Journal: Cancer

Article Title: Colorectal carcinoma cell production of transforming growth factor beta decreases expression of endothelial cell vascular endothelial growth factor receptor 2.

doi: 10.1002/cncr.26247

Figure Lengend Snippet: Figure 6. (A) Representative Western blot shows vascular endothelial growth factor receptor 2 (VEGFR2) and activated Smad protein expression and (B) proportion of VEGFR2 protein levels in vehicle-treated and 5 ng/mL transforming growth factor beta (TGF-b)-treated bovine aortic endothelial cells (BAECs) cultured in atmospheric (21%) O2 and <0.1% O2 (anoxia) over 6 and 24 hours. Analysis of variance, P ¼ .0015; n ¼ 4; *P < .05. (C) VEGFR2 and Smad protein levels in BAECs treated for 24 hours with 0 to 50 ng/mL bone morphogenetic protein 9 (BMP9) are shown. (D) VEGFR2 and Smad protein levels in BAECs treated for 24 hours with combinations of 10 ng/mL BMP9, 5 ng/mL TGF-b1, and 5 lM SB-431542 (SB) are shown. Western blot is representative of 2 independent experiments.

Article Snippet: BAECs were grown until confluent and serum-starved overnight before treatment in serum-free medium with 0.1 to 20 ng/mL of recombinant human TGF-b1; 10 ng/mL recombinant human BMP9 (both R&D Systems, Minneapolis, Minn); 5 lM SB431542, an inhibitor of TGbR-I isoforms ALK5, ALK4, and ALK722; and 2.5 lM SD-208, an inhibitor of ALK5 and ALK4 (both Tocris Bioscience, Ellisville, Mo).23 CRC cells were grown until superconfluent, serumstarved overnight, then incubated in fresh serum-free DMEM for 48 hours.

Techniques: Western Blot, Expressing, Cell Culture

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: bioRxiv

Article Title: BMP9 regulates the endothelial secretome to drive pulmonary hypertension

doi: 10.1101/2025.08.29.673113

Figure Lengend Snippet: (A) Administering ALK1-Fc (6 doses) after the development of PH 3 weeks following exposure to SU-Hx elicited improvements in (B) RVSP and (C) Fulton’s index as compared to vehicle-treated rats, whereas administering ALK1-Fc (2 doses) before exposure to SU-Hx elicited a trend towards improved RVSP. ( n = 8 to 10 per group, mean ± SD, *P<0.05, **P<0.01, One-way ANOVA with Sidak’s test. (D) Administering ALK1-Fc (6 doses) coinciding with exposure to hypoxia elicited improvements in (E) RVSP and (F) Fulton’s index as compared to hypoxia-only exposed rats. ( n = 6 – 12 per group, mean ± SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, One-way ANOVA with Dunnett’s test (RVSP) and Sidak’s Test (Fulton’s index). (G) Immunofluorescence images reveal muscularization of small (<50 μm) pulmonary arterioles in SU-Hx vs. normoxic rats, which was attenuated by ALK1-Fc administered before (Pre-Tx), after (Post-Tx), or during hypoxia. (H) The percentage of fully, partially and non-muscularized vessels in normoxic rats vs. SU-Hx rats pre- or post-treated with ALK1-Fc compared to vehicle control and Hx rats treated with ALK1-Fc (diameter, 10 to 50 µm). Values shown mean ± SD, ** P <0.01, **** P <0.0001, one-way ANOVA with Tukey’s test for the percentage of fully muscularized vessels). (I) Adult SD rats were subjected to SU-Hx and (SUGEN5416 20 mg/kg, sc and FiO 2 = 0.10) for 3 weeks, followed by 3 weeks of treatment in normoxia with rBMP9 (30 µg/kg for low dose, 150 µg/kg for high dose, ip, daily) and the examined for (J) RVSP, (K) Fulton’s index and (L) intimal-medial thickness index of pulmonary arterioles < 50 µm ( n = 5 to 11 per group, means ± SD, * P < 0.05, one-way ANOVA with Dunnett’s test. (M) Immunofluorescence images of muscularization of pulmonary arterioles in rats subjected to normoxia, SU-Hx, treated with vehicle, low or high dose rBMP9. (N) The percentage of fully, partially and non-muscularized vessels (diameter <50 µm, mean ± SD, two-way ANOVA with Dunnett’s test for percentage of fully muscularized vessels). (O) Heatmap showing the lung transcriptomes of Nx rats, SU-Hx rats and SU-Hx rats treated with vehicle, low dose BMP9, or high dose BMP9.

Article Snippet: Monoclonal antibody to BMP9 (MAB3209), and biotinylated affinity purified antibodies to BMP9 (BAF3209) were obtained from R&D Systems.

