Journal: Blood Vessels, Thrombosis & Hemostasis

Article Title: An angiopoietin-2 vaccine improves arteriovenous malformation pathology in hereditary hemorrhagic telangiectasia mice

doi: 10.1016/j.bvth.2026.100155

Figure Lengend Snippet: Immunogenicity of the ANG2-P3:CRM197 vaccine in C57BL/6 mice. (A) Schematic diagram of the vaccination schedule and generation of the BMP9/10ib model (created with biorender.com ). (B) Protein sequence alignment of the C-terminal end of human ANG2 (hANG2), mouse ANG2 (mANG2), human ANG1 (hANG1), and mouse ANG1 (mANG1), along with the peptide sequences of ANG2-P3 and ANG1-P3. (C-D) Serum antibody titers against ANG2-P3 (C) and ANG1-P3 (D) in ANG2-P3:CRM197-vaccinated females (Vac-1 to Vac-5) and controls (injected with saline [Sal-1 and Sal-2] or CRM197-only [CRM-1 to CRM-5]). The vaccinated females with the highest anti-ANG2-P3 titers were identified as “best responders” (marked with a red box). OD, optical density.

Article Snippet: After 3 additional washes with PBST, serial dilutions of individual mouse serum samples were prepared, along with a reference mouse anti-BMP9 antibody (no. MAB3209; R&D Systems) and a reference mouse anti-BMP10 antibody (no. MAB2926; R&D Systems), both diluted in 1% BSA in PBS.

Techniques: Immunopeptidomics, Sequencing, Injection, Saline

Journal: Blood Vessels, Thrombosis & Hemostasis

Article Title: An angiopoietin-2 vaccine improves arteriovenous malformation pathology in hereditary hemorrhagic telangiectasia mice

doi: 10.1016/j.bvth.2026.100155

Figure Lengend Snippet: ANG2-P3:CRM197 vaccine reduces AVM pathology in BMP9/10ib neonates. (A) Serum antibody titers against ANG2-P3 in pups and their corresponding dams vaccinated with ANG2-P3:CRM197 (Vac-1 to Vac-3) or injected with saline (Saline). Vac-1, Vac-2, and Vac-3 pups: n = 7; Saline pups: n = 2. Dams: n = 1. (B) Representative immunofluorescence staining with isolectin B4 (IB4; green) and of SMA (red) in P6 retinas of pups treated with PBS or BMP9/10ib from a dam vaccinated with ANG2-P3:CRM197 or injected with saline. Scale bar: 1.5 mm. (C-D ) AVM count per retina (C), and retinal AVM surface area (D) in BMP9/10ib pups from dams vaccinated with ANG2-P3:CRM197 or injected with saline (Saline). BMP9/10ib + saline: n = 24; BMP9/10ib + ANG2-P3:CRM197: n = 37 (C). BMP9/10ib + saline: n = 24; BMP9/10ib + ANG2-P3:CRM197: n = 34 (D). (E) Spearman rank correlation matrix of the indicated variables (n = 15). (F-H) Retinal artery diameter (F), retinal vein diameter (G), and SMA coverage area (H) in BMP9/10ib pups from dams vaccinated with ANG2-P3:CRM197 or injected with saline (Saline). Data are shown as mean ± standard error of the mean (SEM); unpaired t test with Welch correction for panel C, Mann-Whitney test for panel D, and 1-way analysis of variance with Tukey multiple comparisons test for panels F-H. PBS + saline: n = 15; BMP9/10ib + saline: n = 24; BMP9/10ib + ANG2-P3:CRM197: n = 37 (F,G). PBS + saline: n = 13; BMP9/10ib + saline: n = 21; BMP9/10ib + ANG2-P3:CRM197: n = 29 (H). ∗ P < .05; ∗∗∗ P ≤ .001; ∗∗∗∗ P < .0001. (I) Immunoassay data measuring ANG2 levels in plasma from patients with HHT and healthy controls. Data are shown as mean ± SEM; unpaired t test with Welch correction. Healthy: n = 36; HHT: n = 36. ∗∗ P ≤ .01. a, artery; ns, not significant; v, vein.

Article Snippet: After 3 additional washes with PBST, serial dilutions of individual mouse serum samples were prepared, along with a reference mouse anti-BMP9 antibody (no. MAB3209; R&D Systems) and a reference mouse anti-BMP10 antibody (no. MAB2926; R&D Systems), both diluted in 1% BSA in PBS.

