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:

Article Title: A model for gene therapy of human hereditary lymphedema

doi: 10.1073/pnas.221449198

Figure Lengend Snippet: The VEGFR-3(I1053F) mutant is tyrosine kinase inactive. (A) The alignment of the human and mouse VEGFR-3 amino acid sequences showing the lymphedema-linked human mutations (bold) and the I1053F mutation found in the Chy mice (red). (B) Localization of the I1053F mutation within the catalytic domain of the ligand-bound VEGFR-3 heterodimer. (C) VEGFR-3(I1053F) is kinase inactive. We transfected cells with Mock, WT, or I1053F VEGFR-3 expression vectors and analyzed VEGFR-3 by immunoprecipitation and Western blotting of the cell lysates using phosphotyrosine antibodies (Upper). We also confirmed the expression of similar amounts of VEGFR-3 (Lower).

Article Snippet: We used the biotinylated anti-mouse VEGFR-3 Ab (R&D Systems, Oxon, U.K.) for whole-mount stainings.

Techniques: Mutagenesis, Transfection, Expressing, Immunoprecipitation, Western Blot

Journal:

Article Title: A model for gene therapy of human hereditary lymphedema

doi: 10.1073/pnas.221449198

Figure Lengend Snippet: Defective lymphatic vessels in the Chy mice. (A and B) The Chy mice were recognized by the accumulation of chyous fluid into the abdomen (red arrow). WT littermate is shown as a comparison. The stomach is marked with a dashed line. (C–F) The subserosal lymphatic vessels are enlarged in the Chy mouse intestine as detected by VEGFR-3 IHC (C and D), and by VEGFR-3 wholemount staining (E and F). (G) The feet of the Chy mice are swollen (arrows), when compared with a WT littermate. (H and I) MRI of the mouse feet shows the prominent hyperintensity in the Chy mouse foot, which is absent in a WT mouse. (J and K) The lymphatic endothelium (black arrows) of E15.5 skin was visualized by VEGFR-3 IHC in the Chy and WT mice. Note the absence of the lymphatic vessels in the Chy mice. (L and M) PECAM-1 IHC reveals no differences in the blood vasculature (red arrows). (N and O) VEGFR-3 IHC indicates that the lymphatics surrounding the aorta (a) in the Chy mice are similar to those of the WT mice. (P and Q) The hematoxylin-eosin staining of the back skin shows that the dermis (d) and s.c. adipose tissue (sc) are thickened in the Chy mice when compared with the WT littermate. (R and S) The transport of the Evans blue dye into the collecting lymphatic vessels (arrow) was visualized in the WT but not in the Chy mice after intradermal injection of the dye. [Bars = 5 mm (A and B), 90 μm (C and D), 200 μm (E and F), 5 mm (G), 100 μm (H and I), 70 μm (J–M), 200 μm (N and O), 210 μm (P and Q), and 1.5 mm (R and S).]

Article Snippet: We used the biotinylated anti-mouse VEGFR-3 Ab (R&D Systems, Oxon, U.K.) for whole-mount stainings.

Techniques: Comparison, Staining, Injection

Journal:

Article Title: A model for gene therapy of human hereditary lymphedema

doi: 10.1073/pnas.221449198

Figure Lengend Snippet: NRP-2 binds VEGF-C and is differentially expressed in the visceral organs and in the skin. (A) Labeled VEGF-C is precipitated by VEGFR-3-IgG and by NRP-2-IgG, but not by VEGFR-1-IgG fusion protein. (B and C) The intestinal VEGFR-3-positive lymphatic vessels (C; black arrow) stain also for NRP-2 (B), whereas the blood vessels are not stained (red arrows). (D and E) The VEGFR-3-positive vessels in the skin (E) are not stained by the NRP-2 antibodies (D). (Bars = 35 μm.)

Article Snippet: We used the biotinylated anti-mouse VEGFR-3 Ab (R&D Systems, Oxon, U.K.) for whole-mount stainings.

