Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A Fluorescent Aptasensor Based on Assembled G-Quadruplex and Thioflavin T for the Detection of Biomarker VEGF165

doi: 10.3389/fbioe.2021.764123

Figure Lengend Snippet: (A) Schematic diagram of the construction of the G-quadruplex aptamer. (B,C) Affinity and specificity analysis of the anti-VEGF165 aptamer and the assembled G4-Apt by biolayer interferometry. (D,E) Conformation analysis of the anti-VEGF165 aptamer and the assembled G4-Apt by circular dichroism.

Article Snippet: Recombinant human VEGF165 protein and VEGF165 ELISA kits were purchased from Sino Biological Inc. (Beijing, China).

Techniques:

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A Fluorescent Aptasensor Based on Assembled G-Quadruplex and Thioflavin T for the Detection of Biomarker VEGF165

doi: 10.3389/fbioe.2021.764123

Figure Lengend Snippet: (A) Binding affinity analysis of the assembled G4-Apt and the aptamer to thioflavin T (ThT) by biolayer interferometry. (B) Fluorescence spectra of ThT in the free state (brown curve), ThT with G4-Apt (red curve), and ThT with G4-Apt and VEGF165 (blue curve).

Article Snippet: Recombinant human VEGF165 protein and VEGF165 ELISA kits were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Binding Assay, Fluorescence

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A Fluorescent Aptasensor Based on Assembled G-Quadruplex and Thioflavin T for the Detection of Biomarker VEGF165

doi: 10.3389/fbioe.2021.764123

Figure Lengend Snippet: (A) Optimization of the concentration for the assembled G4-Apt. (B) Optimization of binding time for VEGF165 in the detection system.

Article Snippet: Recombinant human VEGF165 protein and VEGF165 ELISA kits were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Concentration Assay, Binding Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A Fluorescent Aptasensor Based on Assembled G-Quadruplex and Thioflavin T for the Detection of Biomarker VEGF165

doi: 10.3389/fbioe.2021.764123

Figure Lengend Snippet: (A) Fluorescence spectra of the detection system at various concentrations of VEGF165. (B) Linear relationship between the aptasensor signal and the concentration of VEGF165 in the range of 1.56–25 nM. (C) Cross-reactivity experiments of the fluorescent aptasensor with HAS, SA, BSA, ATP, and GTX. The error bars represent the standard deviation of three independent measurements. (D) Comparison of the normalized concentration of VEGF165.

Article Snippet: Recombinant human VEGF165 protein and VEGF165 ELISA kits were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Fluorescence, Concentration Assay, Standard Deviation

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A Fluorescent Aptasensor Based on Assembled G-Quadruplex and Thioflavin T for the Detection of Biomarker VEGF165

doi: 10.3389/fbioe.2021.764123

Figure Lengend Snippet: Comparison of different aptamer-based detection methods for VEGF165.

Article Snippet: Recombinant human VEGF165 protein and VEGF165 ELISA kits were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Fluorescence, Förster Resonance Energy Transfer, SPR Assay

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: Vascular endothelial growth factor confers endothelial resistance to apoptosis through poly(ADP-ribose) polymerase.

doi: 10.1111/j.1538-7836.2011.04368.x

Figure Lengend Snippet: Fig. 2. Vascular endothelial growth factor (VEGF) time-dependently increases resistance to apoptosis. (A) Preincubation of human umbilical vein endothelial cells (HUVECs) grown on vitronectin with 100 pg mL)1 to 100 ng mL)1 (0.1–100 ng mL)1) VEGF-A(165) for 24 h significantly reduced apoptosis induced by 5 lg mL)1 cRGDfV (RGD) by an average of 64.8% ± 4.0% (*P < 0.05) as compared with incubation with cRGDfV only, as demonstrated by flow cytometry. Apoptotic cells were considered annexin V+/PI) cells. Mean ± standard deviation (SD) of three experiments in duplicate. (B) Longer-term preincubation of HUVECs with 100 pg mL)1 to 100 ng mL)1 (0.1–100 ng mL)1) VEGF-A(165) at 48-h intervals for 6 days further reduced cRGDfV-induced apoptosis significantly, by an average of 90.3% ± 3.3% (*P < 0.05). Results were reproduced in hCMEC/D3 and EA.hy.926 cells. Mean ± SD of four experiments in duplicate. (C) Preincubation of HUVECs with VEGF-A(165) for 6 days (48-h intervals) reduced the number of non-vital cells in trypan blue exclusion assays as compared with cRGDfV only, and (D) reduced apoptotic chromatin condensation and fragmentation as analyzed by diamidino-2-phenylindole (DAPI)-stained nuclear morphology, corroborating the results from flow cytometry. Mean ± SD of three experiments in duplicate. The small panel in (D) shows the DAPI staining of a representative experiment: HUVECs incubated with 10 ng mL)1

Article Snippet: They were then incubated with 5% normal serum/PBS, and then with goat-anti-human PARP (1 : 20; R&D Systems), goat anti-human VEGF (1 : 50; R&D Systems), mouse anti-human VEGFR-2 (clone Y-23, 1 lg mL)1; Santa Cruz), rabbit-antihuman NP-1 (5 lg mL)1; Invitrogen) and rabbit-anti-human von Willebrand factor (VWF) (1 : 200; Dako) antibodies for 2 or 16 h at 4 C, and subsequently with biotinylated secondary antibodies (streptavidin–alkaline phosphatase conjugate, Fast Red, Mayer s hemalum counterstaining [all Sigma-Aldrich]), and covered with glycerol gelatine.

