Journal: Angiogenesis

Article Title: Genetic and pharmacological targeting of mTORC1 in mouse models of arteriovenous malformation expose non-cell autonomous signalling in HHT

doi: 10.1007/s10456-024-09961-5

Figure Lengend Snippet: EC-specific overactivation of mTORC1 reduces incidence and thickness of AVMs induced by blocking antibodies. A . Time scheme for administration of tamoxifen, BMP9/10 blocking antibodies (bAbs)/Isotype IgG, and sample collection. B . Representative pictures showing retinal vasculatures of control (Cre-) and Tsc1 iECKO mice treated with IgGs or anti-BMP9/10 antibodies (Abs). Arrows indicate AVMs. C . Quantification of number of AVMs per retina of Control ( n = 5 mice) and Tsc1 iECKO ( n = 5 mice) mice treated with anti-BMP9/10 Abs. D . Quantification of AVM thickness in Control ( n = 5 mice) and Tsc1 iECKO mice ( n = 3) treated with IgGs or anti-BMP9/10 Abs. E . Central retinal vasculatures of control (Cre-) and Tsc1 iECKO mice, treated with anti-BMP9/10 Abs, immunolabelled for CD31, p-RPS6, and YFP (indicative of recombination). F . Quantification of the ratio of the p-RPS6 + area within the CD31 + area to the complete CD31 + area ( F ), as well as the p-RPS6 + area outside the CD31 + area to the complete non-vascular area ( G ) of central retinas of Control ( n = 3 mice) and Tsc1 iECKO ( n = 3 mice) mice treated with anti-BMP9/10 Abs. Note the vascular specific increase of p-RPS6, indicative of EC-specific mTORC1 overactivation. All data was analysed by two-tailed unpaired t-test with Welch’s correction. Bars indicate mean ± s.d. * p < 0.05

Article Snippet: Pups were treated with mouse monoclonal antibodies against BMP9 and BMP10 (15 mg/kg, IgG2b, MAB3209; 15 mg/kg, IgG2a, MAB2926; R&D Systems, respectively) or isotype control antibodies (15 mg/kg, IgG2b, MAB004; 15 mg/kg, IgG2a, MAB003; R&D Systems, respectively) at P3, P4 and P5 and tissues were harvested at P6.

Techniques: Blocking Assay, Control, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: BMP-9 mediates fibroproliferation in fibrodysplasia ossificans progressiva through TGF-β signaling

doi: 10.1038/s44321-024-00174-3

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Human/Mouse/Primate BMP-9 monoclonal antibody , R&D Systems , MAB3209.

Techniques: Transgenic Assay, Recombinant, Cell Counting, Cell Cycle Assay, Binding Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay, Western Blot, Reverse Transcription, Control, Software, Real-time Polymerase Chain Reaction

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) Relative transcript levels of type I (ALK1, ALK2, ALK3, ALK6) and type II (BMPR2, ACVR2A, ACVR2B) BMP receptors in primary human foreskin fibroblasts (HFF-1) were determined by RT-qPCR. ( B ) Schematic representation of the workflow. After 2 h of serum starvation, HFF-1 were stimulated with IFNβ (5 ng/ml), BMP4 (18 nM), BMP6 (18 nM), BMP9 (3 nM), BMP15 (18 nM), or Activin B (4 nM), followed by either immunoblot analysis 1 h post stimulation or RNA extraction from cell lysates and RT-qPCR 6 h post stimulation. GAPDH transcript levels were used for normalization. ( C ) Immunoblot analysis of HFF-1 upon stimulation to determine phosphorylation levels of respective signaling components. ( D ) Transcript levels of the BMP-responsive genes Id1 and Id3 upon stimulation with the indicated ligands. ( E ) Transcript levels of the ISGs Isg15 , Irf9 , Ifi6 , and Stat2 upon stimulation with the indicated ligands. Data information: ( A , B ) The Experiment was performed two independent times, one representative is shown. ( D , E) The experiment was performed three independent times, one representative is shown. Data are shown as mean ± SD. .

Article Snippet: Recombinant human BMP4 (#314-BP-010), human BMP6 (#507-BP-020), human BMP9 (#3209-BP-010), human BMP15 (#5096-BM-005), human Activin B (#659-AB-005), and the human/mouse anti-BMP9 antibody (#AF3209) were purchased from R&D Systems.

