Journal: PLoS ONE

Article Title: Combined Inhibition of Epidermal Growth Factor Receptor and Cyclooxygenase-2 Leads to Greater Anti-tumor Activity of Docetaxel in Advanced Prostate Cancer

doi: 10.1371/journal.pone.0076169

Figure Lengend Snippet: Cells grown in 1% FBS were exposed to 0.01 μmol/L docetaxel (D), 20 μmol/L gefitinib (G) or100 μmol/LNS-398 (N), alone or in combination, or with DMSO as control for 24 h. NF-KB, MMP-9 and VEGF mRNA (A and B) and protein levels (C and D) were measured as described in Materials and Methods. Values are reported as the mean±SD of three independent experiments.

Article Snippet: Antibodies against human VEGF and MMP-9 were obtained from R&D System and Bioworld, respectively.

Techniques: Control

Journal: Cells

Article Title: Effects of Human Deciduous Dental Pulp-Derived Mesenchymal Stem Cell-Derived Conditioned Medium on the Metabolism of HUVECs, Osteoblasts, and BMSCs

doi: 10.3390/cells11203222

Figure Lengend Snippet: Effect of VEGF-A in SHED-CM on the proliferative ability of HUVECs. The cells in the SHED-CM group exhibited significantly higher proliferative ability than those in the SHED-CM + anti-VEGF-A neutralizing antibody-supplemented (hereafter termed SHED-CM + anti-VEGF-A), VEGF-A, and control groups. Cells in the SHED-CM + anti-VEGF-A and VEGF-A groups showed significantly higher proliferative ability than those in the control group. ( n = 12, ** p < 0.01, * p < 0.05). SHED-CM, human deciduous dental pulp-derived mesenchymal stem cell-derived culture supernatant; VEGF-A, vascular endothelial growth factor-A; HUVECs, human umbilical vein endothelial cells.

Article Snippet: HUVECs were treated with SHED-CM in the presence or absence of 10 μg/mL anti-VEGF-A antibody (MAB293; R&D Systems, Minneapolis, MN, USA) in serum-free EBM-2 medium.

Techniques: Control, Derivative Assay

Journal: Cells

Article Title: Effects of Human Deciduous Dental Pulp-Derived Mesenchymal Stem Cell-Derived Conditioned Medium on the Metabolism of HUVECs, Osteoblasts, and BMSCs

doi: 10.3390/cells11203222

Figure Lengend Snippet: Effects of VEGF-A in SHED-CM on HUVEC lumen assembly. ( A ) Luminal view of HUVECs in the four groups. Lumen formation was observed more frequently in the SHED-CM- and VEGF-A-supplemented groups. Scale bar = 300 μm. ( B ) Vascular bifurcation in the four groups. ( C ) Vessel length in the four groups ( n = 4, ** p < 0.01). Black lines indicate vessel length; black arrows indicate joints. SHED-CM, human deciduous dental pulp-derived mesenchymal stem cell-derived culture supernatant; VEGF-A, vascular endothelial growth factor-A; HUVECs, human umbilical vein endothelial cells.

Article Snippet: HUVECs were treated with SHED-CM in the presence or absence of 10 μg/mL anti-VEGF-A antibody (MAB293; R&D Systems, Minneapolis, MN, USA) in serum-free EBM-2 medium.

Techniques: Derivative Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Fibroblast growth factor 2 activation of stromal cell vascular endothelial growth factor expression and angiogenesis.

doi: 10.1038/labinvest.3780212

Figure Lengend Snippet: Figure 2. Temporal induction and regression of the angiogenic response to FGF-2–Matrigel implants. Neovascular basement membrane structures were evaluated in the Matrigel/skin stromal interface using antimouse type IV collagen immunostaining. Matrigel/skin cross-sections were immunostained with anti-type IV collagen to identify stromal neovasculature derived from control implants (MG1hep) or FGF-2 implants (MG1hep1FGF-2) isolated at 4, 7, and 14 days postimplantation. Dermis (D), stromal interface (I), and Matrigel (MG) structures are identified. Arrows depict new vessels in the stromal interface in the FGF-2–containing implant at 4 and 7 days. Note the prominent regression and Matrigel degradation at 14 days postimplantation in the FGF-2–containing implant.

Article Snippet: The capture antibody used was the chicken anti-VEGF polyclonal antibody coated at 5 mg/ml and the goat antimouse VEGF polyclonal antibody (R and D Systems, Minneapolis, Minnesota) used as detection antibody at 5 mg/ml, followed by horseradish peroxidase conjugated antigoat IgG at 0.5 mg/ml (Sigma).