Techniques: Incubation, Cytometry, Standard Deviation, Staining

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: Vascular endothelial growth factor confers endothelial resistance to apoptosis through poly(ADP-ribose) polymerase.

doi: 10.1111/j.1538-7836.2011.04368.x

Figure Lengend Snippet: Fig. 5. Vascular endothelial growth factor (VEGF) induces poly(ADP-ribose) polymerase-1 (PARP) expression through VEGF receptor 2 (VEGFR-2), Akt and c-Jun N-terminal kinase (JNK) activation. (A) Intracellular signaling was analyzed by short-term exposure of human umbilicalvein endothelial cells (HUVECs)to 10and 100 ng mL)1 VEGF-A(165) for15–120 min. Kinase phosphorylationwasdemonstratedbyautomated densitometryof Western blots; phospho-kinases were correlated with the total amount of the respective kinase expressed as ratio of relative optical density (ROD). VEGF significantly induced time-dependent and dose-dependent phosphorylation of JNK and Akt as compared with negative controls (P < 0.05), whereas p38 mitogen- activated protein kinase and extracellular signal-related kinase 1/2 (p42/44) remained uninfluenced. Mean (%) ± standard deviation (SD) of four exper- iments in duplicate. (B) VEGF receptor signal transduction responsible for PARP regulation was analyzed by incubation of HUVECs with 10 ng mL)1

Article Snippet: They were then incubated with 5% normal serum/PBS, and then with goat-anti-human PARP (1 : 20; R&D Systems), goat anti-human VEGF (1 : 50; R&D Systems), mouse anti-human VEGFR-2 (clone Y-23, 1 lg mL)1; Santa Cruz), rabbit-antihuman NP-1 (5 lg mL)1; Invitrogen) and rabbit-anti-human von Willebrand factor (VWF) (1 : 200; Dako) antibodies for 2 or 16 h at 4 C, and subsequently with biotinylated secondary antibodies (streptavidin–alkaline phosphatase conjugate, Fast Red, Mayer s hemalum counterstaining [all Sigma-Aldrich]), and covered with glycerol gelatine.

Techniques: Expressing, Activation Assay, Western Blot, Phospho-proteomics, Standard Deviation, Transduction, Incubation

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: Vascular endothelial growth factor confers endothelial resistance to apoptosis through poly(ADP-ribose) polymerase.

doi: 10.1111/j.1538-7836.2011.04368.x

Figure Lengend Snippet: Fig. 6. Poly(ADP-ribose) polymerase-1 (PARP) expression in an organ culture model. Vascular endothelial growth factor (VEGF) increased endothelial PARP expression in a human vascular organ culture model implemented in internal mammary artery (IMA) segments from nine pa- tients in duplicate or triplicate, as demonstrated by immunohistochemistry. The panels show a representative IMA specimen preculture (Pre), control incubation(Co)andincubationwith10 ng mL)1VEGF-A(165)for6 days. Endothelial PARP expression is indicated by arrowheads. Magnification: · 400. Scale bar: 100 lm. End, endothelium; Int, intima; Med, media.

Article Snippet: They were then incubated with 5% normal serum/PBS, and then with goat-anti-human PARP (1 : 20; R&D Systems), goat anti-human VEGF (1 : 50; R&D Systems), mouse anti-human VEGFR-2 (clone Y-23, 1 lg mL)1; Santa Cruz), rabbit-antihuman NP-1 (5 lg mL)1; Invitrogen) and rabbit-anti-human von Willebrand factor (VWF) (1 : 200; Dako) antibodies for 2 or 16 h at 4 C, and subsequently with biotinylated secondary antibodies (streptavidin–alkaline phosphatase conjugate, Fast Red, Mayer s hemalum counterstaining [all Sigma-Aldrich]), and covered with glycerol gelatine.