Techniques: Quantitative RT-PCR, Western Blot, RNA Extraction, Phospho-proteomics

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) Presence of type I (ALK1, ALK2, ALK3) and type II (BMPR2) receptors in HFF-1 and 293T was verified by immunoblotting with the respective antibodies. Detection of GAPDH protein served as loading control. ( B ) Relative transcript levels of type I (ALK1, ALK2, ALK3, ALK6) and type II (BMPR2, ACVR2A, ACVR2B) BMP receptors in 293T were determined by RT-qPCR. ( C ) 293T were co-transfected with expression plasmids for the BRE-Luciferase reporter and a Renilla luciferase normalization control (EF1α-Renilla). 24 h post transfection, 293T were either stimulated with BMP9 (3 nM), or BMP9 (3 nM) incubated for 15 min at RT with an α-BMP9 antibody (1 µg/ml or 5 µg/ml) for 16 h, followed by a dual-luciferase assay readout. ( D ) 293T were co-transfected as in ( B ). 24 h post transfection, 293 T were either stimulated with BMP4 (18 nM), BMP6 (18 nM), BMP9 (3 nM), BMP15 (18 nM), or Activin B (4 nM), or with the ligands incubated for 15 min at RT with an α-BMP9 antibody (1 µg/ml) for 16 h, followed by a dual-luciferase assay readout. ( E ) HFF-1 were either mock, DMSO, Ruxolitinib (10 µM) or DMH1 (10 µM) treated for 2 h, followed by stimulation with IFNβ (5 ng/ml) or BMP4 (18 nM) for 2 h. Cells were lysed and lysates were subjected to immunoblot analysis with p-STAT1, STAT1, p-SMAD1/5/9, SMAD1, p-p38, p38, p-p44/42, p44/42, and Calnexin-specific antibodies. Data information: ( A – E ) Experiments were performed two independent times, one representative is shown. Luciferase fold induction was calculated by dividing Renilla-normalized values from stimulated samples by the corresponding values from unstimulated samples.

Article Snippet: Recombinant human BMP4 (#314-BP-010), human BMP6 (#507-BP-020), human BMP9 (#3209-BP-010), human BMP15 (#5096-BM-005), human Activin B (#659-AB-005), and the human/mouse anti-BMP9 antibody (#AF3209) were purchased from R&D Systems.

Techniques: Western Blot, Control, Quantitative RT-PCR, Transfection, Expressing, Luciferase, Incubation

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) Schematic representation of the workflow. After 2 h of serum starvation, HFF-1 were stimulated for 6 h with BMP4 (18 nM), BMP6 (18 nM), BMP9 (3 nM), BMP15 (18 nM), or Activin B (4 nM), or co-stimulated with IFNβ (5 ng/ml), followed by HCMV infection (MOI 0.5) for 16 h. Cells were fixed, nuclei were stained and cells were labeled for HCMV IE1 + cells as a readout for infection. ( B ) HCMV IE1 + cells normalized to total cell numbers and the untreated control (white column) in BMP/Activin stimulated samples (left panel) or with IFNβ co-stimulated samples (right panel). ( C ) HCMV IE1 + cells normalized to total cell numbers in cells pre-stimulated with either low (0.25 nM) or high (3 nM) concentrations of BMP9 (green symbols) or IFNβ co-stimulated with low and high concentrations of BMP9 (beige symbols). ( D ) HFF-1 were infected by centrifugal enhancement with HCMV WT (MOI 0.5) and supernatants of infected cells were collected in 6 h increments. 293T were co-transfected with expression plasmids for the BRE-Luciferase reporter and a Renilla luciferase normalization control. Twenty-four hours post transfection, 293T were either stimulated with supernatants from HCMV-infected cells, or supernatants from HCMV-infected cells incubated for 15 min at RT with an α-BMP9 antibody, for 16 h, followed by a dual-luciferase assay readout. Luciferase fold induction was calculated by dividing Renilla-normalized values from stimulated samples by the corresponding values from unstimulated samples. Data information: ( B ) Experiment was performed three independent times, one representative is shown. ( C , D ) Data are combined from two independent experiments. Student’s t test (unpaired, two-tailed), n.s. not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. Data are shown as mean ± SD. .

Article Snippet: Recombinant human BMP4 (#314-BP-010), human BMP6 (#507-BP-020), human BMP9 (#3209-BP-010), human BMP15 (#5096-BM-005), human Activin B (#659-AB-005), and the human/mouse anti-BMP9 antibody (#AF3209) were purchased from R&D Systems.