Techniques: Immunofluorescence, Control

Journal: bioRxiv

Article Title: BMP9 regulates the endothelial secretome to drive pulmonary hypertension

doi: 10.1101/2025.08.29.673113

Figure Lengend Snippet: (A) SDS-PAGE analysis of recombinant pro-complex BMP9 protein from different sources under reducing and non-reducing conditions reveals prodomain of ∼40 Kd, and covalently linked homodimer of ∼25 Kd, and homodimeric from of ∼12.5 Kd. Lanes 1-2 reveal two preparations containing nearly 100% disulfide-linked BMP9 homodimer under non-reducing conditions, both produced in CHO cells; lane 3 reveals ∼25% disulfide-linked homodimer and ∼75% non-disulfide linked BMP9 monomers, also produced in CHO cells and used in reference . (B) BRE-luciferase assay showing relative BMP-mediated transcriptional activity in telomerase-immortalized microvascular endothelial cells stimulated for 24 h with 40 pM of 100% disulfide-linked BMP9 dimer, mixed 70%/30% disulfide-linked/non-disulfide linked BMP9 dimer, and 100% non-disulfide linked mutant BMP9 C329S protein. (C) Immunoblot analysis (biotinylated polyclonal anti- BMP9 BAF3209, followed by streptavidin-HRP) of BMP9 immunoprecipitated from 10 mL pooled human AB donor serum (HS) using anti-BMP9 (MAB3209, α-BMP9, 1 µg/mL, 4C x 12h). Serum immunoprecipitated BMP9 migrated as a ∼25 Kd dimer under non-reducing conditions and as a ∼12.5 Kd monomer under reducing conditions (R), similar to control (CTRL) recombinant mature BMP9 homodimer (1 μg). Anti-VEGFA antibody (α-VEGF) was used as a non-specific immunoprecipitation control antibody. (D) SDS-PAGE analysis under non-reducing conditions of recombinant BMP9 produced as 70%/30% disulfide linked/non-linked BMP9 (“70/30”), 100% non-disulfide linked mutant BMP9 C329S protein (“0/100”), 100% disulfide linked pro-complex BMP9 (“100/0”), and 100% disulfide-linked mature BMP9. (E) Immunoblot demonstrates the ability of different BMP9 preparations to elicit activation of SMAD1 and SMAD3 in cultured TIME cells. A 70%/30% disulfide linked/non-linked BMP9, 100% disulfide linked pro-complex BMP9, and disulfide-linked mature BMP9 elicit similar activation of SMAD1 and SMAD3, whereas non-disulfide linked mutant BMP9 C329S (“0/100”) protein elicited attenuated activation of SMAD1.

Article Snippet: Monoclonal antibody to BMP9 (MAB3209), and biotinylated affinity purified antibodies to BMP9 (BAF3209) were obtained from R&D Systems.

Techniques: SDS Page, Recombinant, Produced, Luciferase, Activity Assay, Mutagenesis, Western Blot, Immunoprecipitation, Control, Activation Assay, Cell Culture

Journal: bioRxiv

Article Title: BMP9 regulates the endothelial secretome to drive pulmonary hypertension

doi: 10.1101/2025.08.29.673113

Figure Lengend Snippet: (A) Adult male SD rats developed severe PH following exposure to SU-Hx for three weeks, and were treated with anti-BMP9 (MAB3209, 8 mg/kg), ALK1-Fc isotype control (8 mg/kg), ACTRIIA-Fc (2.1 mg/kg), or isotype control Ab (8 mg/kg, all i.p, twice weekly) for 1 or 3 weeks under normoxia. (B-E) Treatment with ACTRIIA-Fc, ALK1-Fc, and anti-BMP9 elicited similar improvements in RVSP at 1 week and 3 weeks. ACTRIIA-Fc and anti-BMP9 improved Fulton’s index after 1 week, whereas ACTRIIA-Fc and ALK1-Fc improved Fulton’s index at 3 weeks, and anti-BMP9 elicited a trend (n = 3 - 11 per group, mean ± SD, *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA with Dunnet’s test (RVSP), and Holm-Sidak’s test (Fulton’s Index). (F-G) Immunofluorescence analysis of microvessels (<50 µm) revealed reduced muscularization in SU-Hx rats with anti-BMP9, ALK1-Fc, and ACTRIIA-Fc, shown as mean ± SD, *P<0.05 by one- way ANOVA with Holm-Sidak’s test for the percentage of non-muscularized vessels). (H) Micro- CT angiography of the pulmonary vasculature of SU-Hx rats with or without anti-BMP9 treatment reveals improved vascular density with anti-BMP9 treatment. (I) Single nucleus RNA- Seq analysis of control and SU-Hx exposed rat lungs reveals broad representations of typical cell populations (doublet population not shown). (J) Dot plot of BMP/TGFb receptors across all cell types (doublets and neuroendocrine excluded, expression scaled by column/gene). BMP9/BMP10 receptors Bmpr2, Acvrl1, and Eng are enriched in endothelial lineages; activin receptors Acvr2a and Acvr1b are broadly expressed with enrichment of Acvr2a in fibroblast lineages; TGFβ receptors Tgfbr2 and Tgfbr1 are broadly expressed. (K) Dot plot of SMAD1/5/9 and SMAD2/3 target genes in different treatments and cell types (expression scaled by column/gene, within each cell type). At 1 week after return to normoxia, SU-Hx exposure was associated with increased SMAD1/5/9 and SMAD2/3 transcriptional activity based on Id1/Id2/Id3 and Serpine1/Ccn2 expression, respectively; One week of treatment with anti-BMP9 or ACTRIIA-Fc elicited similar effects in dampening SMAD1/5/9 and SMAD2/3 transcriptional activity in endothelial and fibroblast lineages. (L) Barplots with the number of differentially expressed genes (p adjust < 0.05) for each cluster and contrast. After one week of treatment with anti-BMP9 or ACTRIIA-Fc, the highest number of downregulated genes were in endothelial and fibroblast compartments, whereas the most upregulated genes were found in the endothelial compartment. (M) Within endothelium there was a high correlation of genes upregulated and downregulated by anti-BMP9 and ACTRIIA-Fc (r(Spearman)=0.83, p=2.54e-43). (N) Within endothelial lineages after 1 week of treatment, the significant differentially expressed genes (DEGs) had a high degree of overlap between anti-BMP9 and ACTRIIA-Fc, with 22 of 48 genes downregulated by anti-BMP9 and 69 genes downregulated by ACTRIIA-Fc shared. (O) Heatmap of representative genes (normalized counts, row scaled z-scores) in endothelial cells. Similar samples clusters (columns) are grouped by hierarchical clustering, with representative genes upregulated by exposure to SU-Hx in endothelium and downregulated by treatment with anti-BMP9 and ACTRIIA-Fc shown.