Techniques: Expressing, Organ Culture, Immunohistochemistry, Control, Incubation

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: Vascular endothelial growth factor confers endothelial resistance to apoptosis through poly(ADP-ribose) polymerase.

doi: 10.1111/j.1538-7836.2011.04368.x

Figure Lengend Snippet: Fig. 7. Poly(ADP-ribose) polymerase-1 (PARP) expression in human atherosclerotic and normal arteries. (A) PARP expression was compared in 24 atherosclerotic (11 carotid, 13 femoral/iliac specimens) vs. 10 normal arteries by immunohistochemistry (see also Table 1). The panels show representative atherosclerotic (femoral) and normal (internal mammary artery) specimens demonstrating PARP and von Willebrand factor (VWF) immunostaining (arrowheads). Magnification: · 200. In atherosclerosis, endothelial PARP immunostaining was increased as compared with nor- mal arteries. (B) Local vascular endothelial growth factor (VEGF) expression was found in atherosclerotic arteries (magnification: · 100). Strong VEGF immunostaining in the lamina media (black arrowheads) and thrombocyte aggregates (open arrowhead) is shown in a representa- tive atherosclerotic specimen. In (A) and (B), immunostaining was visu- alized by alkaline phosphatase conjugate, Fast Red staining, and hemalum counterstaining, and documented with the use of a Zeiss Axio Scope microscope, AXIOVISION software (Zeiss), and IMAGE J (freeware). Vas- cular structures are indicated as follows: End, endothelium; Int, intima; Med, media; Neo, atherosclerotic neointima. The scale bars in (A) and (B) represent 100 lm. The results underline the significance of a local vascular VEGF effect for increased endothelial PARP expression in human arteries and in the vascular culture model.

Article Snippet: They were then incubated with 5% normal serum/PBS, and then with goat-anti-human PARP (1 : 20; R&D Systems), goat anti-human VEGF (1 : 50; R&D Systems), mouse anti-human VEGFR-2 (clone Y-23, 1 lg mL)1; Santa Cruz), rabbit-antihuman NP-1 (5 lg mL)1; Invitrogen) and rabbit-anti-human von Willebrand factor (VWF) (1 : 200; Dako) antibodies for 2 or 16 h at 4 C, and subsequently with biotinylated secondary antibodies (streptavidin–alkaline phosphatase conjugate, Fast Red, Mayer s hemalum counterstaining [all Sigma-Aldrich]), and covered with glycerol gelatine.

Techniques: Expressing, Immunohistochemistry, Immunostaining, Staining, Microscopy, Software

Journal: Biofabrication

Article Title: Development of a clay based bioink for 3D cell printing for skeletal application.

doi: 10.1088/1758-5090/aa7e96

Figure Lengend Snippet: Figure 9: Release of model proteins from 0-3-9 and 3-3-3 plotted scaffolds. (a,b) Cumulative release curves of BSA. 0-3-9 showed a high initial burst, while 3-3-3 demonstrated a more sustained release. Data suggest, that a significant amount of BSA was remained inside 3-3-3 blend after 21 days. (c,d) Cumulative release curves of VEGF. Again, high initial burst was observed from 0-3-9 scaffolds, while VEGF from 3-3-3 was released in a sustained manner. After 7 days, 3-3-3 released significantly less VEGF than 0-3-9. However, from day 5 and day 7, absolute release of both scaffolds was roughly the same. (e) Bioactivity of VEGF in supernatants of 3-3-3 scaffolds in comparison to supernatants of negative controls (set as 1). VEGF retained its bioactivity at every time point.

Article Snippet: ELISA for VEGF quantification ELISA using goat anti-human VEGF (Sigma-Aldrich) as capture and biotinylated goat anti-human VEGF (R&D Systems, Minneapolis, USA) as detection antibody was applied as described previously [42].

Techniques: Comparison

Journal: The Journal of clinical endocrinology and metabolism

Article Title: 17Beta-estradiol up-regulates vascular endothelial growth factor receptor-2 expression in human myometrial microvascular endothelial cells: role of estrogen receptor-alpha and -beta.

doi: 10.1210/jc.2001-010588

Figure Lengend Snippet: FIG. 1. Effect of 17-estradiol and progesterone on rhVEGF165 bind- ing to human myometrial MEC. A, Flow cytometry traces showing that (left panel) proliferating MEC bind more rhVEGF165 than qui- escent MEC (basal binding) and (right panel) 17-estradiol (E2), but not progesterone (P4) increased basal rhVEGF165 binding. The neg- ative control represents nonspecific binding. B, Quiescent MEC in- cubated with vehicle, 10 nmol/liter E2, 100 nmol/liter progesterone (P), and 17-estradiol progesterone for 18 h at 37 C in the presence 10 g/liter VEGF were harvested and rhVEGF165 binding measured. Results are mean SEM from four experiments on separate MEC isolates. *, P 0.01.

Article Snippet: Recombinant human (rh) VEGF165, biotinylated rhVEGF165, biotinylated soybean trypsin inhibitor, RDF buffer and avidin-fluorescein isothiocyanate (avidin-FITC) were purchased as a kit from R&D Systems (Minneapolis, MN).

