Journal: bioRxiv

Article Title: Tacrolimus rescues endothelial ALK1 loss-of-function signaling and improves HHT vascular pathology

doi: 10.1101/137737

Figure Lengend Snippet: ( A and B ) AVM number (A) and AVM diameter (B) in the retinal vasculature of P6 pups treated via the transmammary route with BMP9 and BMP10 blocking antibodies [see Methods and Ref. ], and treated with tacrolimus (FK-506, 0.5 mg/kg/d) or vehicle (DMSO). Data represent mean ± s.e.m. per retina ( n = 10-20 pups per group); * P < 0.05; Student’s t -test (A) and Mann Whitney U test (B). ( C-H ) Representative images of retinas stained with fluorescent isolectin B4 from pups treated or not (DMSO) with tacrolimus (FK-506, 0.5 mg/kg/d), and treated via the transmammary route with control IgG2a/b (C and F) or BMP9/10 blocking antibodies (D, E, G, and H). Higher magnifications in (F-H) show retinal vasculature fields (plexus area) between an artery (a) and a vein (v). Arrows in (D) and (G) denote AVMs. ( I ) Scatter plot showing the density of the retinal vascular plexus in pups treated as in (F-H). Data represent mean ± s.e.m. ( n = 6-8); ** P < 0.01, **** P < 0.0001; one-way ANOVA, Tukey’s multiple comparisons test. ( J-O ) Histochemistry analysis of the vascular front of P6 retinas treated as in (C-H) and stained with fluorescent isolectin B4 (J-L, green) and anti-Dll4 antibody (J-O, red). ( P ) Retinal ECs isolated with anti-CD31 microbeads from pups treated as in (A) were analyzed for Id1 mRNA levels by RT-qPCR. The results are expressed as relative levels of the control condition ( n = 3 determinations). ( Q ) Quantification of Dll4 levels in 3 experiments as in (N and O). Data in (P) and (Q) represent mean ± ( n = 3-5); * P < 0.05; Student’s t -test (P) and Mann Whitney U test (Q). Scale bars, 500 μm (C-E), 100 μm (F-H), 30 μm (J-O).

Article Snippet: Briefly, lactating dams were injected i.p. once on P3 with mouse monoclonal isotype control antibodies (15 mg/kg, IgG2b, MAB004; 15 mg/kg, IgG2a, MAB003; R&D Systems) or mouse monoclonal anti-BMP9 and anti-BMP10 antibodies (15 mg/kg, IgG2b, MAB3209; 15 mg/kg, IgG2a, MAB2926; R&D Systems, respectively).

Techniques: Blocking Assay, MANN-WHITNEY, Staining, Control, Isolation, Quantitative RT-PCR

Journal: Cardiovascular Research

Article Title: Bone morphogenetic protein 10 is increased in pre-capillary pulmonary hypertension patients

doi: 10.1093/cvr/cvaf028

Figure Lengend Snippet: Up-regulated local BMP10 expression in the right atrium of precPH patients. ( A ) Quantification of the relative BMP10 mRNA expression in control ( n = 9) and precPH ( n = 5 CTEPH) RA tissues. ( B and C ) Quantification of total RA BMP10 fluorescent area and RA cardiomyocytes BMP10 intensity levels in control ( n = 6) and precPH ( n = 4 PAH) paraffin-embedded RA tissue sections stained against BMP10, respectively. Representative immunofluorescent stainings of BMP10, Ulex-rhodamine (Ulex, endothelium), and cardiac TroponinT (cTnT, myocardium) in the negative control sample for anti-rabbit Alexa488 and anti-mouse Alexa647 ( D ), in the control ( E ), and in the precPH ( F ) RA tissues at 60×-oil magnification. ( D’–F’ ) Alexa488 single-channel images from the stainings in ( D–F ). ( E ′′ and F′′ ) Zoom-in images from ( E′ and F′ ) to appreciate the sarcomeric pattern of the BMP10 staining in the cardiomyocytes and the homogeneous staining in the vessels. Scale bars = 50 μm. Brightness and contrast for the Alexa488 channel have not been modified. Normality of data was checked and transformed if needed, and statistical differences between precPH patients and controls were tested using an independent sample t -test.

Article Snippet: Finally, we could not determine BMP10 activity directly using trap antibody against BMP10 (#MAB2926, R&D Systems), as described, because this antibody did not inhibit BMP10 transcriptional activity in our samples; therefore, we used the ALK1-Fc.

Techniques: Expressing, Control, Staining, Negative Control, Modification, Transformation Assay

Journal: Cardiovascular Research

Article Title: Bone morphogenetic protein 10 is increased in pre-capillary pulmonary hypertension patients

doi: 10.1093/cvr/cvaf028

Figure Lengend Snippet: Up-regulated local BMP10 activity in the right atrium of precPH patients. ( A ) Quantification of positive pSMAD1/5/8 nuclei in vascular and non-vascular cells within the RA tissues from precPH ( n = 4 PAH) and controls ( n = 5). ( B and C ) Representative immunofluorescent staining of positive pSMAD1/5/8 nuclei in vascular and non-vascular cells from control and precPH with rhodamine and Alexa488 single-channel images on the sides. ( D ) Quantification of positive ID3 nuclei in vascular and non-vascular cells within the RA tissues from precPH ( n = 4 PAH) and controls ( n = 5). ( E and F ) Representative immunofluorescent staining of positive ID3 nuclei in vascular and non-vascular cells from control and precPH with rhodamine and Alexa488 single-channel images on the sides. Arrowheads indicate positive pSMAD1/5/8 and ID3 nuclei. Zoom-in images are included within ( B , C , E , and F ). Nuclei were counterstained with Hoechst 33342 and vessels with Ulex-rhodamine ( B , C , E , and F ). Negative control images are shown in , . Scale bars = 50 μm. Vascular and non-vascular measurements are plotted with their own Y -axis on the left or right side, respectively. Normality of data was checked and transformed if needed, and statistical differences between precPH patients and controls were tested using a Wilcoxon rank-sum test (in A and D ).