Techniques: Infection, Staining, Labeling, Control, Transfection, Expressing, Luciferase, Incubation, Two Tailed Test

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) Schematic representation of the workflow. After 2 h of serum starvation, HFF-1 were stimulated for 6 h with either IFNβ (5 ng/ml) or BMP9 (3 nM) alone, or co-stimulated with IFNβ and BMP9, followed by RNA extraction from cell lysates and RT-qPCR. ( B ) Transcript levels of BMP receptors Acvrl1 (ALK1, type I receptor) and Bmpr2 (type II receptor), which are the main receptors for BMP9, and the BMP-responsive gene Id1 . ( C ) Transcript levels of the ISGs Isg15 , Irf7 , Ifi6 , Irf9 , Stat2 , and Irf1 . ( D ) Transcript levels of the negative regulators Usp18 and Smurf1 . ( E ) HFF-1 were incubated with either Ruxolitinib or DMH1 for 1 h, then stimulated for 6 h with either IFNβ (5 ng/ml) or BMP9 (3 nM) alone, or co-stimulated with IFNβ and BMP9, followed by RNA extraction from cell lysates and RT-qPCR for transcript levels of Id3 , Stat2 , and Irf9 . Data information: ( B – D ) Data are combined from three independent experiments with the exception of Ifi6 transcript levels where three independent experiments were performed and data was combined from two independent experiments. ( E ) The experiment was performed two independent times, one representative is shown. Student’s t test (unpaired, two-tailed), n.s. not significant, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are shown as mean ± SD. .

Article Snippet: Recombinant human BMP4 (#314-BP-010), human BMP6 (#507-BP-020), human BMP9 (#3209-BP-010), human BMP15 (#5096-BM-005), human Activin B (#659-AB-005), and the human/mouse anti-BMP9 antibody (#AF3209) were purchased from R&D Systems.

Techniques: RNA Extraction, Quantitative RT-PCR, Incubation, Two Tailed Test

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) After 2 h of serum starvation, HFF-1 were stimulated for either one or 8 h with IFNβ (5 ng/ml) or BMP9 (3 nM) alone, or were co-stimulated with IFNβ and BMP9, followed by cell lysis and immunoblot analysis with antibodies for phospho-STAT1, STAT1, phospho-SMAD1/5/9, and β-Actin. Phospho-STAT1 band intensities were first normalized to corresponding total STAT1 levels, then to IFNβ stimulation only, and are shown below. ( B ) After 2 h of serum starvation, HFF-1 were stimulated for 1 h with either IFNα2 (5 ng/ml), IFNγ (5 ng/ml), or BMP9 (3 nM) alone, or co-stimulated with IFNα2 or IFNγ and BMP9, followed by cell lysis, immunoblot analysis and quantification as in ( A ). ( C ) After 2 h of serum starvation, HFF-1 were stimulated for 6 h with either IFNα2 (5 ng/ml), IFNβ (5 ng/ml), IFNγ (5 ng/ml), or BMP9 (3 nM) alone, or co-stimulated with IFNα2, IFNβ, IFNγ, and BMP9, followed by RNA extraction from cell lysates and RT-qPCR for Irf9 , Stat2 , Irf1 , and Id3 transcripts. Data information: ( A , B ) Experiment was performed three independent times, one representative immunoblot is shown. Quantified data for the STAT1 phosphorylation levels are combined from three independent experiments. ( C ) Data are combined from three independent experiments. Student’s t test (unpaired, two-tailed), n.s. not significant, * P < 0.05, ** P < 0.01, **** P < 0.0001. Data are shown as mean ± SD. .

Article Snippet: Recombinant human BMP4 (#314-BP-010), human BMP6 (#507-BP-020), human BMP9 (#3209-BP-010), human BMP15 (#5096-BM-005), human Activin B (#659-AB-005), and the human/mouse anti-BMP9 antibody (#AF3209) were purchased from R&D Systems.

