Journal: The Journal of Biological Chemistry

Article Title: Rapid Activation of Bone Morphogenic Protein 9 by Receptor-mediated Displacement of Pro-domains *

doi: 10.1074/jbc.M115.680009

Figure Lengend Snippet: Release of mature growth factor from BMP9·prodomain complex. Release of mature BMP9 domain after binding of an anti-pro-domain antibody was confirmed by application of BMP9·pro-domain to the pro-domain ELISA using anti-pro-domain antibodies AF3879 as capture and BAF3879 as detection (A). B, supernatant from A and recombinant mature BMP9 standard were applied to the mature BMP9 ELISA using anti-mature BMP9 antibodies MAB3209 as a capture and BAF3209 as detection. The mature BMP9 ELISA detected both mature BMP9 standard and released mature BMP9 from pro-domain ELISA supernatant.

Article Snippet: Antibodies used included anti-mature BMP9 antibodies MAB3209 and its biotinylated conjugate BAF3209 and anti-pro-domain antibodies AF3879 and its biotinylated conjugate BAF3879 and were purchased from R&D Systems.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Angiogenesis

Article Title: BMP10 functions independently from BMP9 for the development of a proper arteriovenous network

doi: 10.1007/s10456-022-09859-0

Figure Lengend Snippet: Bmp10 -inducible null mutants die prematurely showing enlarged hearts. a Schematic representation of the experimental strategy for Bmp10 global deletion in control (CreER-negative Bmp10 -iKO), Bmp9 -KO (CreER-negative Bmp9/10 -dKO), Bmp10 -inducible KO ( Bmp10 -iKO), and Bmp9/10 -double KO ( Bmp9/10 -dKO) mice using R26 CreER/+ line. Arrowheads indicate intragastric injections of 50 μg tamoxifen at P1, P2, P3, and P4 and the analysis at P6. b Levels of Bmp10 mRNA were detected by RT-qPCR in the hearts, livers, lungs, spleens, and kidneys from tamoxifen-injected P6 littermate control and Bmp10 -iKO mice. Inset indicates the level of Bmp10 mRNA in the livers. The mRNA levels are normalized to Actin . Data are mean ± SEM. Unpaired t test ( n = 6 mice per group). c Representative immunoblots show the levels of BMP10 proteins in the right atrium at P6. Unprocessed monomer (Pro-BMP10) and free prodomain (Free Pro) of BMP10 proteins were determined with anti-BMP10-Pro antibodies. Dimer of growth factor domain (GFD) was blotted using anti-BMP10-GFD antibodies under the non-reducing condition. TNNI3 was used as a loading control. Numbers indicate the molecular mass in kDa. d and e , Plasmatic levels of BMP9 ( d ) and BMP10 ( e ) proteins assessed by ELISA in control, Bmp9 -KO, Bmp10 -iKO, and Bmp9/10 -dKO mice at P1 and P6 as indicated. Data are mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test ( n = 4 to 9 mice per group). f Survival curves for control ( n = 15 mice), Bmp9 -KO ( n = 15), Bmp10 -iKO ( n = 19), and Bmp9/10 -dKO mice ( n = 19) generated by the Kaplan–Meier method. Log-rank test (Match SPSS and SAS) was performed to determine significance. P < 0.0001. To compare the Bmp10 -iKO and Bmp9/10 -dKO survival curves, Mantel-Cox log-rank test was performed. g, Gross morphology ( upper ), and coronal ( middle ) and horizontal ( lower ) slices from 3D diceCT scanned images of control, Bmp9 -KO, Bmp10 -iKO, and Bmp9/10 -dKO P6 hearts. RA right atrium, LA left atrium, RV right ventricle, LV left ventricle. Scale bars, 1 mm. h – j Quantification of heart weight/body weight (HW/BW, h ), heart weight/tibia length (HW/Tibia, i ), and lung weight/tibia length (LW/Tibia, j ) ratios. Data are mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test ( n = 6 to 16 mice per group). k – n , Scatter plots measuring hemoglobin level (Hb, k ), red blood cell count (RBC, l ), hematocrit (HCT, m ), and mean corpuscle volume (MCV, n ) to assess anemia. Data represent individual mice and mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test ( n = 6 to 9 mice per group)

Article Snippet: Control PBS, 100 ng of mouse BMP9 protein (R&D systems, 5566-BP), or 100 ng of mouse BMP10 protein (R&D systems, 6038-BP) was injected intraperitoneally and daily on the opposite side of the milk spot until the sample collection.

