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 deficiency reduces smooth muscle coverage. a Left panel: Serial lung sections immunostained with α-smooth muscle actin (αSMA) or von Willebrand factor (vWF) shown in lower magnification with serial section vessels labelled with arrows and, Right panel: Higher magnification of vessels (labelled with red arrows in the left panel) indicating αSMA and vWF staining. Scale bar = 50 μm. b Quantification of non, partially, or fully-muscularised vessels as a percentage of arteries associated with alveolar ducts in wild type (WT; n = 11) and Bmp9 KO (n = 17) lungs. 20 arteries were counted per animal. c Wall thickness was evaluated by identifying small arteries (< 100 μm) proximal to the terminal epithelial bronchioles. Diameter was measured and then wall thickness measurements were assessed at four different positions of the artery, with a minimum of 10 arteries assessed in each lung section. d RNA was isolated from WT (n = 4) and Bmp9 KO (n = 6) mice lungs. Acta2 , Des and Myh11 gene expression was normalised against Hprt . b Two-way ANOVA. ( c and d ) Unpaired t-test. *P ≤ 0.05. Error bars represent mean ± S.E.M

Article Snippet: Bmp9 KO mice aged 4.5–5.5 weeks were dosed by intraperitoneal injection daily for 28 days with either 0.03mg/kg BMP9 (Recombinant Human BMP-9 Protein CF; R&D Systems) in PBS/0.1% mouse serum albumin (MSA; Sigma-Aldrich).

Techniques: Staining, Isolation, 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: 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: Bmp9 KO mice aged 4.5–5.5 weeks were dosed by intraperitoneal injection daily for 28 days with either 0.03mg/kg BMP9 (Recombinant Human BMP-9 Protein CF; R&D Systems) in PBS/0.1% mouse serum albumin (MSA; Sigma-Aldrich).

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: Differential expression of genes identified in the Bmp9 KO RNA-seq analysis in PAH patients vs healthy controls. a Levels of ANXA8 , COLQ , DNAH1 , ITGA6 and SYT15 measured in the UK PAH cohort study using RNA-seq divided into healthy controls (HC; n = 67), and PAH (n = 356; IPAH = 285; BMPR2 mutations-PAH = 71). TPM = transcript per million). Median and standard deviation values are detailed in Supplementary Table 3. b and c Kaplan–Meier survival analysis for patients stratified by mean gene expression. High and low expression of COLQ and ITGA6 was divided above and below the mean. Statistical analysis was performed using pairwise log-rank test. Time = years. a Unpaired t-test. ** P ≤ 0.01, **** P ≤ 0.0001

Article Snippet: Bmp9 KO mice aged 4.5–5.5 weeks were dosed by intraperitoneal injection daily for 28 days with either 0.03mg/kg BMP9 (Recombinant Human BMP-9 Protein CF; R&D Systems) in PBS/0.1% mouse serum albumin (MSA; Sigma-Aldrich).

Techniques: Quantitative Proteomics, RNA Sequencing, Standard Deviation, 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: Anti-BMP9 treatment causes distinct transcriptional changes. a Schematic of treatment regime. Wild type (WT) mice were administered weekly for 3-weeks with 5 mg/kg BMP9 antibody (anti-BMP9) or equivalent volume of mouse IgG2B (IgG) isotype as a control group. Mice were bled every week to check BMP9 levels. Relevant tissue was collected after 3-weeks. b WT mice were administered weekly for 3-weeks with 5 mg/kg BMP9 antibody (anti-BMP9) or equivalent volume of mouse IgG2B (IgG) isotype as a control group. Mice were bled every week to check BMP9 levels. Mice treated with IgG (n = 8/9) or anti-BMP9 (n = 6–9) were bled every week to check BMP9 levels in serum using a BMP9 specific ELISA. c Quantification of non-, partially, or fully-muscularised vessels as a percentage of arteries associated with alveolar ducts in IgG (n = 9) and anti-BMP9 (n = 9) treated mice. 20 arteries were counted per animal. d RVSP was measured in IgG (n = 7) and anti-BMP9 (n = 9) mice. ( e — h ) RNA was isolated from lungs from WT mice treated with IgG (n = 9) or anti-BMP9 (n = 9) mice. Gene expression was normalised against the housekeeping gene, Hprt . e Anxa8 , Colq , Dnah1 , Itga6 , Syt15 and Tgtp1 expression. f Acta2 , Des and Myh11 expression. g Smad6 expression. h Adm and Edn1 expression. b One-way ANOVA. g and h Unpaired t-test. ** P ≤ 0.01, *** P ≤ 0.001. Error bars represent mean ± S.E.M

Article Snippet: Bmp9 KO mice aged 4.5–5.5 weeks were dosed by intraperitoneal injection daily for 28 days with either 0.03mg/kg BMP9 (Recombinant Human BMP-9 Protein CF; R&D Systems) in PBS/0.1% mouse serum albumin (MSA; Sigma-Aldrich).

Techniques: 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 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: Bmp9 KO mice aged 4.5–5.5 weeks were dosed by intraperitoneal injection daily for 28 days with either 0.03mg/kg BMP9 (Recombinant Human BMP-9 Protein CF; R&D Systems) in PBS/0.1% mouse serum albumin (MSA; Sigma-Aldrich).

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: Bmp9 KO mice aged 4.5–5.5 weeks were dosed by intraperitoneal injection daily for 28 days with either 0.03mg/kg BMP9 (Recombinant Human BMP-9 Protein CF; R&D Systems) in PBS/0.1% mouse serum albumin (MSA; Sigma-Aldrich).

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: Bmp9 KO mice aged 4.5–5.5 weeks were dosed by intraperitoneal injection daily for 28 days with either 0.03mg/kg BMP9 (Recombinant Human BMP-9 Protein CF; R&D Systems) in PBS/0.1% mouse serum albumin (MSA; Sigma-Aldrich).

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: Bmp9 KO mice aged 4.5–5.5 weeks were dosed by intraperitoneal injection daily for 28 days with either 0.03mg/kg BMP9 (Recombinant Human BMP-9 Protein CF; R&D Systems) in PBS/0.1% mouse serum albumin (MSA; Sigma-Aldrich).

Techniques: Enzyme-linked Immunosorbent Assay, Staining