Techniques: Lysis, Western Blot, RNA Extraction, Quantitative RT-PCR, Phospho-proteomics, Two Tailed Test

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) 293T were co-transfected with expression plasmids for empty vector (EV) or V5-tagged US18, US20, or M27 (a known inhibitor of IFNAR signaling), together with either a BRE-Luciferase or MX1-Luciferase reporter and a Renilla luciferase normalization control. Twenty-four hours post transfection, 293T were stimulated for 16 h with either IFNβ (5 ng/ml) or BMP9 (3 nM) alone, co-stimulated with IFNβ and BMP9, or left unstimulated, followed by cell lysis and a Dual-luciferase assay readout. ( B ) Results from the Dual-luciferase assay with the BRE-Luciferase (top panel) and MX1-Luciferase (bottom panel) reporter. ( C ) Cell lysates from ( B ) were analyzed by immunoblot for the expression of M27, US18 and US20 with a V5-specific antibody, β-Actin served as loading control. ( D ) 293T were co-transfected with expression plasmids for the BRE-Luciferase and Renilla reporters as in ( A ), together with V5-tagged US18, US20 or co-transfected with US18 and US20 in combination. 24 h post transfection 293 T were stimulated for 16 h with BMP9 (3 nM), or left unstimulated, followed by cell lysis and a Dual-luciferase assay readout. ( E ) 293T were co-transfected with expression plasmids for either Cherry-STING and cGAS-GFP (left panel), RIG-I N (middle panel), or IRF3-5D (a constitutively activate IRF3 mutant; right panel), together with the murine IFNβ-luciferase reporter (IFNβ-Luc) and the Renilla reporter as normalization control. Cells were additionally transfected with expression plasmids for EV, V5-tagged M35 (a known inhibitor of PRR-mediated signaling pathways, (Chan et al, ), US18 or US20. Twenty hours post-transfection, cells were lysed and a dual-luciferase assay was performed. Immunoblot analysis of cell lysates from the respective experiments for M35, US18, and US20 detected with a V5-specific antibody, and β-Actin as a loading control, are shown below. Data information: ( B – E ) Data are combined from three independent experiments, for the immunoblots one representative is shown. Student’s t test (unpaired, two-tailed), n.s. not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are shown as mean ± SD. Luciferase fold induction was calculated by dividing Renilla-normalized values from stimulated samples by the corresponding values from unstimulated samples. .

Article Snippet: Recombinant human BMP4 (#314-BP-010), human BMP6 (#507-BP-020), human BMP9 (#3209-BP-010), human BMP15 (#5096-BM-005), human Activin B (#659-AB-005), and the human/mouse anti-BMP9 antibody (#AF3209) were purchased from R&D Systems.

Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Control, Lysis, Western Blot, Mutagenesis, Protein-Protein interactions, Two Tailed Test

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) HFF-1 with doxycycline-inducible expression of US18-V5, US20-HA, or US18-V5 and US20-HA with corresponding control cell lines (EV1, EV2, EV1 + 2) were generated. Protein expression was induced with 1 µg/ml doxycycline for 20 h prior stimulation and verified by immunoblot with V5- and HA-specific antibodies, and β-Actin as a loading control. ( B ) HFF-1 US18-V5, HFF-1 US20-HA, or HFF-1 US18-V5 US20-HA were left untreated or protein expression was induced with 1 µg/ml doxycycline to the medium for 20 h. Cell lysates were either left untreated or treated with PNGase F for 3 h at 37 °C, followed by immunoblot analysis with V5-, HA-, and Calnexin-specific antibodies. ( C , D ) Indicated HFF-1 lines were treated with 1 µg/ml doxycycline to induce protein expression for 18 h, followed by 2 h of serum starvation. ( C ) Cells were stimulated for 1 h with BMP9 (3 nM), followed by cell lysis and immunoblot analysis with phospho-SMAD1/5/9, SMAD1, V5, HA, and Calnexin antibodies. ( D ) Cells were stimulated for 6 h with either IFNβ (5 ng/ml) or BMP9 (3 nM) alone, or co-stimulated with IFNβ and BMP9, followed by RNA extraction from cell lysates and RT-qPCR for Id3 and Stat2 transcripts. Data information: ( A – C ) Experiment was performed three independent times, one representative is shown. ( D ) Data are combined from three independent experiments. Student’s t test (unpaired, two-tailed), n.s. not significant, * P < 0.05, ** P < 0.01. Data are shown as mean ± SD. .

Article Snippet: Recombinant human BMP4 (#314-BP-010), human BMP6 (#507-BP-020), human BMP9 (#3209-BP-010), human BMP15 (#5096-BM-005), human Activin B (#659-AB-005), and the human/mouse anti-BMP9 antibody (#AF3209) were purchased from R&D Systems.

