Journal: Hypertension (Dallas, Tex. : 1979)

Article Title: BMP9 Modulates IL-33 Signaling to Mitigate EndMT in Pulmonary Arterial Hypertension

doi: 10.1161/HYPERTENSIONAHA.125.24916

Figure Lengend Snippet: BMP (bone morphogenetic protein) 9 protects from IL (interleukin)-33–induced endothelial-to-mesenchymal transition (EndMT) and induces sST2 (soluble supression of tumorigenicity 2) expression in control pulmonary arterial endothelial cells (PAECs) in vitro. A , Representative immunofluorescent staining of PAEC for CD31 (endothelial marker), SM22α (smooth muscle protein 22-alpha; mesenchymal marker), and 4′,6-diamidino-2-phenylindole (DAPI; nuclei). Cells were treated with BMP9 (1 ng/mL), IL-33 (100 ng/mL), both, or left untreated (control [CTR]) for 3 days. Bar graphs show CD31 and SM22α intensity quantification (n=3). B , sST2 , ST2L , CTGF , and PAI-1 gene expressions in PAECs after 16-hour stimulation with TGF (transforming growth factor)-β (1 ng/mL), activin A (50 ng/mL), or untreated (CTR; technical replicates [t.n.]=3). C , sST2 , ST2L , ID1 , and ID3 gene expressions in PAECs after 3-hour stimulation with BMP4 (50 ng/mL), BMP6 (50 ng/mL), BMP9 (1 ng/mL), BMP10 (1 ng/mL), or untreated (CTR; t.n.=3). Statistical analysis: 1-way ANOVA with the Tukey post hoc test; * P <0.05, ** P <0.01, and **** P <0.0001. Data are shown as mean±SD.

Article Snippet: Human recombinant BMP4 (314-BP-010/CF), BMP6 (507-BP-020/CF), BMP9 (3209-BP-010/CF), BMP10 (2926-BP-025/CF), activin A (338-AC-010/CF), TGF-b1 (240-B-010/CF), and recombinant human soluble ST2/IL-33R Fc (523-ST-100) were obtained from R&D Systems.

Techniques: Expressing, Control, In Vitro, Staining, Marker

Journal: Hypertension (Dallas, Tex. : 1979)

Article Title: BMP9 Modulates IL-33 Signaling to Mitigate EndMT in Pulmonary Arterial Hypertension

doi: 10.1161/HYPERTENSIONAHA.125.24916

Figure Lengend Snippet: BMP (bone morphogenetic protein) 9 protects from IL (interleukin)-33–induced endothelial-to-mesenchymal transition (EndMT) and induces sST2 (soluble supression of tumorigenicity 2) expression in pulmonary arterial endothelial cells (PAECs) from patients with pulmonary arterial hypertension (PAH) in vitro. A , Representative immunofluorescent staining of PAH PAEC for CD31 (endothelial marker), SM22α (smooth muscle protein 22-alpha; mesenchymal marker), and 4′,6-diamidino-2-phenylindole (DAPI). Cells were treated with BMP9 (1 ng/mL), IL-33 (100 ng/mL), both, or left untreated (control [CTR]) for 3 days. Bar graphs show quantification of CD31 and SM22α intensity (technical replicates [t.n.]=3). B , sST2 , ST2L , CTGF , and Pai-1 gene expressions in PAH PAECs s after 16-hour stimulation with TGF (transforming growth factor)-β (1 ng/mL), activin A (50 ng/mL), or untreated (CTR; t.n.=3). C , sST2 , ST2L , ID1 , and ID3 gene expressions in PAH PAECs after 3-hour stimulation with BMP4 (50 ng/mL), BMP6 (50 ng/mL), BMP9 (1 ng/mL), BMP10 (1 ng/mL), or untreated (CTR; t.n.=3). Statistical analysis: 1-way ANOVA with the Tukey post hoc test; P <0.05, * P <0.01, ** P <0.001, and *** P <0.0001. Data are shown as mean±SD.

Article Snippet: Human recombinant BMP4 (314-BP-010/CF), BMP6 (507-BP-020/CF), BMP9 (3209-BP-010/CF), BMP10 (2926-BP-025/CF), activin A (338-AC-010/CF), TGF-b1 (240-B-010/CF), and recombinant human soluble ST2/IL-33R Fc (523-ST-100) were obtained from R&D Systems.

