Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: BMP9 (Bone Morphogenetic Protein-9)/Alk1 (Activin-Like Kinase Receptor Type I) Signaling Prevents Hyperglycemia-Induced Vascular Permeability

doi: 10.1161/atvbaha.118.310733

Figure Lengend Snippet: Figure 1. Hyperglycemia inhibits BMP (bone morphogenetic protein) 9/Alk1 (activin-like kinase receptor type I)/Smad (suppressor of mothers against decapentaplegic) 1, 5, 9 signaling In vivo and in vitro in endothelial cells. A, Human umbilical endothelial cell (HUVECs) were exposed to normoglycemia or hyperglycemia (5 and 11 mmol/L D-glucose) for 18 h before stimulation for ≤60 min with BMP9 followed by immunoblotting for pSmad1, 5, 9, Smad 1, or B-actin. Representative blots are shown (top), and bands were quantified by densitometry (bottom). Total protein Smad1 and internal B-actin reference antibody were used for normalizations, and values are expressed as mean±SEM. Data represent the mean of 3 independent experiments. B, HUVECs were exposed to 5 or 11 mmol/L D-glu- cose for 18 h and processed for immunoblotting against ALK1, BMPR2 (BMP receptor 2), ActRIIb (activin receptor 2B), endoglin, and B-actin. Representative blots are shown (top), and bands were quantified by densitometry (bottom). Internal B-actin reference antibody was used for normalizations, and values are expressed as mean±SEM. Data represent the mean of 4 independent (Continued )

Article Snippet: The following primary antibodies were used: ALK1 (human AF370 or mouse AF770; 0.4 μg/mL) and Eng (AF1320; 0.2 μg/mL) were obtained from R&D Systems. pSmad1, 5, 9 (cs9511; 10 μmol/L), Smad1 (cs6944; 10 μmol/L), pVEGFR2-Y951 (cs4991; 10 μmol/L), Nonstandard Abbreviations and Acronyms ActRIIB activin receptor 2B AGE advanced glycation end products ALK1 activin-like kinase receptor type I BMP bone morphogenetic protein BMPR2 bone morphogenetic protein receptor 2 CRP C-reactive protein DME diabetic macular edema ECGM-2 endothelial cell growth medium 2 EGFR epidermal growth factor receptor eNOS endothelial NO synthase HRMEC human retinal microvascular endothelial cell HUVEC human umbilical endothelial cell rhBMP9 recombinant human bone morphogenetic protein 9 Smad suppressor of mothers against decapentaplegic STZ streptozotocin TNF-α tumor necrosis factor-α VEGF vascular endothelial growth factor VEGFR vascular endothelial growth factor receptor by guest on June 7, 2018 http://atvb.ahajournals.org/ D ow nloaded from Akla et al Alk1 Signaling and Vascular Permeability 3 VEGFR2 (cs55B11 or cs9698; 10 μmol/L), pSrc (cs6943; 10 μmol/L), Src (cs2108; 10 μmol/L), peNOS (cs9570; 10 μmol/L), pβ-catenin/ Ser 675 (cs4176; 10 μmol/L), and eNOS (endothelial NO synthase; cs32027; 10 μmol/L) were acquired from Cell Signaling Technology.

Techniques: In Vivo, In Vitro, Western Blot

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: BMP9 (Bone Morphogenetic Protein-9)/Alk1 (Activin-Like Kinase Receptor Type I) Signaling Prevents Hyperglycemia-Induced Vascular Permeability