Techniques: Control, Quantitative RT-PCR, Injection, Western Blot, Enzyme-linked Immunosorbent Assay, Generated, Cell Counting

Journal: Angiogenesis

Article Title: BMP10 functions independently from BMP9 for the development of a proper arteriovenous network

doi: 10.1007/s10456-022-09859-0

Figure Lengend Snippet: Postnatal Bmp10 deletion leads to delayed progression of retinal vessels. a Schematic representation of the experimental strategy for Bmp10 global deletion in control, Bmp9 -KO, Bmp10 -iKO, and Bmp9/10 -dKO mice. Arrowheads indicate intragastric injections of 50 μg tamoxifen at P1, P2, P3, and P4 and the analysis at P6. b–e CD31, smooth muscle actin (SMA), and ERG1/2/3 staining of whole-mount P6 retinas from control, Bmp9 -KO, Bmp10 -iKO, and Bmp9/10 -dKO mice. Dotted circles represent outgrowth of the control retina ( b ). Scale bars, 500 μm ( b–e ); 100 m ( e ). a artery, v vein. f–h Quantification of the vessel radial length ( f , n = 8 to 16 retinas per group), vessel density in the vascular front ( g , n = 20 to 24 areas from 5 to 6 retinas per group), and total tip cell number ( h , n = 12 to 17 of areas from 5 to 7 retinas per group). Data are mean ± SEM. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test

Article Snippet: Control PBS, 100 ng of mouse BMP9 protein (R&D systems, 5566-BP), or 100 ng of mouse BMP10 protein (R&D systems, 6038-BP) was injected intraperitoneally and daily on the opposite side of the milk spot until the sample collection.

Techniques: Control, Staining

Journal: Angiogenesis

Article Title: BMP10 functions independently from BMP9 for the development of a proper arteriovenous network

doi: 10.1007/s10456-022-09859-0

Figure Lengend Snippet: Bmp10 deficiency leads to the development of arteriovenous malformation in neonatal retinas. a Schematic representation of Bmp10 deletion. Arrowheads indicate intragastric tamoxifen injections at P2 and P3 and the analysis at P7. b CD31 and SMA staining of control, Bmp9 -KO, Bmp10 -iKO, and Bmp9/10 -dKO P7 retinas. a artery, v vein. Scale bars, 200 μm. c–e Quantification of the vessel radial length ( c , n = 8 to 14 retinas per group), artery diameter ( d , n = 26 to 31 arteries from 6 to 8 retinas per group), and vein diameter ( e , n = 25 to 34 veins from 6 to 8 retinas per group). Data are mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test. f Representative images of the latex dye-perfused retinas of control, Bmp9 -KO, Bmp10 -iKO, and Bmp9/10 -dKO mice at P7. Arrows mark arteriovenous shunts. a artery, v vein. Scale bars, 500 μm. g–j Scatter plots measuring Hb ( g ), RBC ( h ), HCT ( i ), and MCV ( j ) to assess hemorrhage. Data represent individual mice and mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test ( n = 6 to 13 mice per group)

Article Snippet: Control PBS, 100 ng of mouse BMP9 protein (R&D systems, 5566-BP), or 100 ng of mouse BMP10 protein (R&D systems, 6038-BP) was injected intraperitoneally and daily on the opposite side of the milk spot until the sample collection.

Techniques: Staining, Control

Journal: Angiogenesis

Article Title: BMP10 functions independently from BMP9 for the development of a proper arteriovenous network

doi: 10.1007/s10456-022-09859-0

Figure Lengend Snippet: Proliferation and size of endothelial cells are increased in Bmp10 -deleted retinas. a CD31, Ki67, and ERG1/2/3 staining in vascular plexus of control, Bmp9 -KO, Bmp10 -iKO, and Bmp9/10 -dKO P7 retinas. a artery, v vein. Scale bars, 100 μm. b Quantification of the number of Ki67 and ERG1/2/3 double positive nuclei of endothelial cells per vascular area involved in AVM. Data are mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test ( n = 7 retinas per group). c IB4, VE-Cad, and ERG1/2/3 staining of control, Bmp9 -KO, Bmp10 -iKO, and Bmp9/10 -dKO P7 retinas. Higher magnified images of vessels in the artery (a), capillary (c), and vein (v) are shown. The arteriovenous shunts are outlined by dotted lines. Scale bars, 100 μm; 10 μm (magnified images in a, c, and v). d and e Quantification of areas ( d ) and numbers ( e ) of endothelial cells in veins. Data are mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test ( n = 4 to 6 retinas per group)

