scholarly journals A Cyclic BMP-2 Peptide Upregulates BMP-2 Protein-Induced Cell Signaling in Myogenic Cells

Polymers ◽  
2021 ◽  
Vol 13 (15) ◽  
pp. 2549
Author(s):  
Vijaya Narasimha Gudivada ◽  
Chen-Ji Huang ◽  
Yueh-Hsia Luo ◽  
Guo-Chung Dong
Keyword(s):  
Active Region ◽  
Binding Affinity ◽  
Cyclic Peptide ◽  
Chip Surface ◽  
Suitable Candidate ◽  
Protein Receptor ◽  
Morphogenetic Protein ◽  
Related Transcription Factor ◽  
Peptide Analogues

In the current study, we designed four cyclic peptide analogues by incorporating two cysteine residues in a BMP-2 linear knuckle epitope in such a way that the active region of the peptide could be either inside or outside the cyclic ring. Bone morphogenetic protein receptor BMPRII was immobilized on the chip surface, and the interaction of the linear and cyclic peptide analogues was studied using surface plasmon resonance (SPR). From the affinity data, the peptides with an active region inside the cyclic ring had a higher binding affinity in comparison to the other peptides. To confirm that our affinity data are in line in vitro, we studied the expression levels of RUNX2 (runt-related transcription factor) and conducted an osteogenic marker alkaline phosphatase (ALP) assay and staining. Based on the affinity data and the in vitro experiments, peptide P-05 could be a suitable candidate for osteogenesis, with higher binding affinity and increased RUNX2 and ALP expression in comparison to the linear peptides.

Crystal Structure Of Tissue Factor Pathway Inhibitor (TFPI) Kunitz Domain 1 and 2 In Complex With An Inhibitory Cyclic Peptide,

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 453-453
Author(s):  
Michael Dockal ◽  
Rudolf Hartmann ◽  
Markus Fries ◽  
Katerina Prohaska ◽  
Robert Pachlinger ◽  
...  
Keyword(s):  
Whole Blood ◽  
Research Funding ◽  
Cyclic Peptide ◽  
Intramolecular Interactions ◽  
Chip Surface ◽  
Activation Assay ◽  
Kunitz Type ◽  
Β Sheet ◽  
Interaction Surface