Article Snippet: Monoclonal antibody to BMP9 (MAB3209), and biotinylated affinity purified antibodies to BMP9 (BAF3209) were obtained from R&D Systems.

Techniques: Control, Immunofluorescence, Micro-CT, RNA Sequencing, Expressing, Activity Assay

Journal: bioRxiv

Article Title: BMP9 regulates the endothelial secretome to drive pulmonary hypertension

doi: 10.1101/2025.08.29.673113

Figure Lengend Snippet: (A) A surface plasmon resonance (SPR) kinetic assay was conducted to measure the affinity of Ab93 to human rBMP9 protein at 37°C via Biacore T200 instrument. Ab93 was adsorbed onto an anti- Fc chip, and rBMP9 was applied with titration series (4 nM to 0.25 nM, with 2-fold dilutions) with 60 s association time and 900 s dissociation time to generate rate constants and affinities, yielding an equilibrium dissociation (K D ) value of rBMP9 for Ab93 of 54.89 pM. ( B-E ) ELISA measurement of the binding mode of Ab93 for rBMP9. Various type II or type I BMP and activin receptor extracellular domains expressed as IgG Fc fusion proteins (ACTRIIA-Fc, ACTRIIB-Fc, BMPR2-Fc, and ALK1-Fc) were adsorbed onto plates, and incubated with biotinylated rBMP9 (rBMP9-biotin) in the presence of varying concentrations of Ab93 or IgG isotype control (Ab8.8), revealing Ab93 competes with binding of rBMP9 to type II but not type I receptors. ( F ) Adult male SD rats developed severe PH following exposure to SU-Hx for three weeks, and were treated with anti-BMP9 (Ab93, 8 mg/kg) or isotype control (8 mg/kg, both ip, twice weekly) for 3 weeks under normoxia. (G) RVSP and (H) Fulton’s index were significantly improved in response to treatment ( n = 6 to 8 per group, mean ± SD, ***P<0.001 by one-way ANOVA with Tukey’s test). (I-J) Immunofluorescence analysis of muscularization based on intimal-medial thickness of pulmonary small vessels (< 50 µm) revealed improved muscularization in Ab93 treatment vs. isotype (n = 5 animals per group with 20-30 vessels counted, mean ± SD, **P<0.01, ***P<0.001 by one-way ANOVA with Dunnett’s test). (K) Principal component analysis (PCA) of bulk RNAseq analysis of whole lung tissues demonstrated distinct clustering of tissues from animals exposed to normoxia, SU-Hx treated with isotype control, and SU-Hx treated with Ab93. (L) Heatmap showing the dysregulated genes in SU-Hx rats’ lung normalized by Ab93.

Article Snippet: Monoclonal antibody to BMP9 (MAB3209), and biotinylated affinity purified antibodies to BMP9 (BAF3209) were obtained from R&D Systems.

Techniques: SPR Assay, Kinetic Assay, Titration, Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation, Control, Immunofluorescence