Article Snippet: Finally, we could not determine BMP10 activity directly using trap antibody against BMP10 (#MAB2926, R&D Systems), as described, because this antibody did not inhibit BMP10 transcriptional activity in our samples; therefore, we used the ALK1-Fc.

Techniques: Activity Assay, Staining, Control, Negative Control, Transformation Assay

Journal: Cardiovascular Research

Article Title: Bone morphogenetic protein 10 is increased in pre-capillary pulmonary hypertension patients

doi: 10.1093/cvr/cvaf028

Figure Lengend Snippet: Higher BMP10 plasma levels in precPH patients compared with controls. ( A and B ) BMP10 protein circulating plasma levels in precPH patients ( n = 48) and subgroups ( n = 48: 22 iPAH, 14 hPAH, and 12 CTEPH), respectively, vs. controls ( n = 16). ( C and D ) BMP9 protein circulating plasma levels in precPH patients ( n = 45) and subgroups ( n = 45: 20 iPAH, 14 hPAH, and 11 CTEPH), respectively, vs. controls ( n = 16). ( E and F ) Correlation between BMP10 and BMP9 plasma levels in precPH patients ( n = 45) or subgroups ( n = 45: 20 iPAH, 14 hPAH, and 11 CTEPH), respectively, vs. controls ( n = 16). Logarithmic Y -axis is used in graphs ( A – D ). Data in ( A and B ) are y + 1 for logarithmic scale transformation. Normality of data was checked and transformed if needed. Statistical differences between precPH patients or precPH subgroups and controls were tested with an independent sample t -test or a one-way ANOVA, respectively. Associations were tested with univariate linear regression analysis.

Article Snippet: Finally, we could not determine BMP10 activity directly using trap antibody against BMP10 (#MAB2926, R&D Systems), as described, because this antibody did not inhibit BMP10 transcriptional activity in our samples; therefore, we used the ALK1-Fc.

Techniques: Clinical Proteomics, Transformation Assay

Journal: Cardiovascular Research

Article Title: Bone morphogenetic protein 10 is increased in pre-capillary pulmonary hypertension patients

doi: 10.1093/cvr/cvaf028

Figure Lengend Snippet: BMP10 transcriptional activity in precPH patients and controls. ( A ) Schematic explanation of the BRE-LUC reporter assay to determine BMP transcriptional activity in venous serum. Specific trap antibodies targeting BMP9 or BMP9 and BMP10 are used to assess BMP10 activity. Created with BioRender.com. B ) Relative BMP transcriptional activity as a luciferase read-out from the HMEC-BRE-LUC, endothelial cells expressing a BMP-specific luciferase reporter, in control ( n = 15) and precPH subgroups ( n = 21 iPAH, n = 13 hPAH, and n = 11 CTEPH) after incubation with phosphate-buffered saline (PBS) (baseline), anti-BMP9, or ALK1-Fc (inhibition of BMP9 and BMP10). ( C ) BMP10 activity in controls and precPH subgroups has been calculated from the subtraction of anti-BMP9 and ALK1-Fc to total BMP activity. Normality of data was checked and transformed if needed. Statistical differences between precPH patients and controls, and between baseline conditions and trap antibodies, were tested with an independent sample t -test or a one-way ANOVA, after which pairwise t -testing with Bonferroni correction was applied, respectively.

Article Snippet: Finally, we could not determine BMP10 activity directly using trap antibody against BMP10 (#MAB2926, R&D Systems), as described, because this antibody did not inhibit BMP10 transcriptional activity in our samples; therefore, we used the ALK1-Fc.

Techniques: Activity Assay, Reporter Assay, Luciferase, Expressing, Control, Incubation, Saline, Inhibition, Transformation Assay

Journal: Cardiovascular Research

Article Title: Bone morphogenetic protein 10 is increased in pre-capillary pulmonary hypertension patients

doi: 10.1093/cvr/cvaf028

Figure Lengend Snippet: Patients with more right atrial dilatation, reduced RV ejection fraction, and higher NT-proBNP have higher levels of circulation BMP10 activity. ( A and B ) BMP10 transcriptional activity in precPH patients with RA or RV dilation, respectively. ( C–E ) BMP10 transcriptional activity in precPH patients with high RAP, reduced RVEF, or high NT-proBNP, respectively. PrecPH patients were stratified according to RA volume (>79 mL/mm 2 for male patients or >69 mL/mm 2 for female patients), RV end-diastolic volume index (≥109 mL/mm 2 for males, and ≥97 mL/mm 2 for females), RAP (>14 mmHg), RVEF (<35%), and NT-proBNP levels (>1100 ng/L). Normality of data was checked and transformed if needed. Statistical differences between both groups were tested with an independent samples t -test.

