alk1 fc  (R&D Systems)

Journal: medRxiv

Article Title: Bone Morphogenetic Protein-9 Controls Pulmonary Vascular Growth and Remodeling

doi: 10.1101/2023.06.02.23290910

Figure Lengend Snippet: ( A ) Transcriptomic profile of each cell (dots) represented by a Uniform Manifold Approximation and Projection (UMAP), noted from C1 to 5 in the basal state and differential expression of BMP receptors in the five clusters (n=5), the clusters can be subdivided on two subgroups based on ALK1 expression: ALK1 high and ALK1 low . (B) TOP 10 GO terms that characterized the ALK1 high PMEC population. (C) Gene set enrichment analysis (GSEA) of differentially expressed genes (DEGs) between ALK1 high versus ALK1 low PMECs. NES: normalized enrichment score; FDR: false discovery rate.

Article Snippet: To suppress BMP-9–ALK1–Smad1,5,8 signaling in human PMECs, studies were performed in the presence of human recombinant ALK1-Fc (R&D Systems), a ligand trap for BMP-9 and BMP-10, or with the ALK1 inhibitor ML347 (Tocris) at the concentrations indicated in the legends.

Techniques: Expressing

Journal: medRxiv

Article Title: Bone Morphogenetic Protein-9 Controls Pulmonary Vascular Growth and Remodeling

doi: 10.1101/2023.06.02.23290910

Figure Lengend Snippet: (A) UMAP illustration showing changes in the transcriptomic profile in ALK1 high and ALK1 low PMEC-clusters 4h after BMP-9 stimulation (10ng/mL). (B) Violin plots of standardized expression of the main BMP-9 receptors across ALK1 high and ALK1 low PMEC-clusters with or without BMP-9 stimulation. TOP 50 Gene Ontology/Biological Process (GO/BP) terms that characterized the BMP-9 responses in ALK1 high (C) and ALK1 high PMEC-clusters ( D) . Data are represented as mean± SEM. Significance was measured using nonparametric Mann-Whitney t-test: ****, p<0.0001 compared to ALK1 high .

Article Snippet: To suppress BMP-9–ALK1–Smad1,5,8 signaling in human PMECs, studies were performed in the presence of human recombinant ALK1-Fc (R&D Systems), a ligand trap for BMP-9 and BMP-10, or with the ALK1 inhibitor ML347 (Tocris) at the concentrations indicated in the legends.

Techniques: Expressing, MANN-WHITNEY

Journal: medRxiv

Article Title: Bone Morphogenetic Protein-9 Controls Pulmonary Vascular Growth and Remodeling

doi: 10.1101/2023.06.02.23290910

Figure Lengend Snippet: (A) Representative images and quantifications of tube formation by human PMECs exposed to non-relevant IgG or ALK1-Fc (300ng/mL). (B) Representative images and quantifications of the surface of wound covered by human PMECs exposed to non-relevant IgG or ALK1-Fc (300ng/mL). (C) Effects of BMP-9 and siALK1 on BrdU incorporation in human PMECs. (D-F) Effects of non-relevant IgG or ALK1-Fc (300ng/mL) on tube formation, migration and proliferation of human PMECs. Data are represented as mean± SEM. Significance was measured using parametric paired t-test or 1-way ANOVA with Tukey post hoc tests: *, p<0.05; **, p<0.01; ****, p<0.0001 versus IgG, Scr sequence or 0.3% fetal calf serum (FCS). #, p<0.05; ##, p<0.01 versus Scr seq. AU: arbitrary unit. ALK1-Fc: soluble chimeric protein consisting of the extracellular part of ALK1 fused to a Fc fragment. IgG: immunoglobulin G. Nbr: number. BrdU: bromodeoxyuridine

Article Snippet: To suppress BMP-9–ALK1–Smad1,5,8 signaling in human PMECs, studies were performed in the presence of human recombinant ALK1-Fc (R&D Systems), a ligand trap for BMP-9 and BMP-10, or with the ALK1 inhibitor ML347 (Tocris) at the concentrations indicated in the legends.

Techniques: BrdU Incorporation Assay, Migration, Sequencing

Journal: medRxiv

Article Title: Bone Morphogenetic Protein-9 Controls Pulmonary Vascular Growth and Remodeling

doi: 10.1101/2023.06.02.23290910

Figure Lengend Snippet: (A) Violin plots showing BMP-9 effects on mRNA levels for VEGF-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor ( PGF ), VEGFR1 ( FLT1 ), and VEGFR2 ( KDR ) in ALK1 High , with or without BMP-9 stimulation and in ALK1 Low . Representative immunoblots and densitometric analysis of ALK1 (B) , VEGFR2 and VEGFR1 expression in human PMECs upon 24h of BMP-9 stimulation (10ng/mL) (C) . (D) Representative immunoblots and densitometric analysis of p-VEGFR2, p-SRC and p-AKT in human PMECs pretreated for 24h with BMP-9 (10ng/mL) and/or stimulated for 30min with VEGF-A (20ng/mL). (E) Representative immunoblots and densitometric analysis of ALK1, VEGFR1, p-VEGFR2, VEGFR2, p-AKT and AKT in Gdf2-/- and Gdf2+/+ rat lungs. (F) Representative immunofluorescent staining of ALK1 (red), CD31 (green) and nuclei (DAPI, blue) in Gdf2-/- and Gdf2+/+ rat lungs. Scale bars=50 μm. Data are represented as mean± SEM. Significance was measured using parametric paired t-test or 1-way ANOVA with Tukey post hoc tests: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 versus basal condition or Gdf2 +/+. AKT = protein kinase B. AU = arbitrary unit. DAPI = 4’,6-diamidino-2-phenylindole. GAPDH = glyceraldehyde-3-phosphate dehydrogenase