Techniques: Expressing, In Vitro, Staining, Marker, Control

Journal: Journal of Biological Chemistry

Article Title: Tbx20 Transcription Factor Is a Downstream Mediator for Bone Morphogenetic Protein-10 in Regulating Cardiac Ventricular Wall Development and Function

doi: 10.1074/jbc.m111.279679

Figure Lengend Snippet: FIGURE 2. In situ hybridization analysis of Tbx20 expression in develop- ing ventricular myocardium. A, Tbx20 expression is down-regulated in BMP10-deficientventricle(blackarrows)comparedwithwild-typeventricular wall (red arrows) at E9.5. B, Tbx20 expression is up-regulated in MHC-BMP10 ventricular myocardium at E13.5. AT, atrium; TA, truncus artery; V, ventricle.

Article Snippet: Primary cardiomyocytes were cultured for 24 h before switching to media containing 50 ng/ml recombinant human BMP10 (R&D Systems) or BMP2 (R&D Systems) for 4 h of incubation.

Techniques: In Situ Hybridization, Expressing

Journal: Journal of Biological Chemistry

Article Title: Tbx20 Transcription Factor Is a Downstream Mediator for Bone Morphogenetic Protein-10 in Regulating Cardiac Ventricular Wall Development and Function

doi: 10.1074/jbc.m111.279679

Figure Lengend Snippet: FIGURE 1. Up-regulation of Tbx20 gene in MHC-BMP10 hearts. A, gene profiling of differentially expressed T-box transcriptional factors in the hearts of MHC-BMP10 transgenic and control littermates (4 weeks old) using Affymetrix mouse exon array. Tbx20 is significantly up-regulated in the trans- genic mouse heart. B, qRT-PCR confirming that Tbx20, but not the other car- diac Tbx members, was up-regulated in MHC-BMP10 hearts.

Article Snippet: Primary cardiomyocytes were cultured for 24 h before switching to media containing 50 ng/ml recombinant human BMP10 (R&D Systems) or BMP2 (R&D Systems) for 4 h of incubation.

Techniques: Transgenic Assay, Control, Quantitative RT-PCR

Journal: Journal of Biological Chemistry

Article Title: Tbx20 Transcription Factor Is a Downstream Mediator for Bone Morphogenetic Protein-10 in Regulating Cardiac Ventricular Wall Development and Function

doi: 10.1074/jbc.m111.279679

Figure Lengend Snippet: FIGURE 3. Up-regulation of Tbx20 expression in MHC-BMP10 transgenic mouse heart. In situ hybridization analysis of BMP10 (A and C), Tbx20 (B and D), Tbx2 (G), and Tbx5 (H) in NTG and MHC-BMP10 TG hearts (3 weeks old), respectively, is shown. The same orientation was applied to each section. A, in postnatal wild-type heart, BMP10 expression is restricted to the right atrium. B, Tbx20 expression in the wild-type heart can be detected throughout all four chambers, whereas the right atrium has a significantly higher level of Tbx20 expression. This finding is further confirmed by qRT-PCR (E and F). C, BMP10 expression expands to the left atrium and both sides of ventricles in MHC-BMP10 transgenic hearts (3 weeks old). D, in MHC-BMP10 transgenic hearts (3 weeks old), Tbx20 expression is significantly increased throughout the myocardium in both the atria and ventricles. E and F, qRT-PCR confirms the correlation of Tbx20 and BMP10 expression patterns in the normal heart (3 weeks old). G and H, expression of Tbx2 and Tbx5 is not different in ventricles between NTG and TGhearts(3weeksold).Theirexpressionismostlyrestrictedtothemesenchymalcellsinvalvesandthesmoothmusclecelllayerofmainarteries.LA,leftatrium; RA, right atrium; LV, left ventricle; RV, right ventricle; VE, ventricle.

Article Snippet: Primary cardiomyocytes were cultured for 24 h before switching to media containing 50 ng/ml recombinant human BMP10 (R&D Systems) or BMP2 (R&D Systems) for 4 h of incubation.

Techniques: Expressing, Transgenic Assay, In Situ Hybridization, Quantitative RT-PCR

Journal: Journal of Biological Chemistry

Article Title: Tbx20 Transcription Factor Is a Downstream Mediator for Bone Morphogenetic Protein-10 in Regulating Cardiac Ventricular Wall Development and Function

doi: 10.1074/jbc.m111.279679

Figure Lengend Snippet: FIGURE 4. BMP10 up-regulates Tbx20 in primary cardiomyocytes. Aa, Smad1 phosphorylation in response to different concentration of rhBMP10 and rhBMP2 in cultured P19 cells. Ab, Smad1 phosphorylation in response to different concentration of rhBMP10 in cultured neonatal cardiac myocytes. B, evaluation of Tbx20, Tbx2, Tbx5, and Tbx18 expression in cultured primary cardiomyocytes treated with rhBMP10 and rhBMP2 (50 ng/ml). *, p 0.01, statistics performed using paired Student’s t test.