Techniques: Expressing, Control, Generated, Western Blot, Lysis, RNA Extraction, Quantitative RT-PCR, Two Tailed Test

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) Schematic representation of the workflow. Recombinant HCMV US18stop, HCMV US20stop, and HCMV US18/20stop were constructed by introducing a 16 base pair (bp) stop cassette within the respective coding region. HFF-1 were infected by centrifugal enhancement with HCMV WT, HCMV US18stop, HCMV US20stop, or HCMV US18/20stop (MOI 4 for the 3 h time point, MOI 0.5 for the 48 h time point). Three or 48 h post infection, cells were stimulated with BMP9 (3 nM) for (1) 2 h, followed by cell lysis and immunoblot analysis, or (2) 6 h, followed by RNA extraction from cell lysates and RT-qPCR. ( B , C ) Cell lysates from ( A ) were subjected to immunoblot analysis with p-SMAD1/5/9, SMAD1, HCMV UL35, and GAPDH-specific antibodies (top panel), and transcript levels of Id3 and Stat2 are shown below. Data information: ( B , C ) Data are combined from three independent experiments. Student’s t test (unpaired, two-tailed), n.s. not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are shown as mean ± SD. .

Article Snippet: Recombinant human BMP4 (#314-BP-010), human BMP6 (#507-BP-020), human BMP9 (#3209-BP-010), human BMP15 (#5096-BM-005), human Activin B (#659-AB-005), and the human/mouse anti-BMP9 antibody (#AF3209) were purchased from R&D Systems.

Techniques: Recombinant, Construct, Infection, Lysis, Western Blot, RNA Extraction, Quantitative RT-PCR, Two Tailed Test

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) HFF-1 were infected by centrifugal enhancement with HCMV WT or HCMV US18/20stop (MOI 4). Three hours post infection, cells were stimulated with IFNβ (1 ng/ml) or BMP9 (3 nM) alone, or co-stimulated with IFNβ and BMP9 for 6 h, followed by RNA extraction from cell lysates and RT-qPCR. ( B – D ) Transcript levels of Id3 ( B ), the ISGs Irf9 and Stat2 ( C ), and HCMV transcripts HCMV IE1 and HCMV UL44 ( D ). Data information: Three independent experiments with similar results were performed and data shown is combined from two of the three independent experiments. Student’s t test (unpaired, two-tailed), n.s. not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are shown as mean ± SD. .

Article Snippet: Recombinant human BMP4 (#314-BP-010), human BMP6 (#507-BP-020), human BMP9 (#3209-BP-010), human BMP15 (#5096-BM-005), human Activin B (#659-AB-005), and the human/mouse anti-BMP9 antibody (#AF3209) were purchased from R&D Systems.

Techniques: Infection, RNA Extraction, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: CDK6-mediated endothelial cell cycle acceleration drives arteriovenous malformations in hereditary hemorrhagic telangiectasia

doi: 10.1101/2023.09.15.554413

Figure Lengend Snippet: ( A ) Schematic representation of the i.p. injection protocol. Arrowheads indicate the postnatal days (P) of injection (AM or PM). Pups were euthanized at P6 for analysis. ( B ) Representative staining using isolectin B4 (IB4, green), and of ERG (blue) and EdU (red) in the peri-optic nerve and mid-plexus regions of retinas from PBS controls, vehicle-treated BMP9/10ib mice, and palbociclib-treated BMP9/10ib mice. Arrows denote AVMs; a, artery; v, vein. Scale bars, 50 μm. ( C and D ) Scatter plots showing the total number of ERG + cells per 200 μm in arteries ( C ) and veins ( D ) in the peri-optic nerve region across three groups: PBS (n=11), vehicle-treated BMP9/10ib (n=9), and palbociclib-treated BMP9/10ib (n=12) mice. ( E and F ) Scatter plots showing ERG + EdU + cells in the peri-optic nerve ( E ) and mid-plexus ( F ) regions across three groups: PBS (n=4-6), vehicle-treated BMP9/10ib (n=4-6), and palbociclib-treated BMP9/10ib (n=6) mice. Data represent individual retinas and mean ± SEM, one-way ANOVA with Tukey’s multiple comparisons test. ns, not significant; *P < 0.05, ***P < 0.001, ****P < 0.0001.

Article Snippet: BMP9 (25 mg/kg; MAB3209, R&D System) and BMP10 (50 mg/kg; MAB2926, R&D System) were injected intraperitoneally (i.p.) at P3 and P4.