Article Snippet: Finally, we could not determine BMP10 activity directly using trap antibody against BMP10 (#MAB2926, R&D Systems), as described, because this antibody did not inhibit BMP10 transcriptional activity in our samples; therefore, we used the ALK1-Fc.

Techniques: Activity Assay, Transformation Assay

Journal: Cardiovascular Research

Article Title: Bone morphogenetic protein 10 is increased in pre-capillary pulmonary hypertension patients

doi: 10.1093/cvr/cvaf028

Figure Lengend Snippet: Effect of pressure unloading on BMP10 activity in precPH patients. ( A ) Serum relative BMP activity at baseline and post-PEA in CTEPH patients. Incubation with anti-BMP9 only blocked BMP9 activity, while ALK1-Fc blocked both BMP9 and BMP10 activities. ( B ) Calculated BMP10 transcriptional activity at baseline and post-PEA ( n = 13), respectively. BMP10 transcriptional activity is calculated by subtracting BMP activity values after incubation with the trap antibodies. Normality of data was checked and transformed if needed. Statistical differences between baseline conditions and trap antibodies, and between baseline and post-PEA, were tested with an independent sample t -test.

Article Snippet: Finally, we could not determine BMP10 activity directly using trap antibody against BMP10 (#MAB2926, R&D Systems), as described, because this antibody did not inhibit BMP10 transcriptional activity in our samples; therefore, we used the ALK1-Fc.

Techniques: Activity Assay, Incubation, Transformation Assay

Journal: Molecular genetics & genomic medicine

Article Title: Homozygous GDF2 nonsense mutations result in a loss of circulating BMP9 and BMP10 and are associated with either PAH or an "HHT-like" syndrome in children.

doi: 10.1002/mgg3.1685

Figure Lengend Snippet: FIGURE 2 Homozygous GDF2 truncation mutations lead to an absence of circulating BMP9 and pBMP10. (a,b) Levels of (a) BMP9 and (b) pBMP10 in plasma were measured with specific ELISAs. (a) Samples were assayed as 25% EDTA-plasma, 0.2% goat-serum, and 0.5% Triton X-100 in BioRad ELISA buffer. (b) Samples were assayed as BMP9, but with inclusion of 4.5 mM EDTA. All samples were assayed in duplicate, each point represents a different individual. The dotted lines indicate the lower limits of quantification and dashed lines indicate the limits of detection for these assays. (c-e) Endothelial serum BMP activity, representing BMP9 and BMP10, was assayed in the stable HMEC1-BRE reporter line. Serum-starved cells were incubated with 2%-10% serum for 6 hr and luciferase activity then measured. Samples were measured in duplicate, and the assay was repeated on three separate occasions. Data are presented as mean of the averaged luciferase values for each individual experiment ± SEM. The dotted line indicates the mean SFM response. Individual graphs show: (c) control adult female samples from three separate individuals (FC1-3), (d) p.[Gln26Ter]:[Gln26Ter] patient sample and control male samples from four separate individuals (MC1-4), and (e) p.[Glu279Ter]:[Glu279Ter] patient sample and samples from unaffected p.[Glu279Ter] heterozygous parents. Data from all individuals were compared by a multiple comparisons two-way ANOVA using GraphPad Prism 9. p < 0.001 is relative to all individual controls

Article Snippet: After washing, biotinylated anti- human BMP10 propeptide (0.04 μg/well BAF3956; R&D Systems) in 1% BSA, 0.2% goat serum was added.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Activity Assay, Incubation, Luciferase, Control

Journal: Cardiovascular Research

Article Title: Bone morphogenetic protein 10 is increased in pre-capillary pulmonary hypertension patients

doi: 10.1093/cvr/cvaf028

Figure Lengend Snippet: Up-regulated local BMP10 expression in the right atrium of precPH patients. ( A ) Quantification of the relative BMP10 mRNA expression in control ( n = 9) and precPH ( n = 5 CTEPH) RA tissues. ( B and C ) Quantification of total RA BMP10 fluorescent area and RA cardiomyocytes BMP10 intensity levels in control ( n = 6) and precPH ( n = 4 PAH) paraffin-embedded RA tissue sections stained against BMP10, respectively. Representative immunofluorescent stainings of BMP10, Ulex-rhodamine (Ulex, endothelium), and cardiac TroponinT (cTnT, myocardium) in the negative control sample for anti-rabbit Alexa488 and anti-mouse Alexa647 ( D ), in the control ( E ), and in the precPH ( F ) RA tissues at 60×-oil magnification. ( D’–F’ ) Alexa488 single-channel images from the stainings in ( D–F ). ( E ′′ and F′′ ) Zoom-in images from ( E′ and F′ ) to appreciate the sarcomeric pattern of the BMP10 staining in the cardiomyocytes and the homogeneous staining in the vessels. Scale bars = 50 μm. Brightness and contrast for the Alexa488 channel have not been modified. Normality of data was checked and transformed if needed, and statistical differences between precPH patients and controls were tested using an independent sample t -test.

Article Snippet: RA paraffin-embedded tissue from controls ( n = 6) and precPH patients ( n = 4 PAH) was stained using an antibody against BMP10 (diluted 1:200; Biorbyt, United Kingdom), cardiac Troponin T (cTnT, diluted 1:500; Abcam, United Kingdom), phosphorylated receptor-regulated Smad1, Smad5, and Smad8 (pSMAD1/5/8, diluted 1:200; Cell Signaling, United States of America), and inhibitor of differentiation 3 (ID3, diluted 1:50; Santa Cruz Biotechnologies, United States of America).