doi: 10.1161/atvbaha.118.310733

Figure Lengend Snippet: Figure 1 Continued. experiments. C, Flow cytometry analysis of ALK1 cell surface expression. Data±SEM on graph represent the quantification of mean fluorescence intensity (MFI) from 4 independent experiments. D, Cell surface internalization of ALK1 in HUVECs cultured in the presence of 5, 11, or 11 mmol/L glucose with or without BMP9. Data±SEM on graphs represent the quantification of MFI from 4 independent experiments. E, Six- to 8-wk-old C57/BL6 mice were injected intraperitoneally with streptozotocin (STZ). Eight weeks after onset of diabetes mellitus, pulmonary tissue was harvested, and homogenates were analyzed with specific antibodies by immunob- lotting. Blots were quantified by densitometry (bottom). n=5 control mice, n=4 diabetic mice. F, Immunofluorescence on retinal cryosec- tions revealed that Alk1 protein (green) shows robust vascular expression in control retinas (top) but is weakly expressed in blood vessels at 8 wk after STZ injection (bottom), as confirmed by colocalization with the vascular marker IsoB4. Representative images of 3 inde- pendent mice (scale bar=40 μm). Differences between means were analyzed using unpaired t test. NS indicates nonsignificant. *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: The following primary antibodies were used: ALK1 (human AF370 or mouse AF770; 0.4 μg/mL) and Eng (AF1320; 0.2 μg/mL) were obtained from R&D Systems. pSmad1, 5, 9 (cs9511; 10 μmol/L), Smad1 (cs6944; 10 μmol/L), pVEGFR2-Y951 (cs4991; 10 μmol/L), Nonstandard Abbreviations and Acronyms ActRIIB activin receptor 2B AGE advanced glycation end products ALK1 activin-like kinase receptor type I BMP bone morphogenetic protein BMPR2 bone morphogenetic protein receptor 2 CRP C-reactive protein DME diabetic macular edema ECGM-2 endothelial cell growth medium 2 EGFR epidermal growth factor receptor eNOS endothelial NO synthase HRMEC human retinal microvascular endothelial cell HUVEC human umbilical endothelial cell rhBMP9 recombinant human bone morphogenetic protein 9 Smad suppressor of mothers against decapentaplegic STZ streptozotocin TNF-α tumor necrosis factor-α VEGF vascular endothelial growth factor VEGFR vascular endothelial growth factor receptor by guest on June 7, 2018 http://atvb.ahajournals.org/ D ow nloaded from Akla et al Alk1 Signaling and Vascular Permeability 3 VEGFR2 (cs55B11 or cs9698; 10 μmol/L), pSrc (cs6943; 10 μmol/L), Src (cs2108; 10 μmol/L), peNOS (cs9570; 10 μmol/L), pβ-catenin/ Ser 675 (cs4176; 10 μmol/L), and eNOS (endothelial NO synthase; cs32027; 10 μmol/L) were acquired from Cell Signaling Technology.

Techniques: Flow Cytometry, Expressing, Fluorescence, Cell Culture, Injection, Control, Immunofluorescence, Marker

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: BMP9 (Bone Morphogenetic Protein-9)/Alk1 (Activin-Like Kinase Receptor Type I) Signaling Prevents Hyperglycemia-Induced Vascular Permeability

doi: 10.1161/atvbaha.118.310733

Figure Lengend Snippet: Figure 4. BMP (bone morphogenetic protein) 9/ALK1 (activin-like kinase receptor type I) inhibits VEGF (vascular endothelial growth factor)-induced permeability via inhibition of VEGF signaling in endothelial cells. A and B, VEGF-induced vascular leakage (45 min) of serum albumin-bound Evans blue in the back skin of control (Cdh5-Cre) or homozygote Alk1ΔEC mice (Alk1−/−) adenovirally over- expressing BMP9 or empty vector (CTRL). A, Spectrophotometric analysis of vascular leakiness by formamide-extracted Evans blue dye content. Evans blue dye quantification of mice skin injected with VEGF (50 ng) overexpressing BMP9 was significantly decreased compared with that of skin injected with VEGF alone in CTRL-CRE mice but not in Alk1−/− mice. The results are expressed number of folds relative to control and are presented by mean±SEM (n=7 mice per group [control mice] or n=4 mice per group [Alk1−/− mice]). B, Representative images of Evans blue extravasation in dorsal skin of control mice. C, Immunoblotting of human umbilical endothelial cell (HUVECs) stimulated with VEGF after pretreatment with D-glucose (5, 11, and 25 mmol/L) and BMP9 with specific (Continued )