Article Snippet: Primary cardiomyocytes were cultured for 24 h before switching to media containing 50 ng/ml recombinant human BMP10 (R&D Systems) or BMP2 (R&D Systems) for 4 h of incubation.

Techniques: Phospho-proteomics, Concentration Assay, Cell Culture, Expressing

Journal: Scientific Reports

Article Title: GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension

doi: 10.1038/s41598-023-33779-8

Figure Lengend Snippet: GATA6 expression is induced via BMP10-BMPR2/ALK1 axis. ( A–F ) HPAECs were transfected with ALK1, BMPR2, Endoglin, or control scr siRNA for 48 h, and then treated with 10 ng/ml BMP10 or vehicle for 6 h for RNA isolation and 24 h for protein isolation. ( A,C,E ): GATA6 mRNA measured by qPCR. Data are means ± SE; each experiment was repeated at least three times. ( B,D,F ): GATA6 protein levels were measured by immunoblot analysis. Data are means ± SE, each experiment was repeated at least three times. Representative blots are shown. *p < 0.05, **p < 0.01, ***p < 0.001 by Kruskal–Wallis test with post hoc Dunn’s test for multiple comparisons. ( G–I ) Human PAH PASMC were treated with 10 ng/ml BMP10 or vehicle ( − ) for 48 h and immunoblot analysis to detect indicated proteins was performed. Data are means ± SE from n = 4 subjects/group. *p < 0.05 by Mann Whitney U test. ( J,K ) Equal amounts of human PAH HPAEC and PASMC were plated at 6-well plates and treated with 10 ng/ml BMP10 or vehicle ( − ). 48 h later cell counts were performed. Data are means ± SE from n = 3 subjects/group, 3 technical repetitions/subject. *p < 0.05 by Mann Whitney U test. ( L,M ) HPAECs were transfected with SMAD1 siRNA, and then treated with BMP10 for 6 h. GATA6 and SMAD1 mRNA levels were measured by qPCR. Data shown as means ± SE. Each experiment was repeated at least three times. *p < 0.05, **p < 0.01 by Kruskal–Wallis test with post-hoc correction for multiple comparisons. ( N,O ) HPAEC were treated for 30 min with diluent ( − ), 5 µM ERK1/2 inhibitor SCH772984 (ERKi), or 5 µM GSK3 inhibitor CHIR99021 (GSK3i) and then stimulated with BMP10 (10 ng/ml) or vehicle for 24 h. Representative immunoblots ( N ) and statistical analysis ( O ) are shown. ( O ): Data represent GATA6/β-actin ratio. Data are means ± SE from five independent experiments. *p < 0.05 by Kruskal–Wallis test with post-hoc Dunn’s correction for multiple comparisons. ( E ) HPAECs were treated with BMP10 in the presence or absence of 10 µM ERK1/2 inhibitor SCH772984 for 24 h. GATA6 mRNA levels were measured by qPCR. Data shown as means ± SE. Each experiment was repeated six times. *p < 0.05 by Kruskal–Wallis test with post-hoc Dunn’s correction for multiple comparisons. The original blots are presented in Supplementary Fig. .

Article Snippet: BMP9 (GDF-2) and BMP10 were purchased from PeproTech (Cat.no 120–07 and 120–40, respectively).

Techniques: Expressing, Transfection, Control, Isolation, Western Blot, MANN-WHITNEY

Journal: Scientific Reports

Article Title: GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension

doi: 10.1038/s41598-023-33779-8

Figure Lengend Snippet: Graphical representation of the role of GATA6 in coordinating cross-talk between BMP10 and oxidative stress axis in PAH. GATA6 is an activator of anti-oxidant enzymes and its deficiency in PAEC and PASMC induces oxidative stress and mitochondrial dysfunction. BMP10 induces expression of GATA6 through the ALK1, BMPRII, ENG and ERK pathway. GATA6, in turn, transcriptionally activates BMP receptors in PAEC. Endothelial GATA6 regulates PASMC function via paracrine factors. TGFβ2 secreted by GATA6 deficient PAEC induces PASMC proliferation. Administration of dimethyl fumarate (DMF) to mice with endothelial Gata6 loss restores expression of BMP receptors, resolves oxidative stress, and reverses PH.

Article Snippet: BMP9 (GDF-2) and BMP10 were purchased from PeproTech (Cat.no 120–07 and 120–40, respectively).

Techniques: Expressing