-9 during culture expansion. All the groups were evaluated for population-doubling, cell cycle analysis, surface marker and gene expression for chondrogenesis, hypertrophy, multilineage differentiation and GAG (glycosaminoglycan)/DNA following chondrogenic differentiation.Concerning growth kinetics, the BMP-9 group exhibited a significantly lower S-phase and population-doubling when compared to the other two groups. Qualitative analysis for chondrogenic potential (Alcian blue, Safranin O staining and Toluidine blue for GAG) revealed that the BMP-9 group exhibited the highest uptake. The BMP-9 group also showed significantly higher COL2A1 expression than the control group, with no change in the hypertrophy marker expression.BMP-9 can potentially be used as an additive for CPCs expansion, to enhance their chondrogenic potential without affecting their low hypertrophic tendency. The mitigating effects of 1-34PTH on hypertrophy would benefit further investigation when used in combination with BMP-9 to enhance chondrogenesis whilst reducing hypertrophy.

Extracranial arteriovenous malformations (AVMs) are characterized by anomalous arterial-to-venous connections, aberrant angiogenesis, local inflammation and hypoxia, and disorganized histological architecture; however, the precise molecular perturbations leading to this phenotype remain elusive. We hypothesized that extracranial AVM tissue would demonstrate deregulation of the TGF-β/BMP signaling pathway, which may serve as a potential target in the development of molecular-based therapies for AVMs. AVM tissue was harvested during resection from 10 patients with AVMs and compared to control tissue. Blood was collected from 14 AVM patients and 10 patients without AVMs as controls. Expression of TGF-β/BMP pathway components was analyzed using RT-PCR, western blotting, and immunohistochemistry. Circulating levels of TGF-β1 were analyzed by ELISA. Paired t tests were utilized to perform statistical analysis. The mRNA levels of TGF-β1, ALK1, Endoglin (ENG), Smad6, Smad7, and Smad8 were significantly elevated in AVM tissue when compared to controls. Protein levels of TGF-β1 and Smad3 were elevated in AVM tissue while protein levels of BMP-9, ALK1, Smad1, Smad6, and Smad8 were significantly decreased in AVMs. Immunohistochemistry demonstrated increased TGF-β1 in the perivascular cells of AVMs compared to normal controls, and circulating levels of TGF-β1 were significantly higher in AVM patients. Patients with AVMs demonstrate aberrant TGF-β/BMP expression in AVM tissue and blood compared to controls. Targeting aberrantly expressed components of the TGF-β/BMP pathway in extracranial AVMs may be a viable approach in the development of novel molecular therapies, and monitoring circulating TGF-β1 levels may be a useful indicator of treatment success.

Bone morphogenetic protein-9 (BMP-9) is a novel cytokine which is cloned from the fetal mouse liver cDNA library and belongs to the member of the transforming growth factor-β (TGF-β) superfamily. BMP-9 is mainly secreted by the liver and exerts a variety of physiological functions. In this review, we present the latest knowledge on the biochemistry of BMP-9 and its role in glucose metabolism and lipid homeostasis. We introduced the expression site, structure, synthesis, and secretion of BMP-9, as well as BMP-9 signaling pathway. We also discuss the effects of BMP-9 on glucose metabolism and lipid metabolism in different organs. BMP-9 can regulate glucose and lipid homeostasis in the body by inhibiting liver gluconeogenesis, transforming white adipose tissue to brown adipose tissue, promoting muscle glycogen synthesis, increasing the uptake and utilization of glucose by muscle tissue, increasing liver and adipose tissue insulin sensitivity, promoting insulin synthesis and secretion, inhibiting liver lipid deposition, and playing a leptin-like role. Finally, through the results of animal intervention studies and human clinical studies in the review, we deeply understand the association of BMP-9 with obesity, insulin resistance (IR), type 2 diabetes, and non-alcoholic fatty liver disease (NAFLD), which provides new ideas for the prevention and treatment of diseases.