Journal: bioRxiv

Article Title: BMP9 regulates the endothelial secretome to drive pulmonary hypertension

doi: 10.1101/2025.08.29.673113

Figure Lengend Snippet: (A-B) Biolayer interferometry was used to analyze the ligand specificity of Ab93 and ACTRIIA-Fc. At ligand concentrations of 100 nM, associations of rBMP9 and commercial (R&D) rBMP9 were detected for Ab93 (A) , but not other ligands tested (BMP10, activin A, GDF8, GDF11). Under the same conditions, (B) interactions for all other ligands tested, rBMP9, BMP10, activin A, GDF8 and GDF11, were found with ACTRIIA-Fc. (C-D) Circulating levels of BMP9 and BMP10 were measured by a sensitive LC-MS method in plasma obtained from control adult male SD rats, and those undergoing MCT-induced PH for 3 weeks and treated with Ab93 (or isotype control Ab (10 mg/kg i.p. twice weekly) revealing (C) markedly increased levels of circulating BMP9, consistent with trapping and metabolic protection of circulating BMP9, whereas (D) levels of BMP10 were not increased but slightly diminished. Values shown are mean ± S.D., *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001, comparisons based on one-way ANOVA with Sidak’s test. (E-H) Bulk RNAseq analysis of lungs from SU-Hx exposed rats treated with ALK1-Fc versus Ab93 were compared. (E) Heatmap showing DEGs upregulated by SU-Hx in comparison to Normoxic rats and downregulated by ALK1-Fc. (F) H eatmap showing DEGs downregulated by SU-Hx in comparison to Normoxic rats and upregulated by ALK1-Fc. (G) Comparison of numbers of upregulated DEGs in SU-Hx animals that ere downregulated by Ab93 and ALK1-Fc revealed overlapping sets of genes including Cxcl12, Igfbp4, Ccl21, and Grem1 . (H) Genes that were downregulated in SU-Hx rat lungs that were upregulated by treatment of Ab93 or ALK1-Fc included Spn and Rnase6 .

Article Snippet: Monoclonal antibody to BMP9 (MAB3209), and biotinylated affinity purified antibodies to BMP9 (BAF3209) were obtained from R&D Systems.

Techniques: Liquid Chromatography with Mass Spectroscopy, Clinical Proteomics, Control, Comparison

Journal: bioRxiv

Article Title: BMP9 regulates the endothelial secretome to drive pulmonary hypertension

doi: 10.1101/2025.08.29.673113

Figure Lengend Snippet: (A) Uniform manifold approximation and projection (UMAP) plot showing identified endothelial cell types in lungs from 3 IPAH patients and 6 donor controls (GSE169471). (B) Venn diagram showing the overlap of upregulated genes in SU-Hx rat lung (SU-Hx–Isotype control vs. Nx), downregulated genes in Ab93-treated SU-Hx rat lung (SU-Hx–Ab93 vs. SU-Hx–Isotype), and upregulated genes in IPAH pulmonary general capillary endothelial cells (gCap) (IPAH vs. Control, GSE169471). (C) Venn diagram showing the overlap of upregulated genes in BMP9-treated HPMVEC (BMP9-1.5h vs. control) and upregulated genes in IPAH pulmonary gCap ECs (IPAH vs. Control, GSE169471). (D-G) Volcano plots showing changes and significance of candidate driver genes across different datasets, including (D) upregulation of ID1 , BMPR2 , CXCL12, EDN1, COL18A1 , and IGFBP4 in IPAH vs. control lungs; (E) upregulation of ID1, BMPR2 , EDN1, IGFBP4, VEGFA, PDGFA, PDGFB, VWA1 , and SOX18 in BMP9-treated PMVEC; (F) upregulation of Edn1, Cald1, Vwa1, Col18a1 , and Igfbp4 in Su-Hx treated rats vs. normoxia; and (G) downregulation of Cxcl12, Igfbp4, Col18a1, Vwa1 , and Cald1 in Ab93-treated vs. isotype control treated SU-Hx rats. (H-L) Violin plots showing increased expression of CXCL12 , IGFBP4 , EDN1 , ENG , and BMPR2 in various subsets of endothelial cells from IPAH patients (GSE169471). (M) BMP9 (40 pM) increased CXLC12 mRNA expression in HPMVEC in a time-dependent manner. (N-R) BMP9 (40 pM) increased secretion of CXCL12, IGFBP4, ET-1, PDGF-BB and CCL2 in cultured HPMVEC supernatants. ( n = 3 per group, means ± SD. *P<0.05 as compared to 0 h or all the other groups, Dunnett’s test) (S-U) BMP9 (40 pM) treatment of HPMVEC increased mRNA expression of CXCL12 , which was diminished when BMPR2 , ACVRL1 or ENG were knocked down. ( n = 3 per group, mean ± SD, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 by one-way ANOVA with Sidak’s test).

Article Snippet: Monoclonal antibody to BMP9 (MAB3209), and biotinylated affinity purified antibodies to BMP9 (BAF3209) were obtained from R&D Systems.