Article Snippet: Control PBS, 100 ng of mouse BMP9 protein (R&D systems, 5566-BP), or 100 ng of mouse BMP10 protein (R&D systems, 6038-BP) was injected intraperitoneally and daily on the opposite side of the milk spot until the sample collection.

Techniques: Staining, Control

Journal: Angiogenesis

Article Title: BMP10 functions independently from BMP9 for the development of a proper arteriovenous network

doi: 10.1007/s10456-022-09859-0

Figure Lengend Snippet: Bmp10 deletion results in brain AVMs and disrupts blood–brain barrier integrity in neonatal mice. a Schematic representation of Bmp10 deletion in neonatal mice. Arrowheads indicate intragastric tamoxifen injections at P2 and P3 and the analysis at P7 ( upper ). Representative images of the latex dye-injected cerebrovasculature in the brains of control, Bmp9 -KO, Bmp10 -iKO, Bmp9/10 -dKO, and L1Cre- Alk1 mice ( lower ). Magnified images of the boxes are shown in right and left hemispheres. Arrows mark arteriovenous shunts. a, artery; v, vein. Scale bars, 1 mm. b Percentage of brain AVMs of control ( n = 4 mice), Bmp9 -KO ( n = 7), Bmp10 -iKO ( n = 10), and Bmp9/10 -dKO mice ( n = 10). c Percentage of visible bleeding in brains. d Representative images of the brains showing internal visible hemorrhage after the perfusion. A magnified image of the box in the Bmp10 -iKO brain is shown in right panel. An arrow in the Bmp9/10 -dKO brain indicates coronally dissected position showing internal bleeding and the image of coronal dissected brain slice is shown in right panel. Scale bars, 1 mm. e and f Fluorescent whole brain ( e ) and retina ( f ) images injected with Alexa Fluor 555-cadaverine (Cad-A555) at P8. Scale bars, 1 mm. g and h , Quantification of fluorescence intensity in brains ( g , n = 4 to 6 mice per group) and retinas ( h , n = 8 to 12 retinas per group). Data are mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test

Article Snippet: Control PBS, 100 ng of mouse BMP9 protein (R&D systems, 5566-BP), or 100 ng of mouse BMP10 protein (R&D systems, 6038-BP) was injected intraperitoneally and daily on the opposite side of the milk spot until the sample collection.

Techniques: Injection, Control, Slice Preparation, Fluorescence

Journal: Angiogenesis

Article Title: BMP10 functions independently from BMP9 for the development of a proper arteriovenous network

doi: 10.1007/s10456-022-09859-0

Figure Lengend Snippet: Bmp10 deletion induces de novo AVM formation in response to skin wounding in adult mice. a and b Bmp10 gene deletion was induced by intraperitoneal injection of tamoxifen at 100 μg/g body weight for three consecutive days from the day of wounding (day 0) in 6 ~ 8-week-old adult mice. Skin vasculatures were examined by latex dye injection at day 7 (control, Bmp9 -KO, and Bmp10 -iKO) or at day 6 ( Bmp9/10 -dKO). Plasmatic levels of BMP9 ( a ) and BMP10 ( b ) proteins in control, Bmp9 -KO, Bmp10 -iKO, and Bmp9/10 -dKO mice were assessed by ELISA at Day 6 or Day 7 as indicated. Data are mean ± SEM. Kruskal–Wallis test followed by Dunn’s post hoc test ( n = 8 to 13 mice per group). c Survival curves for control ( n = 9 mice), Bmp9 -KO ( n = 9), Bmp10 -iKO ( n = 7), and Bmp9/10 -dKO mice ( n = 5) generated by the Kaplan–Meier method. Log-rank test (Match SPSS and SAS) was performed to determine significance. P < 0.0001. To compare the Bmp10 -iKO and Bmp9/10 -dKO survival curves, Mantel-Cox log-rank test was performed. d Percentile change of body weight from Day 0. Data are mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test ( n = 8 to 14 mice per group). e Hemoglobin levels in the beginning and end of the study period. Data are mean ± SEM. Two-way ANOVA followed by Tukey’s post hoc test ( n = 8 to 14 mice per group). f and g Scatter plots measuring RBC ( f ) and HCT ( g ) at the end day of the study. Data represent mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test ( n = 6 to 10 mice per group). h Percentage of skin wound-induced AVMs ( n = 8 to 14 mice per group). i–n Latex dye-injected vasculature surrounding the wound in the dorsal skin of control ( i ), Bmp9 -KO ( j ), Bmp10 -iKO ( k , l ), and Bmp9/10 -dKO ( m , n ) mice. The magnified images of boxed area in the left panels are shown in the right panels. The wound sites are indicated by asterisks. Some arteriovenous shunts are outlined by dotted lines as examples. a artery, v vein. Scale bars, 1 mm

