Journal: Circulation Research

Article Title: Bone Morphogenetic Protein 4 Promotes Pulmonary Vascular Remodeling in Hypoxic Pulmonary Hypertension

doi: 10.1161/01.res.0000181152.65534.07

Figure Lengend Snippet: Figure 5. Attenuation of hypoxia-induced peripheral vascular muscularization in Bmp4lacZ/ mice. A, Degree of peripheral musculariza- tion in wild-type and Bmp4 mutant mice (data represented as meanSEM; wild type: normoxia, n11, 3 weeks hypoxia, n7, and 5 weeks hypoxia, n8; Bmp4lacZ/: normoxia, n10, 3 weeks hypoxia, n9, and 5 weeks hypoxia, n7). Two-way ANOVA, P0.05: *for all degrees of muscularization in wild-type normoxia vs hypoxia, **3-week hypoxic wild type vs Bmp4lacZ/, ***5-week hypoxic wild type vs Bmp4lacZ/. B through E, Representative immunofluorescence staining of lung sections for von Willebrand Factor (red) and -actin (green) and merged (yellow) under normoxic conditions (B and D) and on exposure to hypoxia for 3 weeks (C and E) in wild-type (B and C) and Bmp4lacZ/ mutant (D and E) mice. Small arrowheads indicate representative partially muscularized arteries; larger arrowheads, fully muscularized arteries; and small arrows, representative nonmuscularized arteries. F through I, Immunohistochemical peroxidase- based staining for -actin demonstrating normal alveolar structure under normoxic conditions (F and H) and on exposure to hypoxia for 3 weeks (G and I) in wild-type (F and G) and Bmp4lacZ/ mutant (H and I) mice. Images taken at 200 magnification.

Article Snippet: After 48-hour serum starvation, media were treated with either BMP ligands, a 1:2 dilution of 24-hour 1% oxygen, or normoxic conditionally immortalized mPMVEC-conditioned media with or without recombinant Noggin (Alpha Diagnostics) or neutralizing anti-BMP4 monoclonal antibody (R&D systems, catalogue No. MAB757), and cells were cultured for an additional 24 hours at 1% oxygen while labeling with 3H-thymidine.

Techniques: Mutagenesis, Staining, Immunohistochemical staining

Journal: Circulation Research

Article Title: Bone Morphogenetic Protein 4 Promotes Pulmonary Vascular Remodeling in Hypoxic Pulmonary Hypertension

doi: 10.1161/01.res.0000181152.65534.07

Figure Lengend Snippet: Figure 7. Activation of BMP4 signaling by hypoxia in mouse PMVECs. A, Conditionally immortalized mPMVECs were cul- tured under serum-free conditions under normoxia or 1% oxy- gen for 24 and 72 hours. BMP4 protein secreted into the media was pulled down on heparin-sepharose columns and detected by Western blot. Concurrent expression of BMP4, phospho- Smad1/5/8, Id1, and HIF-1 were determined in the cell lysates. B, Primary mouse PMVECs were isolated and cultured under normoxia or 1% oxygen for 24 and 72 hours, and BMP4 protein secretion into the conditioned media was determined as before.

Article Snippet: After 48-hour serum starvation, media were treated with either BMP ligands, a 1:2 dilution of 24-hour 1% oxygen, or normoxic conditionally immortalized mPMVEC-conditioned media with or without recombinant Noggin (Alpha Diagnostics) or neutralizing anti-BMP4 monoclonal antibody (R&D systems, catalogue No. MAB757), and cells were cultured for an additional 24 hours at 1% oxygen while labeling with 3H-thymidine.

Techniques: Activation Assay, Western Blot, Expressing, Isolation, Cell Culture

Journal: JBMR plus

Article Title: Bone Morphogenetic Protein 4 Gene Therapy in Mice Inhibits Myeloma Tumor Growth, But Has a Negative Impact on Bone.

doi: 10.1002/jbm4.10247

Figure Lengend Snippet: Fig. 1. Overview of experimental set up. In brief, 14 weeks old RAG2−/−γC−/−mice were implanted with calcium phosphate scaffolds containing human MSCs. AAV8-BMP4 or AAV8-CTRL were administered 8 weeks post scaffold implantation by tail-vein injection (1012 viral particles/100 μL saline/mouse). 10 weeks post implantation 106 fluorescently labeled KJON myeloma cells were injected into 3 out of 4 scaffolds. Empty scaffold refers to scaffold with MSCs, but without tumor cells.