Techniques: Expressing, Control, Staining, Negative Control, Modification, Transformation Assay

Journal: Cardiovascular Research

Article Title: Bone morphogenetic protein 10 is increased in pre-capillary pulmonary hypertension patients

doi: 10.1093/cvr/cvaf028

Figure Lengend Snippet: Up-regulated local BMP10 activity in the right atrium of precPH patients. ( A ) Quantification of positive pSMAD1/5/8 nuclei in vascular and non-vascular cells within the RA tissues from precPH ( n = 4 PAH) and controls ( n = 5). ( B and C ) Representative immunofluorescent staining of positive pSMAD1/5/8 nuclei in vascular and non-vascular cells from control and precPH with rhodamine and Alexa488 single-channel images on the sides. ( D ) Quantification of positive ID3 nuclei in vascular and non-vascular cells within the RA tissues from precPH ( n = 4 PAH) and controls ( n = 5). ( E and F ) Representative immunofluorescent staining of positive ID3 nuclei in vascular and non-vascular cells from control and precPH with rhodamine and Alexa488 single-channel images on the sides. Arrowheads indicate positive pSMAD1/5/8 and ID3 nuclei. Zoom-in images are included within ( B , C , E , and F ). Nuclei were counterstained with Hoechst 33342 and vessels with Ulex-rhodamine ( B , C , E , and F ). Negative control images are shown in , . Scale bars = 50 μm. Vascular and non-vascular measurements are plotted with their own Y -axis on the left or right side, respectively. Normality of data was checked and transformed if needed, and statistical differences between precPH patients and controls were tested using a Wilcoxon rank-sum test (in A and D ).

Article Snippet: RA paraffin-embedded tissue from controls ( n = 6) and precPH patients ( n = 4 PAH) was stained using an antibody against BMP10 (diluted 1:200; Biorbyt, United Kingdom), cardiac Troponin T (cTnT, diluted 1:500; Abcam, United Kingdom), phosphorylated receptor-regulated Smad1, Smad5, and Smad8 (pSMAD1/5/8, diluted 1:200; Cell Signaling, United States of America), and inhibitor of differentiation 3 (ID3, diluted 1:50; Santa Cruz Biotechnologies, United States of America).

Techniques: Activity Assay, Staining, Control, Negative Control, Transformation Assay

Journal: Cardiovascular Research

Article Title: Bone morphogenetic protein 10 is increased in pre-capillary pulmonary hypertension patients

doi: 10.1093/cvr/cvaf028

Figure Lengend Snippet: Higher BMP10 plasma levels in precPH patients compared with controls. ( A and B ) BMP10 protein circulating plasma levels in precPH patients ( n = 48) and subgroups ( n = 48: 22 iPAH, 14 hPAH, and 12 CTEPH), respectively, vs. controls ( n = 16). ( C and D ) BMP9 protein circulating plasma levels in precPH patients ( n = 45) and subgroups ( n = 45: 20 iPAH, 14 hPAH, and 11 CTEPH), respectively, vs. controls ( n = 16). ( E and F ) Correlation between BMP10 and BMP9 plasma levels in precPH patients ( n = 45) or subgroups ( n = 45: 20 iPAH, 14 hPAH, and 11 CTEPH), respectively, vs. controls ( n = 16). Logarithmic Y -axis is used in graphs ( A – D ). Data in ( A and B ) are y + 1 for logarithmic scale transformation. Normality of data was checked and transformed if needed. Statistical differences between precPH patients or precPH subgroups and controls were tested with an independent sample t -test or a one-way ANOVA, respectively. Associations were tested with univariate linear regression analysis.

Article Snippet: RA paraffin-embedded tissue from controls ( n = 6) and precPH patients ( n = 4 PAH) was stained using an antibody against BMP10 (diluted 1:200; Biorbyt, United Kingdom), cardiac Troponin T (cTnT, diluted 1:500; Abcam, United Kingdom), phosphorylated receptor-regulated Smad1, Smad5, and Smad8 (pSMAD1/5/8, diluted 1:200; Cell Signaling, United States of America), and inhibitor of differentiation 3 (ID3, diluted 1:50; Santa Cruz Biotechnologies, United States of America).

Techniques: Clinical Proteomics, Transformation Assay

Journal: Cardiovascular Research

Article Title: Bone morphogenetic protein 10 is increased in pre-capillary pulmonary hypertension patients

doi: 10.1093/cvr/cvaf028

Figure Lengend Snippet: BMP10 transcriptional activity in precPH patients and controls. ( A ) Schematic explanation of the BRE-LUC reporter assay to determine BMP transcriptional activity in venous serum. Specific trap antibodies targeting BMP9 or BMP9 and BMP10 are used to assess BMP10 activity. Created with BioRender.com. B ) Relative BMP transcriptional activity as a luciferase read-out from the HMEC-BRE-LUC, endothelial cells expressing a BMP-specific luciferase reporter, in control ( n = 15) and precPH subgroups ( n = 21 iPAH, n = 13 hPAH, and n = 11 CTEPH) after incubation with phosphate-buffered saline (PBS) (baseline), anti-BMP9, or ALK1-Fc (inhibition of BMP9 and BMP10). ( C ) BMP10 activity in controls and precPH subgroups has been calculated from the subtraction of anti-BMP9 and ALK1-Fc to total BMP activity. Normality of data was checked and transformed if needed. Statistical differences between precPH patients and controls, and between baseline conditions and trap antibodies, were tested with an independent sample t -test or a one-way ANOVA, after which pairwise t -testing with Bonferroni correction was applied, respectively.