TFPI is a Kunitz-type protease inhibitor which efficiently regulates the extrinsic coagulation pathway. It is composed of three flexible linked Kunitz-type domains (KD) where KD1 and KD2 are involved in efficient inhibition of TF/FVIIa and FXa. TFPI inhibition has been shown to improve coagulation and hemostasis in hemophilia models in vitro and in vivo. Recombinant KD1-KD2 (residues 22-150) produced by E. coli and complexed to JBT-B5, a cyclic peptide composed of 23 amino acids, was co-crystallized in 20% w/v PEG6000 and 50mM imidazole, pH8.0. JBT-B5 binding to TFPI was verified by BiaCore experiments with TFPI immobilized on a chip surface. Functional inhibition of TFPI by JBT-B5 was tested in model assays including TFPI inhibition of FXa, FX activation by TF/FVIIa, inhibition of TFPI released upon platelet activation, and by global hemostatic assays including calibrated automated thrombography in FVIII-inhibited plasma and rotational thomboelastometry (ROTEM) using FVIII-inhibited whole blood. Inhibition of cell surface TFPI was analyzed in an FX activation assay performed on HUVECs. TFPI KD1-KD2 bound to JBT-B5 formed a crystal containing two independent complexes in the asymmetric unit. The complexes belong to the orthorhombic spacegroup P212121 and diffracted to a maximum resolution of 1.95 Å. To our knowledge, this is the first TFPI structure consisting of KD1, KD2 and their linker. The KD1-KD2 structure is fully defined in the electron density. Both domains show a Kunitz-type structure, where only ∼1/3 of the structure is engaged in secondary structure elements. These are two short α-helical elements at Ser24-Ala27(KD1)/Asp95-Phe98(KD2) (α1/α3) and Leu69-Met75/Leu140-Glu148 (α2/α4) and a two-stranded β sheet comprising Met39-Asn45/Ile110-Asn116 (β1/β3) and Arg49-Ile55/Lys120-Lys126 (β2/β4). These elements form the topological framework that is stabilized by the three canonical disulfide bonds involving Cys26-Cys76, Cys35-Cys59, and Cys51-Cys72 in KD1 and Cys95-Cys147, Cys106-Cys130, and Cys122-Cys143 in KD2. The 23mer TFPI inhibitor, JBT-B5, is sandwiched between the two Kunitz domains of TFPI and assumes a β-hairpin-like structure. It can be segmented into (i) a two-stranded β sheet comprising Tyr2-Ala8 and Thr17-Phe23; (ii) and a long β-turn loop comprising Met9-Met16. The β-sheet is stabilized by a disulfide bridge (Cys7 and Cys18) and a hydrophobic zipper comprising the side chains of Tyr3, Trp5 and Trp20. Strikingly, JBT-B5 locks KD1-KD2 in a distinct conformational state in which both Kunitz-domains are related via a two-fold symmetry and reactive center loops (RCL) from each KD are forced to opposite sides. The interactions between KD1-KD2 and JBT-B5 are extensive, as are the intramolecular interactions within JBT-B5. Analyzing the interactions between KD1-KD2 and JBT-B5 with the PISA server resulted in a total interaction surface of 1340Å2. More than 2/3 of the interaction surface consists of a hydrophobic anchor in JBT-B5 which interact with residues spread all over TFPI including KD1, KD2 and their linker. In addition, several polar interactions stabilize the KD1-KD2/JBT-B5 complex, explaining JBT-B5´s exlusive binding to human TFPI. Interaction studies revealed high affinity binding to TFPI (KD: 0.5nM). The highly complex and extensive interaction of JBT-B5 with TFPI translates to a highly efficient inhibition of recombinant TFPI and;TFPI released from activated platelets which is demonstrated in model assays on FXa (EC50: 1.3nM) and FXa generation by TF/FVIIa (EC50: 0.2nM). HUVE cell-based FX activation and global hemostasis assays such as thrombin generation in hemophilia plasma (EC50: 4nM) and ROTEM in FVIII-inhibited whole blood confirm the inhibition of all physiologic TFPI forms. For the first time, a TFPI structure comprising KD1, KD2 and their linker in complex with a TFPI- inhibitory cyclic peptide was solved. This structure provides atomic details explaining the inhibitory mode of action of this efficient TFPI antagonist, and will guide the design of efficient inhibitors for use in hemophilia treatment. Disclosures: Dockal: Baxter Innovations GmbH, Vienna, Austria: Employment. Hartmann:Baxter Innovations GmbH, Vienna, Austria: Employment. Fries:Baxter Innovations GmbH, Vienna, Austria: Employment. Prohaska: Baxter Innovations GmbH, Vienna, Austria: Employment. Pachlinger:Baxter Innovations GmbH, Vienna, Austria: Employment. Polakowski:3B Pharmaceuticals, Berlin, Germany: Employment. Brandstetter: Baxter Innovations GmbH, Vienna, Austria: Research Funding. Rosing:Baxter Innovations GmbH, Vienna, Austria: Consultancy, Research Funding. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.

scholarly journals Expression of Bone Morphogenetic Protein Receptor (BMPR) during Perinatal Ovary Development and Primordial Follicle Formation in the Hamster: Possible Regulation by FSH

Endocrinology ◽  
2008 ◽  
Vol 150 (4) ◽  
pp. 1886-1896 ◽  
Author(s):  
Cheng Wang ◽  
Shyamal K. Roy
Keyword(s):  
Bone Morphogenetic Protein ◽  
Somatic Cells ◽  
Primordial Follicle ◽  
Ovary Development ◽  
Mrna Levels ◽  
Primordial Follicles ◽  
Bone Morphogenetic Protein Receptor ◽  
Protein Receptor ◽  
Morphogenetic Protein