Techniques: Labeling, Staining

Journal:

Article Title: A model for gene therapy of human hereditary lymphedema

doi: 10.1073/pnas.221449198

Figure Lengend Snippet: Gene therapy by using viral VEGF-C overexpression. (A and B) Adenovirally induced VEGF-C overexpression in a Chy mouse ear induces formation of functional lymphatic vessels, as analyzed by fluorescent microlymphography (A) or by VEGFR-3 IHC (B). The dye depot is marked with a dashed line. (C and D) Microlymphography of the lymphatic vessels 7 weeks after AAV infection reveals functional lymphatic vessels (white arrows) in the AAV-VEGF-C-infected Chy ear (C) when compared with the control ear (D). (E and F) VEGFR-3 IHC shows that the AAV-VEGF-C-infected Chy ears contain VEGFR-3-positive vessels (E, black arrows), whereas no staining is detected in the AAV-EGFP-infected ears (F). (G and H) The fluorescent dextran was collected by the draining lymphatic vessel (white arrow) in the AAV-VEGF-C-treated Chy ear (G), unlike in the noninfected ear of the same mouse (H). Red arrows mark the blood vessels, and an arrowhead marks the cartilage of the ear. (I and J) In WT mice, there are no major changes in lymphatic vasculature after AAV-VEGF-C infection (I), when compared with the control ears (J). (K and L) The same is also confirmed by VEGFR-3 IHC. [Bars = 200 μm (A), 150 μm (B, E, F, K, and L), 300 μm (C, D, I, and J), and 5 mm (G and H).]

Article Snippet: We used the biotinylated anti-mouse VEGFR-3 Ab (R&D Systems, Oxon, U.K.) for whole-mount stainings.

Techniques: Over Expression, Functional Assay, Infection, Staining

Journal:

Article Title: A model for gene therapy of human hereditary lymphedema

doi: 10.1073/pnas.221449198

Figure Lengend Snippet: Analysis of the AAV-VEGF-C expression. (A) AAV-produced VEGF-C polypeptides were metabolically labeled and subjected to immunoprecipitation with VEGF-C antibodies (IP) or to binding assay with soluble VEGFR-3 and NRP-2 IgG fusion proteins. AAV-EGFP-infected cells were used as a negative control and VEGF-C produced in 293T cells as a positive control (p-C). (B) Northern blotting of total RNA from AAV-VEGF-C-infected and control ears of a Chy mouse.

Article Snippet: We used the biotinylated anti-mouse VEGFR-3 Ab (R&D Systems, Oxon, U.K.) for whole-mount stainings.

Techniques: Expressing, Produced, Metabolic Labelling, Labeling, Immunoprecipitation, Binding Assay, Infection, Negative Control, Positive Control, Northern Blot

Journal:

Article Title: A model for gene therapy of human hereditary lymphedema

doi: 10.1073/pnas.221449198

Figure Lengend Snippet: Lymphatic vessel growth in the Chy × K14-VEGF-C156S mice. (A–C) Staining by using VEGFR-3 antibodies shows lymphatic vessels (black arrows) in the ear of the Chy × K14-VEGF-C156S mice, in comparison with the aplastic lymphatic vessels in the Chy mouse ear (B), or with the WT littermate (C). (D–F) PECAM IHC confirms that the blood vasculature is normal in all mice. (G–I) VEGF-C IHC shows the VEGF-C156S transgene expression in the basal cells of the hair follicles (red arrows). (J–L) Fluorescent microlymphography shows the functional lymphatic capillary network in the Chy × K14-VEGF-C156S ear (J), resembling that of the WT mouse (L). The FITC-dextran is not collected into the lymphatic vessels in the Chy mouse ear (K). [Bars = 70 μm (A–F), 25 μm (G–I), and 600 μm (J–L).]

Article Snippet: We used the biotinylated anti-mouse VEGFR-3 Ab (R&D Systems, Oxon, U.K.) for whole-mount stainings.