Article Snippet: RA paraffin-embedded tissue from controls ( n = 6) and precPH patients ( n = 4 PAH) was stained using an antibody against BMP10 (diluted 1:200; Biorbyt, United Kingdom), cardiac Troponin T (cTnT, diluted 1:500; Abcam, United Kingdom), phosphorylated receptor-regulated Smad1, Smad5, and Smad8 (pSMAD1/5/8, diluted 1:200; Cell Signaling, United States of America), and inhibitor of differentiation 3 (ID3, diluted 1:50; Santa Cruz Biotechnologies, United States of America).

Techniques: Activity Assay, Reporter Assay, Luciferase, Expressing, Control, Incubation, Saline, Inhibition, Transformation Assay

Journal: Cardiovascular Research

Article Title: Bone morphogenetic protein 10 is increased in pre-capillary pulmonary hypertension patients

doi: 10.1093/cvr/cvaf028

Figure Lengend Snippet: Patients with more right atrial dilatation, reduced RV ejection fraction, and higher NT-proBNP have higher levels of circulation BMP10 activity. ( A and B ) BMP10 transcriptional activity in precPH patients with RA or RV dilation, respectively. ( C–E ) BMP10 transcriptional activity in precPH patients with high RAP, reduced RVEF, or high NT-proBNP, respectively. PrecPH patients were stratified according to RA volume (>79 mL/mm 2 for male patients or >69 mL/mm 2 for female patients), RV end-diastolic volume index (≥109 mL/mm 2 for males, and ≥97 mL/mm 2 for females), RAP (>14 mmHg), RVEF (<35%), and NT-proBNP levels (>1100 ng/L). Normality of data was checked and transformed if needed. Statistical differences between both groups were tested with an independent samples t -test.

Article Snippet: RA paraffin-embedded tissue from controls ( n = 6) and precPH patients ( n = 4 PAH) was stained using an antibody against BMP10 (diluted 1:200; Biorbyt, United Kingdom), cardiac Troponin T (cTnT, diluted 1:500; Abcam, United Kingdom), phosphorylated receptor-regulated Smad1, Smad5, and Smad8 (pSMAD1/5/8, diluted 1:200; Cell Signaling, United States of America), and inhibitor of differentiation 3 (ID3, diluted 1:50; Santa Cruz Biotechnologies, United States of America).

Techniques: Activity Assay, Transformation Assay

Journal: Cardiovascular Research

Article Title: Bone morphogenetic protein 10 is increased in pre-capillary pulmonary hypertension patients

doi: 10.1093/cvr/cvaf028

Figure Lengend Snippet: Effect of pressure unloading on BMP10 activity in precPH patients. ( A ) Serum relative BMP activity at baseline and post-PEA in CTEPH patients. Incubation with anti-BMP9 only blocked BMP9 activity, while ALK1-Fc blocked both BMP9 and BMP10 activities. ( B ) Calculated BMP10 transcriptional activity at baseline and post-PEA ( n = 13), respectively. BMP10 transcriptional activity is calculated by subtracting BMP activity values after incubation with the trap antibodies. Normality of data was checked and transformed if needed. Statistical differences between baseline conditions and trap antibodies, and between baseline and post-PEA, were tested with an independent sample t -test.

Article Snippet: RA paraffin-embedded tissue from controls ( n = 6) and precPH patients ( n = 4 PAH) was stained using an antibody against BMP10 (diluted 1:200; Biorbyt, United Kingdom), cardiac Troponin T (cTnT, diluted 1:500; Abcam, United Kingdom), phosphorylated receptor-regulated Smad1, Smad5, and Smad8 (pSMAD1/5/8, diluted 1:200; Cell Signaling, United States of America), and inhibitor of differentiation 3 (ID3, diluted 1:50; Santa Cruz Biotechnologies, United States of America).

Techniques: Activity Assay, Incubation, Transformation Assay

Journal: Angiogenesis

Article Title: BMP9 knockout impairs pulmonary vessel muscularisation and confers aberrant tamoxifen sensitivity