Techniques: Flow Cytometry, Binding Assay, Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: 17Beta-estradiol up-regulates vascular endothelial growth factor receptor-2 expression in human myometrial microvascular endothelial cells: role of estrogen receptor-alpha and -beta.

doi: 10.1210/jc.2001-010588

Figure Lengend Snippet: FIG. 2. 17-estradiol increased rhVEGF165 binding to myometrial MEC in a time- and dose-dependent manner. A, Time course of rh- VEGF165 binding to MEC incubated with 10 nmol/liter 17-estradiol. B, Concentration-response curve of MEC incubated with or without 17-estradiol for 18 h at 37 C. MFI of rhVEGF165 binding is reported as percentage of control (vehicle-treated cells). Results are mean

Article Snippet: Recombinant human (rh) VEGF165, biotinylated rhVEGF165, biotinylated soybean trypsin inhibitor, RDF buffer and avidin-fluorescein isothiocyanate (avidin-FITC) were purchased as a kit from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Incubation, Concentration Assay, Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: 17Beta-estradiol up-regulates vascular endothelial growth factor receptor-2 expression in human myometrial microvascular endothelial cells: role of estrogen receptor-alpha and -beta.

doi: 10.1210/jc.2001-010588

Figure Lengend Snippet: FIG. 5. 17-estradiol increases VEGFR-2 expression in ER ex- pressing but not in ER myometrial MEC. Quiescent MEC in hu- man serum-free medium were incubated for 18 h at 37 C in the presence of 0 (vehicle), 1 and 10 nmol/liter 17-estradiol and rhVEGF165 binding (f) and VEGFR-2 expression () then measured by flow cytometry. A, Representative flow cytometry histograms of VEGFR-2-FITC fluorescence for ER and ER samples. Negative control is mouse IgG1. B, ER expressing MEC. C, ER MEC. Results are expressed as percentage of control (vehicle only) rhVEGF165 binding or MFI of VEGFR-2 expression. Means SEM from n 4 (B) and n 6 (C) separate MEC isolates. *, P 0.05; **, P 0.01.

Article Snippet: Recombinant human (rh) VEGF165, biotinylated rhVEGF165, biotinylated soybean trypsin inhibitor, RDF buffer and avidin-fluorescein isothiocyanate (avidin-FITC) were purchased as a kit from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Incubation, Binding Assay, Flow Cytometry, Fluorescence, Negative Control, Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: 17Beta-estradiol up-regulates vascular endothelial growth factor receptor-2 expression in human myometrial microvascular endothelial cells: role of estrogen receptor-alpha and -beta.

doi: 10.1210/jc.2001-010588

Figure Lengend Snippet: FIG. 6. Antiestrogen ICI 182,780 inhibits 17-estradiol up-regula- tion of rhVEGF165 binding to myometrial MEC and 17-estradiol enhancement of VEGF-stimulated MEC proliferation. A, Quiescent MEC in serum-free medium containing 10 g/liter VEGF were incu- bated for 18 h at 37 C with either 0.1 or 10 nmol/liter 17-estradiol in the presence or absence of 0.01 or 1 mol/liter ICI 182,780 respec- tively and rhVEGF165 binding was then measured. Results are mean SEM from three experiments on separate MEC isolates. *, P 0.01; **, P 0.05. B, Quiescent myometrial MEC (4 103) seeded in triplicate were incubated with 1 mol/liter ICI 182,780 in the pres- ence or absence of 10 nmol/liter 17-estradiol for 6 d at 37 C in phenol-red free M199 medium containing 2 g/liter VEGF and 5% ch-HS and 15% ch-FCS, then MTS reagent added and absorbance measured. Results are presented as the change in absorbance over 6 d expressed as percentage of control MEC (vehicle only). Means ( SEM) from five experiments on separate MEC isolates are shown. *, P 0.01; **, P 0.05.

Article Snippet: Recombinant human (rh) VEGF165, biotinylated rhVEGF165, biotinylated soybean trypsin inhibitor, RDF buffer and avidin-fluorescein isothiocyanate (avidin-FITC) were purchased as a kit from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Incubation, Control

Journal: Development (Cambridge, England)

Article Title: Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis.

doi: 10.1242/dev.100495

Figure Lengend Snippet: Fig. 2. ccbe1, vegfc and vegfr3 genetically interact in double and triple heterozygous animals. (A-H) Confocal projections of Tg(fli1a:EGFP; kdr- l:mCherry) show grossly unaltered overall morphology and blood vasculature in ccbe1hu3613 (B), vegfr3hu4602 (E) and vegfchu5055 (F) mutants compared with wild type (A). The TD (C, arrows) is absent in ccbe1hu3613 (D), vegfr3 hu4602 (G) and vegfc hu5055 (H) mutants (asterisks). (I-K) ccbe1, vegfc and vegfr3 genetically interact in double heterozygote embryos, which display lymphatic defects. Offspring from vegfc+/−and vegfr3+/−carriers give rise to 28% of embryos (n=28/99) with a TD length of ≤50%. This population is significantly enriched (71%; n=20/28; P<0.0001) in double heterozygotes (I). Similarly in ccbe1+/−and vegfr3+/−

Article Snippet: Western blotting was performed with antibodies targeting VEGFC (R&D Systems, BAF752) or CCBE1 (Abcam, ab101967).