Human amniotic mesenchymal stem cells (hAMSCs) are multiple potent progenitor cells (MPCs) that can differentiate into different lineages (osteogenic, chondrogenic, and adipogenic cells) and have a favorable capacity for angiogenesis. Schnurri-3 (Shn3) is a large zinc finger protein related to Drosophila Shn, which is a critical mediator of postnatal bone formation. Bone morphogenetic protein 9 (BMP9), one of the most potent osteogenic BMPs, can strongly upregulate various osteogenesis- and angiogenesis-related factors in MSCs. It remains unclear how Shn3 is involved in BMP9-induced osteogenic differentiation coupled with angiogenesis in hAMSCs. In this investigation, we conducted a comprehensive study to identify the effect of Shn3 on BMP9-induced osteogenic differentiation and angiogenesis in hAMSCs and analyze the responsible signaling pathway. The results from in vitro and in vivo experimentation show that Shn3 notably inhibits BMP9-induced early and late osteogenic differentiation of hAMSCs, expression of osteogenesis-related factors, and subcutaneous ectopic bone formation from hAMSCs in nude mice. Shn3 also inhibited BMP9-induced angiogenic differentiation, expression of angiogenesis-related factors, and subcutaneous vascular invasion in mice. Mechanistically, we found that Shn3 prominently inhibited the expression of BMP9 and activation of the BMP/Smad and BMP/MAPK signaling pathways. In addition, we further found activity on runt-related transcription factor 2 (Runx2), vascular endothelial growth factor (VEGF), and the target genes shared by BMP and Shn3 signaling pathways. Silencing Shn3 could dramatically enhance the expression of Runx2, which directly regulates the downstream target VEGF to couple osteogenic differentiation with angiogenesis. To summarize, our findings suggested that Shn3 significantly inhibited the BMP9-induced osteogenic differentiation and angiogenesis in hAMSCs. The effect of Shn3 was primarily seen through inhibition of the BMP/Smad signaling pathway and depressed expression of Runx2, which directly regulates VEGF, which couples BMP9-induced osteogenic differentiation with angiogenesis.

Background The purpose of present study was to identify the differentially expressed genes (DEGs) associated with BMP-9-induced osteogenic differentiation of mesenchymal stem cells (MSCs) by using bioinformatics methods. Methods Gene expression profiles of BMP-9-induced MSCs were compared between with GFP-induced MSCs and BMP-9-induced MSCs. GSE48882 containing two groups of gene expression profiles, 3 GFP-induced MSC samples and 3 from BMP-9-induced MSCs, was downloaded from the Gene Expression Omnibus (GEO) database. Then, DEGs were clustered based on functions and signaling pathways with significant enrichment analysis. Pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) demonstrated that the identified DEGs were potentially involved in cytoplasm, nucleus, and extracellular exosome signaling pathway. Results A total of 1967 DEGs (1029 upregulated and 938 downregulated) were identified from GSE48882 datasets. R/Bioconductor package limma was used to identify the DEGs. Further analysis revealed that there were 35 common DEGs observed between the samples. GO function and KEGG pathway enrichment analysis, among which endoplasmic reticulum, protein export, RNA transport, and apoptosis was the most significant dysregulated pathway. The result of protein-protein interaction (PPI) network modules demonstrated that the Hspa5, P4hb, Sec61a1, Smarca2, Pdia3, Dnajc3, Hyou1, Smad7, Derl1, and Surf4 were the high-degree hub nodes. Conclusion Taken above, using integrated bioinformatical analysis, we have identified DEGs candidate genes and pathways in BMP-9 induced MSCs, which could improve our understanding of the key genes and pathways for BMP-9-induced osteogenic of MSCs.