doi: 10.1007/s10456-025-10017-5

Figure Lengend Snippet: RNA sequencing of Gdf2 −/− lungs identifies genes associated with BMP9 loss. a RNA was isolated from wild type (WT; n = 4) and Bmp9 KO (n = 6) mice lungs. Following RNA libraries preparation, samples were analysed for 50 bp paired end reads on a Novaseq 6000 sequencer (Illumina). Volcano plot of differentially expressed genes in Bmp9 KO versus WT after fitting linear models and adjusting P values for multiple testing. b Schematic of treatment regime. WT and Bmp9 KO mice were administered daily for 3-weeks with 0.03 mg/kg recombinant human BMP9 or vehicle control. Mice were bled at the beginning and end of treatment regime to check BMP9 levels. c Lung sections were immunostained with α-smooth muscle actin (αSMA). Quantification of non-, partially, or fully-muscularised vessels as a percentage of arteries associated with alveolar ducts in WT (n = 11), Bmp9 KO plus vehicle (n = 7) and Bmp9 KO plus BMP9 (n = 8) mice. 20 arteries were counted per animal. d RNA was isolated from WT (n = 11), Bmp9 KO plus vehicle (n = 6/7) and Bmp9 KO plus BMP9 (n = 6/8) mice lungs. Gene expression Anxa8 , Colq , Dnah1 , Itga6 , Rbp3 , Syt15 and Tgtp1 was normalised against the housekeeping gene, Hprt . e and f Human pulmonary microvascular cells (PMVECs; n = 4 biological replicates) were serum-starved (0.1%) overnight prior to treatment with BMP9 or BMP10 (0.1, 0.3, 1 ng/ml) for 8 h. Gene expression of ITGA6 ( e ) and SYT15 ( f ) was measured using qPCR, normalised to 2 housekeeping genes ( B2M and HPRT ). ( c ) Two-way ANOVA. ( d , e, and f ) One-way ANOVA. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Error bars represent mean ± S.E.M

Article Snippet: After washing, biotinylated anti‐human BMP10 detection antibody (0.1 μg/well; R&D Systems) was added in PBS/1% BSA containing 0.2% GS.

Techniques: RNA Sequencing, Isolation, Recombinant, Control, Gene Expression

Journal: Angiogenesis

Article Title: BMP9 knockout impairs pulmonary vessel muscularisation and confers aberrant tamoxifen sensitivity

doi: 10.1007/s10456-025-10017-5

Figure Lengend Snippet: Bmp9 KO and double knockout mice treated with tamoxifen exhibit reduced smooth muscle associated gene expression. a Schematic of treatment regime. Bmp10 fl/fl (WT), Bmp10 fl/fl x Gdf2 −/− ( Bmp9 KO), Bmp10 fl/fl xRosa26 Cre−ERT ( Bmp10 cKO) and Bmp10 fl/fl xRosa26 Cre−ERT x Gdf2 −/− (dKO) mice were treated with tamoxifen once a day for five days with a two-day recovery period followed by a further 5 days at a dose of 40 mg/kg. As a vehicle control, WT mice were administered corn oil for the same period. Mice then underwent right heart catheterisation on day 56. Mice were also bled at day -3, 21 and 56 to assess BMP9 levels. Right atrium was also taken at day 56 to generate BMP10 conditioned media (RACM). Relevant tissue was collected on day 56. b–l RNA was isolated on day 56 from lungs of WT (corn oil; n = 8), WT (tamoxifen; n = 8), Bmp9 KO (tamoxifen; n = 8), Bmp10 cKO (tamoxifen; n = 8) and dKO (tamoxifen; n = 8). Gene expression was normalised against the housekeeping gene, Hprt . Acta2 ( b ), Des ( c ), Myh11 ( d ), Anxa8 ( e ), Colq ( f ), Rbp3 ( g ), Itga6 ( h ), Tgtp1 ( i ), Syt15 ( j ), Bmpr2 ( k ), Eng ( l ) and Smad6 ( m ) gene expression. b – m One-way ANOVA. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Error bars represent mean ± S.E.M

Article Snippet: After washing, biotinylated anti‐human BMP10 detection antibody (0.1 μg/well; R&D Systems) was added in PBS/1% BSA containing 0.2% GS.

Techniques: Double Knockout, Gene Expression, Control, Isolation

Journal: Angiogenesis

Article Title: BMP9 knockout impairs pulmonary vessel muscularisation and confers aberrant tamoxifen sensitivity

doi: 10.1007/s10456-025-10017-5

Figure Lengend Snippet: Transcriptional changes in conditional knockout mice treated with anti-BMP9. a Schematic of treatment regime. Bmp10 fl/fl xRosa26 Cre−ERT ( Bmp10 -cKO) were treated with tamoxifen once a day for five days with a two-day recovery period followed by a further 5 days at a dose of 40 mg/kg. As a vehicle control Bmp10 fl/fl (WT) mice were administered corn oil for the same period. On day 21 mice were dosed weekly for 2-weeks with 5mg/kg BMP9 antibody (anti-BMP9) or equivalent volume of mouse IgG2B (IgG) isotype as a control. Mice then underwent right heart catheterisation on day 42. Mice were also bled at day -3, 21 and 42 to assess BMP9 levels. Right atrium was also taken at day 42 to generate BMP10 conditioned media (RACM). Relevant tissue was collected on day 42. b Conditioned media from right atria collected at day 42 from WT (corn oil; n = 6), Bmp10 cKO – IgG (tamoxifen; n = 3) and Bmp10 cKO—anti-BMP9 (tamoxifen; n = 3) mice was assayed for BMP10 levels using a BMP10 growth factor domain (GFD) specific ELISA. c Serum from WT (corn oil; n = 6), Bmp10 cKO—IgG (n = 9) and Bmp10 cKO—anti-BMP9 ( Bmp10 cKO—anti-BMP9; n = 9) mice bled at day -3, 21 and 42 were assayed for BMP9 levels using a BMP9 specific ELISA. d – l RNA was isolated on day 45 from lungs of Bmp10 cKO (IgG; n = 8) and Bmp10 cKO (anti-BMP9; n = 9). Gene expression was normalised against the housekeeping gene, Hprt . Anxa8 ( d ), Colq ( e ), Dnah1 ( f ), Itga6 ( g ) Syt15 ( h ), Tgtp1 ( i ), Edn1 ( j ), Adm ( k ) and Smad6 ( l ) expression. ( e , g , h , i , j , k, and l ) Unpaired t-test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Error bars represent mean ± S.E.M

Article Snippet: After washing, biotinylated anti‐human BMP10 detection antibody (0.1 μg/well; R&D Systems) was added in PBS/1% BSA containing 0.2% GS.

