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: Nature Communications

Article Title: Endothelial progenitor cells stimulate neonatal lung angiogenesis through FOXF1-mediated activation of BMP9/ACVRL1 signaling

doi: 10.1038/s41467-022-29746-y

Figure Lengend Snippet: a Immunostaining shows an increase in phosphorylated SMAD1 (pSMAD1) after treatment of endothelial MFLM-91U cells with BMP9. Nuclear pSMAD1 staining (red) is indicated by arrows. Endothelial cells are counterstained with DAPI (blue) and ENG (green). The experiment was repeated 3 times with similar results. Scale bars are 10 μm. b Western blot shows that BMP9 treatment increases pSMAD1 and ACVRL1 protein levels but does not affect FOXF1 or total SMAD1 in MFLM-91U cells ( n = 3). Inhibition of FOXF1 by siRNA decreases pSMAD1 and ACVRL1 proteins in BMP9-treated cells. c Dual luciferase assay shows that BMP9 increases activity of the BRE-LUC reporter plasmid in MFLM-91U cells ( n = 3). siRNA-mediated inhibition of FOXF1, ACVRL1 or ENG decreases BRE-LUC reporter activity in BMP9-treated cells. b , c Data were shown as mean ± SD, and one-way ANOVA followed by Tukey’s test (two-tailed) were performed. ** p < 0.01, * p < 0.05 ns is not significant. d – f In vitro angiogenesis assay shows that inhibition of FOXF1 by siRNA decreases the formation of endothelial sprouts in MFLM-91U cells. BMP9 treatment partially restores the in vitro angiogenesis in FOXF1-deficient endothelial cells. Treatment of cells with ALK1-Fc chimeric protein inhibits the effect of BMP9. Scale bars are 20 μm. The complexity of the vascular network in Matrigel is quantitated by measurements of sprout length e and counts of the sprout junctions f ( n = 8 per each group). e , f Data were shown as mean ± SD, and one-way ANOVA (two-tailed) followed by Tukey’s multiple comparison test was performed. ** p < 0.01, *p < 0.05, ns is not significant.

Article Snippet: Alk1-Fc protein (R&D, 770-MA) was used to inhibit the effect of BMP9/ACVRL1 signaling.

Techniques: Immunostaining, Staining, Western Blot, Inhibition, Luciferase, Activity Assay, Plasmid Preparation, Two Tailed Test, In Vitro, Angiogenesis Assay

Journal: bioRxiv

Article Title: Decreased Colonic Activin Receptor-Like Kinase 1 Disrupts Epithelial Barrier integrity and is associated with a poor clinical outcome in Crohn’s disease

doi: 10.1101/2020.02.21.960070

Figure Lengend Snippet: (A) Correlation of expression in the colonic mucosa of CD patients (N=10) between miR-31-5p (reads per million miRNAs mapped, RPMMM) and predicted targets of miR-31-5p ( E2F2, ALK1, PRKAB1 , and DCBLD2 ; DESeq normalized). (B) Association between the expression of miR-31-5p and the expression of ALK1 or E2F2 in isolated colonic epithelial cells (N=27). Gene expression was quantified by qPCR and samples were split into 3 equally sized groups (N=9 per group) according to the relative miR-31-5p expression levels. (C) Representative blot of ALK1 expression in the colonic tissue of NIBD and CD patients. ( D ) Correlation between ALK1 protein expression and miR-31-5p in the colonic mucosa (N=18). All correlation values were calculated by Spearman’s correlation coefficient. Each gene expression was normalized to GAPDH ( ALK1, E2F2 ) or RNU48 (miR-31-5p). *P<0.05. P-values were determined by Kruskal-Wallis test followed by Dunn’s multiple comparison test.

Article Snippet: In several experiments, cells were expanded in EM for 4 days and subsequently stimulated with 10 ng/ml bone morphogenetic protein (BMP) 9 (BioLegend, CA, USA) and 100 ng/ml ALK1-Fc chimera protein (R&D Systems, MN, USA), a soluble chimeric protein consisting of the extracellular part of ALK1 fused to a Fc fragment, which inhibits BMP9-ALK1 interaction and signaling .