The biocompatibility of cationic liposomes has led to their clinical translation in gene delivery and their application apart from cancer to cardiovascular diseases, osteoporosis, metabolic diseases, and more. We have prepared PEGylated stearyl amine (pegSA) lipoplexes meticulously considering the physicochemical properties and formulation parameters to prepare single unilamellar vesicles (SUV) of

Bone morphogenetic protein 9 (BMP9)/BMP10-ALK1 receptor signaling is essential for endothelial differentiation and vascular morphogenesis. Mutations in ALK1/ACVRL1 and other signal-related genes are implicated in human vascular diseases, and the Alk1/Acvrl1 deletion in mice causes severe impairment of vascular formation and embryonic lethality. In the microarray screen to search for novel downstream genes of ALK1 signaling, we found that the mRNA and protein expression of serum/glucocorticoid-regulated kinase 1 (SGK1) was rapidly up-regulated by the BMP9 stimulation of cultured human endothelial cells. The increase in SGK1 mRNA was completely blocked by the transcriptional inhibitor actinomycin D and significantly suppressed by the siRNA treatment against the co-SMAD transcription factor SMAD4. Upon the BMP9 treatment of endothelial cells, phosphorylated SMAD1/5/9 bound to a consensus site upstream of the SGK1 gene, which was necessary for BMP9-dependent increment of the luciferase reporter activity driven by the SGK1 proximal enhancer. The Sgk1 mRNA expression in mouse embryos was enriched in vascular endothelial cells at embryonic day 9.0-9.5, at which Sgk1 null mice showed embryonic lethality due to abnormal vascular formation, and its mRNA as well as protein expression was clearly reduced in Alk1/Acvrl1 null embryos. These results indicate that SGK1 is a novel target gene of BMP9/BMP10-ALK1 signaling in endothelial cells and further suggest a possibility that down-regulation of the Sgk1 expression may be involved in the mechanisms of vascular defects by the ALK1 signaling deficiency.

To investigate the roles of miR-149 in the progression of human osteosarcoma (OS). miR-149 level was upregulated in tissues from OS patients more than in normal subjects. Cell proliferation and apoptosis assays revealed that miR-149 increased cell proliferation and inhibited cell apoptosis in OS cell line (MG63). An increase of Bcl-2 gene expression and a decrease of cleaved-caspase-3, and cleaved-PARP expression were observed in MG63 cells with transfection of miR-149. Additionally, bone morphogenetic protein 9 (BMP9) was identified as a target of miR-149 in MG63 cells, and BMP9 expression was negatively correlated with miR149 level in OS clinical samples. Co-overexpression of BMP9 with miR-149 in MG63 cells prohibited miR-149-mediated promotive effects on OS progression. Importantly, overexpression of miR-149 conferred chemoresistance in MG63 cells. miR-149 promotes OS progression via targeting BMP9.

It has been reported that bone morphogenetic protein (BMP)-9 has potent osteoinductive properties among the BMP family by adenovirus-transfection experiments. We very recently reported that absorbable collagen sponge (ACS) as a carrier for recombinant human (rh) BMP-9, compared with chitosan sponge, was suitable for inducing bone healing/regeneration by BMP-9 in a rat calvarial defect model. The aim of this study was to evaluate different doses of rhBMP-9/ACS on new bone formation in rat critical size calvarial defects. Bilateral calvarial defects (n = 32) were surgically created in 16 wistar rats and randomly filled with one of the following materials: (1) absorbable collagen sponge (ACS) alone; (2) 1 μg-rhBMP-9/ACS (L-rhBMP-9/ACS); (3) 5 μg-rhBMP-9/ACS (H-rhBMP-9/ACS); and (4) blank defects (control). The animals were sacrificed 8 weeks postsurgery for radiographic and histomorphometric analyses. Bone volume and defect closure were statistically higher in the rhBMP-9/ACS-implanted (L-rhBMP-9/ACS and H-rhBMP-9/ACS) groups when compared with ACS-alone group (p