Article Snippet: Recombinant murine (rm) BMP4 (Cat# 5020-BP), recombinant human (rh) BMP4 (Cat# 314-BP), rhM-CSF (Cat# 216-MC), rhRANKL (Cat# 390-TN), neutralizing BMP4 antibody (Cat# MAB50201), and rat isotype control (MAB006) were from R&D Systems (Bio-Techne, Abingdon, UK).

Techniques: Injection, Saline, Labeling

Journal: JBMR plus

Article Title: Bone Morphogenetic Protein 4 Gene Therapy in Mice Inhibits Myeloma Tumor Growth, But Has a Negative Impact on Bone.

doi: 10.1002/jbm4.10247

Figure Lengend Snippet: Fig. 2. BMP4 inhibited myeloma cell growth in vivo. (A) the myeloma cell line KJON was treated with different doses of rmBMP-4. After 48 h, the numbers of viable cells were determined by labeling with annexin V-FITC and propidium iodide (PI). The amount of annexin V/PI negative cells was plotted, n = 3 independent experiments. ****; p < .0001, 1-way ANOVA, Dunnett’s multiple comparisons test. (B) the figure shows representative dot plots of the results presented in a. cells in the lower left quadrant, which were negative for both annexin V and PI, were considered viable. (C) To estimate tumor burden, the amount of near-infrared fluorescent protein (iRFP) in each scaffold was measured weekly using the pearl imager system in AAV8-CTRL mice (n = 30) and AAV8-BMP4 mice (n = 27), p < .01, 2-way ANOVA, Bonferroni post test. (D) Representative images of tumor burden in AAV8-CTRL (top) and AAV8-BMP4 (bottom) treated mice are shown. (E) Amount of BMP4 in the serum was estimated at end point by semi-quantitative western blotting. (F) Serum from AAV8-CTRL or AAV8-BMP4 mice, 4% final serum concentration, was added to cultures of NS0 murine myeloma cells and incubated for 48 h. the graph shows relative ATP-levels compared to medium control (not shown) as a measure of reduced cell viability. The reduction in cell viability was counteracted by a BMP4 neutralizing antibody, p < .0005, Bonferroni post test. all error bars represent SEM.

Article Snippet: Recombinant murine (rm) BMP4 (Cat# 5020-BP), recombinant human (rh) BMP4 (Cat# 314-BP), rhM-CSF (Cat# 216-MC), rhRANKL (Cat# 390-TN), neutralizing BMP4 antibody (Cat# MAB50201), and rat isotype control (MAB006) were from R&D Systems (Bio-Techne, Abingdon, UK).

Techniques: In Vivo, Labeling, Western Blot, Concentration Assay, Incubation, Control

Journal: Blood

Article Title: In anemia of multiple myeloma, hepcidin is induced by increased bone morphogenetic protein 2

doi: 10.1182/blood-2010-03-274571

Figure Lengend Snippet: Effect of cytokine inhibitors on the induction of HAMP promoter activity by MM patient sera. HuH7 cells were cotransfected with WT-HAMP promoter-firefly luciferase construct and pTK-RL construct. Next, cells were treated with indicated doses of cytokines or 10% serum with or without cytokine inhibitors. After treatment, cells were lysed and luciferase activity was measured. (A) Specificity array of the cytokine inhibitors. All results are expressed as percentage promoter activity. Bars represent mean ± SD of at least 3 independent experiments executed in duplicate. Statistical significance was determined with the Student t test or Mann-Whitney rank-sum test; *P < .05, and †P < .001, compared with the cytokine-only group (cytokine/vehicle). (B) The effect of cytokine inhibitors on the induction of the WT-HAMP promoter by MM or healthy sera. Anti–BMP-2/4 and noggin-Fc significantly reversed the stimulatory effect of MM sera. Results are expressed as fold induction over untreated control (vehicle/optimem). Bars represent mean ± SD. Statistical significance was determined with 1-way ANOVA to compare the same sera for the different cytokine inhibitors. To compare MM patient sera with normal sera within the same group, the Mann-Whitney rank-sum test was used; *P < .05, compared with untreated control (vehicle/optimem), and †P < .05, compared with serum only group (vehicle/serum).