To understand whether bone morphogenetic protein plays any role in the formation of primordial follicles in the hamster, we examined the temporal and spatial expression of bone morphogenetic protein receptor (BMPR) mRNA and protein in embryonic (E) 13 through postnatal day (P) 15 ovarian cells and a possible regulation by FSH during the formation of primordial follicles on P8. BMPRIA and BMPRII mRNA levels were significantly higher than that of BMPR1B throughout ovary development. BMPRIA and BMPRII mRNA levels increased significantly on E14 and declined by P5 through P6. Whereas BMPRII mRNA increased again by P7, BMPRIA mRNA levels increased through P8 concurrent with primordial follicle formation. In contrast, BMPRIB mRNA levels increased greater than 10-fold on P7-9, with a further 3-fold increase by P10. BMPR proteins were low in the somatic cells and oocytes on E13 but increased progressively during postnatal development. BMPR expression in somatic cells increased markedly on P8. Whereas BMPRII expression declined by P10 and remained steady thereafter, BMPRIA protein expression fluctuated until P15 when it became low and steady. Overall, BMPRIB immunoreactivity also declined by P10 and then remained low in the interstitial cells through P15. FSH antiserum treatment on E12 significantly attenuated receptor mRNA and protein levels by P8, but equine chorionic gonadotropin replacement on P1 reversed the inhibition. Furthermore, FSH in vitro up-regulated BMPR levels in P4 ovaries. This unique pattern of BMPR expression in the oocytes and somatic cells during perinatal ovary development suggests that BMP may play a regulatory role in primordial follicle formation. Furthermore, FSH may regulate BMP action by modulating the expression of its receptors.

Abstract 445: Endothelial Inflammation and Loss of Bone Morphogenetic Protein Receptor 2 in Oscillatory Shear Stress Model

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Collins Ezeuka
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Laminar Flow ◽  
Bone Morphogenetic Protein ◽  
Aortic Endothelial Cells ◽  
Bone Morphogenetic Protein Receptor ◽  
Endothelial Inflammation ◽  
Protein Receptor ◽  
Morphogenetic Protein

Background: Bone Morphogenetic Protein Receptor II (BMPR2) plays an unexpected role as a critical anti-inflammatory and anti-atherogenic protein in endothelial cells (ECs) via a reactive oxygen species (ROS) and NFκB-dependent mechanism. Pro-atherogenic stimuli such as disturbed laminar flow, angiotensin II, hypercholesterolemia and the pro-inflammatory cytokine TNFα, significantly downregulate BMPR2 expression in endothelium, while anti-atherogenic stimuli such as laminar flow and statins upregulate BMPR2’s expression in vivo and in vitro. These findings suggest that there may be a common mechanism by which pro-atherogenic factors downregulate BMPR2 expression and that protecting or restoring its expression could be a novel therapeutic approach for prevention and treatment of atherosclerosis. Our preliminary studies have identified microRNAs that possibly play a causative role in the loss of BMPR2, by binding to its 3’-UTR, leading to degradation of BMPR2, endothelial dysfunction, inflammation, and subsequent atherosclerosis. Hypothesis: Rescuing loss of BMPR2 will decrease endothelial inflammation and atherosclerosis Methods: Our in vitro model of disturbed blood flow is characterized by a cone and plate system, wherein mouse aortic endothelial cells are subjected to unidirectional laminar shear (LS 15 dyn/cm2) or oscillatory shear (OS, +/1 5 dyn/cm2 at 1 Hz frequency) for 24 hours. Endothelial cell inflammatory markers, BMPR2, and specific microRNA mRNA transcript fold changes, were then assessed via qPCR. Results: Under oscillatory flow conditions, in our in vitro shear stress system, BMPR2 is lost and mouse aortic endothelial cells acquire an inflammation phenotype, with a corresponding increase in the fold change of mRNA for microRNAs-17, 21, 25, and 181. Conclusion: We have identified microRNAs that may target BMPR2, leading to its degradation, and subsequent onset of endothelial inflammation. Blocking the aforementioned microRNAs may represent a novel therapy in the treatment of endothelial inflammation and subsequent atherosclerosis.

