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R&D Systems vegf a
Chemotactic Migration of EPCs toward mRNA-Engineered EPCs 1 × 10 5 murine EPCs were cultivated overnight, and then they were transfected with 1.6 μg <t>ANG-1,</t> 0.8 μg <t>VEGF-A,</t> or 0.5 μg SDF-1α mRNA or with an mRNA cocktail containing 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α mRNA. Migration behavior of untreated EPCs (5 × 10 4 ) seeded on transwell inserts toward mRNA-transfected EPCs was analyzed using a chemotactic migration assay. As a control, EPCs incubated with medium or medium containing transfection reagent (TR) were used. After 6 hr, migrated EPCs through 8-μm transwell inserts were stained with DAPI, and cell numbers were calculated using ImageJ software. Scale bars represent 100 μm. Results are shown as mean + SEM (n = 4). Statistical differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test (***p
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1) Product Images from "Improving the Angiogenic Potential of EPCs via Engineering with Synthetic Modified mRNAs"

Article Title: Improving the Angiogenic Potential of EPCs via Engineering with Synthetic Modified mRNAs

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2018.09.005

Chemotactic Migration of EPCs toward mRNA-Engineered EPCs 1 × 10 5 murine EPCs were cultivated overnight, and then they were transfected with 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with an mRNA cocktail containing 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α mRNA. Migration behavior of untreated EPCs (5 × 10 4 ) seeded on transwell inserts toward mRNA-transfected EPCs was analyzed using a chemotactic migration assay. As a control, EPCs incubated with medium or medium containing transfection reagent (TR) were used. After 6 hr, migrated EPCs through 8-μm transwell inserts were stained with DAPI, and cell numbers were calculated using ImageJ software. Scale bars represent 100 μm. Results are shown as mean + SEM (n = 4). Statistical differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test (***p
Figure Legend Snippet: Chemotactic Migration of EPCs toward mRNA-Engineered EPCs 1 × 10 5 murine EPCs were cultivated overnight, and then they were transfected with 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with an mRNA cocktail containing 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α mRNA. Migration behavior of untreated EPCs (5 × 10 4 ) seeded on transwell inserts toward mRNA-transfected EPCs was analyzed using a chemotactic migration assay. As a control, EPCs incubated with medium or medium containing transfection reagent (TR) were used. After 6 hr, migrated EPCs through 8-μm transwell inserts were stained with DAPI, and cell numbers were calculated using ImageJ software. Scale bars represent 100 μm. Results are shown as mean + SEM (n = 4). Statistical differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test (***p

Techniques Used: Migration, Transfection, Incubation, Staining, Software

Analysis of Wound-Healing Capacity of mRNA-Engineered EPCs via Wound Scratch Migration Assay 1 × 10 5 murine EPCs were cultivated overnight, and then they were transfected with 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with an mRNA cocktail containing 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α mRNA. The next day, cells were detached, and 28,000 EPCs with or without mRNA transfection were seeded in each chamber of Culture-Insert 3 wells in μ-dishes. After 5 hr, when the cells completely attached and covered the surface, an open wound field was generated. Immediately after the generation of wound areas (0 hr) and after 12, 24, and 36 hr, phase-contrast images were taken and closed wound areas were calculated using Tscratch software. Scale bar represents 500 μm. Results are shown as mean + SD (n = 8). Statistical differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test (*p
Figure Legend Snippet: Analysis of Wound-Healing Capacity of mRNA-Engineered EPCs via Wound Scratch Migration Assay 1 × 10 5 murine EPCs were cultivated overnight, and then they were transfected with 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with an mRNA cocktail containing 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α mRNA. The next day, cells were detached, and 28,000 EPCs with or without mRNA transfection were seeded in each chamber of Culture-Insert 3 wells in μ-dishes. After 5 hr, when the cells completely attached and covered the surface, an open wound field was generated. Immediately after the generation of wound areas (0 hr) and after 12, 24, and 36 hr, phase-contrast images were taken and closed wound areas were calculated using Tscratch software. Scale bar represents 500 μm. Results are shown as mean + SD (n = 8). Statistical differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test (*p

Techniques Used: Migration, Transfection, Generated, Software

Expression of ANG-1, VEGF-A, and SDF-1α after the Transfection of Synthetic mRNA into Murine EPCs 1 × 10 5 EPCs were seeded and transfected the next day with (A) 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with (B) an mRNA cocktail containing 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α mRNA. The protein expression was analyzed in supernatants 24 hr after the transfection using ELISA. Cells treated with only medium or medium and transfection reagent (TR) served as negative controls. Results are shown as mean + SEM (n = 4). Statistical differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test (****p
Figure Legend Snippet: Expression of ANG-1, VEGF-A, and SDF-1α after the Transfection of Synthetic mRNA into Murine EPCs 1 × 10 5 EPCs were seeded and transfected the next day with (A) 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with (B) an mRNA cocktail containing 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α mRNA. The protein expression was analyzed in supernatants 24 hr after the transfection using ELISA. Cells treated with only medium or medium and transfection reagent (TR) served as negative controls. Results are shown as mean + SEM (n = 4). Statistical differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test (****p