Techniques: Membrane, Immunostaining, Derivative Assay, Control, Isolation

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Fibroblast growth factor 2 activation of stromal cell vascular endothelial growth factor expression and angiogenesis.

doi: 10.1038/labinvest.3780212

Figure Lengend Snippet: Figure 3. Correlation of VEGF and PECAM-1 immunostaining with FGF-2–containing Matrigel implants. A, Matrigel implants containing Matrigel alone (MG), Matrigel with heparin (MG1hep), and MG with heparin and FGF-2 (MG1hep1FGF-2) were evaluated for the expression of vascular endothelial growth factor (VEGF) and platelet-endothelial cell adhesion molecule 1 (PECAM-1) at 7 days postimplantation. Note the prominent detection of VEGF (anti-VEGF) in the FGF-2–containing implant stromal interface (arrows). Very little VEGF was detected in the heparin control and none in the Matrigel control. The detection of PECAM-1 (anti-PECAM-1) was also selectively restricted to the highly angiogenic FGF-2–induced stromal interface with both lumenal vascular capillary structures and individual cells invading into the Matrigel matrix (arrows). B, Higher power view of anti-PECAM-1 staining (blue signal) in Matrigel with heparin (MG 1 hep) and Matrigel with heparin and FGF-2 (MG1hep1FGF-2) (original magnification, 3900). Dermal, interface, and Matrigel regions indicated as D, I, and MG, respectively. Arrowheads show PECAM-1 positive capillaries in dermal regions and arrows show PECAM-1 positive vessels in the interface regions. C, Quantification of VEGF and PECAM-1 immunostaining in the FGF-2 and control Matrigel implants at Day 7 postimplantation. The number of positive cells per high-power field were averaged as described in “Materials and Methods.” Statistical significance of the FGF-2 implants compared with the Matrigel 1 heparin control was performed by Student’s t test for n 5 4. * p , 0.05; ** p , 0.001.

Article Snippet: The capture antibody used was the chicken anti-VEGF polyclonal antibody coated at 5 mg/ml and the goat antimouse VEGF polyclonal antibody (R and D Systems, Minneapolis, Minnesota) used as detection antibody at 5 mg/ml, followed by horseradish peroxidase conjugated antigoat IgG at 0.5 mg/ml (Sigma).

Techniques: Immunostaining, Expressing, Control, Staining

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Fibroblast growth factor 2 activation of stromal cell vascular endothelial growth factor expression and angiogenesis.

doi: 10.1038/labinvest.3780212

Figure Lengend Snippet: Figure 4. Left panel, VEGF and VEGF receptor-2/flk-1 expression in FGF-2–induced angiogenesis detected by in situ hybridization analysis. VEGF was detected with antisense probe (VEGF AS) and sense control (VEGF S) in FGF-2–containing Matrigel implants (7 days). Bright (B.F.) and dark inverted (D.F.) field photographs are presented. Arrows indicate strong focal cell staining. VEGF receptor-2/flk-1 was detected with antisense probe (FLK-1 AS) and sense control (FLK-1 S). Arrows indicate regions of positive staining. Dermis (D), stromal interface (I), and Matrigel (MG) are indicated. Right panel, Quantification of silver grains in the individual regions for VEGF antisense and sense and FLK-1 antisense and sense probes. The average of three fields in each region was measured and antisense compared with sense control for each region by Student’s t test. * p , 0.005.

Article Snippet: The capture antibody used was the chicken anti-VEGF polyclonal antibody coated at 5 mg/ml and the goat antimouse VEGF polyclonal antibody (R and D Systems, Minneapolis, Minnesota) used as detection antibody at 5 mg/ml, followed by horseradish peroxidase conjugated antigoat IgG at 0.5 mg/ml (Sigma).

Techniques: Expressing, In Situ Hybridization, Control, Staining

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Fibroblast growth factor 2 activation of stromal cell vascular endothelial growth factor expression and angiogenesis.

doi: 10.1038/labinvest.3780212

Figure Lengend Snippet: Figure 5. FGF-2 induces VEGF mRNA and protein in Balb/c 3T3 embryonic fibroblasts. A, Serum-restricted fibroblasts were treated with FGF-2 (10 ng/ml) (F), hypoxia (2% O2) (H), or untreated control (C). Northern blot analysis for VEGF (VEGF) and ribosome-associated protein (36B4) was performed (exposure time: 24 hours). B, VEGF protein secretion from control (2FGF-2) or FGF-2–treated (1FGF-2) Balb/c 3T3 fibroblasts over an 8-hour time period. Triplicate plate conditioned media samples were analyzed by ELISA and represented as average VEGF (ng/ml) 6 SEM. Statistically significant difference from untreated samples (* p , 0.05). C, Transient transfection and transcriptional activation of VEGF-promoter-luciferase reporter construct. Balb/c 3T3 cells were trans- fected in triplicate with either the VEGF promoter-luciferase construct or the pGL3 control vector. Postrecovery (18 hours), cells were treated with FGF-2 (FGF-2), hypoxia, or nothing (Control) for 8 hours. Cell lysates were analyzed for luciferase activity and subtracted from the pGL3 control levels. Data is presented as average light units per mg protein 6 SEM.