Abstract 1636: Adverse Effect of Estrogen on Bone Morphogenetic Protein Receptor Signal Pathway in Pulmonary Arterial Endothelial Cells

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Hiroaki Ichimori ◽  
Shigetoyo Kogaki ◽  
Hidekazu Ishida ◽  
Jun Narita ◽  
Toshiki Uchikawa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Estrogen Receptor ◽  
Bone Morphogenetic Protein ◽  
Signal Pathway ◽  
Bone Morphogenetic Protein Receptor ◽  
Protein Receptor ◽  
Morphogenetic Protein ◽  
Pulmonary Arterial ◽  
Arterial Endothelial Cells

Gender differences in the development of Pulmonary Artery Hypertension (PAH) have been documented in both human and animal studies. In human, idiopathic PAH is predominantly a disease of young women in their child-bearing years, which suggests a role of female sex hormones in the pathogenesis of PAH. However, the effect of sex hormones on pulmonary vasculatures and the development of PAH has not been fully understood. Recent researches have revealed genetic predisposition such as BMPR (Bone Morphogenetic Protein Receptor). The aim of the present study is to investigate the effect of β-estradiol (E2) and oxygen concentration upon BMPR signal pathway in pulmonary arterial endothelial cells (PAEC) in vitro. Human and rat PAEC were cultured and we examined the expression of BMPR2, BMP-regulated Smads, and Id1 under 21% or 1% O 2 with BMP2 stimulation. Then, we investigate changes in the expression of these molecules in the presence of E2 with or without estrogen receptor antagonist (ICI 182.780.). First, we confirmed that estrogen receptor α and β were expressed in both PAECs. Second, we demonstrated that the expression of mRNA transcripts for BMPR2 and Id1 in PAEC was reduced after exposure to 24 hours’ hypoxia. In addition, E2 decreased the expression of phosphorylated Smad (p-Smad)1/5/8 in a dose-dependent manner (10 −10 M-10 −7 M) and p-Smad1/5/8 expression were decreased about 80% by 10 −7 M of E2. These attenuation of p-Smad1/5/8 expression were rescued by ICI182.780. Third, under normoxic condition with cobalt chloride or deferoxamine to prevent the degradation of HIF (hypoxia-inducible factor)-1α, the presence of E2 decreased the expression of p-Smad1/5/8 like under hypoxia. Conversely, administration of HIF-1α inhibitor (YC-1) canceled the reduced expression of p-Smad1/5/8 like under normoxia. Under hypoxia, the presence of E2 attenuates the BMPR signal pathway in PAEC in vitro. Our data indicated that the advance effect of E2 on BMPR signal pathway was associated with HIF-1α and estrogen receptor. Our observations provide the first evidence that female sex hormone affects on BMPR signal pathway, which can offer new strategies for the treatment of PAH.

scholarly journals Bioactivity of a Novel Polycaprolactone-Hydroxyapatite Scaffold Used as a Carrier of Low Dose BMP-2: An In Vitro Study

Polymers ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 466
Author(s):  
Pawornwan Rittipakorn ◽  
Nuttawut Thuaksuban ◽  
Katanchalee Mai-ngam ◽  
Satrawut Charoenla ◽  
Warobon Noppakunmongkolchai
Keyword(s):  
In Vitro Study ◽  
Bone Morphogenetic Protein 2 ◽  
Morphogenetic Protein ◽  
Cell Scaffold ◽  
Related Transcription Factor ◽  
Group A ◽  
In Vitro Response ◽  
Hydroxyapatite Scaffold ◽  
Group B

Scaffolds of polycaprolactone-30% hydroxyapatite (PCL-30% HA) were fabricated using melt stretching and multilayer deposition (MSMD), and the in vitro response of osteoblasts to the scaffolds was assessed. In group A, the scaffolds were immersed in 10 µg/mL bone morphogenetic protein-2 (BMP-2) solution prior to being seeded with osteoblasts, and they were cultured in the medium without BMP-2. In group B, the cell-scaffold constructs without BMP-2 were cultured in medium containing 10 µg/mL BMP-2. The results showed greater cell proliferation in group A. The upregulation of runt-related transcription factor 2 and osteocalcin genes correlated with the release of BMP-2 from the scaffolds. The PCL-30% HA MSMD scaffolds appear to be suitable for use as osteoconductive frameworks and BMP-2 carriers.