Techniques: Injection, Saline, Immunofluorescence, Staining, MANN-WHITNEY, Clinical Proteomics

Journal: International Dental Journal

Article Title: Pulpitis Transiently Affect Hepatic Bone Morphogenetic Protein 9 Expression by Lipopolysaccharide

doi: 10.1016/j.identj.2026.109435

Figure Lengend Snippet: Changes of BMP9 expression in liver after pulp opening in rats/mice. (A) Haematoxylin–eosin (HE) staining of mice pulpitis tissues (black arrows: inflammatory cell). (B) Expression of BMP9 in rat liver occurred mainly in the peripheral cells of the sinusoid (blue arrows); it decreased significantly at D1 and returned to normal at D3 and D7 (IHC). (C) At D1 after pulp opening, the expression of BMP9 in mice liver of the PO group decreased significantly, but there was no significant change in the PC or AN groups (blue arrows) (IHC). (D) At D3 after pulp opening, the expression of BMP9 in liver tissue of mice in PO group increased, returning to the normal level, whereas there was no significant change in other groups (IHC). (E) RT-qPCR results showed that BMP9 gene expression began to decline at 6 hours after pulp opening and reached its lowest level at D1, before returning to normal levels. (F and G) WB results showed that the expression of BMP9 protein decreased at D1 after pulp opening and increased at D3. (H) ELISA showed that BMP9 levels in the blood of mice decreased at D1 and rose at D3. AN, anaesthetic; D, day; NS, normal sample; PC, pulp capping; PO, pulp opening. Data are presented as mean ± SD; n = 6; * P < .05. Black scale: 100 μm, red scale: 50 μm; green scale: 150 μm; white scale: 25 μm.

Article Snippet: The slides were incubated overnight at 4°C with antibodies against BMP9 (1:500, sc-514211, Santa Cruz Biotechnology) and LPS (1:100, KL20041-01, KangLang Biological).

Techniques: Expressing, Staining, Quantitative RT-PCR, Gene Expression, Enzyme-linked Immunosorbent Assay

Journal: International Dental Journal

Article Title: Pulpitis Transiently Affect Hepatic Bone Morphogenetic Protein 9 Expression by Lipopolysaccharide

doi: 10.1016/j.identj.2026.109435

Figure Lengend Snippet: Changes of LPS concentration in liver and blood of mice after pulp opening and BMP9 expression in mHSCs stimulated by P.g LPS. The concentration of LPS in the blood (A) and liver (B) began to rise at 12 hours, peaking at D1 and dropping to basal level at D3 (ELISA and IHC) ( n = 6). (C) The expression sites of BMP9 (yellow) and demsin (green) overlapped in liver tissue (IF) ( n = 3). (D and E) P.g LPS inhibited the expression of BMP9 in mHSCs cultured in vitro for a short time (WB) ( n = 3). D, day; NS, normal sample. Data are presented as mean ± SD; * P < .05; ** P < .01. Black scale: 100 μm, red scale: 50 μm; white scale: 50 μm.

Article Snippet: The slides were incubated overnight at 4°C with antibodies against BMP9 (1:500, sc-514211, Santa Cruz Biotechnology) and LPS (1:100, KL20041-01, KangLang Biological).

Techniques: Concentration Assay, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, In Vitro

Journal: International Dental Journal

Article Title: Pulpitis Transiently Affect Hepatic Bone Morphogenetic Protein 9 Expression by Lipopolysaccharide

doi: 10.1016/j.identj.2026.109435

Figure Lengend Snippet: UNC0642 inhibited the downregulation of BMP9 expression in P.g LPS-stimulated mHSCs in vitro. (A) P.g LPS inhibited the expression of BMP9 and increased the H3K9me2 levels. UNC0642 at different molar concentrations restored the expression of BMP9 and inhibited H3K9me2 levels (WB). (B) The changes in BMP9 expression. (C) The changes in H3K9me2 levels. (D-I) P.g LPS reduced the fluorescence intensity of BMP9 and increased that of H3K9me2. 1 μM UNC0642 restored the fluorescence intensity of BMP9 and inhibited that of H3K9me2 (ICF). me2: dimethylation. Data are presented as mean ± SD; n = 3; ** P < .01.