Article Snippet: The capture antibody used was the chicken anti-VEGF polyclonal antibody coated at 5 mg/ml and the goat antimouse VEGF polyclonal antibody (R and D Systems, Minneapolis, Minnesota) used as detection antibody at 5 mg/ml, followed by horseradish peroxidase conjugated antigoat IgG at 0.5 mg/ml (Sigma).

Techniques: Control, Northern Blot, Enzyme-linked Immunosorbent Assay, Transfection, Activation Assay, Luciferase, Construct, Plasmid Preparation, Activity Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Fibroblast growth factor 2 activation of stromal cell vascular endothelial growth factor expression and angiogenesis.

doi: 10.1038/labinvest.3780212

Figure Lengend Snippet: Figure 6. FGF-2–induced VEGF expression is independent of cell adherence to attach- ment matrices. A, Serum-free Balb/c 3T3 cells were plated onto serum- (Plastic), Matrigel- (ECM), and fibronectin (Fn)-coated plates in the presence (FGF-2) or absence (control) of FGF-2 (10 ng/ml) for 8 hours. Phase photographs denote attachment to all three matrices and activation by FGF-2 (increase in rounded cell morphology). B, Top panel, Northern blot analysis for VEGF and 36B4 control from cells on the indicated matrices in the presence (F) or absence (C) of FGF-2 (10 ng/ml) for 8 hours. Bottom panel, Phosphorimage quantification of the VEGF mRNA signal normalized to the 36B4 control for Balb/c 3T3 cells plated and treated on each indicated matrix. Note the nearly identical FGF-2–induced VEGF mRNA levels for each matrix.

Article Snippet: The capture antibody used was the chicken anti-VEGF polyclonal antibody coated at 5 mg/ml and the goat antimouse VEGF polyclonal antibody (R and D Systems, Minneapolis, Minnesota) used as detection antibody at 5 mg/ml, followed by horseradish peroxidase conjugated antigoat IgG at 0.5 mg/ml (Sigma).

Techniques: Expressing, Control, Activation Assay, Northern Blot

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Fibroblast growth factor 2 activation of stromal cell vascular endothelial growth factor expression and angiogenesis.

doi: 10.1038/labinvest.3780212

Figure Lengend Snippet: Figure 7. Effect of systemic anti-VEGF antibodies on FGF-2–induced angiogenesis in vivo. A, Gross photographs of three representative Matrigel implants containing FGF-2 combined with systemic treatment with nonimmune chicken IgY (Control IgY) and anti-VEGF (Anti-VEGF IgY). Note the lack of associated skin vessel dilation and Matrigel coloration in the anti-VEGF group. B, Histologic evaluation of the stromal and angiogenic response induced by FGF-2 with control and anti-VEGF systemic treatments. Hematoxylin and eosin (H&E) and antimouse type IV collagen (collagen IV) are presented for control IgY (a and b) and anti-VEGF IgY (c and d). Matrigel (MG), stromal interface (I), and dermis (D) are indicated. Inset, Quantification of the angiogenic response in the control and anti-VEGF groups (n 5 10) is expressed as average vessels per field 6 SEM. Statistical difference from control IgG: * p , 0.001.

Article Snippet: The capture antibody used was the chicken anti-VEGF polyclonal antibody coated at 5 mg/ml and the goat antimouse VEGF polyclonal antibody (R and D Systems, Minneapolis, Minnesota) used as detection antibody at 5 mg/ml, followed by horseradish peroxidase conjugated antigoat IgG at 0.5 mg/ml (Sigma).

Techniques: In Vivo, Control

Journal: Stem cells translational medicine

Article Title: External Application of Human Umbilical Cord-Derived Mesenchymal Stem Cells in Hyaluronic Acid Gel Repairs Foot Wounds of Types I and II Diabetic Rats Through Paracrine Action Mode.

doi: 10.1093/stcltm/szad050

Figure Lengend Snippet: Figure 2. Histopathological features of diabetic foot wound closure in type I diabetic (T1DM) rats on day 21. (A) Representative micrographs of HE staining, scale bar = 500 μm (40×) and 50 μm (400×). (B) Representative micrographs of Masson’s trichrome stained sections, scale bar = 500 μm (40×) and 50 μm (400×). (C) Histogram of collagen deposition from Masson’s trichrome. (D) Representative micrographs of VEGFA antibody immunohistochemistry and blood vessels labeled by yellow allows, scale bar = 500 μm (40×) and 50 μm (400×). (E) Quantification of immunohistochemical staining intensity (VEGFA antibody). (F) Quantification of average number of blood vessels. Data were mean ± SD. *P < .05 and **P < .01 vs the control group, #P < .05 and ##P < .01 vs the model group, $P < .05 and $$P < .01 vs the HA group.

Article Snippet: The antibodies against PCNA (NB500-106) and VEGFA (NB100-664) were purchased from Novus Biologicals, Inc., the antibodies against β-actin (A3854), p-p38 (Thr180/Tyr182, 4511), total p38 (87869), total JNK (9252S), p-JNK (Thr183/Tyr185, 9255S), p-ERK1/2 (p-p44/42, Thr202/Tyr204, 4370), total ERK1/2 (p44/42, 4695), p-Akt (Ser473, 4060T), and total Akt (4691T) were purchased from Cell Signaling Technology, Inc., and horseradish peroxidase–conjugated antibody was purchased from Zhongshan Jinqiao Biotechnology Co., Ltd.

Techniques: Staining, Immunohistochemistry, Labeling, Immunohistochemical staining, Control

Journal: Stem cells translational medicine

Article Title: External Application of Human Umbilical Cord-Derived Mesenchymal Stem Cells in Hyaluronic Acid Gel Repairs Foot Wounds of Types I and II Diabetic Rats Through Paracrine Action Mode.

doi: 10.1093/stcltm/szad050

Figure Lengend Snippet: Figure 3. Histopathological features of diabetic foot wounds closure in type II diabetic (T2DM) rats on day 21. (A) Representative micrographs of H&E staining, scale bar = 500 μm (40×) and 50 μm (400×). (B) Representative micrographs of Masson’s trichrome stained sections, scale bar = 500 μm (40×) and 50 μm (400×). (C) Histogram of collagen deposition from Masson’s trichrome. (D) Representative micrographs of VEGFA antibody immunohistochemistry and blood vessels labeled by yellow allows, scale bar = 500 μm (40×) and 50 μm (400×). (E) Quantification of immunohistochemical staining intensity (VEGFA antibody). (F) Quantification of average number of blood vessels. Data were mean ± SD. *P < .05 and **P < .01 vs the control group, #P < .05 and ##P < .01 vs the model group, $P < .05 and $$P < .01 vs the HA group.

Article Snippet: The antibodies against PCNA (NB500-106) and VEGFA (NB100-664) were purchased from Novus Biologicals, Inc., the antibodies against β-actin (A3854), p-p38 (Thr180/Tyr182, 4511), total p38 (87869), total JNK (9252S), p-JNK (Thr183/Tyr185, 9255S), p-ERK1/2 (p-p44/42, Thr202/Tyr204, 4370), total ERK1/2 (p44/42, 4695), p-Akt (Ser473, 4060T), and total Akt (4691T) were purchased from Cell Signaling Technology, Inc., and horseradish peroxidase–conjugated antibody was purchased from Zhongshan Jinqiao Biotechnology Co., Ltd.

Techniques: Staining, Immunohistochemistry, Labeling, Immunohistochemical staining, Control

Journal: PLoS ONE

Article Title: Let It Flow: Morpholino Knockdown in Zebrafish Embryos Reveals a Pro-Angiogenic Effect of the Metalloprotease Meprin α 2

doi: 10.1371/journal.pone.0008835

Figure Lengend Snippet: (A) The cleavage of recombinant human VEGF-A 165 (CON, untreated, 24 kDa) by recombinant meprin α and β (each 85 nM) for 1 to 30 min resulted in two fragments of different molecular weight (19.6 kDa in case of meprin α, 20.5 kDa by meprin β), visualized by western blot analysis. In wild type zebrafish whole lysates (FL), VEGF-A could be detected using specific antibodies indicating a fragment similar to that produced by meprin processing. (B) Domain structure of human VEGF-A 165 (P15692-4). The lightning indicates the cleavage site between Ala4 and Glu5, identified after incubation with recombinant human meprin α. PDGF (platelet-derived growth factor). In addition, the N-terminal cleavage site in human VEGF-A 165 between Ala30 and Glu31 due to recombinant human meprin α activity could be identified by N-terminal sequencing.

Article Snippet: In zebrafish whole lysate (from adult fish), VEGF was detected by western blotting using a monoclonal anti-zebrafish VEGF antibody (R&D Systems, Wiesbaden, Germany).

Techniques: Recombinant, Molecular Weight, Western Blot, Produced, Incubation, Derivative Assay, Activity Assay, Sequencing