Promotion of rhBMP2 and rhBMP7 for the routine use to support fracture healing has been hampered by high costs, safety concerns and reasonable failure rates, imposing restrictions in its clinical use. Since there is little debate regarding its treatment potential, there is rising need for a better understanding of the mode of action of these BMPs to overcome its drawbacks and promote more efficacious treatment strategies for bone regeneration. Recently, BMP9, owing to its improved osteogenic potential, is gaining attention as a promising therapeutic alternative. Our study aimed at identifying specific gene expression patterns which may predict and explain individual responses to rhBMP7 and rhBMP9 treatments. Therefore, we investigated the effect of rhBMP7 and rhBMP9 on primary human osteoblasts from 110 donors and corresponding THP-1-derived osteoclasts. This was further compared with each other and our reported data on rhBMP2 response. Based on the individual donor response, we found three donor groups profiting from rhBMP treatment either directly via stimulation of osteoblast function or viability and/or indirectly via inhibition of osteoclasts. The response on rhBMP7 treatment correlated with expression levels of the genes BAMBI, SOST, Noggin, Smad4 and RANKL, while the response of rhBMP9 correlated to the expression levels of Alk6, Endoglin, Smurf1, Smurf2, SOST and RANKL in these donors. Noteworthy, rhBMP9 treatment showed significantly increased osteogenic activity (AP activity and Smad nuclear translocation) when compared to the two clinically used rhBMPs. Based on patient's respective expression profiles, clinical application of rhBMP9 either solely or in combination with rhBMP2 and/or rhBMP7 can become a promising new approach to fit the patient's needs to promote fracture healing.

Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor-β superfamily, regulate a wide range of cellular responses including cell proliferation, differentiation, adhesion, migration, and apoptosis. BMP9, the latest BMP to be discovered, is reportedly expressed in a variety of human carcinoma cell lines, but the role of BMP9 in breast cancer has not been fully clarified. In a previous study, BMP9 was found to inhibit the growth, migration, and invasiveness of MDA-MB-231 breast cancer cells. In the current study, the effect of BMP9 on the bone metastasis of breast cancer cells was investigated. After absent or low expression of BMP9 was detected in the MDA-MB-231 breast cancer cells and breast non-tumor adjacent tissues using Western blot and immunohistochemistry, In our previous study, BMP9 could inhibit the proliferation and invasiveness of breast cancer cells MDA-MB-231 in vitro and in vivo. This paper shows that BMP9 inhibit the bone metastasis of breast cancer cells by activating the BMP/Smad signaling pathway and downregulating connective tissue growth factor (CTGF); however, when CTGF expression was maintained, the inhibitory effect of BMP9 on the MDA-MB-231 cells was abolished. Together, these observations indicate that BMP9 is an important mediator of breast cancer bone metastasis and a potential therapeutic target for treating this deadly disease.

OBJECTIVE: Despite adequate treatment 5-30 % of bone fracture patients experience delayed union. During normal fracture union, bone morphogenetic proteins (BMPs) induce healing through a sequential cascade of events. Improved fracture healing after BMP-2 or -7 supplementation in patients with impaired fracture union suggests a deficiency of one or more of these factors. We postulated that low levels of circulating BMPs may result in delayed bone healing. The aim of this study was to quantify differences in levels of circulating BMP-2, -4, -6, -7, and -9 in patients that have demonstrated normal or delayed fracture healing. PATIENTS AND METHODS: Blood samples were collected from an unselected cohort of 65 patients that had been treated for a diaphyseal tibia or femur fracture. Patients were divided into a group with fracture healing within nine months after injury and a group with delayed fracture union. BMP plasma concentrations were quantified using ELISAs and compared between these two groups. RESULTS: Circulating plasma levels of BMP-2, -4, -6, and -7 did not differ between 34 patients with normal fracture healing and 31 patients with delayed fracture healing. Also the median BMP-9 plasma levels were not statistically different between the two groups of patients. However, the distribution in the patients with normal union showed a wider range (72-2496 pg/ml) compared with the delayed union group (120-816 pg/ml). CONCLUSION: In general, circulating BMP concentrations are not statistically different between patients who demonstrated normal or delayed fracture healing. High circulating BMP-9 levels seem to be associated with faster fracture healing, but are apparently not decisive.