Techniques: Control, Expressing, Cell Culture

Journal: bioRxiv

Article Title: BMP9 regulates the endothelial secretome to drive pulmonary hypertension

doi: 10.1101/2025.08.29.673113

Figure Lengend Snippet: ( A ) Dot plot highlighting log 10 average expression of selected marker genes used to identify endothelial clusters. The dot size corresponds to the percentage of cells expressing a gene in a given cluster. ( B ) Venn diagram showing the overlap of upregulated genes in SU-Hx rat lung (SU-Hx– Isotype control vs Nx), downregulated genes in Ab93-treated SU-Hx rat lungs (SU-Hx–Ab93 vs SU-Hx–Isotype), and upregulated genes in IPAH pulmonary artery endothelial cells (AEC, IPAH vs Control, GSE169471). ( C ) Venn diagram showing the overlap of upregulated genes in BMP9-treated HPMVEC (BMP9-1.5h vs control) and upregulated genes in IPAH pulmonary AECs (IPAH vs Control, GSE169471). ( D ) Volcano plot showing potential candidate genes upregulated in IPAH pulmonary AEC (IPAH vs Control, GSE169471). BMP9 (40 pM) increased CXLC12 mRNA ( E ) and ( F ) protein expression in HPAEC at varying intervals up to 24h. Stimulation of cultured HPAEC with BMP9 (40 pM) increased ( G ) IGFBP4 mRNA and ( H ) protein expression, as well as protein expression of ( I ) Endothelin-1, ( J ) PDGF-BB, and ( K ) CCL2 protein in supernatants at varying intervals over 24h. ( n = 3 per group, mean ± SD, *P<0.05 as compared to 0 h, one-way ANOVA with Dunnett’s multiple comparisons test).

Article Snippet: Monoclonal antibody to BMP9 (MAB3209), and biotinylated affinity purified antibodies to BMP9 (BAF3209) were obtained from R&D Systems.

Techniques: Expressing, Marker, Control, Cell Culture

Journal: bioRxiv

Article Title: BMP9 regulates the endothelial secretome to drive pulmonary hypertension

doi: 10.1101/2025.08.29.673113

Figure Lengend Snippet: (A) Heatmap of endothelial DEGs between various treatments: anti-BMP9//MAB3209 (8 mg/kg), ACTRIIA-Fc (2.1 mg/kg), or isotype control Ab (8 mg/kg, all i.p, twice weekly) in SU-Hx rats, and normoxic rats. Single-cell expression is aggregated on a sample level, normalized, scaled to z-scores and similar genes (rows) and treatments (columns) aggregated by hierarchical clustering. Row label (‘Specific’) indicates genes that are statistically significant in anti-BMP9 only (red) in ACTRIIA-Fc (blue) only or both treatments (purple). (A) Panel denotes endothelial genes downregulated with treatments compared to isotype control. (B) Panel denotes endothelial genes upregulated with treatments compared to isotype control.

Article Snippet: Monoclonal antibody to BMP9 (MAB3209), and biotinylated affinity purified antibodies to BMP9 (BAF3209) were obtained from R&D Systems.

Techniques: Control, Expressing

Journal: bioRxiv

Article Title: BMP9 regulates the endothelial secretome to drive pulmonary hypertension

doi: 10.1101/2025.08.29.673113

Figure Lengend Snippet: (A) Violin plot showing expression of ACVRL1 in various subpopulations of human pulmonary endothelial cells (GSE169471). (B-D) BMP9 (40 pM) treatment of HPMVEC increased mRNA expression of ENG and BMPR2 , but not ACVRL1 , each of which were diminished by treatment with specific siRNA ( n = 3 per group, mean ± SD, ** P <0.01, *** P <0.001, **** P <0.0001 by one-way ANOVA with Sidak’s test). (E) BMP9 (40 pM) induced expression of CXCL12 mRNA in TIME cells in a manner requiring expression of BMPR2 , ACVRL1 , and ENG based on treatment with specific siRNA. (F) BMP9 (40 pM) induced expression of CXCL12 mRNA in TIME cells in a manner requiring expression of SMAD1 and SMAD5 based on treatment with specific siRNA. Values are shown as mean ± S.D., * P <0.05, ** P <0.01, *** P <0.001, *** P <0.0001, or as indicated by one-way ANOVA with Sidak’s test. (G-M) BrdU incorporation assay showing that CXCL12 inhibited the proliferation of PMVEC from healthy (G) and to a lesser degree PAH donors ( H, I, J) as well as PAEC (K) . CXCL12 and PDGF-BB increased the proliferation of HPASMC ( L and M ). ( n = 8 per group). Values are shown as mean ± S.D., *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, or as indicated by t-test.

Article Snippet: Monoclonal antibody to BMP9 (MAB3209), and biotinylated affinity purified antibodies to BMP9 (BAF3209) were obtained from R&D Systems.

Techniques: Expressing, BrdU Incorporation Assay

Journal: bioRxiv

Article Title: BMP9 regulates the endothelial secretome to drive pulmonary hypertension