Article Snippet: The following primary antibodies were used: ALK1 (human AF370 or mouse AF770; 0.4 μg/mL) and Eng (AF1320; 0.2 μg/mL) were obtained from R&D Systems. pSmad1, 5, 9 (cs9511; 10 μmol/L), Smad1 (cs6944; 10 μmol/L), pVEGFR2-Y951 (cs4991; 10 μmol/L), Nonstandard Abbreviations and Acronyms ActRIIB activin receptor 2B AGE advanced glycation end products ALK1 activin-like kinase receptor type I BMP bone morphogenetic protein BMPR2 bone morphogenetic protein receptor 2 CRP C-reactive protein DME diabetic macular edema ECGM-2 endothelial cell growth medium 2 EGFR epidermal growth factor receptor eNOS endothelial NO synthase HRMEC human retinal microvascular endothelial cell HUVEC human umbilical endothelial cell rhBMP9 recombinant human bone morphogenetic protein 9 Smad suppressor of mothers against decapentaplegic STZ streptozotocin TNF-α tumor necrosis factor-α VEGF vascular endothelial growth factor VEGFR vascular endothelial growth factor receptor by guest on June 7, 2018 http://atvb.ahajournals.org/ D ow nloaded from Akla et al Alk1 Signaling and Vascular Permeability 3 VEGFR2 (cs55B11 or cs9698; 10 μmol/L), pSrc (cs6943; 10 μmol/L), Src (cs2108; 10 μmol/L), peNOS (cs9570; 10 μmol/L), pβ-catenin/ Ser 675 (cs4176; 10 μmol/L), and eNOS (endothelial NO synthase; cs32027; 10 μmol/L) were acquired from Cell Signaling Technology.

Techniques: Permeability, Inhibition, Control, Expressing, Plasmid Preparation, Injection, Western Blot

Journal: Disease Models & Mechanisms

Article Title: The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN

doi: 10.1242/dmm.024448

Figure Lengend Snippet: Comparison of inhibition effects of crizotinib and PF-06463922 on WT and neuroblastoma gain-of-function mutant TKDs by in vitro kinase assay. (A,B) Different ALK TKD proteins were incubated with either PF-06463922 (A) or crizotinib (B) in the presence of ATP (0.1 mM) and substrate peptides (0.2 mM). The incorporation of labelled γ- 32 P was detected under different conditions. Background counts from no-enzyme controls were subtracted, and the data were normalized to the 0 nM inhibitor reactions. (C) IC 50 values from A,B were calculated by fitting data to a log (inhibitor) versus normalized response (variable slope) equation in GraphPad Prism 6.0. All data are shown as mean±s.d. from at least two independent experiments. (D) Crystal structures of ALK kinase domain in complex with PF-06463922 (top) or crizotinib (bottom). Compounds indicated in black. Gain-of-function ALK mutations F1174, R1192P, F1245, G1269 and Y1278 are shown as red spheres. The ribbon diagram displays αC helix (1157-1173; orange), catalytic loop (1246-125; magenta), activation loop (1271-1288; cyan) with DFG motif marked in blue. Figures were generated with PyMol using published coordinates (Protein data bank code: 4CLI and 2XP2).

Article Snippet: This was followed by incubation with primary antibodies for detection of total ALK (1:2000; 3633) and pALK Y1586 (1:2000; 3348) (both from Cell Signaling Technology, Danvers, MA) subsequent to incubation with a secondary antibody (1 μg/ml; SULFO-tag, R32AB-1, Meso Scale Diagnostics) for assay readout with a SECTORTM 6000 instrument.

Techniques: Comparison, Inhibition, Mutagenesis, In Vitro, Kinase Assay, Incubation, Activation Assay, Generated

Journal: Disease Models & Mechanisms

Article Title: The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN

doi: 10.1242/dmm.024448

Figure Lengend Snippet: Preclinical efficacy of PF-06463922 in a murine model of Th- ALK F1174L /MYCN -driven neuroblastoma. (A) Waterfall plots of tumoral response in Th- ALK F1174L /MYCN mice treated with vehicle, crizotinib (100 mg/kg, once daily) or PF-06463922 (10 mg/kg, twice daily). Each bar indicates percent change in the volume of an individual tumor, as assessed by T 2 -weighted MRI, on day 7 compared with day 0 of treatment. (B) Representative MRI of each treatment group on day 0 and day 7 after treatment of indicated vehicle or drug. Hashed white line indicates tumor border. (C) Immunoblot analysis of phosphorylated ALK (phospho-ALK-Y1278), total ALK, and MYCN of tumors treated for 2 days with vehicle or PF-06463922. GAPDH was employed as loading control. Arrow indicates pALK-Y1278. (D) Quantification of band intensity of MYCN relative to GAPDH (left) and ratio of band intensity of pALK over total ALK (right) comparing vehicle versus treated samples. (E) Meso Scale Discovery assay depicted as electrochemiluminescence signal of treated samples relative to the respective vehicle controls for total ALK, phospho-ALK-Y1568, and the ratio of pALK to ALK. (F) Representative H&E (top) and immunohistochemical images for Ki67 (bottom) of vehicle and treated samples. Quantification of overall percentage of area positive for Ki67 (right). Error bars represent s.d. between individual animals (per group: n =4 in A, n =5 in C-F). P -values equal unpaired t -test comparison between vehicle and treatment groups.