Techniques: Staining, Comparison, Expressing, Functional Assay

Journal: Bioscience Reports

Article Title: Direct binding of hepatocyte growth factor and vascular endothelial growth factor to CD44v6

doi: 10.1042/BSR20150093

Figure Lengend Snippet: ( A ) Serum-starved Bsp73AS (AS) or Bsp73ASs6 (ASs6) cells were incubated with a CD44v6 antibody (1.1ASML) , a v6 peptide (14mer) or a control peptide prior to induction with biotinylated HGF and subsequent detection of binding using FITC–streptavidin. All samples were analysed in a FACScan flow cytometer. The FITC–streptavidin negative control is shown in blue, HGF-induced samples either non-treated or treated with CD44v6-specific blocking reagents are shown in green. Representative pictures from one experiment are shown. Three independent experiments per condition were performed giving the same results. ( B ) Serum-starved T47D and T47D/Met cells were pretreated either with a CD44v6 antibody (VFF18), or a v6 peptide (14mer) prior to induction with biotinylated HGF. Expression of CD44v6 and CD44s.ectodomains (labelled as Ecd) was achieved by transfection of the expression constructs using Lipofectamine. The cells were then subjected to FACS analysis 48 h after transfection. Binding of biotinylated HGF was detected using FITC labelled streptavidin. All samples were analysed in a FACScan flow cytometer. Cells exclusively incubated with FITC–streptavidin were used as a negative control (purple). Cells treated with HGF alone, or pretreated with the peptides, antibody or transfected with the ectodomains are represented in green. Representative pictures from one experiment are shown. Three independent experiments were performed giving the same results. ( C ) Serum-starved HUVEC and ASs6 cells were pre-incubated either with a v6 peptide (5mer) or a control peptide as indicated and induced with VEGF 165 . Binding of VEGF 165 to the cells was detected by means of a specific antibody against VEGF and a secondary PE-labelled antibody and subsequently analysed in a FACScan flow cytometer. Cells exclusively incubated with the PE-labelled antibody were used as a negative control (purple). Cells treated with VEGF 165 or additionally pre-incubated with the peptides and probed with the antibodies are represented in green. Representative pictures from one experiment are shown. Three independent experiments were performed giving the same results.

Article Snippet: The antibodies used in the present study were directed against rat CD44v6 (1.1ASML, described in [ ]) and human CD44v6 (VFF18; Boehringer Ingelheim), Erk-1 (K-23; Santa Cruz), phospho-p44/42 MAPK (Thr202/Tyr204; Cell Signaling Technology), penta-His (Qiagen), human HGF (AF-294-NA; R&D Systems), rat Met (B2; Santa Cruz), human Met (25H2; Cell Signaling Technology) phospho-Met (Tyr1234/1235; Cell Signaling Technology), human TGF-α (transforming growth factor alpha; Peprotech), VEGFR-2 (A3; Santa Cruz) and human VEGF-A165 (VEGF 165 ) (AF-293-NA; R&D Systems).

Techniques: Incubation, Control, Binding Assay, Flow Cytometry, Negative Control, Blocking Assay, Expressing, Transfection, Construct

Journal: Bioscience Reports

Article Title: Direct binding of hepatocyte growth factor and vascular endothelial growth factor to CD44v6

doi: 10.1042/BSR20150093

Figure Lengend Snippet: ( A ) For the cellular ELISA serum-starved AS, ASs6 and HT29 cells were induced with biotinylated HGF. The binding of the growth factor to the cells was detected by means of an avidin–HRP conjugate and ABTS solution and subsequently analysed in an ELISA reader. Experimental data are reported as mean ± S.D. of three independent experiments (* P < 0.05, ** P < 0.005; Student's t -test). ASs6 cells were in addition treated with a v6 (5mer) and a control peptide as indicated and induced with biotinylated HGF. ( B ) The AS and ASs6 cells were transfected with a VEGFR-2 expression construct using FuGene. 48 h after the transfection the cellular ELISA was performed with AS, ASs6 and HUVEC cells as described above and the cells were induced with biotinylated VEGF 165 .