Techniques Used: Expressing, Transfection, Enzyme-linked Immunosorbent Assay

Analysis of In Vivo Angiogenetic Potential of mRNA-Engineered EPCs in Chick Embryo Chorioallantoic Membrane Assay (A) Schematic representation of the chorioallantoic membrane (CAM) assay. ANG-1, SDF-1α, and VEGF-A mRNA-transfected and untreated cells (medium or medium containing transfection reagent [TR]) were applied in silicone rings (8-mm inner diameter) onto the CAMs at the ninth day of incubation at 37°C and 60% humidity. At the fourth day of incubation, the CAMs were fixed and excised. (B) Analysis of the region of the inner ring circle with Wimasis WimCAM image software to quantify angiogenesis. Scale bar of photographs (upper) and analyzed pictures (lower) represents 2.7 mm. (C) Quantification of angiogenesis using Wimasis WimCAM web-based service. The vessel density, total branching points, total vessel network length, and total segments were quantified and compared to the medium control. Results are shown as mean + SD (n = 3). Statistical differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test (**p
Figure Legend Snippet: Analysis of In Vivo Angiogenetic Potential of mRNA-Engineered EPCs in Chick Embryo Chorioallantoic Membrane Assay (A) Schematic representation of the chorioallantoic membrane (CAM) assay. ANG-1, SDF-1α, and VEGF-A mRNA-transfected and untreated cells (medium or medium containing transfection reagent [TR]) were applied in silicone rings (8-mm inner diameter) onto the CAMs at the ninth day of incubation at 37°C and 60% humidity. At the fourth day of incubation, the CAMs were fixed and excised. (B) Analysis of the region of the inner ring circle with Wimasis WimCAM image software to quantify angiogenesis. Scale bar of photographs (upper) and analyzed pictures (lower) represents 2.7 mm. (C) Quantification of angiogenesis using Wimasis WimCAM web-based service. The vessel density, total branching points, total vessel network length, and total segments were quantified and compared to the medium control. Results are shown as mean + SD (n = 3). Statistical differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test (**p

Techniques Used: In Vivo, Chick Chorioallantoic Membrane Assay, Transfection, Incubation, Software

Influence of mRNA Transfection on the Viability of Murine EPCs 1 × 10 5 EPCs were transfected with 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with an mRNA cocktail containing 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α mRNA. Viability was determined 24 hr post-transfection using PrestoBlue assay. The viability of cells incubated with Opti-MEM (medium) was set to 100%, and the viability of samples was expressed relative to these cells. The data are shown as mean + SEM (n = 3). No statistically significant differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test.
Figure Legend Snippet: Influence of mRNA Transfection on the Viability of Murine EPCs 1 × 10 5 EPCs were transfected with 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with an mRNA cocktail containing 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α mRNA. Viability was determined 24 hr post-transfection using PrestoBlue assay. The viability of cells incubated with Opti-MEM (medium) was set to 100%, and the viability of samples was expressed relative to these cells. The data are shown as mean + SEM (n = 3). No statistically significant differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test.

Techniques Used: Transfection, Prestoblue Assay, Incubation

Quality Control of the Generated PCR Products and In Vitro -Transcribed mRNAs ANG-1-, SDF-1α-, and VEGF-A-encoding mRNAs were synthesized and modified with 3′-PolyA 120 tail, 5′-ARCA, 100% m5CTP, 100% Ψ-UTP, and post-transcriptional phosphatase treatment. The specific lengths of the amplified DNA and the synthetic mRNA were detected using 1% agarose gel electrophoresis and GelRed staining at approximately 1.8 kb for ANG-1, 0.6 kb for SDF-1α, and 0.9 kb for VEGF-A. The 0.08- to 10-kb range mix DNA ladder and the 0.5- to 10-kb RNA ladder were used as length markers (Ms).
Figure Legend Snippet: Quality Control of the Generated PCR Products and In Vitro -Transcribed mRNAs ANG-1-, SDF-1α-, and VEGF-A-encoding mRNAs were synthesized and modified with 3′-PolyA 120 tail, 5′-ARCA, 100% m5CTP, 100% Ψ-UTP, and post-transcriptional phosphatase treatment. The specific lengths of the amplified DNA and the synthetic mRNA were detected using 1% agarose gel electrophoresis and GelRed staining at approximately 1.8 kb for ANG-1, 0.6 kb for SDF-1α, and 0.9 kb for VEGF-A. The 0.08- to 10-kb range mix DNA ladder and the 0.5- to 10-kb RNA ladder were used as length markers (Ms).