Article Snippet: The slides were incubated overnight at 4°C with antibodies against BMP9 (1:500, sc-514211, Santa Cruz Biotechnology) and LPS (1:100, KL20041-01, KangLang Biological).

Techniques: Expressing, In Vitro, Fluorescence

Journal: International Dental Journal

Article Title: Pulpitis Transiently Affect Hepatic Bone Morphogenetic Protein 9 Expression by Lipopolysaccharide

doi: 10.1016/j.identj.2026.109435

Figure Lengend Snippet: UNC0642 restored BMP9 expression in C57 mice and binding of H3K9me2 to the promoter region of BMP9 gene. (A) The expression of BMP9 in the DMSO-PO group was decreased, whereas that in the UNC0642-PO group was upregulated and similar to that in the DMSO control group (WB). (B) The RT-qPCR results are consistent with the IHC results. (C and D) The WB results were consistent with the IHC results ( n = 6). The groups 1 to 3 correspond to the DMSO group, DMSO-PO group, and UNC0642-PO group, respectively. (E) After stimulation of mHSCs with P.g LPS, the enrichment rate of H3K9me2 in the promoter region of BMP9 gene was significantly increased (ChIP) ( n = 3). (F) Full-text pattern diagram. LPS from pulpitis reached the liver through the bloodstream, stimulating hepatic stellate cells and regulating the expression of BMP9 in the liver via H3K9 dimethylation. DMSO represents the working fluid of inhibitors in vivo: DMSO + 40% PEG300 + 5% Tween80 + double-distilled H2O; LPS, samples stimulated with P.g LPS; me2, dimethylation; NS, normal sample; PO, pulp opening. Data are presented as mean ± SD; * P < .05. Black scale: 100 μm, white scale: 50 μm.

Article Snippet: The slides were incubated overnight at 4°C with antibodies against BMP9 (1:500, sc-514211, Santa Cruz Biotechnology) and LPS (1:100, KL20041-01, KangLang Biological).

Techniques: Expressing, Binding Assay, Control, Quantitative RT-PCR, In Vivo

Journal: bioRxiv

Article Title: BMP9-mediated regulation of endothelin-1 requires integrated SMAD1/5 and SMAD2/3 signaling

doi: 10.64898/2025.12.24.696445

Figure Lengend Snippet: (A) UMAP plots depict distinct cell clusters in control (Ctrl) versus PAH explanted lungs identified by single cell RNAseq. EDN1 expression is highly enriched in the endothelial cluster of cells in both control and PAH lungs, and is increased in the lungs of PAH versus control lungs (GSE169471). (B) Volcano plot depicts upregulated and downregulated genes in explanted lungs from patients with PAH versus non-diseased controls. EDN1 is one of the most highly and significantly upregulated genes (GSE169471). (C) ET-1 levels were measured in plasma from normoxic (Nx) adult rats, and rats injected with SU5416 (40 mg/kg s.c.) and exposed to 3 weeks of hypoxia (FIO2=0.1) followed by 3 weeks of normoxia (SU-Hx). (D-E) Bovine aortic ECs (BAECs) were deprived of serum for 16 h and treated with varying concentrations of BMP9 (0.1, 1, 10 ng/mL) or TGFβ1 (5 ng/mL), and supernatants analyzed at (D) 6 h or (E) 24 h for presence of ET-1 peptide by ELISA, revealing dose-dependent effects. (F) Analysis of Edn1 mRNA by qRT-PCR in BAECs stimulated with BMP9 (25 ng/mL) revealed time-dependent effects. (G) Immunoblotting of BAECs stimulated with varying concentrations (0-50 ng/mL) of BMP9 for 25 min demonstrated activation of SMADs 1/5/9 in a dose-dependent manner. (H) Immunoblotting of BAECs stimulated with BMP9 or TGFβ1 revealed kinetics of SMAD2 and SMAD3 activation. Values are mean ± SEM, n=5, * p ≤0.05 and ** p <0.01 as compared to basal transcriptional levels, 1-Way ANOVA, Kruskal-Wallis Test.

Article Snippet: Recombinant ALK1 extracellular domain expressed as a soluble fusion protein with IgG Fc domain (ALK1-Fc), a neutralizing anti-BMP9 monoclonal antibody (MAB3209), and recombinant BMP inhibitor, Noggin, were obtained from R&D Systems.