Techniques: Expressing, Isolation, Comparison

Journal: bioRxiv

Article Title: Decreased Colonic Activin Receptor-Like Kinase 1 Disrupts Epithelial Barrier integrity and is associated with a poor clinical outcome in Crohn’s disease

doi: 10.1101/2020.02.21.960070

Figure Lengend Snippet: (A) 3’UTR reporter assay for ALK1 in the presence or absence of 30nM miRNA mimics for hsa-miR-31-5p (m31), hsa-miR-122a-5p (m122), or hsa-miR-215-5p (m215) or negative control mimics (NC). (B) Site-directed mutagenesis assay with 10nM of m31 or NC mimics. *P<0.05. P-values were determined by Kruskal-Wallis test followed by Dunn’s multiple comparison test. N=6 per group for all experiments.

Article Snippet: In several experiments, cells were expanded in EM for 4 days and subsequently stimulated with 10 ng/ml bone morphogenetic protein (BMP) 9 (BioLegend, CA, USA) and 100 ng/ml ALK1-Fc chimera protein (R&D Systems, MN, USA), a soluble chimeric protein consisting of the extracellular part of ALK1 fused to a Fc fragment, which inhibits BMP9-ALK1 interaction and signaling .

Techniques: Reporter Assay, Negative Control, Mutagenesis, Comparison

Journal: bioRxiv

Article Title: Decreased Colonic Activin Receptor-Like Kinase 1 Disrupts Epithelial Barrier integrity and is associated with a poor clinical outcome in Crohn’s disease

doi: 10.1101/2020.02.21.960070

Figure Lengend Snippet: (A) EdU assay in NIBD patient-derived colonic epithelial cell monolayers (N=4-8 per group). Expanded cells were cultured in EM in the presence or absence of BMP9 and ALK1-Fc chimera protein. Red, EdU; Blue, Hoechst 33342. (B) Proliferation- and stemness-related gene expression in NIBD patient-derived colonic epithelial cell monolayers (N=6 per group). Each gene expression was normalized to RPLP0 . *P<0.05, **P<0.01. P-values were determined by Kruskal-Wallis test (A) or Friedman test (B) followed by Dunn’s multiple comparison test. EM, expansion media; DM, differentiation media.

Article Snippet: In several experiments, cells were expanded in EM for 4 days and subsequently stimulated with 10 ng/ml bone morphogenetic protein (BMP) 9 (BioLegend, CA, USA) and 100 ng/ml ALK1-Fc chimera protein (R&D Systems, MN, USA), a soluble chimeric protein consisting of the extracellular part of ALK1 fused to a Fc fragment, which inhibits BMP9-ALK1 interaction and signaling .

Techniques: EdU Assay, Derivative Assay, Cell Culture, Expressing, Comparison

Journal: bioRxiv

Article Title: Decreased Colonic Activin Receptor-Like Kinase 1 Disrupts Epithelial Barrier integrity and is associated with a poor clinical outcome in Crohn’s disease

doi: 10.1101/2020.02.21.960070

Figure Lengend Snippet: (A) Lineage-specific gene expression in NIBD patient-derived colonic epithelial cell monolayers. Expanded cells were cultured in expansion media in the presence or absence of BMP9 and ALK1-Fc chimera protein. Each gene expression was normalized to RPLP0 (N=6 per group). (B) CA1 protein expression in NIBD patient-derived colonic epithelial monolayers (N=4 per group). *P<0.05, **P<0.01. P-values were determined by Friedman test followed by Dunn’s multiple comparison test.

Article Snippet: In several experiments, cells were expanded in EM for 4 days and subsequently stimulated with 10 ng/ml bone morphogenetic protein (BMP) 9 (BioLegend, CA, USA) and 100 ng/ml ALK1-Fc chimera protein (R&D Systems, MN, USA), a soluble chimeric protein consisting of the extracellular part of ALK1 fused to a Fc fragment, which inhibits BMP9-ALK1 interaction and signaling .