Techniques:

Journal: Development (Cambridge, England)

Article Title: Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis.

doi: 10.1242/dev.100495

Figure Lengend Snippet: Fig. 3. Phenotypes driven by ectopic Vegfc/Vegfr3 signaling are suppressed in ccbe1-deficient embryos. (A) At 72 hpf, dll4 morphants display an arterial hyperbranching phenotype (arrow) driven by increased Vegfc/Vegfr3 signaling in the transgenic Tg(fli1a:EGFP) line. This phenotype was suppressed in ccbe1hu3613 mutants. Eighty-one per cent of MO-dll4 injected embryos displaying wild-type or mild phenotypes were ccbe1 mutants (n=17/21), whereas the population displaying the most severe phenotype was mainly composed of wild-type or heterozygous siblings (83%; n=139/168). (B) In dll4 morphants, arteries are sensitized to increased vegfc expression during primary sprouting. Arteries in MO-dll4, vegfc mRNA-injected embryos display aberrant, ectopic turning (arrow) as early as 30 hpf. Embryos from ccbe1 carrier incrosses, injected with 100 ng vegfc mRNA and 5 ng MO-dll4, were sorted into the phenotypic categories ‘wild type’ and ‘severe’. Genotyping revealed that 70% of the embryos displaying wild-type morphology were ccbe1 mutants (n=19/27). By contrast, the population affected by the most severe phenotype was composed of 83% wild-type or heterozygous siblings (n=122/147). (C) Confocal projections of Tg(fli1a:EGFP; flt1:tomato; hsp70l:Gal;4XUAS:vegfc) embryos show that endothelial cells in heat-shocked embryos display aberrant ectopic branching at 72 hpf. The ectopic endothelial cells are venous derived (flt1:tomato negative, arrow in Ciii). Heat-shocked embryos that were injected with 2.5 ng of MO-ccbe1 do not show this phenotype (asterisks). Scoring of the number of aberrant vISVs per heat-shocked embryo showed a significant rescue (0.12 in MO-ccbe1 injected n=22, versus 4.76 in uninjected controls n=25; P<0.0001) of the phenotype.

Article Snippet: Western blotting was performed with antibodies targeting VEGFC (R&D Systems, BAF752) or CCBE1 (Abcam, ab101967).

Techniques: Transgenic Assay, Injection, Expressing, Derivative Assay

Journal: Development (Cambridge, England)

Article Title: Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis.

doi: 10.1242/dev.100495

Figure Lengend Snippet: Fig. 4. Vegfr3-dependent Erk signaling requires ccbe1 during the induction of secondary sprouting in zebrafish. (A) Analysis of phospho-Erk (P-Erk) expression in 32 hpf embryos. P-Erk (green) and fli1a:EGFP (white) images (lateral view) show P-Erk detected broadly in whole-mount and cross-sectioned (right-hand panels, merge upper, P-Erk lower) control embryos. Signal was increased in Vegfc-induced (dll4 MO + vegfc mRNA-injected) embryos in the posterior cardinal vein (n=8/8; Vegfc-induced embryos all showed ectopic expression in the ventral wall of the PCV). Cross section merged channel images shown in a and b, P-Erk only in c and d. Treatment with the Erk inhibitor PD98059 led to a reduction in all P-Erk staining. Arrows indicate P-Erk expression in the dorsal PCV. DA (dorsal aorta) and PCV (posterior cardinal vein) are indicated. (B) Comparison of P-Erk staining in control uninjected (left), MO-vegfr3 and MO-ccbe1 embryos. Upper panels are merged images and lower P-Erk only, viewed laterally (left) and cross-sectioned (right). Cross sections (right) are from separate embryos. Arrows indicate P-Erk expression in the dorsal PCV. DA and PCV are indicated. (C) Quantification of P-Erk-positive cells in the cardinal vein located in the dorsal compared with ventral wall (left-hand graph). Scores through individual sections of z-stack images from 12 control embryos, scored laterally across three somites in the trunk. Quantification of P-Erk-positive cells in the cardinal vein in control and MO-injected conditions (right-hand graph) (scores from n=10 control embryos, n=13 MO-vegfr3-injected and n=15 MO-ccbe1-injected embryos). (D) Immunoprecipitation (IP) and western blot (IB) detection of phosphorylated Vegfr3 at 32 hpf in wild type and in ccbe1, vegfr3, vegfc morphant and vegfc mRNA-injected embryos. The level of phosphorylated Vegfr3 is markedly reduced in ccbe1, vegfr3 and vegfc morphants, but is increased in vegfc-mRNA injected (500 ng) embryos compared with wild type (D, upper blot, IP for phospho-Vegfr3 and IB detection with phospho-Vegfr3). Loading controls were: the IgG light chain [IgG(l)] present in all blots after IP (D, middle blot), and Myosin to monitor protein input in IPs (D, lower blot). Quantification of Vegfr3 phosphorylation (relative to the loading control) based on three independent experiments is shown in right-hand panel. The decrease in MO-ccbe1 compared with uninjected controls is statistically significant (P<0.05). (E) qPCR analysis of the expression of ccbe1, vegfr3, vegfc, kdr and kdrl in uninjected control and MO-ccbe1-, MO-vegfc-, and MO-vegfr3-injected embryos. Error bars represent s.d. (C) or s.e.m. (D,E).