Article Snippet: The antibodies used in the present study were directed against rat CD44v6 (1.1ASML, described in [ ]) and human CD44v6 (VFF18; Boehringer Ingelheim), Erk-1 (K-23; Santa Cruz), phospho-p44/42 MAPK (Thr202/Tyr204; Cell Signaling Technology), penta-His (Qiagen), human HGF (AF-294-NA; R&D Systems), rat Met (B2; Santa Cruz), human Met (25H2; Cell Signaling Technology) phospho-Met (Tyr1234/1235; Cell Signaling Technology), human TGF-α (transforming growth factor alpha; Peprotech), VEGFR-2 (A3; Santa Cruz) and human VEGF-A165 (VEGF 165 ) (AF-293-NA; R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Avidin-Biotin Assay, Control, Transfection, Expressing, Construct

Journal: Bioscience Reports

Article Title: Direct binding of hepatocyte growth factor and vascular endothelial growth factor to CD44v6

doi: 10.1042/BSR20150093

Figure Lengend Snippet: ( A – C ') The nickel pull-down assays were performed with the histidine-tagged ectodomains of CD44 (CD44s and CD44v6). The CD44 ectodomains were coupled to Ni-NTA beads (labelled as NiB) and incubated with the growth factors HGF, VEGF 165 and TGF-α as indicated. ( A , B , C ) Binding of the ligands to the histidine-tagged CD44 ectodomains was analysed in Western blots by means of specific antibodies against HGF, VEGF and TGF-α. As loading controls, the respective growth factors as well as the CD44 ectodomains were loaded directly on the gel. ( A ', B ', C ') To control the equal loading of the ectodomains the membranes were stripped and reprobed with a specific antibody against histidine for detection of the histidine-tagged CD44 proteins. The Western blots were performed at least three times and gave similar results. A representative blot is shown.

Article Snippet: The antibodies used in the present study were directed against rat CD44v6 (1.1ASML, described in [ ]) and human CD44v6 (VFF18; Boehringer Ingelheim), Erk-1 (K-23; Santa Cruz), phospho-p44/42 MAPK (Thr202/Tyr204; Cell Signaling Technology), penta-His (Qiagen), human HGF (AF-294-NA; R&D Systems), rat Met (B2; Santa Cruz), human Met (25H2; Cell Signaling Technology) phospho-Met (Tyr1234/1235; Cell Signaling Technology), human TGF-α (transforming growth factor alpha; Peprotech), VEGFR-2 (A3; Santa Cruz) and human VEGF-A165 (VEGF 165 ) (AF-293-NA; R&D Systems).

Techniques: Incubation, Binding Assay, Western Blot, Control

Journal: Bioscience Reports

Article Title: Direct binding of hepatocyte growth factor and vascular endothelial growth factor to CD44v6

doi: 10.1042/BSR20150093

Figure Lengend Snippet: ( A – F ) Using MST the K d was determined whenever binding was observed (fitting curves in blue). ( A ) Fluorescently labelled VEGF 165 was titrated against varying concentrations of the CD44v6 ectodomain ( K d =51±9 nM) or ( B ) the CD44s ectodomain. ( C ) Titration of serial dilutions of the CD44v6 ectodomain ( K d =610±90 nM) or ( D ) the CD44s ectodomain ( K d =130±300 nM) to fluorescently labelled HGF. ( E ) Fluorescently labelled CD44v6 ectodomain was titrated against serial dilutions of TGF-α or ( F ) against the Met ectodomain (Met 928 ). Binding affinities were calculated from at least three different experiments.

Article Snippet: The antibodies used in the present study were directed against rat CD44v6 (1.1ASML, described in [ ]) and human CD44v6 (VFF18; Boehringer Ingelheim), Erk-1 (K-23; Santa Cruz), phospho-p44/42 MAPK (Thr202/Tyr204; Cell Signaling Technology), penta-His (Qiagen), human HGF (AF-294-NA; R&D Systems), rat Met (B2; Santa Cruz), human Met (25H2; Cell Signaling Technology) phospho-Met (Tyr1234/1235; Cell Signaling Technology), human TGF-α (transforming growth factor alpha; Peprotech), VEGFR-2 (A3; Santa Cruz) and human VEGF-A165 (VEGF 165 ) (AF-293-NA; R&D Systems).