ALK1 (ACVRL1) is a member of the TGFβ receptor family and is expressed predominantly by arterial endothelial cells (EC). Mutations in ACVRL1 are responsible for Hereditary Hemorrhagic Telangiectasia Type 2 (HHT2), a disease manifesting as fragile vessels, capillary overgrowth, and numerous arterio-venous malformations (AVMs). Arterial EC also express EphrinB2, which has multiple roles in vascular development and angiogenesis and is known to be reduced in ACVRL1 knockout mice. Using an in vitro angiogenesis model we find that the Alk1 ligand BMP9 induces EphrinB2 in EC, and this is entirely dependent on expression of Alk1 and at least one of the co-receptors BMPRII or ActRII. BMP9 induces both ID1 and ID3, and both are necessary for full induction of EphrinB2. Loss of Alk1 or EphrinB2 results in increased arterial-venous anastomosis, while loss of Alk1 but not EphrinB2 results in increased VEGFR2 expression and enhanced capillary sprouting. Conversely, BMP9 blocks EC sprouting and this is dependent on Alk1, BMPRII/ActRII and ID1/ID3. Finally, notch signaling overcomes the loss of Alk1 – restoring EphrinB2 expression in EC, and curbing excess sprouting. Thus, in an in vitro model of HHT2, loss of Alk1 blocks BMP9 signaling, resulting in reduced EphrinB2 expression, enhanced VEGFR2 expression, and misregulated EC sprouting and anastomosis.

Bone morphogenetic proteins (BMPs) and 45S5 Bioglass® microspheres (bioactive GM) can increase the differentiation of osteoblasts. Recombinant human BMP-2 (rhBMP-2) is presently the BMP most frequently used in delivery systems and it has already been used in clinical bone healing studies. We have developed a delivery system that combines a collagen Type I gel, BMP and bioactive GM. Since BMP-9 seems to be more osteogenic than BMP-2, we compared the differentiation of MC3T3-E1 preosteoblasts induced by our delivery system containing either a peptide derived from BMP-9 (pBMP-9), or rhBMP-2, both at 100 ng/mL. After 5 days, alkaline phosphatase staining showed that pBMP-9 induced more differentiation than rhBMP-2 in all experimental conditions. Also, bioactive GM increased this BMP effect. Since preosteoblasts secreted matrix metalloproteinases (MMPs) that can degrade collagen, we then studied the influence of the delivery system on MMPs production. We observed that MMP-2 was the major MMP involved in all experimental conditions. In addition, pBMP-9 with bioactive GM generated less MMP-2 than did rhBMP-2 on days 3 and 5. Thus, a delivery system using collagen Type I gel with pBMP-9 and bioactive GM seems to be a promising system for bone regeneration.

This study was designed to see if immunosuppression achieved using local application of cyclosporine A (Cs. A) or CD4 and CD8 antibodies would improve bone formation following intramuscular injections of human BMP-4 and BMP-9 adenoviral vectors (ADhBMP4 and ADhBMP9) in Sprague-Dawley rats. Cs. A was injected into the thigh muscle. After 2 days, ADhBMP4, ADhBMP9, and the antibodies were separately injected into the left and right rear legs. At this time, the number of CD4+/CD3+ cells was significantly lower and the number of CD8+/CD3+ cells higher in the Cs. A group than in the control group (P < 0.01). The total number of white blood cells 3 days following injection of CD4 and CD8 antibodies was significantly lower than that before the injection (P < 0.01). At 4 weeks after the viral and antibody injections, mean bone volumes at the ADhBMP9 treatment sites were 0.29 +/- 0.01 cm3 in the viral control group, 0.17 +/- 0.03 cm3 in the Cs. A-ADhBMPs group, and 0.59 +/- 0.07 cm3 in the antibodies-ADhBMPs group. ADhBMP4 did not induce new bone formation in any group. This study demonstrates that local immunomodulation may improve the osteogenic potential of bone morphogenetic protein gene therapy in the clinical setting.