Article Snippet: Western blotting was performed with antibodies targeting VEGFC (R&D Systems, BAF752) or CCBE1 (Abcam, ab101967).

Techniques: Expressing, Control, Injection, Staining, Comparison, Immunoprecipitation, Western Blot, Phospho-proteomics

Journal: Development (Cambridge, England)

Article Title: Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis.

doi: 10.1242/dev.100495

Figure Lengend Snippet: Fig. 5. Ccbe1 enhances Vegfc-driven sprouting and regulates levels of bioactive VEGFC in vitro. (A) Confocal projections at 32 hpf of Tg(shh:ccbe1), Tg(shh:vegfc) and Tg(shh:ccbe1;shh:vegfc) in a Tg(fli1a:EGFP) background. Co- overexpression of ccbe1 and vegfc in the floorplate leads to aberrant ectopic turning of the ISVs at 32 hpf (upper panels; n=32/36; P<0.0001). At 48 hpf, ccbe1 overexpression in the floorplate does not result in any phenotype, whereas vegfc-overexpressing animals display hyperbranching of the ISVs, and enhanced endothelial cell accumulation at the horizontal myoseptum (arrowhead). ccbe1 and vegfc co-overexpression in the floorplate also leads to hyperbranching ISVs, and to a marked accumulation of endothelial cells at dorsal aspects of the embryo (arrow). (B) Western blot of 293EBNA-1 cells (stably expressing VEGFC) indicate that CCBE1 is detected in the lysate of cells transfected with CCBE1 plasmid, but not in controls. (C) An increase in the levels of all forms of VEGFC is detected in the medium of CCBE1- transfected cells, compared with control cells. The mature form of VEGFC (detected at ~23 kDa) is predominant. (C′) Relative intensity (split axis) of the different processed forms of VEGFC presented in C based on multiple exposures. Note the saturation of the mature form in C. (D) qPCR showing that CCBE1 transfection does not affect VEGFC mRNA levels in vitro in 293EBNA-1 cells stably expressing VEGF-C (D, left panel). Consistent with this, in zebrafish embryos the injection of vegfc or ccbe1 mRNA does not affect the endogenous levels of the other (D, right panel). Error bars represent s.e.m.

Article Snippet: Western blotting was performed with antibodies targeting VEGFC (R&D Systems, BAF752) or CCBE1 (Abcam, ab101967).

Techniques: In Vitro, Over Expression, Western Blot, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Control, Injection

Journal: Development (Cambridge, England)

Article Title: Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis.

doi: 10.1242/dev.100495

Figure Lengend Snippet: Fig. 6. Ectopic expression of mature VEGFC rescues secondary sprouting in ccbe1 morphants. (A) Confocal projections of Tg(fli1a:EGFP) at 54 hpf. Knock down of ccbe1 or vegfr3 leads to a loss of PLs at the horizontal myoseptum (arrowheads and asterisks). Ectopic expression of the mature form of VEGFC strongly rescues PL formation in ccbe1 morphants but not in vegfr3 morphants. Arrows indicate hyperbranched ISVs. (B) Quantification of PL formation at 54 hpf. In wild type, 98% (n=54/55) of embryos develop PLs, whereas in MO- ccbe1-injected embryos <4% (n=2/52) do. PL development is rescued to 74% (n=29/39) in ccbe1 morphants transiently overexpressing ΔNΔCVEGFC (P<0.0001). This rescue was never observed in vegfr3 morphants with all embryos devoid of PLs (n=23/23). (C) Quantification of ISV hypersprouting at 54 hpf. ISV hypersprouting was observed in wild-type embryos (93%; n=40/43), with mild reductions in ccbe1 morphants (79%; n=31/39) and vegfr3 morphants (65%; n=15/23) after ΔNΔCVEGFC overexpression.

Article Snippet: Western blotting was performed with antibodies targeting VEGFC (R&D Systems, BAF752) or CCBE1 (Abcam, ab101967).

Techniques: Expressing, Knockdown, Injection, Over Expression

Journal: Development (Cambridge, England)

Article Title: Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis.

doi: 10.1242/dev.100495

Figure Lengend Snippet: Fig. 7. Ccbe1 activates Vegfc to induce Vegfr3 signaling. Proposed model for coordination of angiogenesis by Ccbe1, Vegfc and Vegfr3 in the developing embryo. Vegfc is produced in a largely inactive full-length form that is processed and released from the cell surface/ECM in a Ccbe1- dependent manner to generate the mature, highly active form. Downstream, arteries respond in a manner dampened by Dll4-dependent suppression of Vegfr3 signaling (Hogan et al., 2009b), whereas Vegfr3 signaling in veins induces secondary angiogenesis, which produces both intersegmental veins and lymphatic vascular precursor cells.

Article Snippet: Western blotting was performed with antibodies targeting VEGFC (R&D Systems, BAF752) or CCBE1 (Abcam, ab101967).