Article Snippet: This was followed by incubation with primary antibodies for detection of total ALK (1:2000; 3633) and pALK Y1586 (1:2000; 3348) (both from Cell Signaling Technology, Danvers, MA) subsequent to incubation with a secondary antibody (1 μg/ml; SULFO-tag, R32AB-1, Meso Scale Diagnostics) for assay readout with a SECTORTM 6000 instrument.

Techniques: Western Blot, Control, Electrochemiluminescence, Immunohistochemical staining, Comparison

Journal: Frontiers in Cellular Neuroscience

Article Title: Activin Receptor-Like Kinase 1 Combined With VEGF-A Affects Migration and Proliferation of Endothelial Cells From Sporadic Human Cerebral AVMs

doi: 10.3389/fncel.2018.00525

Figure Lengend Snippet: mRNA expression of gene Alk1 in HA-ECs and cAVM-ECs in patients with different ages. Levels of VEGF-A mRNA expression (0.986 ± 0.134) in the HA-ECs were lower than that in cAVM-ECs (8 years) (4.183 ± 0.238) and cAVM-ECs (14 years) (3.834 ± 0.451) (all ∗ p < 0.05). In addition, the levels of VEGF-A mRNA expression were (2.92 ± 0.58) in cAVM-ECs (17 years), (3.04 ± 0.79) in cAVM-ECs (29 years), (3.08 ± 0.37) in cAVM-ECs (32 years), (2.08 ± 0.28) in cAVM-ECs (42 years), (1.97 ± 0.24) in cAVM-ECs (44 years), and (1.97 ± 0.25) in cAVM-ECs (51 years), all of that were higher than that (0.986 ± 0.134) in HA-ECs (all ∗ p < 0.05).

Article Snippet: After blocking with 5% non-fat dried milk for 2 h at RT, membranes were incubated with the primary antibodies overnight at 4°C: Primary antibodies against Alk1 (1:1,000, Cell Signaling Technology, Cat No. 31278, United States) and GAPDH (1:5,000, Cell Signaling Technology, Cat No. 51332, United States), each primary antibody was diluted in blocking buffer and then incubated overnight at 4°C.

Techniques: Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: Activin Receptor-Like Kinase 1 Combined With VEGF-A Affects Migration and Proliferation of Endothelial Cells From Sporadic Human Cerebral AVMs

doi: 10.3389/fncel.2018.00525

Figure Lengend Snippet: Expression of Alk1 proteins in HA-ECs and cAVM-ECs. (A–D) Immunofluorescent staining to detect the Alk1 protein expressed in HA-ECs and cAVM-ECs. (A–C) In HA-ECs, (A) Alk1 protein was stained by Green fluorescent, (B) nuclei was stained by DAPI in Green fluorescent; (C) merge; (D–F) In cAVM-ECs, (D) Alk1 protein was stained by Green fluorescent, (E) nuclei was stained by DAPI in Green fluorescent; the fluorescence intensity in cAVM-ECs was obviously lower than in HA-ECs; (F) merge. Scale bar = 200 μm; (G) Detect the expression of Alk1 by Western blot, (H) expression of Alk1 proteins in the HA-ECs cultures were significantly higher than that in cAVM-ECs in vitro ( ∗ p < 0.05).

Article Snippet: After blocking with 5% non-fat dried milk for 2 h at RT, membranes were incubated with the primary antibodies overnight at 4°C: Primary antibodies against Alk1 (1:1,000, Cell Signaling Technology, Cat No. 31278, United States) and GAPDH (1:5,000, Cell Signaling Technology, Cat No. 51332, United States), each primary antibody was diluted in blocking buffer and then incubated overnight at 4°C.