Bone morphogenetic protein (BMP) adenoviral vectors for the induction of osteogenesis are being developed for the treatment of bone pathology. However, it is still unknown which BMP adenoviral vector has the highest potential to stimulate bone formation in vivo. In this study, the osteogenic activities of recombinant human BMP-2, BMP-4, BMP-6, BMP-7, and BMP-9 adenoviruses were compared in vitro, in athymic nude rats, and in Sprague-Dawley rats. In vitro osteogenic activity was assessed by measuring the alkaline phosphatase activity in C2C12 cells transduced by the various BMP vectors. The alkaline phosphatase activity induced by 2 x 10(5) PFU/well of BMP viral vector was 4890 x 10(-12) U/well for ADCMVBMP-9, 302 x 10(-12) U/well for ADCMVBMP-4, 220 x 10(-12) U/well for ADCMVBMP-6, 45 x 10(-12) U/well for ADCMVBMP-2, and 0.43 x 10(-12) U/well for ADCMVBMP-7. The average volume of new bone induced by 10(7) PFU of BMP vector in athymic nude rats was 0.37+/-0.03 cm(3) for ADCMVBMP-2, 0.89+/-0.07 cm(3) for ADCMVBMP-4, 1.02+/-0.07 cm(3) for ADCMVBMP-6, 0.24+/-0.05 cm(3) for ADCMVBMP-7, and 0.63+/-0.07 cm(3) for ADCMVBMP-9. In immunocompetent Sprague-Dawley rats, no bone formation was demonstrated in the ADCMVBMP-2, ADCMVBMP-4, and ADCMVBMP-7 groups. ADCMVBMP-6 at a viral dose of 10(8) PFU induced 0.10+/-0.03 cm(3) of new bone, whereas ADCMVBMP-9 at a lower viral dose of 10(7) PFU induced more bone, with an average volume of 0.29+/-0.01 cm(3).

A coordinated functional genomics program was implemented to identify secreted polypeptides with therapeutic applications in the treatment of diabetes. Secreted factors were predicted from a diverse expressed-sequence tags (EST) database, representing1,000 cDNA libraries, using a combination of bioinformatic algorithms. Subsequently, approximately 8,000 human proteins were screened in high-throughput cell-based assays designed to monitor key physiological transitions known to be centrally involved in the physiology of type 2 diabetes. Bone morphogenetic protein-9 (BMP-9) gave a positive response in two independent assays: reducing phosphoenolpyruvate carboxykinase (PEPCK) expression in hepatocytes and activating Akt kinase in differentiated myotubes. Purified recombinant BMP-9 potently inhibited hepatic glucose production and activated expression of key enzymes of lipid metabolism. In freely fed diabetic mice, a single subcutaneous injection of BMP-9 reduced glycemia to near-normal levels, with maximal reduction observed 30 hours after treatment. BMP-9 represents the first hepatic factor shown to regulate blood glucose concentration. Using a combination of bioinformatic and high-throughput functional analyses, we have identified a factor that may be exploited for the treatment of diabetes.

Development of anti-angiogenic therapy including the vascular endothelial growth factor (VEGF) antibodies and VEGF-tyrosine kinase receptors has been a major landmark in cancer therapy leading improvement in survival in several cancers. While anti-angiogenic therapy is effective in some settings, resistance often develops owing to evasive, alternative pathways. Novel targets for anti-angiogenic therapy are urgently required to provide treatment alternatives in patients whose tumors are unresponsive to approved anti-angiogenic agents; one such pathway is the bone morphogenetic proteins (BMP 9 and BMP 10) that activate the type I activin receptor-like kinase-1 (ALK1), which has been implicated in the development of functional vasculature. Dalantercept (ACE-041) is a novel anti-angiogenic agent, which is a soluble form of ALK1, and acts as a ligand trap for BMP 9 and BMP 10, inhibiting their interaction with ALK1, which further disrupts the process of vascular development. This review will discuss the preclinical and clinical development of dalantercept as a novel anti-angiogenic therapy in treating a variety of cancers and its distinct safety profile compared to other anti-VEGF agents. We will also discuss the ongoing and completed studies of dalantercept, including combination studies with other VEGF-directed therapies.