Techniques: Control, Expressing, Clinical Proteomics, Injection, Peptide ELISA, Quantitative RT-PCR, Western Blot, Activation Assay

Journal: bioRxiv

Article Title: BMP9-mediated regulation of endothelin-1 requires integrated SMAD1/5 and SMAD2/3 signaling

doi: 10.64898/2025.12.24.696445

Figure Lengend Snippet: (A) Immunoblot of BAECs treated with BMP9 (5 ng/mL) revealed differential sensitivity of SMAD1 vs. SMAD3 activation ( p -SMAD1, p -SMAD3) to ALK1/2/3/6 kinase inhibition with varying concentrations of LDN-193189, vs. total SMAD1 ( t -SMAD1). (B) Immunoblot of matched ALK1 wild-type and knockout ( Acvrl1 flox/flox and Acvrl1 -/- ) mouse microvascular lung ECs (MLECs) treated with BMP9 (5 ng/mL) demonstrate absence of BMP9-mediated SMAD3 activation in ALK1 knockout cells, diminished SMAD1 activation, and more potent inhibition of SMAD1 activation by LDN-193189 in the absence of ALK1. (C) Immunoblot of BAECs stimulated with BMP9 (25 ng/mL ) or TGFβ1 (5 ng/mL) revealed high sensitivity of BMP9-mediated SMAD3 activation and partial sensitivity of SMAD1 activation in response to ALK1/2/3/6 inhibition (250 nM LDN-193189), and modest sensitivity of BMP9-mediated SMAD3 activation and no sensitivity of SMAD1 activation in response to ALK4/5/7 inhibition (5 μM SB-431542), while activation of SMAD3 or SMAD1 by TGFβ1 was sensitive to ALK4/5/7 inhibition. (D) Edn1 mRNA expression measured by RT-PCR in BAECs stimulated with BMP9 (25 ng/mL) or TGFβ1 (5 ng/mL) revealed differential sensitivity of BMP9- vs. TGFβ1-medated Edn1 expression to ALK1/2/3/6 (250 nM LDN-193189) or ALK4/57 inhibition (5 µM SB-431542) inhibition. (E) Treatment of BAECs with SMAD3 activation inhibitor SIS3 (5 μM) prevented both BMP9- and TGFβ1-induced expression of Edn1 mRNA by RT-CR. (F) BMP9-mediated expression of Edn1 mRNA in BAECs was inhibited by pretreatment with siRNA specific for SMAD3 but not SMAD1 by RT-PCR. (G) BMP9-induced expression of EDN1 mRNA in TIME cells was inhibited by pre-treatment with siRNA specific for SMAD2 or SMAD3, but not siRNA specific for SMAD1, SMAD5, or SMAD9. Bars represent mean ± SEM, n=3-4 as shown, * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001, 2-Way ANOVA with Holm-Sidak Test (D, G) or Fisher’s LSD test (E, F).

Article Snippet: Recombinant ALK1 extracellular domain expressed as a soluble fusion protein with IgG Fc domain (ALK1-Fc), a neutralizing anti-BMP9 monoclonal antibody (MAB3209), and recombinant BMP inhibitor, Noggin, were obtained from R&D Systems.

Techniques: Western Blot, Activation Assay, Inhibition, Knock-Out, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: BMP9-mediated regulation of endothelin-1 requires integrated SMAD1/5 and SMAD2/3 signaling