Techniques: Binding Assay, Titration

Journal: Bioscience Reports

Article Title: Direct binding of hepatocyte growth factor and vascular endothelial growth factor to CD44v6

doi: 10.1042/BSR20150093

Figure Lengend Snippet: (A–D) Dissociation constants were determined via FCS by titrating the respective CD44 isoform against the fluorophore-labelled growth factor. The study on VEGF 165 -ATTO 647N binding to ( A ) CD44v6 and ( B ) CD44s revealed dissociation constants of 65±8 nM and 1300±600 nM, respectively. The same experiment was conducted with HGF-ATTO 647N yielding a ( C ) K d =1830±60 nM for CD44v6 and ( D ) K d =5000±3000 nM for CD44s.

Article Snippet: The antibodies used in the present study were directed against rat CD44v6 (1.1ASML, described in [ ]) and human CD44v6 (VFF18; Boehringer Ingelheim), Erk-1 (K-23; Santa Cruz), phospho-p44/42 MAPK (Thr202/Tyr204; Cell Signaling Technology), penta-His (Qiagen), human HGF (AF-294-NA; R&D Systems), rat Met (B2; Santa Cruz), human Met (25H2; Cell Signaling Technology) phospho-Met (Tyr1234/1235; Cell Signaling Technology), human TGF-α (transforming growth factor alpha; Peprotech), VEGFR-2 (A3; Santa Cruz) and human VEGF-A165 (VEGF 165 ) (AF-293-NA; R&D Systems).

Techniques: Binding Assay

Journal: Bioscience Reports

Article Title: Direct binding of hepatocyte growth factor and vascular endothelial growth factor to CD44v6

doi: 10.1042/BSR20150093

Figure Lengend Snippet: Overview of binding affinities as determined with MST, FCS and FA The dissociation constants ( K d ) shown are the average values of N experiments carried out for each pair of binding partners.

Article Snippet: The antibodies used in the present study were directed against rat CD44v6 (1.1ASML, described in [ ]) and human CD44v6 (VFF18; Boehringer Ingelheim), Erk-1 (K-23; Santa Cruz), phospho-p44/42 MAPK (Thr202/Tyr204; Cell Signaling Technology), penta-His (Qiagen), human HGF (AF-294-NA; R&D Systems), rat Met (B2; Santa Cruz), human Met (25H2; Cell Signaling Technology) phospho-Met (Tyr1234/1235; Cell Signaling Technology), human TGF-α (transforming growth factor alpha; Peprotech), VEGFR-2 (A3; Santa Cruz) and human VEGF-A165 (VEGF 165 ) (AF-293-NA; R&D Systems).

Techniques: Binding Assay, Protein Binding

Journal: The Journal of surgical research

Article Title: Revascularization and muscle adaptation to limb demand ischemia in diet-induced obese mice.

doi: 10.1016/j.jss.2016.06.001

Figure Lengend Snippet: Fig. 1 e Effect of demand ischemia or sedentary ischemia on capillary density in hind limb skeletal muscle. Representative images (A, B, and C) are an immunostaining of CD31 in cross section of the TA muscles from control, demand ischemia, and sedentary ischemia groups (n [ 7 in each group) pinpointing capillaries in the field indicated by the black arrows. Demand ischemia muscle (B) showed extensive CD31 staining (dark brown color) compared with both control and sedentary animals. Quantitative data, given as averages of capillaries counted per 2003 field ± standard error of the mean, are summarized in graph (D). Demand ischemia had substantially higher capillary density compared with control (***P < 0.001) or sedentary ischemic muscles (**P < 0.01). (Color version of figure is available online.)

Article Snippet: Cross sections of 7 mm thickness were generated from the mid belly of each muscle and incubated overnight with rabbit anti-PECAM-1 (CD31) polyclonal IgG (M20, SC-1506, Santa Cruz Biotechnology, Santa Cruz, CA) followed by incubation with goat anti-rabbit biotin-conjugated IgG and avidin-biotin complex.

Techniques: Immunostaining, Muscles, Control, Staining