doi: 10.1101/2025.08.29.673113

Figure Lengend Snippet: (A) Venn diagram of RNA-Seq analysis of whole lung tissues reveals the overlap of upregulated genes in SU-Hx rat lung vs. normoxic rat lungs (SU-Hx up), downregulated genes in Ab93- vs. isotype control treated SU-Hx rat lung (Ab93 down), and upregulated genes in IPAH pulmonary SMC/pericytes (SMC/Pericyte up, GSE169471). (B) Volcano plots depict the expression levels of 6 potentially secreted products of 30 overlapping genes in pulmonary SMC/Pericytes of IPAH patients (GSE169471). (C) Violin plot showing significantly increased Calponin1 (CNN1) expression in pulmonary SMC/pericyte of IPAH patients (GSE169471). (D-H) Treatment with CXCL12 (100 ng/mL) increased expression of contractile phenotype marker genes CNN1 and TAGLN , as well as PAH related genes CPE , THY1 , LTBP2 in monocultured PASMCs. ( n = 4 per group, mean ± SD. ** P <0.01, *** P <0.001, **** P <0.0001 by t-test). (I) PASMCs did not exhibit increased CNN1 expression in response to BMP9 (40 pM) when cultured with other PASMC, but when cocultured with PMVEC exhibited increased CNN1 expression when exposed to BMP9 ( n = 3 to 4 per group, mean ± SD, ** P <0.01 by t-test. (J-K) Treatment of co-cultured PMVEC and PASMC with BMP9 induced CNN1 expression in PASMC. BMP9 treatment-induced expression of CNN1 in PASMC cocultured with PMVEC (J) , which was partially blocked by CXCL12 neutralizing antibody (5 nM, n = 3 per group, mean ± SD. * P <0.05, ** P <0.01, **** P <0.0001 by one-way ANOVA with Holm-Sidak test). (L) Treatment of co-cultured PMVEC and PASMC with 5% human serum induced CNN1 expression in HPASMCs, which was partially neutralized by treatment with anti-BMP9 (MAB3209, 5 mM, n = 3 per group, ** P <0.01, **** P <0.0001 by one- way ANOVA with Dunnett’s test). (M) BMP9 treatment-induced expression of CNN1 in PASMC that were co-cultured with PMVEC was inhibited by treatment with CXCR4 inhibitor AMD3100 (10 µM, n = 4 per group, mean ± SD. ** P <0.01, *** P <0.001 by one-way ANOVA with Dunnett’s test).

Article Snippet: Monoclonal antibody to BMP9 (MAB3209), and biotinylated affinity purified antibodies to BMP9 (BAF3209) were obtained from R&D Systems.

Techniques: RNA Sequencing, Control, Expressing, Marker, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Bone Morphogenetic Protein 9 (BMP9) by Redox-dependent Proteolysis *

doi: 10.1074/jbc.M114.579771

Figure Lengend Snippet: Expression, activity, and purification of recombinant BMP9. A , schematic diagram of BMP9 production and processing. B , schematic diagram of the generation of HBMP9. A His tag ( filled star ) is introduced at the beginning of the mature BMP9. C , conditioned medium from HEK-EBNA cells transfected with prepro-BMP9 ( lane 1 ) or prepro-HBMP9 ( lane 2 ) were separated on a non-reducing SDS-PAGE and probed with anti-BMP9 antibody (MAB3209). The diagram on the right shows the schematic drawing of the BMP9 molecules. D denotes dimer on non-reducing SDS-PAGE, and M denotes monomer on SDS-PAGE. D- and M-forms migrate slower in the HBMP9 due to the addition of the His 6 tag at the N terminus of the mature ligand as depicted in B. D , HEK cell-produced BMP9 and HBMP9 have comparable activity with BMP9 from R&D Systems. Conditioned media containing pBMP9 (because it is very likely to be present as a prodomain bound complex in the conditioned media) or pHBMP9 were quantified by ELISA using R&D BMP9 as a standard and subjected to signaling assay using C2C12 cells transfected with ALK1. Plasmid containing Renilla was co-transfected with reporter plasmid containing BMP response element-luciferase, and the luciferase activity induced by BMP9 signaling was read using the Promega Dual-Luciferase system. E , purified pBMP9 and HBMP9 were fractionated on a 12% non-reducing SDS-PAGE and stained by Coomassie Blue. F , identical samples of R&D Systems BMP9, pBMP9, and HBMP9 were run in parallel on three 12% non-reducing SDS-PAGE, then blotted separately against the following: anti-BMP9 antibody ( left ), anti-BMP9 prodomain antibody ( middle ), and anti-His tag antibody ( right ). A single asterisk indicates a minor band that could not be seen on the SDS-PAGE in E but reacted very strongly with anti-BMP9 antibody. This may be a species of partially processed BMP9. Double asterisks indicate nonspecific carrier protein from R&D Systems BMP9. Pro , prodomain.

Article Snippet: Anti-BMP9 antibody (MAB3209 and AF3209), anti-BMP9 prodomain antibody (AF3879), and control BMP9 were purchased from R&D Systems, Inc. Anti-His tag antibody (37–2900) was purchased from Invitrogen.