Techniques: Produced

Journal: Circulation

Article Title: Important Role of Endogenous Erythropoietin System in Recruitment of Endothelial Progenitor Cells in Hypoxia-Induced Pulmonary Hypertension in Mice

doi: 10.1161/circulationaha.105.583732

Figure Lengend Snippet: Figure 1. EpoR deficiency accelerates hypoxia-induced PH. Shown are effects of EpoR deficiency and chronic hypoxia on RVSP. A, RVH (weight ratio of right ventricle to left ventricle plus septum [RV/LVS], B and C), and hematocrit (D) in mice (n10 each for panels A through C, and n5 each for panel D). WT indicates wild-type mice; EpoR/, EpoR/ rescued mice; control, normoxic mice; and hypoxia, mice exposed to 3 weeks of hypoxia (10% O2). Results are expressed as meanSD. E, Survival curves under chronic hypoxia (n15 each).

Article Snippet: Endothelium-like cells were identified by the uptake of DiI-AcLDL and Flk-1 expression.12 The expression of EpoR on endothelium-like cells was confirmed by double staining with biotinylated anti-mouse EpoR antibodies (1:50, R&D Systems) and anti-Flk-1 (VEGF receptor-2, 1:200, Santa Cruz Biochemicals).26 Endothelium-like cells (1 106 cells per mouse) were labeled with CellTracker CM-DiI (Molecular Probes, Eugene, Ore),26 suspended in 100 L calcium- and magnesium-free phosphate-buffered saline with 2% FBS, and injected into the tail vein of hypoxic mice.

Techniques: Control

Journal: Circulation

Article Title: Important Role of Endogenous Erythropoietin System in Recruitment of Endothelial Progenitor Cells in Hypoxia-Induced Pulmonary Hypertension in Mice

doi: 10.1161/circulationaha.105.583732

Figure Lengend Snippet: Figure 2. EpoR deficiency accelerates hypoxia-induced pulmonary vascular remodeling. Sections stained with hematoxylin and eosin (H&E) at low magnification and elastica Masson (EM) staining at intermediate magnification show the morphological changes in lungs. At high- power magnification, the development of pulmonary vascular remodeling is shown by EM staining or by immunostaining for SMA.

Article Snippet: Endothelium-like cells were identified by the uptake of DiI-AcLDL and Flk-1 expression.12 The expression of EpoR on endothelium-like cells was confirmed by double staining with biotinylated anti-mouse EpoR antibodies (1:50, R&D Systems) and anti-Flk-1 (VEGF receptor-2, 1:200, Santa Cruz Biochemicals).26 Endothelium-like cells (1 106 cells per mouse) were labeled with CellTracker CM-DiI (Molecular Probes, Eugene, Ore),26 suspended in 100 L calcium- and magnesium-free phosphate-buffered saline with 2% FBS, and injected into the tail vein of hypoxic mice.

Techniques: Staining, Immunostaining

Journal: Circulation

Article Title: Important Role of Endogenous Erythropoietin System in Recruitment of Endothelial Progenitor Cells in Hypoxia-Induced Pulmonary Hypertension in Mice

doi: 10.1161/circulationaha.105.583732

Figure Lengend Snippet: Figure 3. EpoR deficiency and hypoxia accelerate the degree of muscularization in pulmonary small vessels in mice. In each animal, 60 vessels were counted per lung section (n6 each). Representa- tive pictures of muscularized small vessels with double elastic lamina are shown in each panel. Results are expressed as meanSD.

Article Snippet: Endothelium-like cells were identified by the uptake of DiI-AcLDL and Flk-1 expression.12 The expression of EpoR on endothelium-like cells was confirmed by double staining with biotinylated anti-mouse EpoR antibodies (1:50, R&D Systems) and anti-Flk-1 (VEGF receptor-2, 1:200, Santa Cruz Biochemicals).26 Endothelium-like cells (1 106 cells per mouse) were labeled with CellTracker CM-DiI (Molecular Probes, Eugene, Ore),26 suspended in 100 L calcium- and magnesium-free phosphate-buffered saline with 2% FBS, and injected into the tail vein of hypoxic mice.

Techniques:

Journal: Circulation

Article Title: Important Role of Endogenous Erythropoietin System in Recruitment of Endothelial Progenitor Cells in Hypoxia-Induced Pulmonary Hypertension in Mice

doi: 10.1161/circulationaha.105.583732

Figure Lengend Snippet: Figure 4. In vivo recruitment of EPCs into the cir- culation is impaired in EpoR/ mice. A and B, Hypoxia (10% O2 for 7 days) significantly increased the number of Flk-1/CD133 cells in the PBMCs (n8 each, A) and the number of DiI-AcLDL/ Flk-1 double-positive cells (endothelium-like cells) in WT mice, but not in EpoR/ mice (n6 each, B). HPF indicates high-power field. C, Endotheli- um-like cells were identified by the uptake of DiI- labeled AcLDL (red) and the staining with Flk-1 (green). The expression of EpoR (red) was observed only in the cells from WT mice but not in those from EpoR/ mice. D, Shown is the incor- poration of injected cells (1106 cells per mouse) into the pulmonary endothelium on day 7 of hypoxic exposure (arrow). E, The number of incor- porated cells was significantly reduced in EpoR/ mice. In each animal, 10 different sections were observed. Results are expressed as meanSD.