Techniques: Expressing, Staining, Fluorescence, Western Blot, In Vitro

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Crizotinib-induced antitumour activity in human alveolar rhabdomyosarcoma cells is not solely dependent on ALK and MET inhibition

doi: 10.1186/s13046-015-0228-4

Figure Lengend Snippet: ALK and MET expression in RMS cell lines and tumour samples. a RT-PCR analysis showing the expression of ALK and MET mRNAs in RH4 and RH30, two ARMS cell lines, but faintly in RD and RD18, two ERMS cell lines. GAPDH expression used as the internal control. b Western blotting experiments showing higher levels of both total ALK and MET proteins in ARMS than in ERMS cells. The activated forms of both ALK and MET proteins were observed in ARMS samples using the specific p-ALK (Tyr1604) and p-MET (Tyr1234/Tyr1235) antibodies, respectively. Tubulin expression was used as the internal control. c Expression of ALK and MET mRNAs measured by RT-PCR in a panel of ARMS and ERMS tumour samples

Article Snippet: Blots were blocked in 5 % not-fat milk or BSA and incubated over-night at +4 °C with the following primary antibodies: phospho (p)-ALK (1:500), p-MET (1:500), p-ERK (1:3000), p-AKT (1:2000), p-IGF1R (1:1000), ALK-XP (1:2000), AKT (1:2000), ERK (1:2000), IGF1R (1:1000), cleaved caspase-3 (1:200), cleaved PARP (1:1000) by Cell Signalling Technology; MET (1:1000), Cyclin B1 (1:1000), Cyclin D3 (1:250), p-Cdc2 (1:300), Cdc25A (1:1000), Cdc25C (1:1000), p27 (1:500) and Cdk4 (1:1000) by Santa Cruz Biotechnology; p62/sequestosome 1 (1:1000, BD Biosciences); LC3 (1:1000, Sigma).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Crizotinib-induced antitumour activity in human alveolar rhabdomyosarcoma cells is not solely dependent on ALK and MET inhibition

doi: 10.1186/s13046-015-0228-4

Figure Lengend Snippet: Effects of crizotinib on ALK/MET phosphorylation, AKT and ERK activation and cell growth through IGF-1 signalling in RH4 and RH30 cell lines. a Immunoprecipitation and western blot showing expression of p-ALK, total ALK and p-MET and total MET in RH4 and RH30 cells treated with or without 1.5 μM crizotinib (Crz) for 24 h. Control cells (Ctrl) were treated with DMSO alone. b Western blotting analysis of phosphorylation levels of AKT and ERK proteins and downstream molecules GSKβ and P70S6K in RH4 and RH30 cells treated with or without 1.5 μM crizotinib (Crz) for 24 h. Control cells (Ctrl) were treated with DMSO alone. c Western blotting analysis of phosphorylation levels of IGF1R and AKT proteins in IGF1-treated cells with or without crizotinib. RH4 and RH30 cells were starved in serum deficient medium for 24 h prior to induce with IGF1 (50 ng/ml) for 30 min. Crizotinib (1.5 μM) was added 4 h before ligand stimulation. d MTT proliferation assay in ARMS cells grown in the presence or absence of IGF1 (50 ng/ml) and crizotinib (1.5 μM) for 48 h. Bars represent the mean values for nine replicate wells ± SD and are representative of three independent experiments

Article Snippet: Blots were blocked in 5 % not-fat milk or BSA and incubated over-night at +4 °C with the following primary antibodies: phospho (p)-ALK (1:500), p-MET (1:500), p-ERK (1:3000), p-AKT (1:2000), p-IGF1R (1:1000), ALK-XP (1:2000), AKT (1:2000), ERK (1:2000), IGF1R (1:1000), cleaved caspase-3 (1:200), cleaved PARP (1:1000) by Cell Signalling Technology; MET (1:1000), Cyclin B1 (1:1000), Cyclin D3 (1:250), p-Cdc2 (1:300), Cdc25A (1:1000), Cdc25C (1:1000), p27 (1:500) and Cdk4 (1:1000) by Santa Cruz Biotechnology; p62/sequestosome 1 (1:1000, BD Biosciences); LC3 (1:1000, Sigma).

Techniques: Phospho-proteomics, Activation Assay, Immunoprecipitation, Western Blot, Expressing, Control, Proliferation Assay