scholarly journals Stimulation of Human Osteoblast Differentiation in Magneto-Mechanically Actuated Ferromagnetic Fiber Networks

2019 ◽  
Vol 8 (10) ◽  
pp. 1522 ◽  
Author(s):  
Galit Katarivas Levy ◽  
Mark A. Birch ◽  
Roger A. Brooks ◽  
Suresh Neelakantan ◽  
Athina E. Markaki
Keyword(s):  
Porous Scaffold ◽  
Bone Ingrowth ◽  
Bone Morphogenetic Protein 2 ◽  
Morphogenetic Protein ◽  
Related Transcription Factor ◽  
Implantable Biomedical Devices ◽  
Experimental Use ◽  
Osteogenesis In Vitro

There is currently an interest in “active” implantable biomedical devices that include mechanical stimulation as an integral part of their design. This paper reports the experimental use of a porous scaffold made of interconnected networks of slender ferromagnetic fibers that can be actuated in vivo by an external magnetic field applying strains to in-growing cells. Such scaffolds have been previously characterized in terms of their mechanical and cellular responses. In this study, it is shown that the shape changes induced in the scaffolds can be used to promote osteogenesis in vitro. In particular, immunofluorescence, gene and protein analyses reveal that the actuated networks exhibit higher mineralization and extracellular matrix production, and express higher levels of osteocalcin, alkaline phosphatase, collagen type 1α1, runt-related transcription factor 2 and bone morphogenetic protein 2 than the static controls at the 3-week time point. The results suggest that the cells filling the inter-fiber spaces are able to sense and react to the magneto-mechanically induced strains facilitating osteogenic differentiation and maturation. This work provides evidence in support of using this approach to stimulate bone ingrowth around a device implanted in bone and can pave the way for further applications in bone tissue engineering.

scholarly journals Repercussion of nonsteroidal anti-inflammatory drugs on the gene expression of human osteoblasts

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5415 ◽  
Author(s):  
Lucia Melguizo-Rodríguez ◽  
Víctor J. Costela-Ruiz ◽  
Francisco J. Manzano-Moreno ◽  
Rebeca Illescas-Montes ◽  
Javier Ramos-Torrecillas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transforming Growth Factor ◽  
Bone Morphogenetic Protein 2 ◽  
Morphogenetic Protein ◽  
Related Transcription Factor ◽  
Inflammatory Drugs ◽  
Anti Inflammatory ◽  
And Function ◽  
Osteoblast Maturation

Background Nonsteroidal anti-inflammatory drugs (NSAIDs) are frequently used in clinical practice, which can have adverse effects on the osteoblast. The objective of this study was to determine the effect of NSAIDs on the osteoblast by analyzing the gene expression of different markers related to osteoblast maturation and function when treated in vitro with different NSAIDs. Methods Three human osteoblast lines from bone samples of three healthy volunteers were treated with 10 µM acetaminophen, indomethacin, ketoprofen, diclofenac, ibuprofen, ketorolac, naproxen, and piroxicam. The gene expression of different markers (run related transcription factor 2 [RUNX-2], type 1 collagen [COL-I], osterix [OSX], osteocalcin [OSC], bone morphogenetic protein 2 [BMP-2] and 7 [BMP-7], transforming growth factor β1 [TGF-β1], and TGFβ receptors [TGFβR1, TGFβR2; TGFBR3]) were analyzed by real-time PCR at 24 h of treatment. Results Expression of RUNX-2, COL-I, OSX, was reduced by treatment with all studied NSAIDs, OSC expression was reduced by all NSAIDs except for ketoprofen, naproxen, or piroxicam. Expression of BMP-7 was reduced by all NSAIDs; BMP-2 was reduced by all except for naproxen. In general, NSAID treatment increased the expression of TGF-β1, but not of its receptors (TGFβ-R1, TGFβ-R2, andTFGβ-R3), which was either unchanged or reduced by the treatment. Conclusion These data confirm that NSAIDs can affect osteoblast physiology, suggesting their possible impact on bone.

