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: For measurement of BMP10 in conditioned right atrial medium, a commercial BMP10 ELISA kit was used (DY2926; R&D Systems) plates were coated with 0.2 μg/well of anti‐human BMP10 (MAB2926; R&D Systems) and blocked as above.

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: For measurement of BMP10 in conditioned right atrial medium, a commercial BMP10 ELISA kit was used (DY2926; R&D Systems) plates were coated with 0.2 μg/well of anti‐human BMP10 (MAB2926; R&D Systems) and blocked as above.

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: For measurement of BMP10 in conditioned right atrial medium, a commercial BMP10 ELISA kit was used (DY2926; R&D Systems) plates were coated with 0.2 μg/well of anti‐human BMP10 (MAB2926; R&D Systems) and blocked as above.

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: For measurement of BMP10 in conditioned right atrial medium, a commercial BMP10 ELISA kit was used (DY2926; R&D Systems) plates were coated with 0.2 μg/well of anti‐human BMP10 (MAB2926; R&D Systems) and blocked as above.

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: For measurement of BMP10 in conditioned right atrial medium, a commercial BMP10 ELISA kit was used (DY2926; R&D Systems) plates were coated with 0.2 μg/well of anti‐human BMP10 (MAB2926; R&D Systems) and blocked as above.

Techniques: Enzyme-linked Immunosorbent Assay, Staining