Techniques: Knock-Out, Control, Enzyme-linked Immunosorbent Assay, Isolation, Gene Expression, Expressing

Journal: Angiogenesis

Article Title: BMP9 knockout impairs pulmonary vessel muscularisation and confers aberrant tamoxifen sensitivity

doi: 10.1007/s10456-025-10017-5

Figure Lengend Snippet: Bmp9 KO and double knockout mice treated with tamoxifen exhibit extensive tissue remodelling. ( a – g ) Bmp10 fl/fl (WT), Bmp10 fl/fl x Gdf2 −/− ( Bmp9 KO), Bmp10 fl/fl xRosa26 Cre−ERT ( Bmp10 cKO) and Bmp10 fl/fl xRosa26 Cre−ERT x Gdf2 −/− (dKO) mice were treated with tamoxifen once a day for five days with a two-day recovery period followed by a further 5 days at a dose of 40 mg/kg. As a vehicle control, WT mice were administered corn oil for the same period. Mice then underwent right heart catheterisation on day 56. Relevant tissue was collected on day 56. a Heart weight was assessed as a ratio of femur length in WT (corn oil; n = 12), WT (tamoxifen; n = 20), Bmp9 KO (tamoxifen; n = 20), Bmp10 cKO (tamoxifen; n = 15) and dKO (tamoxifen; n = 13). b Spleen weight was assessed as a ratio of femur length in WT (corn oil; n = 12), Bmp10 fl/fl (tamoxifen; n = 20), Bmp9 KO (tamoxifen; n = 20), Bmp10 cKO (tamoxifen; n = 15), and dKO (tamoxifen; n = 13). c Ratio of right ventricle (RV) thickness and left ventricle thickness (LV) in WT (corn oil; n = 6), WT (tamoxifen; n = 10), Bmp9 KO (tamoxifen; n = 8), Bmp10 cKO (tamoxifen; n = 8) and dKO (tamoxifen; n = 6). d Heart rate was measured in WT (corn oil; n = 12), WT (tamoxifen; n = 20), Bmp9 KO (tamoxifen; n = 20), Bmp10 cKO (tamoxifen; n = 14) and dKO (tamoxifen; n = 13). e Measurement of cardiac output in WT (corn oil; n = 12), WT (tamoxifen; n = 19), Bmp9 KO (tamoxifen; n = 19), Bmp10 cKO (tamoxifen; n = 13) and dKO (tamoxifen; n = 10). f Right ventricular systolic pressure (RVSP) was measured in WT (corn oil; n = 12), WT (tamoxifen; n = 20), Bmp9 KO (tamoxifen; n = 20), Bmp10 cKO (tamoxifen; n = 14) and dKO (tamoxifen; n = 13). g Lung sections were immunostained with α-smooth muscle actin (αSMA). Quantification of non, partially, or fully-muscularised vessels as a percentage of arteries associated with alveolar ducts in WT (corn oil; n = 12), WT (tamoxifen; n = 15), Bmp9 KO (tamoxifen; n = 15), Bmp10 -cKO (tamoxifen; n = 15) and dKO (tamoxifen; n = 13) mice. 20 arteries were counted per animal. h Alveoli area was assessed in haematoxylin and eosin-stained lung sections. Percentage of counterstained tissue versus no staining of the whole lung area in WT (corn oil; n = 12), WT (tamoxifen; n = 15), Bmp9 KO (tamoxifen; n = 15), Bmp10 -cKO (tamoxifen; n = 15) and dKO (tamoxifen; n = 13) mice. ( i ) Lung sections were stained with Perl’s iron stain. Percentage of Perl’s positive cells in whole lung area from WT (corn oil; n = 12), WT (tamoxifen; n = 15), Bmp9 KO (tamoxifen; n = 15), Bmp10 -cKO (tamoxifen; n = 15) and dKO (tamoxifen; n = 13) mice. Scale bar = 100 μm. ( a , b , c , d , e , f , h and i ) One-way ANOVA. g Two-way ANOVA. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Error bars represent mean ± S.E.M

Article Snippet: After washing, biotinylated anti‐human BMP10 detection antibody (0.1 μg/well; R&D Systems) was added in PBS/1% BSA containing 0.2% GS.