scholarly journals Mechanisms of Palmitate-Induced Lipotoxicity in Human Osteoblasts

Endocrinology ◽  
2014 ◽  
Vol 155 (1) ◽  
pp. 108-116 ◽  
Author(s):  
Krishanthi Gunaratnam ◽  
Christopher Vidal ◽  
Jeffrey M. Gimble ◽  
Gustavo Duque
Keyword(s):  
Confocal Microscopy ◽  
Bone Cells ◽  
Bone Morphogenetic Protein 2 ◽  
Nodule Formation ◽  
Human Osteoblasts ◽  
Morphogenetic Protein ◽  
Negative Effect ◽  
Related Transcription Factor ◽  
The Relationship

The interest in the relationship between fat and bone has increased steadily during recent years. Fat could have a lipotoxic effect on bone cells through the secretion of fatty acids. Palmitate is the most prevalent fatty acid secreted by adipocytes in vitro. Considering that palmitate has shown a high lipotoxic effect in other tissues, here we characterized the lipotoxic effect of palmitate on human osteoblasts (Obs). Initially we tested for changes in palmitoylation in this model. Subsequently we compared the capacity of Obs to differentiate and form bone nodules in the presence of palmitate. From a mechanistic approach, we assessed changes in nuclear activity of β-catenin and runt-related transcription factor 2 (Runx2)/phosphorylated mothers against decapentaplegic (Smad) complexes using Western blotting and confocal microscopy. Quantitative real-time PCR showed negative changes in gene expression of palmitoyltransferase genes. Furthermore, palmitate negatively affected differentiation and bone nodule formation and mineralization by Obs. Although the expression of β-catenin in palmitate-treated cells was not affected, there was a significant reduction in the transcriptional activities of both β-catenin and Runx2. Confocal microscopy showed that whereas Runx2 and Smad-4 and -5 complex formation was increased in bone morphogenetic protein-2-treated cells, palmitate had a negative effect on protein expression and colocalization of these factors. In summary, in this study we identified potential mechanisms of palmitate-induced lipotoxicity, which include changes in palmitoylation, defective mineralization, and significant alterations in the β-catenin and Runx2/Smad signaling pathways. Our evidence facilitates the understanding of the relationship between fat and bone and could allow the development of new potential therapies for osteoporosis in older persons.

scholarly journals Stimulation of Human Osteoblast Differentiation in Magneto-Mechanically Actuated Ferromagnetic Fibre Networks

2019 ◽  
Author(s):  
Galit Katarivas Levy ◽  
Mark A. Birch ◽  
Roger A. Brooks ◽  
Suresh Neelakantan ◽  
Athina E. Markaki
Keyword(s):  
Porous Scaffold ◽  
Bone Ingrowth ◽  
Bone Morphogenetic Protein 2 ◽  
Morphogenetic Protein ◽  
Related Transcription Factor ◽  
Implantable Biomedical Devices ◽  
Experimental Use ◽  
Osteogenesis In Vitro

There is currently an interest in “active” implantable biomedical devices that include mechanical stimulation as an integral part of their design. This paper reports the experimental use of a porous scaffold made of interconnected networks of slender ferromagnetic fibres that can be actuated in vivo by an external magnetic field applying strains to in-growing cells. Such scaffolds have been previously characterized in terms of their mechanical and cellular responses. In this study, it is shown that the shape changes induced in the scaffolds can be used to promote osteogenesis in vitro. In particular, immunofluorescence, gene and protein analyses reveal that the actuated networks exhibit higher mineralization and extracellular matrix production, and express higher levels of osteocalcin, alkaline phosphatase, collagen type 1a1, runt-related transcription factor 2 and bone morphogenetic protein 2 than the static controls at the 3-week time point. The results suggest that the cells filling the inter-fibre spaces are able to sense and react to the magneto-mechanically induced strains facilitating osteogenic differentiation and maturation. This work provides evidence in support of using this approach to stimulate bone ingrowth around a device implanted in bone and can pave the way for further applications in bone tissue engineering.

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