Techniques: Injection, Staining

Journal: bioRxiv

Article Title: CDK6-mediated endothelial cell cycle acceleration drives arteriovenous malformations in hereditary hemorrhagic telangiectasia

doi: 10.1101/2023.09.15.554413

Figure Lengend Snippet: ( A and B ) Representative flow cytometry plots ( A ) and quantification ( B ) of cell cycle phase distribution of ECs isolated from livers of P6 and P8 PBS (n=14) and BMP9/10ib (n=11) mice. ( C and D ) Representative flow cytometry ( C ) and quantification ( D ) of cells analyzed as in ( A ) showing S phase duration measured as intra-S phase of EdU incorporation [intra-S EdU mean fluorescence intensity (MFI)]. Data in ( B ) and ( D ) represent mean ± SEM, unpaired t-test. ( E ) Schematic representation of the i.p. injection protocol. Arrowheads indicate the postnatal days (P) of injection (AM or PM). Pups were euthanized at P8 for analysis. ( F and G ) Representative flow cytometry plots of cell cycle speed analysis in iH2B-FT ECs ( F ) and quantification of fast-cycling iH2B-FT ECs ( G ) isolated from P8 livers of PBS (n=7), vehicle-treated BMP9/10ib (n=8), and palbociclib-treated BMP9/10ib (n=7) mice. Data in ( G ) represent mean ± SEM, one-way ANOVA with Tukey’s multiple comparisons test. ns, not significant; **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: BMP9 (25 mg/kg; MAB3209, R&D System) and BMP10 (50 mg/kg; MAB2926, R&D System) were injected intraperitoneally (i.p.) at P3 and P4.

Techniques: Flow Cytometry, Isolation, Fluorescence, Injection

Journal: bioRxiv

Article Title: CDK6-mediated endothelial cell cycle acceleration drives arteriovenous malformations in hereditary hemorrhagic telangiectasia

doi: 10.1101/2023.09.15.554413

Figure Lengend Snippet: ( A ) Heatmap analysis of a high-throughput cell cycle protein ELISA of ECs isolated from livers of P6 BMP9/10ib mice vs. PBS control littermates. Data represent n=2 independent analyses (n represents one litter of pups combined for each condition; PBS, n=6 mice; BMP9/10ib, n=7 mice). ( B ) qPCR analysis of ECs isolated from livers of P6 PBS and BMP9/10ib mice (n=5/condition). Data represent mean ± SEM, Mann-Whitney test ( Cdk2 ), and unpaired t-test ( Cdk4 and Cdk6 ). ( C ) Flow cytometry quantification of p-RB1 fluorescence intensity in ECs isolated from livers of P6 PBS and BMP9/10ib mice (n=3/condition). Data represent mean ± SEM, unpaired t-test. *P < 0.05. ( D and E ) Representative IF staining of p-RB1 (green) and IB4 (red) in the peri-optic nerve and mid-plexus regions ( D ) and corresponding quantification of the peri-optic nerve region ( E ) of retinas from vehicle-treated PBS (n=6), palbociclib-treated PBS (n=5), vehicle-treated BMP9/10ib (n=6), and palbociclib-treated BMP9/10ib (n=6) mice. Data in ( E ) represent individual retinas and mean ± SEM, one-way ANOVA with Tukey’s multiple comparisons test. ns, not significant; *P < 0.05, **P < 0.01. Scale bars in ( D ), 50 μm. ( F ) Representative H&E and IHC staining of p-RB1 in 4 μm skin sections of HHT2 patients. Te, telangiectasia; N, normal vessel; Ba, basilar cells.

Article Snippet: BMP9 (25 mg/kg; MAB3209, R&D System) and BMP10 (50 mg/kg; MAB2926, R&D System) were injected intraperitoneally (i.p.) at P3 and P4.