doi: 10.64898/2025.12.24.696445

Figure Lengend Snippet: (A-C) Immunoblot of HPAECs following siRNA-mediated knockdown of various BMP type I ( ALK1, ALK2, ALK3 ), type II receptors ( BMPR2, ACVR2A ) and co-receptors ( ENG ) revealed that ALK1 and BMPR2 are the primary mediators of SMAD3 activation in response to BMP9 (25 ng/mL), with little contribution from ALK2, ALK3, ALK5, ENG, or ACVR2A, and revealed ALK1 and the primary mediator of SMAD1 activation. (D) RT-PCR of HPAECs pre-treated with specific siRNA revealed inhibition of incremental BMP9-induced EDN1 expression by si ALK1 and si ENG , but only reduction of basal EDN1 expression by si ALK5 . (E) BMPR2 knockdown in HPAECs abrogated BMP9-mediated EDN1 expression by RT-PCR. (F) RT-PCR analysis of MLEC revealed loss of BMP9-induced (25 ng/mL) expression of Edn1 mRNA in Bmpr2 KO ( Bmpr2 -/- ) MLEC vs. wild-type MLECs ( Bmpr2 flox/flox ). (G) RT-PCR analysis of TIME cells pre-treated with specific siRNA revealed loss of BMP9-induced (1 ng/mL) expression of EDN1 mRNA following knockdown of BMPR2 and ALK1, but not ACVR2a or ALK2. (H-J) Pre-incubation of HPAECs with receptor extracellular domains fusion proteins (E) ALK1-Fc, (F) Eng-Fc, but not (G) ALK5-Fc inhibited BMP9-induced expression of EDN1 mRNA. Values are mean ± SEM, n=3-4, * p <0.05, ** p <0.01, **** p <0.0001, 2-Way ANOVA with Sidak’s Test.

Article Snippet: Recombinant ALK1 extracellular domain expressed as a soluble fusion protein with IgG Fc domain (ALK1-Fc), a neutralizing anti-BMP9 monoclonal antibody (MAB3209), and recombinant BMP inhibitor, Noggin, were obtained from R&D Systems.

Techniques: Western Blot, Knockdown, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Inhibition, Expressing, Incubation

Journal: bioRxiv

Article Title: BMP9-mediated regulation of endothelin-1 requires integrated SMAD1/5 and SMAD2/3 signaling

doi: 10.64898/2025.12.24.696445

Figure Lengend Snippet: EDN1 promoter activity induced by BMP9 versus TGFβ1 in BAECs was assayed using either wild type human EDN1 promoter of varying lengths or using a full-length promoter with mutations in BMP- and TGFβ-specific binding elements. (A) Previously described SMAD2/SMAD3 binding element (SBE1/SBE2), activator protein-1 (AP-1) binding site, and a putative NF-1 site with homology to a consensus SMAD1/5 binding element (“MEME2”) were required for EDN1 transcription in response to BMP9, whereas TGFβ only required the SBE1/SBE2 and AP-1 sites. (B-C) Mutant constructs were generated within a -650 bp version of the EDN1 promoter reporter construct and compared with the signaling of the wild-type construct by luciferase assay. Cells were deprived of fetal calf serum (FCS) for 16 h, and then treated with 25 ng/mL of BMP9 or 5 ng/mL of TGFβ1 for 12 h. Values are mean ± SEM, n=3-5, * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001, 2-Way ANOVA with Sidak’s Test, or in the case of promoter mutants, by Fisher’s exact test for each mutant / wild-type pair.

Article Snippet: Recombinant ALK1 extracellular domain expressed as a soluble fusion protein with IgG Fc domain (ALK1-Fc), a neutralizing anti-BMP9 monoclonal antibody (MAB3209), and recombinant BMP inhibitor, Noggin, were obtained from R&D Systems.

Techniques: Activity Assay, Binding Assay, Mutagenesis, Construct, Generated, Luciferase

Journal: bioRxiv

Article Title: BMP9-mediated regulation of endothelin-1 requires integrated SMAD1/5 and SMAD2/3 signaling

doi: 10.64898/2025.12.24.696445

Figure Lengend Snippet: The schematic illustrates the proposed mechanism demonstrated in this study. BMP9 initiates the formation of a membrane receptor complex consisting of two type I receptors (ALK1) with two type II receptors (BMPR2) and two ENG coreceptors, promoting transphosphorylation of the ALK1 kinase. Activated ALK1 then phosphorylates SMAD 2/3 and SMAD 1/5/9. Subsequently, pSMAD 2/3 and pSMAD 1/5/9 translocate to the nucleus where they regulate EDN1 transcription via binding to SBE1/SBE2 and NF1 promoter sites, respectively, in microvascular and macrovascular endothelial cells, thereby promoting PAH.

Article Snippet: Recombinant ALK1 extracellular domain expressed as a soluble fusion protein with IgG Fc domain (ALK1-Fc), a neutralizing anti-BMP9 monoclonal antibody (MAB3209), and recombinant BMP inhibitor, Noggin, were obtained from R&D Systems.

Techniques: Membrane, Binding Assay