Background The development of chemical refolding of transforming growth factor-beta (TGF-β) superfamily ligands has been instrumental to produce the recombinant proteins for biochemical studies and exploring the potential of protein therapeutics. The osteogenic human bone morphogenetic protein-2 (hBMP-2) and its Drosophila DPP homolog were the early successful cases of refolding into functional form. Despite the similarity in their three dimensional structure and amino acid sequences, several other TGF-β superfamily ligands could not be refolded readily by the same methods. Results Here, we report a comprehensive study on the variables of a rapid-dilution refolding method, including the concentrations of protein, salt, detergent and redox agents, pH, refolding duration and the presence of aggregation suppressors and host-cell contaminants, in order to identify the optimal condition to refold human BMP-9 (hBMP-9). To produce a recombinant form of hBMP-9 in E. coli cells, a synthetic codon-optimized gene was designed to encode the mature domain of hBMP-9 (Ser320 – Arg429) directly behind the first methionine, which we herein referred to as MB109. An effective purification scheme was also developed to purify the refolded MB109 to homogeneity with a final yield of 7.8 mg from 100 mg of chromatography-purified inclusion bodies as a starting material. The chemically refolded MB109 binds to ALK1, ActRIIb and BMPRII receptors with relatively high affinity as compared to other Type I and Type II receptors based on surface plasmon resonance analysis. Smad1-dependent luciferase assay in C2C12 cells shows that the MB109 has an EC50 of 0.61 ng/mL (25 pM), which is nearly the same as hBMP-9. Conclusion MB109 is prone to be refolded as non-functional dimer and higher order multimers in most of the conditions tested, but bioactive MB109 dimer can be refolded with high efficiency in a narrow window, which is strongly dependent on the pH, refolding duration, the presence of aggregation suppressors and the concentrations of protein, salt and detegent. These results add to the current understanding of producing recombinant TGF-β superfamily ligands in the microbial E. coli system. An application of the technique to produce a large number of synthetic TGF-β chimeras for activity screen is also discussed.

Bone morphogenetic proteins (BMPs) are multifunctional cytokines of the transforming growth factor β (TGFβ) superfamily with potential therapeutic applications due to their broad biological functionality. Designing BMP mimetics with specific activity will contribute to the translational potential of BMP-based therapies. Here, we report a BMP9 peptide mimetic, P3, designed from the type I receptor binding site, which showed millimolar binding affinities for the type I receptor activin receptor like kinase 1 (ALK1), ALK2 and ALK3. Although showing no baseline activity, P3 significantly enhanced BMP9-induced Smad1/5 phosphorylation as well as ID1, BMPR2, HEY1 and HEY2 gene expression in pulmonary artery endothelial cells (hPAECs), and this activity is dependent on its alpha helix propensity. However, in human dermal microvascular endothelial cells, P3 did not affect BMP9-induced Smad1/5 phosphorylation, but potently inhibited ALK3-dependent BMP4-induced Smad1/5 phosphorylation and gene expression. In C2C12 mouse myoblast cells, P3 had no effect on BMP9-induced osteogenic signalling, which is primarily mediated by ALK2. Interestingly, a previously published peptide from the knuckle region of BMP9 was found to inhibit BMP4-induced Smad1/5 phosphorylation. Together, our data identify a BMP9-derived peptide that can selectively enhance ALK1-mediated BMP9 signalling in hPAECs and modulate BMP9 and BMP4 signalling in a cell type-specific manner.