Article Snippet: Neutralization experiments Mouse antibodies against human IL-6, IL-6R, BMP-2/4, -4, -6, -9, as well as BMP antagonist noggin-Fc chimera were all obtained from R&D Systems.

Techniques: Activity Assay, Luciferase, Construct, MANN-WHITNEY

Journal: Blood

Article Title: In anemia of multiple myeloma, hepcidin is induced by increased bone morphogenetic protein 2

doi: 10.1182/blood-2010-03-274571

Figure Lengend Snippet: BMP-2 is present in MM sera. (A) MM sera (n = 3) were immunodepleted with anti–BMP-2/4 or isotype-control antibody. Depleted sera were used to treat HuH7 cells that had been transfected with WT-HAMP promoter-luciferase construct. After treatment, cells were lysed and luciferase activity was measured. Bars represent mean ± SD. Statistical significance was determined with the Student t test; *P < .001. (B) ELISA assay was used to measure BMP-2 values in normal (n = 9) and MM sera (n = 23). As expected, MM sera contained higher amounts of BMP-2, then normal sera. Bars represent mean ± SD. The Mann-Whitney rank-sum test was used to determine statistical significance, and *P < .001.

Article Snippet: Neutralization experiments Mouse antibodies against human IL-6, IL-6R, BMP-2/4, -4, -6, -9, as well as BMP antagonist noggin-Fc chimera were all obtained from R&D Systems.

Techniques: Transfection, Luciferase, Construct, Activity Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Bone morphogenetic protein 4 gene therapy in mice inhibits myeloma tumor growth, but has a negative impact on bone

doi: 10.1101/575159

Figure Lengend Snippet: Overview of experimental set up. In brief, 14 weeks old RAG2 −/− γC −/− mice were implanted with calcium phosphate scaffolds containing human MSCs. AAV8-BMP4 or AAV8-CTRL were administered 8 weeks post scaffold implantation by tail-vein injection (10 12 viral particles/100 μL saline/mouse). 10 weeks post implantation 10 6 fluorescently labeled KJON myeloma cells were injected into 3 out of 4 scaffolds. Empty scaffold refers to scaffold with MSCs, but without tumor cells.

Article Snippet: Recombinant murine (rm) BMP4 (Cat# 5020-BP), recombinant human (rh) BMP4 (Cat# 314-BP), rhM-CSF (Cat# 216-MC), neutralizing BMP4 antibody (Cat# MAB50201), and rat isotype control (MAB006) were from R&D Systems (Bio-Techne, Abingdon, UK).

Techniques: Injection, Saline, Labeling

Journal: bioRxiv

Article Title: Bone morphogenetic protein 4 gene therapy in mice inhibits myeloma tumor growth, but has a negative impact on bone

doi: 10.1101/575159

Figure Lengend Snippet: (A) The myeloma cell line KJON was treated with different doses of rmBMP-4. After 48 h, the numbers of viable cells were determined by labeling with annexin V-FITC and propidium iodide (PI). (B) The figure shows representative dot plots of the results presented in A. Cells in the lower left quadrant, which were negative for both annexin V and PI, were considered viable. (C) To estimate tumor burden, the amount of near-infrared fluorescent protein (iRFP) in each scaffold was measured weekly using the Pearl Imager System in AAV8-CTRL mice (n=30) and AAV8-BMP4 mice (n=27), p<0.01, 2-way ANOVA, Bonferroni post test. (D) Representative images of tumor burden in AAV8-CTRL (top) and AAV8-BMP4 (bottom) treated mice are shown. (E) Amount of BMP4 in the serum was estimated at end point by semi-quantitative western blotting. (F) Serum from AAV8-CTRL or AAV8-BMP4 mice, 4% final serum concentration, was added to cultures of NS0 murine myeloma cells and incubated for 48 h. Reduced cell proliferation, as measured by ATP-levels, was counteracted by a BMP4 neutralizing antibody, p<0.0005, Bonferroni post test.