Techniques: Double Knockout, Control, Staining

Journal: Angiogenesis

Article Title: BMP9 knockout impairs pulmonary vessel muscularisation and confers aberrant tamoxifen sensitivity

doi: 10.1007/s10456-025-10017-5

Figure Lengend Snippet: Bmp9 KO mice treated with tamoxifen have cardiomegaly and splenomegaly. Wild type (WT; n = 9), Bmp9 KO (n = 7) and Bmp10 fl/fl xRosa26 Cre−ERT ( Bmp10 cKO; n = 2) mice were treated with tamoxifen once a day for five days with a two-day recovery period followed by a further 5 days at a dose of 40 mg/kg. Mice were also bled at day -3, 21 and 42 to assess BMP9 levels. Right atria were collected at day 42 for conditioned media culture. a Serum from WT (n = 8), Bmp9 KO (n = 7) and Bmp10 cKO (n = 2) mice bled at day -3, 21 and 42 were assayed for BMP9 levels using a BMP9 specific ELISA. BMP9 was undetectable in Bmp9 KO mice. b Conditioned media from right atria collected at day 42 from WT (n = 8), Bmp9 KO (n = 7) and Bmp10 cKO (n = 2) mice were assayed for BMP10 levels using a BMP10 growth factor domain (GFD) specific ELISA. c Heart weight was assessed as a ratio of femur length in WT (n = 9), Bmp9 KO (n = 7) and Bmp10 -cKO (n = 2). d Spleen weight was assessed as a ratio of femur length in WT (n = 9), Bmp9 KO (n = 7) and Bmp10 cKO (n = 2). e Alveoli area was assessed using haematoxylin and eosin and lung sections were stained with Perl’s iron stain. Scale bar = 100 μm. f Percentage of Perl’s positive cells in whole lung area from WT (n = 9), Bmp9 KO (n = 7) and Bmp10 -cKO (n = 2) mice. b , c, and d One-way ANOVA. f Unpaired t-test. * P ≤ 0.05, ** P ≤ 0.01. Error bars represent mean ± S.E.M

Article Snippet: After washing, biotinylated anti‐human BMP10 detection antibody (0.1 μg/well; R&D Systems) was added in PBS/1% BSA containing 0.2% GS.

Techniques: Enzyme-linked Immunosorbent Assay, Staining

Journal: JCI Insight

Article Title: Semaphorin 3E/PlexinD1 signaling is required for cardiac ventricular compaction

doi: 10.1172/jci.insight.125908

Figure Lengend Snippet: Real-time qPCR analysis of Notch pathway genes and their downstream targets using RNA isolated from E10.5, E12.5, and E14.5 control and Plxnd1–/– hearts (A). Expression levels of Nrg1, ErbB2, and ErbB4, as measured by qPCR using RNA isolated from E10.5, E12.5, and E14.5 control and Plxnd1–/– hearts. n = 3 for each genotype/time point (B). Representative Western blot of Notch1 and NICD on E12.5 and E14.5 control and Plxnd1–/– heart lysates. Gapdh was used as loading control (C). Immunostaining for NICD on E14.5 control and Plxnd1–/– heart sections (D). Representative Western blot analysis of ErbB2 and pErbB2 using E12.5 and E14.5 control and Plxnd1–/– heart lysates. Actin was used as loading control (E). Partial rescue of hypertrabeculation and noncompaction defects displayed by Plxnd1–/– embryos after treatment with the γ-secretase inhibitor DBZ. H&E staining of E13.5 control and Plxnd1–/– hearts isolated after vehicle or DBZ treatment (F). Quantification of the thickness of the compact and trabecular layer (G). n = 3 for each genotype. Real-time qPCR analysis of Notch1, Bmp10, and Adamts15 using RNA isolated from E13.5 control and Plxnd1–/– hearts isolated after vehicle or DBZ treatment (H). LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. Scale bars: 100 μm.

Article Snippet: The following antibodies were used for Western blotting and immunohistochemistry: Notch1 (Santa Cruz, catalog sc-6014), Cleaved Notch1 (Val1744) (Cell Signaling, catalog 4147S), ErbB2 (Cell Signaling, catalog 2242), pErbB2 (Cell Signaling, catalog 2247), Bmp10 (R&D System, catalog MAB6038), Vinculin (MilliporeSigma, catalog V9131), Gapdh (Santa Cruz, catalog sc-20357), Actin (Santa Cruz, catalog sc-47778), MF20-C (DSHB), Ki-67 (Abcam, catalog ab15580), Sema3E (MilliporeSigma, catalog HPA029419), Varsican (Thermo Fisher Scientific, catalog PA11748A), Adamts1 (Santa Cruz, catalog sc-47727), Fibronectin (Santa Cruz, catalog sc-8422), and PlexinD1 (R&D System, catalog AF4160).

Techniques: Isolation, Control, Expressing, Western Blot, Immunostaining, Staining

Journal: The FASEB Journal

Article Title: Prodomain processing controls BMP ‐10 bioactivity and targeting to fibrillin‐1 in latent conformation

doi: 10.1096/fj.202401694R

Figure Lengend Snippet: SPR affinity data for interactions between immobilized BMP‐9, ‐10, and ‐9/10 fusion PDs and flowed over BMP‐10 GF.

Article Snippet: For western blot and sandwich ELISAs the following antibodies were used: anti‐His 6 ‐HRP (#130‐092‐785, Miltenyi Biotec, Germany), goat anti‐human BMP‐10 propeptide (#AF3956‐SP, R&D Systems, Minneapolis, MN, USA), mouse anti‐human BMP‐10 GF (#MAB2926, R&D Systems), rabbit anti‐human phospho‐SMAD 1/5/9 (pSMAD1/5/9) (#13820, Cell Signaling Technology, Danvers, MA, USA), mouse anti‐rabbit GAPDH (#ab8245, Abcam, Waltham, MA, USA), and rabbit anti‐human BMP‐7 GF (#500‐P198, PeproTech, Rocky Hill, NJ).

Techniques: