Journal:

Article Title: Vascular endothelial growth factor overproduced by tumour cells acts predominantly as a potent angiogenic factor contributing to malignant progression

doi: 10.1046/j.1365-2613.1999.00122.x

Figure Lengend Snippet: (a) VEGF and (b) bFGF productivity of cancer cells transfected with VEGF cDNA. ▪ parental cells; □ transfected clones. The levels of VEGF and bFGF contained in conditioned medium and cell extract, respectively, were determined by sandwich ELISA. Sample values were plotted against a recombinant VEGF or bFGF standard curve (mean ± SE, n = 3). ND: not detectable. QG90, RPMI4788, MCF-7 and their transfected clones were cultured in RPMI1640 containing 10% FCS (normal). Hormones-stripped FCS with (E2+) or without (E2−) 10−8 M 17 β-oestradiol was used for MCF-7 and its transfected clone, M2–24-G10.

Article Snippet: For determination of VEGF or bFGF activity in test samples by neutralizing antibodies, rabbit neutralizing polyclonal antibody against VEGF (PeproTech) or bFGF (R & D Systems), respectively, was added simultaneously ( Aonuma et al . 1998 ).

Techniques: Transfection, Clone Assay, Sandwich ELISA, Recombinant, Cell Culture

Journal:

Article Title: Vascular endothelial growth factor overproduced by tumour cells acts predominantly as a potent angiogenic factor contributing to malignant progression

doi: 10.1046/j.1365-2613.1999.00122.x

Figure Lengend Snippet: HUVE cell growth stimulatory activities in conditioned media of VEGF-transfected clones of (a) ○ QG90; □ Q1-1-C1; • Q1-24-E6; ▪ Q1-24-F6; ▴ Q1-24-H2, (b) ○ RPMI4788; • S0A12; ▪ S2G2 and (c) ○ MCF-7; • M2-24-D10; ▪ M2-24-E11; □ M2-24-G10 cells. HUVE cells were cultured in the presence of various concentrations of conditioned medium from each clone, indicated in Figure 1. After 4 days, HUVE cell growth was determined by MTT assay. The HUVE cell growth induced by 2.5 ng/ml of human recombinant VEGF165 was used as a positive control (♦). Results are presented as the percentage of the control without any growth factor. Each point indicates the mean of duplicate determinations.

Article Snippet: For determination of VEGF or bFGF activity in test samples by neutralizing antibodies, rabbit neutralizing polyclonal antibody against VEGF (PeproTech) or bFGF (R & D Systems), respectively, was added simultaneously ( Aonuma et al . 1998 ).

Techniques: Transfection, Clone Assay, Cell Culture, MTT Assay, Recombinant, Positive Control, Control

Journal:

Article Title: Vascular endothelial growth factor overproduced by tumour cells acts predominantly as a potent angiogenic factor contributing to malignant progression

doi: 10.1046/j.1365-2613.1999.00122.x

Figure Lengend Snippet: Neutralization by anti-VEGF rabbit polyclonal antibodies (pAb) of the HUVE cell growth stimulatory activity secreted by transfected clones. Conditioned medium from each clone was added to HUVE cells at a concentration of 50% in the absence (□) or presence (▪) of anti-VEGF pAb. After the incubation for 4 days, HUVE cell growth was determined by MTT assay. Results are presented as the percentage growth relative to the fresh medium-treated control and are the average of triplicate determinations (mean ± SE). Additional control cells were incubated with 2.5 ng/ml of human recombinant VEGF165 (hr-VEGF165) or 4 ng/ml of human recombinant bFGF (hr-bFGF).

Article Snippet: For determination of VEGF or bFGF activity in test samples by neutralizing antibodies, rabbit neutralizing polyclonal antibody against VEGF (PeproTech) or bFGF (R & D Systems), respectively, was added simultaneously ( Aonuma et al . 1998 ).

Techniques: Neutralization, Activity Assay, Transfection, Clone Assay, Concentration Assay, Incubation, MTT Assay, Control, Recombinant

Journal:

Article Title: Vascular endothelial growth factor overproduced by tumour cells acts predominantly as a potent angiogenic factor contributing to malignant progression

doi: 10.1046/j.1365-2613.1999.00122.x

Figure Lengend Snippet: Growth curves of VEGF-transfected clones of (a) ○ QG90; □ Q1-1-C1; • Q1-24-E6; ▪ Q1-24-F6; ▴ Q1-24-H2, (b) ○ RPMI4788; • S0A12; ▪ S2G2 and (c) ○ MCF-7; • M2-24-D10; ▪ M2-24-E11; □ M2-24-G10 cells. The number of cells was counted with a Coulter Counter (mean ± SE, n = 3).

Article Snippet: For determination of VEGF or bFGF activity in test samples by neutralizing antibodies, rabbit neutralizing polyclonal antibody against VEGF (PeproTech) or bFGF (R & D Systems), respectively, was added simultaneously ( Aonuma et al . 1998 ).

Techniques: Transfection, Clone Assay

Journal:

Article Title: Vascular endothelial growth factor overproduced by tumour cells acts predominantly as a potent angiogenic factor contributing to malignant progression

doi: 10.1046/j.1365-2613.1999.00122.x

Figure Lengend Snippet: Effects of oestrogen on in vitro growth of the VEGF-overproducing M2–24-G10 clone (□,▪) and parental MCF-7 cells (○,•). The cells grown in the absence (○,□) or presence (•,▪) of 10−8 M 17β-oestradiol (E2) were counted with a Coulter Counter (mean ± SE, n = 3).

Article Snippet: For determination of VEGF or bFGF activity in test samples by neutralizing antibodies, rabbit neutralizing polyclonal antibody against VEGF (PeproTech) or bFGF (R & D Systems), respectively, was added simultaneously ( Aonuma et al . 1998 ).

Techniques: In Vitro

Journal:

Article Title: Vascular endothelial growth factor overproduced by tumour cells acts predominantly as a potent angiogenic factor contributing to malignant progression

doi: 10.1046/j.1365-2613.1999.00122.x

Figure Lengend Snippet: In vivo angiogenic activity of VEGF-transfected clones of (a) QG90 and (b) RPMI4788 cells. A Millipore chamber containing each clone or HBSS was implanted s.c. into a dorsal air sac of a BALB/c mouse (three to six mice/group). After 4 days, the area of new capillarization induced by each clone was determined with an image analyser. Results (mean ± SE) are presented as percentages of the mean area of the control group treated with a HBSS-containing chamber. * P < 0.01 vs. the control group; ns, not significant (by Student's t-test).

Article Snippet: For determination of VEGF or bFGF activity in test samples by neutralizing antibodies, rabbit neutralizing polyclonal antibody against VEGF (PeproTech) or bFGF (R & D Systems), respectively, was added simultaneously ( Aonuma et al . 1998 ).

Techniques: In Vivo, Activity Assay, Transfection, Clone Assay, Control

Journal: Stem Cell Research & Therapy

Article Title: Development of a surrogate potency assay to determine the angiogenic activity of Stempeucel®, a pooled, ex-vivo expanded, allogeneic human bone marrow mesenchymal stromal cell product

doi: 10.1186/s13287-017-0488-3

Figure Lengend Snippet: Activity of VEGF secreted from phBMMSCs is mediated by VEGFR2. a Proliferation of HUVECs using 50% CM from phBMMSCs and 50% CM with Axitinib at concentrations of 0.25 nM, 0.29 nM and 1.2 nM to block VEGFR2, VEGFR2 and VEGFR3, and VEGFR2, VEGFR3 and VEGFR1 respectively. Significant reduction in HUVEC proliferation was observed in the CM with all Axitinib treatment groups, ***p = 0.0001. EGM was used as a positive control for HUVEC proliferation. Analysis of VEGFR1 ( b ), VEGFR2 ( c ) and VEGFR3 ( d ) fold expression in HUVECs treated with phBMMSC CM and phBMMSC CM with anti-VEGF mAb by qRT-PCR. Significant reduction only in VEGFR2 expression was observed in the phBMMSC CM with anti-VEGF mAb group compared with the CM alone group, *p = 0.01. e Analysis of VEGF pathway-dependent markers PI3K and AKT1 in HUVECs cultured in the presence of phBMMSC CM by qRT-PCR; mRNA expression levels were normalized with housekeeping gene β-Actin as an internal standard and subsequently normalized to expression levels in HUVECs cultured in EGM as control. Data generated using two batches of phBMMSC CM, assayed in triplicates and presented as mean ± SEM. f Schematic diagram to depict the possible signal transduction pathway of VEGF secreted by phBMMSCs in HUVECs . SEMs shown as error bars. HUVEC Human umbilical vein endothelial cell, EGM endothelial growth medium, CM conditioned medium, VEGFR vascular endothelial growth factor receptor, mAb monoclonal antibody, Ang-1 angiopoietin-1, SDF-1 stromal-derived factor-1, HGF hepatocyte growth factor, IL interleukin, TGF-β transforming growth factor beta

Article Snippet: The serum starvation medium was replaced with equal volumes of EGM, control medium, CM, CM containing anti-VEGF neutralizing antibody (10 μg/ml) or IgG isotype control (10 μg/ml), SFM or Axitinib (0.25 nM, 0.29 nM and 1.2 nM) (Cayman Chemical, MI, USA).

Techniques: Activity Assay, Blocking Assay, Positive Control, Expressing, Quantitative RT-PCR, Cell Culture, Generated, Transduction, Derivative Assay

Journal: Stem Cell Research & Therapy

Article Title: Development of a surrogate potency assay to determine the angiogenic activity of Stempeucel®, a pooled, ex-vivo expanded, allogeneic human bone marrow mesenchymal stromal cell product

doi: 10.1186/s13287-017-0488-3

Figure Lengend Snippet: Functional in vitro HUVEC migration, proliferation and tube formation with rhVEGF and phBMMSC CM, followed by inhibition of VEGF activity. a Transwell migration of HUVECs in response to rhVEGF serially diluted from 8 ng/ml to 31.2 pg/ml in SFM. Significant inhibition of HUVEC migration was observed in 2 ng/ml rhVEGF with anti-VEGF mAb (10 μg/ml) compared to migration observed with 2 ng/ml rhVEGF concentration, **p = 0.001. b Transwell migration of HUVECs in response to phBMMSC CM serially diluted with SFM from 100% (4 ng/ml of secreted VEGF) to 0.19% (7.8 pg/ml of secreted VEGF). The maximum migration of HUVECs was observed at 50% dilution comprising 2 ng/ml secreted VEGF which was significantly inhibited by anti-VEGF mAb (10 μg/ml) in comparison with phBMMSC CM + IgG isotype, * p = 0.02, and phBMMSC CM, **p = 0.0009. Threshold for HUVEC proliferation was observed at 3.125% CM dilution with 125 pg/ml VEGF. c Proliferation of HUVECs in response to rhVEGF serially diluted starting from 8 ng/ml in SFM. rhVEGF did not effectively induce proliferation and therefore no measurable effect was observed with the anti-VEGF mAb. d HUVEC proliferation with phBMMSC CM serially diluted from 100% to 0.19% in SFM. Addition of anti-VEGF mAb to the 50% CM significantly reduced proliferation, **p = 0.001, in comparison with CM containing IgG isotype, *p = 0.04. Proliferation in HUVECs initiated at 0.78% CM dilution with 31.2 pg/ml VEGF. e – q HUVEC tube formation in response to phBMMSC CM serially diluted from 100% to 0.19%. Maximum tube formation was observed at 50% dilution containing 2 ng/ml secreted VEGF. Significant reduction in tube formation was observed when anti-VEGF mAb was used in 50% diluted CM, while IgG isotype did not alter the tube formation of HUVECs. r Graphical representation of tube formation induced by phBMMSC CM at various concentrations. Tube formation seemed to initiate at 0.78% CM dilution. Statistical significance was determined using Student’s t test. Data presented as mean ± SEM. SEMs shown as error bars. CM conditioned medium, phBMMSC pooled human bone marrow derived mesenchymal stromal cell, SFM serum-free medium, VEGF vascular endothelial growth factor, HPF high-power field

Article Snippet: The serum starvation medium was replaced with equal volumes of EGM, control medium, CM, CM containing anti-VEGF neutralizing antibody (10 μg/ml) or IgG isotype control (10 μg/ml), SFM or Axitinib (0.25 nM, 0.29 nM and 1.2 nM) (Cayman Chemical, MI, USA).

Techniques: Functional Assay, In Vitro, Migration, Inhibition, Activity Assay, Concentration Assay, Derivative Assay

Journal: Stem Cell Research & Therapy

Article Title: Development of a surrogate potency assay to determine the angiogenic activity of Stempeucel®, a pooled, ex-vivo expanded, allogeneic human bone marrow mesenchymal stromal cell product

doi: 10.1186/s13287-017-0488-3

Figure Lengend Snippet: In vitro pro-angiogenic function of secreted VEGF from nine batches of phBMMSCs. a Evaluation of HUVEC migration under various culture conditions. CM ( n = 9) induced significant migration of HUVECs. Migration was unaffected by an isotype-matched antibody ( p = 0.1964), while addition of anti-VEGF mAb caused significant (* p = 0.0001) inhibition of cell migration. Migration of HUVECs in the CM group was significantly higher than that of control medium, ***p = 0.0004. b Correlation of VEGF concentration in phBMMSC CM ( n = 9) with HUVEC migration was observed, r 2 = 0.78, ** p = 0.0014. c HUVEC proliferation in response to various culture conditions; phBMMSC CM ( n = 9) induced HUVEC proliferation similar to that of EGM and complete medium, although significant reduction of cell proliferation was observed in the CM group with anti-VEGF mAb in comparison to CM with isotype IgG, **p = 0.0075. Addition of IgG isotype in the CM did not alter HUVEC proliferation compared with CM alone, p = 0.2985. d Correlation of VEGF concentration-dependent proliferation of HUVECs was observed with phBMMSC CM ( n = 9) with a high correlation coefficient, r 2 = 0.75, ** p = 0.002. Representative images of tube formations in e EGM, f control medium, g phBMMSC CM, h IgG isotype, i phBMMSC CM with anti-VEGF mAb, 4× objective were used for magnification. j phBMMSC CM ( n = 9) induces significant tube formation in HUVECs compared with EGM and control medium (** p = 0.0063). Tube formation was greatly reduced with the addition of anti-VEGF mAb in the CM in comparison with the isotype-matched control group (** p = 0.0084). Tube formation in HUVECs was unaffected with the addition of IgG isotype ( p = 0.2985). k Concentration of VEGF in nine batches of CM highly correlated to the tube lengths, r 2 = 0.89, *** p = 0.0001. Data presented as mean ± SEM. SEMs shown as error bars . CM conditioned medium, phBMMSC pooled human bone marrow derived mesenchymal stromal cell, EGM endothelial growth medium, VEGF vascular endothelial growth factor, HPF high-power field

Article Snippet: The serum starvation medium was replaced with equal volumes of EGM, control medium, CM, CM containing anti-VEGF neutralizing antibody (10 μg/ml) or IgG isotype control (10 μg/ml), SFM or Axitinib (0.25 nM, 0.29 nM and 1.2 nM) (Cayman Chemical, MI, USA).

Techniques: In Vitro, Migration, Inhibition, Concentration Assay, Derivative Assay