Article Snippet: Recombinant murine (rm) BMP4 (Cat# 5020-BP), recombinant human (rh) BMP4 (Cat# 314-BP), rhM-CSF (Cat# 216-MC), neutralizing BMP4 antibody (Cat# MAB50201), and rat isotype control (MAB006) were from R&D Systems (Bio-Techne, Abingdon, UK).

Techniques: Labeling, Western Blot, Concentration Assay, Incubation

Journal: bioRxiv

Article Title: Bone morphogenetic protein 4 gene therapy in mice inhibits myeloma tumor growth, but has a negative impact on bone

doi: 10.1101/575159

Figure Lengend Snippet: (A) Amount of bone/scaffold perimeter is presented for both empty and tumor scaffold for AAV8-CTRL treated mice (n=10) and AAV8-BMP4 treated mice (n=9). The left femur from each mouse, AAV8-CTRL (n=10) and AAV8-BMP4 (n=9), was harvested and examined by ex vivo μCT. *; p<0.05, 1-way ANOVA, Dunn’s multiple comparisons test. (B) Trabecular volume as a proportion of tissue volume (BV/TV, %), (C) trabecular number (Tb. N, mm −1 ) and (D) trabecular separation (Tb.Sp, mm) was assessed. Error bars represent SEM. *; p<0.05, ***; p<0.005, two-tailed unpaired t-test. (E) Representative images for an AAV8-CTRL mouse and an AAV8-BMP4 mouse are shown. Femur cDNA from AAV8-CTRL mice (n=10) and AAV8-BMP4 mice (n=8) was used for comparative RT-PCR using TaqMan Assays for the osteoclast specific markers Ctsk (F) and Nfatc1 (G). The relative gene expression was analyzed using the ΔΔCt method with Gapdh as housekeeping gene. *; p<0.05, two-tailed unpaired t-test. (H) Cells were differentiated with M-CSF (30 ng/mL) in the presence or absence of rmBMP4 as indicated. TRAP positive cells with more than 2 nuclei were counted as osteoclasts. Presented is the mean of three independent experiments and error bars represent SEM. *; p<0.05, 1-way ANOVA, Dunn’s multiple comparisons test.

Article Snippet: Recombinant murine (rm) BMP4 (Cat# 5020-BP), recombinant human (rh) BMP4 (Cat# 314-BP), rhM-CSF (Cat# 216-MC), neutralizing BMP4 antibody (Cat# MAB50201), and rat isotype control (MAB006) were from R&D Systems (Bio-Techne, Abingdon, UK).

Techniques: Ex Vivo, Two Tailed Test, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: bioRxiv

Article Title: Bone morphogenetic protein 4 gene therapy in mice inhibits myeloma tumor growth, but has a negative impact on bone

doi: 10.1101/575159

Figure Lengend Snippet: Overview of experimental set up. In brief, 14 weeks old RAG2 −/− γC −/− mice were implanted with calcium phosphate scaffolds containing human MSCs. AAV8-BMP4 or AAV8-CTRL were administered 8 weeks post scaffold implantation by tail-vein injection (10 12 viral particles/100 μL saline/mouse). 10 weeks post implantation 10 6 fluorescently labeled KJON myeloma cells were injected into 3 out of 4 scaffolds. Empty scaffold refers to scaffold with MSCs, but without tumor cells.

Article Snippet: Recombinant murine (rm) BMP4 (Cat# 5020-BP), recombinant human (rh) BMP4 (Cat# 314-BP), rhM-CSF (Cat# 216-MC), neutralizing BMP4 antibody (Cat# MAB50201), and rat isotype control (MAB006) were from R&D Systems (Bio-Techne, Abingdon, UK).