Techniques: Expressing, Activity Assay, Purification, Recombinant, Transfection, SDS Page, Produced, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Luciferase, Staining

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Bone Morphogenetic Protein 9 (BMP9) by Redox-dependent Proteolysis *

doi: 10.1074/jbc.M114.579771

Figure Lengend Snippet: M-form BMP9 is a non-covalently linked dimer. A , HBMP9 was loaded onto a Superdex 75 10/30 gel filtration column pre-equilibrated in 50 m m Tris·HCl, pH 7.4, containing 150 m m NaCl. Non-reducing SDS-PAGE of the peak fractions (B3 to B5) revealed the D- and M-forms of HBMP9 co-elute under the same peak. The doublet in the M-form on SDS-PAGE was probably due to a partial reduction of intramolecular disulfide bonds. B , a representative of the HBMP9 crystal ( left ) and two examples of washed single crystals ran on a non-reducing SDS-PAGE ( right ) demonstrated that each single crystal contains a mixture of D- and M-forms of HBMP9. C , crystal structure of HBMP9 ( left ), colored according to the B-factors (spectrum, blue to white to red , from 20 to 100) with Cys-73 in two conformations shown in sticks . Electron density (2 F o − F c map at 1σ) clearly shows two conformations of Cys-73 ( right ). D , HBMP9 was overlaid with the published BMP9 structures (Protein Data Bank codes 1ZKZ and 4FAO , all colored as described in C ). In the semi-transparent schematic, type I receptor ALK1 is shown in yellow , and activin receptor type 2B is shown in cyan as in BMP9·ALK1·activin receptor type 2B complex (Protein Data Bank code 4FAO ).

Article Snippet: Anti-BMP9 antibody (MAB3209 and AF3209), anti-BMP9 prodomain antibody (AF3879), and control BMP9 were purchased from R&D Systems, Inc. Anti-His tag antibody (37–2900) was purchased from Invitrogen.

Techniques: Filtration, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Bone Morphogenetic Protein 9 (BMP9) by Redox-dependent Proteolysis *

doi: 10.1074/jbc.M114.579771

Figure Lengend Snippet: M-form BMP9 can bind to ALK1 and prodomain as the D-form. A , M- and D- forms of HBMP9 co-migrate on native PAGE, and both can form complexes with ALK1 ECD. B , M- and D-forms of BMP9 co-migrate, both as free form and as prodomain-bound form. In A and B , bands 1 to 6 from native PAGE were cut out, boiled in 2× SDS-loading buffer for 20 min before loading onto a non-reducing SDS-PAGE to confirm the identities. For SDS-PAGE in A , two parts of the same gel are shown.

Article Snippet: Anti-BMP9 antibody (MAB3209 and AF3209), anti-BMP9 prodomain antibody (AF3879), and control BMP9 were purchased from R&D Systems, Inc. Anti-His tag antibody (37–2900) was purchased from Invitrogen.

Techniques: Clear Native PAGE, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Bone Morphogenetic Protein 9 (BMP9) by Redox-dependent Proteolysis *

doi: 10.1074/jbc.M114.579771

Figure Lengend Snippet: Intermolecular disulfide bond is not required for BMP9 signaling activity. A , conditioned media (6 μl) from HEK-EBNA cells transfected with transfection reagent alone ( lane 1 ), empty vector ( lane 2 ), pro-BMP9 ( lane 3 ), pro-HBMP9 ( lane 4 ), or pro-HBMP9 C73S ( lane 5 ) were fractionated on a 12% SDS-PAGE and probed with anti-BMP9 prodomain antibody. The intensities of bands were quantified using ImageJ, and the ratio of HBMP9 C73S to HBMP9 obtained. HBMP9 C73S concentration in the conditioned medium was normalized to HBMP9 using the above ratio. B , left : hPAECs were serum-restricted in EGM-2, 0.1% FBS overnight, and stimulated with HBMP9 or HBMP9 C73S (both 0.25–3 ng/ml) for 1 h. Cells were harvested, and total cell protein was immunoblotted with anti-pSmad1/5/8 antibody. Band intensities of pSmad1/5/8 blots were analyzed using ImageJ, corrected by ratios obtained from the α-tubulin blot, and normalized to a 0.25 ng/ml wild type sample. Data of the mean ± S.E. from three repeats are shown on the right. C , after quiescence overnight in EGM-2, 0.1% FBS, hPAECs were treated with 1 ng/ml of HBMP9 or HBMP9 C73S. Samples were harvested at 1, 2, 6, and 24 h, and immunoblotting was carried out as described in B . Band intensity of pSmad1/5/8 blots were analyzed using ImageJ, corrected by ratios obtained from the α-tubulin blot and normalized to a wild type 1-h treatment sample. Data of the mean ± S.E. from three repeats are shown on the right. NS , not significant.

Article Snippet: Anti-BMP9 antibody (MAB3209 and AF3209), anti-BMP9 prodomain antibody (AF3879), and control BMP9 were purchased from R&D Systems, Inc. Anti-His tag antibody (37–2900) was purchased from Invitrogen.

Techniques: Activity Assay, Transfection, Plasmid Preparation, SDS Page, Concentration Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Bone Morphogenetic Protein 9 (BMP9) by Redox-dependent Proteolysis *

doi: 10.1074/jbc.M114.579771

Figure Lengend Snippet: BMP9 is regulated by redox potential. Purified HBMP9 ( A ), pBMP9 ( B ), or BMP6 ( C ) were incubated at room temperature overnight with PBS alone or redox buffer containing 0.1 m m GSSG and 0–20 m m GSH. Proteins were then run on a non-reducing SDS-PAGE and detected by Coomassie Blue. M * is the fully reduced form of BMP9.