Article Snippet: To suppress BMP-9–ALK1–Smad1,5,8 signaling in human PMECs, studies were performed in the presence of human recombinant ALK1-Fc (R&D Systems), a ligand trap for BMP-9 and BMP-10, or with the ALK1 inhibitor ML347 (Tocris) at the concentrations indicated in the legends.

Techniques: Western Blot, Expressing, Staining

Journal: BMC Biology

Article Title: Atheroprone fluid shear stress-regulated ALK1-Endoglin-SMAD signaling originates from early endosomes

doi: 10.1186/s12915-022-01396-y

Figure Lengend Snippet: Differential impact of HSS and LSS on ALK1-mediated BMP signaling. A Immunoblot using antibodies specific against pSMAD1/5 shows different levels of SMAD1/5 phosphorylation in HSS versus LSS after 24 h of FSS exposure (upper panel). Densiometric quantification of SMAD1/5 phosphorylation relative to GAPDH expression. Data is presented as mean ± SD from 5 independent experiments. Statistical significance against static control was calculated by unpaired, two-sided Student’s T -test. B Volcano plot depicting –log 10 (adjusted p -value) against log 2 (fold change) from RNAseq data of BMP target genes compiled from . Highlighted genes are commonly used BMP target genes. Red indicates upregulation in LSS, and blue indicates upregulation in HSS. Genes depicted in gray are not significantly regulated. C Immunoblot using antibodies specific against pSMAD1/5 showing responses of 24-h exposure to FSS in the absence/presence of 50 ng/mL ALK1-Fc (left panel). Densiometric quantification of SMAD1/5 phosphorylation relative to GAPDH expression. Data is presented as mean ± SD from 4 independent experiments. Statistical significance against HSS control was calculated using 2-way ANOVA and Šídák’s post hoc test (right panel). D Quantitative PCR showing gene expression of selected markers after 24-h FSS application in the absence/presence of 50 ng/mL ALK1-Fc. Data is presented as mean ± SD from 3 independent experiments. Statistical significance against HSS control was calculated using 2-way ANOVA and Šídák’s post hoc test, **** p < 0.0001,*** p < 0.001, ** p < 0.01, * p < 0.05

Article Snippet: For inhibitor experiments, ALK1-Fc (50 ng/mL, R&D Biosystems) was added to a full medium and incubated at 37°C for 30 min prior to application on the cells.

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: BMC Biology

Article Title: Atheroprone fluid shear stress-regulated ALK1-Endoglin-SMAD signaling originates from early endosomes

doi: 10.1186/s12915-022-01396-y

Figure Lengend Snippet: Endoglin regulates FSS-induced, ALK1-mediated BMP signaling depending on serum concentration. A , B Immunoblots using antibodies specific against pSMAD1/5 and Endoglin in cells exposed to 30 min of FSS in presence of A 0.2% or B 20% FCS after Endoglin knock-down or scrambled siRNA controls (upper panels). Densiometric quantifications of SMAD1/5 phosphorylation relative to GAPDH expression (lower panels). Data is presented as mean ± SD from 3 independent experiments. Statistical significance against HSS control was calculated using 2-way ANOVA and Šídák’s post hoc test. Statistical significance in between groups (HSS vs. LSS) was calculated using 2-way ANOVA and Šídák’s post hoc test, ns means p >0.05. C Immunoblot using antibodies specific against pSMAD1/5 and Endoglin in cells exposed to 24 h of FSS after Endoglin knock-down or scrambled siRNA controls in the absence/presence of 50 ng/mL ALK1-Fc (left panel). Densiometric quantification of SMAD1/5 phosphorylation relative to GAPDH expression (right panel). Data is presented as mean ± SD from 3–5 independent experiments. D Quantitative PCR showing gene expression of selected markers after 24-h FSS application in Endoglin knock-down or scrambled siRNA control cells in the absence/presence of 50 ng/mL ALK1-Fc. Data is presented as mean ± SD from 4 independent experiments. Statistical significance was calculated using 2-way ANOVA and Šídák’s post hoc test, **** p < 0.0001,*** p < 0.001, ** p < 0.01, * p < 0.05. E Phase-contrast and immunofluorescence images of leukocyte adhesion assay on HUVECs transfected with siRNA against Endoglin or scrambled control and subjected to HSS for 24h. F Quantification of leukocyte (THP-1) cell adhesion shown in E . Data is shown as percentage of mean scrambled control adhesion. Statistical significance was calculated using Mann-Whitney test. * p <0.05. G Quantitative PCR showing gene expression of SELP after 24-h HSS application in Endoglin knock-down or scrambled siRNA controls cells in the absence/presence of 50 ng/mL ALK1-Fc. Statistical significance against HSS control was calculated using 2-way ANOVA and Šídák’s post hoc test, **** p < 0.0001,*** p < 0.001, ** p < 0.01, * p < 0.05

Article Snippet: For inhibitor experiments, ALK1-Fc (50 ng/mL, R&D Biosystems) was added to a full medium and incubated at 37°C for 30 min prior to application on the cells.

