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: 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: 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: