Journal: Journal of Neuro-Oncology

Article Title: Identification of two distinct mesenchymal stromal cell populations in human malignant glioma

doi: 10.1007/s11060-016-2302-y

Figure Lengend Snippet: a VEGF and b PGE 2 production in vitro by two different cell populations from each of two GBMs. The CD90 − populations from GBM-47 and GBM-48 produce higher levels of both VEGF and PGE 2 compared to the CD90 + populations from the same tumors. 1 × 10 5 cells were grown in 1 ml MSC Expansion Media for 24 h and the supernatants were analyzed with ELISA. Each experiment was performed three times in duplicate and VEGF data from both tumors was pooled together

Article Snippet: VEGF levels were determined using the Human VEGF DuoSet kit (R&D Systems).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay

Journal: International journal of oncology

Article Title: The oncometabolite d‑2‑hydroxyglutarate induces angiogenic activity through the vascular endothelial growth factor receptor 2 signaling pathway.

doi: 10.3892/ijo.2018.4649

Figure Lengend Snippet: Figure 2. Cytotoxicity of d‑2HG in BAECs. (A and B) BAECs were treated with various concentrations of d‑2HG (25 µM to 50 mM) for 24 h (A) or 48 h (B). Cell viability was measured by CCK‑8 assays. VEGF (40 ng/ml) was used as a positive control. *P<0.05 versus the control. BAECs, bovine aortic endothelial cells; d‑2HG, d‑2‑hydroxyglutarate; VEGF, vascular endothelial growth factor.

Article Snippet: The amount of VEGF protein secreted by the A549 human lung cancer cells into the medium was determined using a VEGF ELISA kit (R&D Systems, Wiesbaden, Germany).

Techniques: Positive Control, Control

Journal: International journal of oncology

Article Title: The oncometabolite d‑2‑hydroxyglutarate induces angiogenic activity through the vascular endothelial growth factor receptor 2 signaling pathway.

doi: 10.3892/ijo.2018.4649

Figure Lengend Snippet: Figure 3. d‑2HG increases VEGF secretion and the proliferation of BAECs. (A) A549 cells were starved for 16 h in 0.5% FBS‑containing medium, treated with

Article Snippet: The amount of VEGF protein secreted by the A549 human lung cancer cells into the medium was determined using a VEGF ELISA kit (R&D Systems, Wiesbaden, Germany).

Techniques:

Journal: International journal of oncology

Article Title: The oncometabolite d‑2‑hydroxyglutarate induces angiogenic activity through the vascular endothelial growth factor receptor 2 signaling pathway.

doi: 10.3892/ijo.2018.4649

Figure Lengend Snippet: Figure 4. d‑2HG increased migration and tube formation ability in BAECs. (A) BAECs (90% confluence) were starved with 0.5% FBS in DMEM/low glucose for 16 h and treated with mitomycin C (1 µg/ml) for 1 h. The cells were scratched with a 1‑ml micropipette tip and incubated in starvation medium with VEGF (40 ng/ml) or d‑2HG (250 µM). Wound closure from the original location was monitored and graphed. *P<0.05 versus the control. (B) Transwell migration assays were performed in 24‑Transwell plates (6.5 mm diameter, 8 µm pore size membrane). BAECs were seeded in the upper chamber, and VEGF (40 ng/ml) and d‑2HG (250 µM) were added to the lower chambers for 24 h. Migrated cells were fixed with 100% methanol, stained with hematoxylin and eosin, and counted. *P<0.05 versus the control. (C) BAECs were starved in 0.5% FBS in DMEM/low glucose for 16 h, seeded (3x104 cells) on the surface of the Matrigel, and incubated for 8‑16 h with or without VEGF (40 ng/ml) or d‑2HG (250 µM) in DMEM/low glucose containing 0.5% FBS. Morphological changes were observed under a microscope and photographed at x100 magnification. The results are presented as the mean number of branching points. BAECs, bovine aortic endothelial cells; d‑2HG, d‑2‑hydroxyglutarate; VEGF, vascular endothelial growth factor.

Article Snippet: The amount of VEGF protein secreted by the A549 human lung cancer cells into the medium was determined using a VEGF ELISA kit (R&D Systems, Wiesbaden, Germany).

Techniques: Migration, Incubation, Control, Pore Size, Membrane, Staining, Microscopy

Journal: International journal of oncology

Article Title: The oncometabolite d‑2‑hydroxyglutarate induces angiogenic activity through the vascular endothelial growth factor receptor 2 signaling pathway.

doi: 10.3892/ijo.2018.4649

Figure Lengend Snippet: Figure 6. d‑2HG induces VEGFR2 signaling pathway activation and MMP2 activity. (A) BAECs were treated with VEGF (40 ng/ml) or d‑2HG (250 µM) for 10 min, and protein lysates was obtained for western blot analysis. (B) Western blot analysis and gelatin zymography analyses for MMP2 and pro‑MMP2, respectively, were performed with medium obtained from the same samples as described in (A). The relative expression pattern of MMP2 was quantified and graphed below the gel images. *P<0.05 versus the control. (C) Schematic summary of the mechanism through which d‑2HG enhances angiogenesis.

Article Snippet: The amount of VEGF protein secreted by the A549 human lung cancer cells into the medium was determined using a VEGF ELISA kit (R&D Systems, Wiesbaden, Germany).

Techniques: Activation Assay, Activity Assay, Western Blot, Zymography, Expressing, Control

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: European radiology

Article Title: Magnetic resonance imaging for monitoring the effects of thalidomide on experimental human breast cancers

doi: 10.1007/s00330-008-1111-x

Figure Lengend Snippet: Representative immunohistochemically stained human breast cancer sections (MDA-MB-435) showing leakage of macromolecular contrast medium (streptavidin-biotin reaction), vascular richness (lectin and RECA-1 antibody), and VEGF in tumor cells. Thalidomide treatment reduces the extravasation of albumin-(Gd-DTPA)27-(biotin)11 without reducing the abundance of tumor blood vessels. a Tumor section from the saline-control group administered albumin-(Gd-DTPA)27-(biotin)11. The strong red signal indicates extravascular 1-h accumulation of biotin-labeled contrast medium surrounding the yellow-green tumor microvessels. b After 7 days of treatment with thalidomide, the density of red-fluorescent, biotin-labeled contrast agent extravasated over 1 h is strongly reduced compared with a, indicating a reduction in leakage and extravascular accumulation of the macromolecules. Confocal microscopic images of tumor vessels in MDA-MB-435 tumors after treatment with saline (c, d) or thalidomide (e, f) for 7 days show no noticeable change in the area density of perfused blood vessels (green lectin-stained) or total blood vessels (red, RECA-1 stained). No difference is observable (c–f) between saline-control and thalidomide-treated groups with regard to tumor vascularity. (g, h). Representative MDA-MB-435 tumor sections after immunohistochemical staining for human VEGF after a 7-day, three-injection treatment protocol with saline (g) or thalidomide (h). No difference in amount VEGF immunoreactivity was detected in the two groups. Scale bar 115 μm in c–f and 120 μm in a, b

Article Snippet: VEGF immunoreactivity of the human tumor cells was detected using goat polyclonal antibody anti-human VEGF (1:500, R&D Systems, Minneapolis, Minn.).

Techniques: Staining, Saline, Control, Labeling, Immunohistochemical staining, Injection

Journal: European radiology

Article Title: Magnetic resonance imaging for monitoring the effects of thalidomide on experimental human breast cancers

doi: 10.1007/s00330-008-1111-x

Figure Lengend Snippet: Measurement of RECA-1 and VEGF area density in tumors treated with thalidomide or saline control. Values obtained after 7 days of therapy

Article Snippet: VEGF immunoreactivity of the human tumor cells was detected using goat polyclonal antibody anti-human VEGF (1:500, R&D Systems, Minneapolis, Minn.).

Techniques: Saline, Control