Article Snippet: Control PBS, 100 ng of mouse BMP9 protein (R&D systems, 5566-BP), or 100 ng of mouse BMP10 protein (R&D systems, 6038-BP) was injected intraperitoneally and daily on the opposite side of the milk spot until the sample collection.

Techniques: Injection, Control, Enzyme-linked Immunosorbent Assay, Generated

Journal: Angiogenesis

Article Title: BMP10 functions independently from BMP9 for the development of a proper arteriovenous network

doi: 10.1007/s10456-022-09859-0

Figure Lengend Snippet: Administration of BMP10, but not BMP9, prevents AVM formation in Bmp9/10 -dKO. a Schematic representation of BMP protein administration and Bmp10 gene deletion into control and Bmp9/10 -dKO mice. Arrowheads indicate injection of 50 μg tamoxifen at P1–P4 and BMP9 or BMP10 proteins daily from P1. b–d CD31 and SMA staining on retinas isolated from P8 control (PBS-treated CreER-negative Bmp10 -iKO, b , BMP9-treated Bmp9/10 -dKO ( c ), and BMP10-treated Bmp9/10 -dKO ( d ) mice. Arrows mark arteriovenous shunts. a artery, v vein. Scale bars, 500 μm. e and f Quantification of retinal vascular outgrowth ( e , n = 5 to 12 retinas from 5 to 6 mice per group) and AVM number ( f , n = 6 to 12 retinas from 5 to 6 mice per group). Data are mean ± SEM. Statics Kruskal–Wallis test followed by Dunn’s post hoc test. g and h Hemoglobin levels ( g , n = 5 to 6 mice per group) and HW/BW ratio ( h , n = 3 to 5 mice per group) at the end day of the study. Data are mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test

Article Snippet: Control PBS, 100 ng of mouse BMP9 protein (R&D systems, 5566-BP), or 100 ng of mouse BMP10 protein (R&D systems, 6038-BP) was injected intraperitoneally and daily on the opposite side of the milk spot until the sample collection.

Techniques: Control, Injection, Staining, Isolation

Journal: Angiogenesis

Article Title: BMP10 functions independently from BMP9 for the development of a proper arteriovenous network

doi: 10.1007/s10456-022-09859-0

Figure Lengend Snippet: BMP10, but not BMP9, suppresses the development of AVMs caused by ENG-deficiency. a – e CD31 and SMA immunofluorescence staining on retinas isolated from P7 control (PBS-treated Scl- CreER-negative Eng -iKO, a , n = 28 retinas), PBS-treated Scl- CreER; Eng -iKO ( b , n = 16), BMP9-treated Scl- CreER; Eng -iKO ( c , n = 8), BMP10-treated Scl- CreER; Eng -iKO ( d , n = 8), and BMP9/BMP10-treated Scl- CreER; Eng -iKO ( e , n = 10) mice. Arrows mark arteriovenous shunts. a artery, v vein. Scale bars, 500 μm. f Quantification of the number of AVMs. Data are mean ± SD. One-way ANOVA followed by Tukey’s post hoc test

Article Snippet: Control PBS, 100 ng of mouse BMP9 protein (R&D systems, 5566-BP), or 100 ng of mouse BMP10 protein (R&D systems, 6038-BP) was injected intraperitoneally and daily on the opposite side of the milk spot until the sample collection.

Techniques: Immunofluorescence, Staining, Isolation, Control