Article Snippet: Endothelium-like cells were identified by the uptake of DiI-AcLDL and Flk-1 expression.12 The expression of EpoR on endothelium-like cells was confirmed by double staining with biotinylated anti-mouse EpoR antibodies (1:50, R&D Systems) and anti-Flk-1 (VEGF receptor-2, 1:200, Santa Cruz Biochemicals).26 Endothelium-like cells (1 106 cells per mouse) were labeled with CellTracker CM-DiI (Molecular Probes, Eugene, Ore),26 suspended in 100 L calcium- and magnesium-free phosphate-buffered saline with 2% FBS, and injected into the tail vein of hypoxic mice.

Techniques: In Vivo, Labeling, Staining, Expressing, Injection

Journal: Circulation

Article Title: Important Role of Endogenous Erythropoietin System in Recruitment of Endothelial Progenitor Cells in Hypoxia-Induced Pulmonary Hypertension in Mice

doi: 10.1161/circulationaha.105.583732

Figure Lengend Snippet: Figure 5. Bone marrow–derived EPCs incorporate into pulmo- nary endothelium and inhibit the development of hypoxia- induced PH. A, Bone marrow–derived endothelial cells were identified by the expression of endothelium-specific expression of Tie2-GFP (green) and staining with CD31 (red). High-power magnification revealed incorporated cells that were positive for both GFP and CD31 (arrows). B, Percentages of the number of GFP cells per CD31 endothelial cells in the lung field were assessed. In each animal, 5 different sections were observed. C, After 3 weeks of hypoxia, hemodynamic analysis revealed the amelioration of PH assessed by RVSP and RVH in EpoR/

Article Snippet: Endothelium-like cells were identified by the uptake of DiI-AcLDL and Flk-1 expression.12 The expression of EpoR on endothelium-like cells was confirmed by double staining with biotinylated anti-mouse EpoR antibodies (1:50, R&D Systems) and anti-Flk-1 (VEGF receptor-2, 1:200, Santa Cruz Biochemicals).26 Endothelium-like cells (1 106 cells per mouse) were labeled with CellTracker CM-DiI (Molecular Probes, Eugene, Ore),26 suspended in 100 L calcium- and magnesium-free phosphate-buffered saline with 2% FBS, and injected into the tail vein of hypoxic mice.

Techniques: Derivative Assay, Expressing, Staining

Journal: Circulation

Article Title: Important Role of Endogenous Erythropoietin System in Recruitment of Endothelial Progenitor Cells in Hypoxia-Induced Pulmonary Hypertension in Mice

doi: 10.1161/circulationaha.105.583732

Figure Lengend Snippet: Figure 6. Hypoxia enhances the expression of EpoR on pulmonary endothelial cells. A, Repre- sentative lung sections from normoxic and hypoxic mice. Hypoxia enhanced the expres- sion of EpoR (green) in WT mice but not in EpoR/ mice. Bar, 50 m. B, Western blot analysis of EpoR in lung homogenates of WT mice after 3 weeks of hypoxia (10% O2, n10 each). Results are expressed as meanSD.

Article Snippet: Endothelium-like cells were identified by the uptake of DiI-AcLDL and Flk-1 expression.12 The expression of EpoR on endothelium-like cells was confirmed by double staining with biotinylated anti-mouse EpoR antibodies (1:50, R&D Systems) and anti-Flk-1 (VEGF receptor-2, 1:200, Santa Cruz Biochemicals).26 Endothelium-like cells (1 106 cells per mouse) were labeled with CellTracker CM-DiI (Molecular Probes, Eugene, Ore),26 suspended in 100 L calcium- and magnesium-free phosphate-buffered saline with 2% FBS, and injected into the tail vein of hypoxic mice.

Techniques: Expressing, Western Blot

Journal: Circulation

Article Title: Important Role of Endogenous Erythropoietin System in Recruitment of Endothelial Progenitor Cells in Hypoxia-Induced Pulmonary Hypertension in Mice

doi: 10.1161/circulationaha.105.583732

Figure Lengend Snippet: Figure 7. Western blot analysis of eNOS in the lung. The expression of eNOS was upregulated in WT but not EpoR/ mice after 3 weeks of hypoxia (10% O2, n10 each). Results are expressed as meanSD.

Article Snippet: Endothelium-like cells were identified by the uptake of DiI-AcLDL and Flk-1 expression.12 The expression of EpoR on endothelium-like cells was confirmed by double staining with biotinylated anti-mouse EpoR antibodies (1:50, R&D Systems) and anti-Flk-1 (VEGF receptor-2, 1:200, Santa Cruz Biochemicals).26 Endothelium-like cells (1 106 cells per mouse) were labeled with CellTracker CM-DiI (Molecular Probes, Eugene, Ore),26 suspended in 100 L calcium- and magnesium-free phosphate-buffered saline with 2% FBS, and injected into the tail vein of hypoxic mice.

Techniques: Western Blot, Expressing