Techniques: Expressing, Derivative Assay, Cell Culture, Comparison

Journal: bioRxiv

Article Title: Decreased Colonic Activin Receptor-Like Kinase 1 Disrupts Epithelial Barrier integrity and is associated with a poor clinical outcome in Crohn’s disease

doi: 10.1101/2020.02.21.960070

Figure Lengend Snippet: (A) Epithelial permeability assay in NIBD patient-derived colonic epithelial cells cultured on transwells. Trans-epithelial electrical resistance (TEER) was measured over time (N=3 per group). (B) Cells were stimulated with BMP9 in expansion media (EM) in the presence or absence of ALK1-Fc chimera protein or cultured in differentiation media (DM) at Day 4. The changes in TEER between Day 4 and Day 6 are shown as ΔTEER% (N=4-8 per group). *P<0.05. P-values determined by Kruskal-Wallis test followed by Dunn’s multiple comparison test.

Article Snippet: In several experiments, cells were expanded in EM for 4 days and subsequently stimulated with 10 ng/ml bone morphogenetic protein (BMP) 9 (BioLegend, CA, USA) and 100 ng/ml ALK1-Fc chimera protein (R&D Systems, MN, USA), a soluble chimeric protein consisting of the extracellular part of ALK1 fused to a Fc fragment, which inhibits BMP9-ALK1 interaction and signaling .

Techniques: Permeability, Derivative Assay, Cell Culture, Comparison

Journal: bioRxiv

Article Title: Decreased Colonic Activin Receptor-Like Kinase 1 Disrupts Epithelial Barrier integrity and is associated with a poor clinical outcome in Crohn’s disease

doi: 10.1101/2020.02.21.960070

Figure Lengend Snippet: (A) The impact of colonic ALK1 expression on clinical outcome. ALK1 expression was quantified in colonic biopsy samples obtained from NIBD controls (N=10) and CD patients (N=28) by qPCR. ALK1 expression was normalized to GAPDH . ***P<0.001. P-value was determined by Mann-Whitney test. (B) Kaplan-Meier survival analysis to evaluate the impact of colonic ALK1 expression on the endoscopic relapse in patients with CD (N=12 for Low-ALK1 and 9 for Hi-ALK1 CD subgroups). P-value determined by Log-rank test.

Article Snippet: In several experiments, cells were expanded in EM for 4 days and subsequently stimulated with 10 ng/ml bone morphogenetic protein (BMP) 9 (BioLegend, CA, USA) and 100 ng/ml ALK1-Fc chimera protein (R&D Systems, MN, USA), a soluble chimeric protein consisting of the extracellular part of ALK1 fused to a Fc fragment, which inhibits BMP9-ALK1 interaction and signaling .

Techniques: Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Decreased Colonic Activin Receptor-Like Kinase 1 Disrupts Epithelial Barrier integrity and is associated with a poor clinical outcome in Crohn’s disease

doi: 10.1101/2020.02.21.960070

Figure Lengend Snippet: (A) ALK1 expression in the colonic mucosa of CD patients at the time of surgery and after surgery (N=5 per group). ALK1 expression was normalized to GAPDH . P-value was determined by Wilcoxon test. (B) Percentages of CD patients who were diagnosed as CD before and after 40 years old in Low-ALK1 (N=15) and Hi-ALK1 (N=13) CD subsets.

Article Snippet: In several experiments, cells were expanded in EM for 4 days and subsequently stimulated with 10 ng/ml bone morphogenetic protein (BMP) 9 (BioLegend, CA, USA) and 100 ng/ml ALK1-Fc chimera protein (R&D Systems, MN, USA), a soluble chimeric protein consisting of the extracellular part of ALK1 fused to a Fc fragment, which inhibits BMP9-ALK1 interaction and signaling .

Techniques: Expressing