Techniques: High Throughput Screening Assay, Enzyme-linked Immunosorbent Assay, Isolation, Control, MANN-WHITNEY, Flow Cytometry, Fluorescence, Staining, Immunohistochemistry

Journal: bioRxiv

Article Title: CDK6-mediated endothelial cell cycle acceleration drives arteriovenous malformations in hereditary hemorrhagic telangiectasia

doi: 10.1101/2023.09.15.554413

Figure Lengend Snippet: ( A ) Representative staining using IB4 (green) and of α-smooth muscle actin (SMA, red) in whole petals (first two rows), th vein front (third row), and peri-optic nerve (forth row) and mid-plexus (last two rows) regions of retinas from mice treated as indicated. Arrows denote AVMs; a, artery; v, vein. Scale bars, 1 mm (whole petal images), 100 μm (vein front), and 50 μm (mid-plexus and peri-optic nerve areas). ( B-F ) Scatter plots showing retinal AVM number ( B ), vein diameter ( E ), and mid-plexus vascular density ( F ) following palbociclib treatment, and retinal AVM number ( C ) and AVM diameter ( D ) following ribociclib treatment. Data represent individual retinas and mean ± SEM; vehicle-treated PBS (n=6 mice), palbociclib-treated PBS (n=7), vehicle-treated BMP9/10ib (n=10-12), and palbociclib-treated BMP9/10ib (n=9-13). Data represent individual retinas and mean ± SEM; unpaired t-test ( B-D ), one-way ANOVA with Tukey’s multiple comparisons test ( E and F ). *P 0.05, **P 0.01, ****P 0.0001.

Article Snippet: BMP9 (25 mg/kg; MAB3209, R&D System) and BMP10 (50 mg/kg; MAB2926, R&D System) were injected intraperitoneally (i.p.) at P3 and P4.

Techniques: Staining

Journal: bioRxiv

Article Title: CDK6-mediated endothelial cell cycle acceleration drives arteriovenous malformations in hereditary hemorrhagic telangiectasia

doi: 10.1101/2023.09.15.554413

Figure Lengend Snippet: ( A ) Representative images of tdTomato-stained retinal vasculature from vehicle-treated and palbociclib-treated P6 Eng iECKO mice. Arrows denote AVMs. Scale bars, 1 mm. ( B-D ) Scatter plot showing AVM number ( B ), AVM diameter ( C ), and mid-plexus vascular density ( D ) in retinas of mice treated as in ( A ). Data represent individual retinas and mean ± SEM; vehicle-treated Eng iECKO (n=6 mice), palbociclib-treated Eng iECKO (n=7); unpaired t-test. **P 0.01, ***P 0.001. ( E ) Representative bright field ( a, b, e, f, i, j ) and BABB-cleared ( c, d, g, h, k, l ) images of blue latex bead-perfused P8 PBS control and BMP9/10ib brains, treated or not (vehicle) with palbociclib. First row, dorsal views; second row, ventral views. Arrows in ( d ) denote the two positions where basilar artery (BA) diameter wa measured. ( F ) Quantification of BA diameter across three groups: PBS (n=4), vehicle-treated BMP9/10ib (n=6), and palbociclib-treated BMP9/10ib (n=4) mice. Data represent two measurements per brain and mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. **P 0.01, ***P 0.001.

Article Snippet: BMP9 (25 mg/kg; MAB3209, R&D System) and BMP10 (50 mg/kg; MAB2926, R&D System) were injected intraperitoneally (i.p.) at P3 and P4.

Techniques: Staining, Control

Journal: bioRxiv

Article Title: CDK6-mediated endothelial cell cycle acceleration drives arteriovenous malformations in hereditary hemorrhagic telangiectasia

doi: 10.1101/2023.09.15.554413

Figure Lengend Snippet: ( A ) Representative staining using IB4 (green) and of SMA (red) in whole petals (first two rows), the vein front (third row), and peri-optic nerve (forth row) and mid-plexus (last two rows) regions of retinas from Cdk6 f/f controls and Cdk6 iECKO mice challenged or not (PBS) with BMP9/10ib. Arrows denote AVMs; a, artery; v, vein. Scale bars, 1 mm (whole petal images), 100 μm (vein front), and 50 μm (mid-plexus and peri-optic nerve areas). ( B-D ) Scatter plots showing retinal AVM number ( B ), vein diameter ( C ), and mid-plexus vascular density ( D ) in Cdk6 f/f ;BMP9/10ib controls (n=11-14) and Cdk6 iECKO ;BMP9/10ib (n=8-12) mice. Data represent individual retinas and mean ± SEM, unpaired t-test. **P 0.01, ***P 0.001. ( D ) Schematic illustration of the proposed mechanism of control of the cell cycle in ECs by ALK1 signaling and its relevance for HHT pathogenesis.

Article Snippet: BMP9 (25 mg/kg; MAB3209, R&D System) and BMP10 (50 mg/kg; MAB2926, R&D System) were injected intraperitoneally (i.p.) at P3 and P4.

Techniques: Staining, Control