Techniques: Concentration Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Cell Adhesion Assay, Transfection, MANN-WHITNEY

Journal: BMC Biology

Article Title: Atheroprone fluid shear stress-regulated ALK1-Endoglin-SMAD signaling originates from early endosomes

doi: 10.1186/s12915-022-01396-y

Figure Lengend Snippet: Caveolin-1-positive early endosomes are signaling hotspots for FSS-induced BMP signaling. Confocal images of Endoglin, Caveolin-1, and DAPI in cells exposed to A HSS or B LSS. Insets show magnified regions. Arrows indicate Endoglin-positive Caveolin vesicles. Scale bars 10 μM. C Quantification of the number of Caveolin-1 vesicles (left) and Endoglin-positive Caveolin-1 vesicles (right). Counting was performed using a self-written ImageJ script (see the “Methods” section for details). D Confocal image of Endoglin, Caveolin-1, and EEA1. Scale bar 1 μM. Insets show single channels in gray scale. The white line indicates the area of intensity profile shown in E . E Intensity profile of Endoglin, Caveolin-1, and EEA1 staining shown in D . F Quantification of EEA1 vesicles (left), Cav-1-positive EEA1 vesicles (middle), and Cav-1 and Endoglin double-positive EEA1 vesicles (right). G Confocal image of Caveolin-1 and PLA of Endoglin and overexpressed ALK1-meGFP. Scale bar 1 μM. The white line indicates the area of intensity profile shown in H . H Intensity profile of Caveolin-1 and Endoglin-ALK1-meGFP PLA staining shown in G . I Confocal image of Caveolin-1, deuterated silicon-rhodamine-labeled BMP9 (BMP9-siR), and DAPI. Scale bars 500 nm. The lower panel schematically depicts the labeling process of BMP9 with deuterated silicone-rhodamine (see the “Methods” section). J Inverted confocal images of SMAD1 show the nuclear accumulation of SMAD1 in LSS over HSS. Scale bar is 10 μM. K Confocal image of Caveolin-1, Endoglin, and SMAD1 shows the location of SMAD1 at Endoglin-positive Caveolin-1 vesicles, indicated by arrows. Scale bar 5 μM. L Quantification of SMAD1-positive Endoglin/Cav-1 vesicles. M Immunoblot using antibodies specific against pSMAD1/5 and Caveolin-1 in cells exposed to 24 h of FSS after Caveolin-1 knock-down in the absence/presence of 50 ng/mL ALK1-Fc. N Densiometric quantification of SMAD1/5 phosphorylation relative to GAPDH expression from immunoblots in M . Data is presented as mean ± SD from 3–5 independent experiments. Statistical significance against HSS control was calculated using 2-way ANOVA and Šídák’s post hoc test, **** p < 0.0001,*** p < 0.001

Article Snippet: For inhibitor experiments, ALK1-Fc (50 ng/mL, R&D Biosystems) was added to a full medium and incubated at 37°C for 30 min prior to application on the cells.

Techniques: Staining, Labeling, Western Blot, Expressing

Journal: BMC Biology

Article Title: Atheroprone fluid shear stress-regulated ALK1-Endoglin-SMAD signaling originates from early endosomes

doi: 10.1186/s12915-022-01396-y

Figure Lengend Snippet: Summary scheme. Under atheroprone LSS conditions (left), circulating BMP9 gets sequestrated by Endoglin, expressed on endothelial cells. Endoglin binds to the type II receptor interface, thereby blocking BMP type I/II receptor complex formation. Nonetheless, residual BMP9-ALK1-type II receptor signaling occurs at the plasma membrane. Upon internalization via caveolae, Endoglin gets substituted by type II receptors in EEs and exacerbated pSMAD1/5 signaling is induced which leads to the expression of mesenchymal and atheroprone genes. Under atheroprotective HSS conditions (right), BMP9 is bound by ALK1 and type II receptors as well as by Endoglin, the latter suppressing BMP9-type I/II receptor formation. Caveolin-mediated endocytosis is markedly reduced when compared to LSS and residual pSMAD1/5 signaling is originating from the plasma membrane rather than from CAV1 + EEs. Ultimately, HSS leads to the expression of genes responsible for EC identity and quiescence

Article Snippet: For inhibitor experiments, ALK1-Fc (50 ng/mL, R&D Biosystems) was added to a full medium and incubated at 37°C for 30 min prior to application on the cells.

Techniques: Blocking Assay, Expressing