Article Snippet: Anti-BMP9 antibody (MAB3209 and AF3209), anti-BMP9 prodomain antibody (AF3879), and control BMP9 were purchased from R&D Systems, Inc. Anti-His tag antibody (37–2900) was purchased from Invitrogen.

Techniques: Purification, Incubation, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Bone Morphogenetic Protein 9 (BMP9) by Redox-dependent Proteolysis *

doi: 10.1074/jbc.M114.579771

Figure Lengend Snippet: M-form BMP9 is more susceptible to redox-dependent proteolysis. HBMP9 ( A ) or pBMP9 ( B ) was incubated in PBS or redox buffer containing 0.1/1 m m GSSG/GSH (0.1/1) or 0.1/4 m m GSSG/GSH (0.1/4) at room temperature overnight. The following day, an aliquot of each treatment was subjected to limited trypsin digestion at 37 °C, 3 h for HBMP9 or overnight for pBMP9. Reactions were stopped by addition of SDS-loading buffer and boiling at 100 °C for 10 min. Cleavage was monitored by fractionating samples on a 12% non-reducing SDS-PAGE and Coomassie Blue staining.

Article Snippet: Anti-BMP9 antibody (MAB3209 and AF3209), anti-BMP9 prodomain antibody (AF3879), and control BMP9 were purchased from R&D Systems, Inc. Anti-His tag antibody (37–2900) was purchased from Invitrogen.

Techniques: Incubation, SDS Page, Staining

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Bone Morphogenetic Protein 9 (BMP9) by Redox-dependent Proteolysis *

doi: 10.1074/jbc.M114.579771

Figure Lengend Snippet: M-form BMP9 is preferentially cleaved in serum and model of BMP9 regulation by redox-dependent proteolysis. A , pBMP9 (0.3 μg) was reconstituted into 10 μl of serum (10 healthy human controls), and equal aliquots of samples were taken at 0 h ( lane 1 ) and after overnight incubation at 37 °C ( lane 2 ). Samples were then fractionated on a 12% SDS-PAGE under non-reducing conditions and detected by anti-BMP9 antibody (AF3209). The D- and M-forms of BMP9 at 0 h and overnight were quantified using ImageJ, and the % BMP9 remaining after overnight incubation was calculated and plotted using GraphPad Prism. Data are mean ± S.E. ( n = 10). B , model of the regulation of BMP9 concentration by redox-dependent proteolysis.

Article Snippet: Anti-BMP9 antibody (MAB3209 and AF3209), anti-BMP9 prodomain antibody (AF3879), and control BMP9 were purchased from R&D Systems, Inc. Anti-His tag antibody (37–2900) was purchased from Invitrogen.

Techniques: Incubation, SDS Page, Concentration Assay

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Characterization of GDF2 Mutations and Levels of BMP9 and BMP10 in Pulmonary Arterial Hypertension

doi: 10.1164/rccm.201906-1141oc

Figure Lengend Snippet: Figure 1: Characterisation of Expressed BMP9 Mutant Proteins. (a) Schematic of

Article Snippet: After washing, 0.04 μg/well of anti-human BMP9 detection antibody (R&D Systems BAF3209) was added in Copyright © 2019 by the American Thoracic Society 6 PBS/1% BSA containing 0.2% GS.

Techniques: Mutagenesis

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Characterization of GDF2 Mutations and Levels of BMP9 and BMP10 in Pulmonary Arterial Hypertension

doi: 10.1164/rccm.201906-1141oc

Figure Lengend Snippet: Figure 2: Loss of Activity in Pro:BMP9 Mutants Predicted to be Pathogenic. (a,b)

Article Snippet: After washing, 0.04 μg/well of anti-human BMP9 detection antibody (R&D Systems BAF3209) was added in Copyright © 2019 by the American Thoracic Society 6 PBS/1% BSA containing 0.2% GS.

Techniques: Activity Assay

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Characterization of GDF2 Mutations and Levels of BMP9 and BMP10 in Pulmonary Arterial Hypertension

doi: 10.1164/rccm.201906-1141oc

Figure Lengend Snippet: Figure 3: Loss of active BMP9 in PAH patients carrying putatively pathogenic GDF2

Article Snippet: After washing, 0.04 μg/well of anti-human BMP9 detection antibody (R&D Systems BAF3209) was added in Copyright © 2019 by the American Thoracic Society 6 PBS/1% BSA containing 0.2% GS.

Techniques:

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Characterization of GDF2 Mutations and Levels of BMP9 and BMP10 in Pulmonary Arterial Hypertension

doi: 10.1164/rccm.201906-1141oc

Figure Lengend Snippet: Figure 4: Plasma BMP9 and pBMP10 levels are not reduced in PAH, but a subset of

Article Snippet: After washing, 0.04 μg/well of anti-human BMP9 detection antibody (R&D Systems BAF3209) was added in Copyright © 2019 by the American Thoracic Society 6 PBS/1% BSA containing 0.2% GS.

Techniques: Clinical Proteomics