Techniques: Injection, Saline, Labeling

Journal: bioRxiv

Article Title: Bone morphogenetic protein 4 gene therapy in mice inhibits myeloma tumor growth, but has a negative impact on bone

doi: 10.1101/575159

Figure Lengend Snippet: (A) The myeloma cell line KJON was treated with different doses of rmBMP-4. After 48 h, the numbers of viable cells were determined by labeling with annexin V-FITC and propidium iodide (PI). (B) The figure shows representative dot plots of the results presented in A. Cells in the lower left quadrant, which were negative for both annexin V and PI, were considered viable. (C) To estimate tumor burden, the amount of near-infrared fluorescent protein (iRFP) in each scaffold was measured weekly using the Pearl Imager System in AAV8-CTRL mice (n=30) and AAV8-BMP4 mice (n=27), p<0.01, 2-way ANOVA, Bonferroni post test. (D) Representative images of tumor burden in AAV8-CTRL (top) and AAV8-BMP4 (bottom) treated mice are shown. (E) Amount of BMP4 in the serum was estimated at end point by semi-quantitative western blotting. (F) Serum from AAV8-CTRL or AAV8-BMP4 mice, 4% final serum concentration, was added to cultures of NS0 murine myeloma cells and incubated for 48 h. Reduced cell proliferation, as measured by ATP-levels, was counteracted by a BMP4 neutralizing antibody, p<0.0005, Bonferroni post test.

Article Snippet: Recombinant murine (rm) BMP4 (Cat# 5020-BP), recombinant human (rh) BMP4 (Cat# 314-BP), rhM-CSF (Cat# 216-MC), neutralizing BMP4 antibody (Cat# MAB50201), and rat isotype control (MAB006) were from R&D Systems (Bio-Techne, Abingdon, UK).

Techniques: Labeling, Western Blot, Concentration Assay, Incubation

Journal: bioRxiv

Article Title: Bone morphogenetic protein 4 gene therapy in mice inhibits myeloma tumor growth, but has a negative impact on bone

doi: 10.1101/575159

Figure Lengend Snippet: (A) Amount of bone/scaffold perimeter is presented for both empty and tumor scaffold for AAV8-CTRL treated mice (n=10) and AAV8-BMP4 treated mice (n=9). The left femur from each mouse, AAV8-CTRL (n=10) and AAV8-BMP4 (n=9), was harvested and examined by ex vivo μCT. *; p<0.05, 1-way ANOVA, Dunn’s multiple comparisons test. (B) Trabecular volume as a proportion of tissue volume (BV/TV, %), (C) trabecular number (Tb. N, mm −1 ) and (D) trabecular separation (Tb.Sp, mm) was assessed. Error bars represent SEM. *; p<0.05, ***; p<0.005, two-tailed unpaired t-test. (E) Representative images for an AAV8-CTRL mouse and an AAV8-BMP4 mouse are shown. Femur cDNA from AAV8-CTRL mice (n=10) and AAV8-BMP4 mice (n=8) was used for comparative RT-PCR using TaqMan Assays for the osteoclast specific markers Ctsk (F) and Nfatc1 (G). The relative gene expression was analyzed using the ΔΔCt method with Gapdh as housekeeping gene. *; p<0.05, two-tailed unpaired t-test. (H) Cells were differentiated with M-CSF (30 ng/mL) in the presence or absence of rmBMP4 as indicated. TRAP positive cells with more than 2 nuclei were counted as osteoclasts. Presented is the mean of three independent experiments and error bars represent SEM. *; p<0.05, 1-way ANOVA, Dunn’s multiple comparisons test.

Article Snippet: Recombinant murine (rm) BMP4 (Cat# 5020-BP), recombinant human (rh) BMP4 (Cat# 314-BP), rhM-CSF (Cat# 216-MC), neutralizing BMP4 antibody (Cat# MAB50201), and rat isotype control (MAB006) were from R&D Systems (Bio-Techne, Abingdon, UK).

Techniques: Ex Vivo, Two Tailed Test, Reverse Transcription Polymerase Chain Reaction, Expressing