Techniques Used: Generated, Polymerase Chain Reaction, In Vitro, Synthesized, Modification, Amplification, Agarose Gel Electrophoresis, Staining, Mass Spectrometry

Analysis of Angiogenic Potential of mRNA-Engineered EPCs by Tube Formation Assay 1 × 10 5 murine EPCs were cultivated overnight, and then they were transfected with 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with an mRNA cocktail containing 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α mRNA. After 24 hr, EPCs were detached and 1 × 10 4 EPCs were seeded on Matrigel-coated angiogenesis slides. After 4 hr of incubation at 37°C, the formation of tubes was examined by phase-contrast microscopy. Microscopic images were analyzed using NIH ImageJ software with Angiogenesis Analyzer plugin, and segments are shown in magenta, master segments in orange, branches in green, and meshes in blue. The numbers (Nb) of nodes, segments, and master segments and the total (Tot.) segment length, total mesh area, and branching interval were quantified and compared to the medium control. The unit of area and length is pixel (px). Scale bars represent 100 μm. Results are shown as mean + SD (n = 3). Statistical differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test (*p
Figure Legend Snippet: Analysis of Angiogenic Potential of mRNA-Engineered EPCs by Tube Formation Assay 1 × 10 5 murine EPCs were cultivated overnight, and then they were transfected with 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with an mRNA cocktail containing 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α mRNA. After 24 hr, EPCs were detached and 1 × 10 4 EPCs were seeded on Matrigel-coated angiogenesis slides. After 4 hr of incubation at 37°C, the formation of tubes was examined by phase-contrast microscopy. Microscopic images were analyzed using NIH ImageJ software with Angiogenesis Analyzer plugin, and segments are shown in magenta, master segments in orange, branches in green, and meshes in blue. The numbers (Nb) of nodes, segments, and master segments and the total (Tot.) segment length, total mesh area, and branching interval were quantified and compared to the medium control. The unit of area and length is pixel (px). Scale bars represent 100 μm. Results are shown as mean + SD (n = 3). Statistical differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test (*p

Techniques Used: Tube Formation Assay, Transfection, Incubation, Microscopy, Software

2) Product Images from "Vascular endothelial growth factor is elevated in ocular fluids of eyes harbouring uveal melanoma: identification of a potential therapeutic window"

Article Title: Vascular endothelial growth factor is elevated in ocular fluids of eyes harbouring uveal melanoma: identification of a potential therapeutic window

Journal: The British Journal of Ophthalmology

doi:

(A) New vessels (arrows) close to the anterior surface of the pupillary margin of the iris in an eye with NVI (von Willebrand factor), and (B) the same eye showing bFGF expression in some of the stromal cells (arrows). (C) Positivity of the ciliary body epithelium (arrowheads) and smooth muscle (arrow) for VEGF-A. (D) Attached retina adjacent to uveal melanoma showing mild positivity for VEGF-A, with strong positivity in ganglion cells. (E) Strong VEGF-A positivity in detached retina with considerable atrophy. (F) bFGF expression in atrophic retina. Artefactual detachment of the retina is easily determined histologically by the presence of pigment granules in retinal pigment epithelium processes stripped with the retina forming a line along the base of the photoreceptor outer segments: the detachments shown in both (E) and (F) were tumour related. (G–I) Negative controls for von Willebrand factor, VEGF-A, and bFGF respectively. (All original magnifications ×400 unless otherwise stated: scale bars = 100 μm.)
Figure Legend Snippet: (A) New vessels (arrows) close to the anterior surface of the pupillary margin of the iris in an eye with NVI (von Willebrand factor), and (B) the same eye showing bFGF expression in some of the stromal cells (arrows). (C) Positivity of the ciliary body epithelium (arrowheads) and smooth muscle (arrow) for VEGF-A. (D) Attached retina adjacent to uveal melanoma showing mild positivity for VEGF-A, with strong positivity in ganglion cells. (E) Strong VEGF-A positivity in detached retina with considerable atrophy. (F) bFGF expression in atrophic retina. Artefactual detachment of the retina is easily determined histologically by the presence of pigment granules in retinal pigment epithelium processes stripped with the retina forming a line along the base of the photoreceptor outer segments: the detachments shown in both (E) and (F) were tumour related. (G–I) Negative controls for von Willebrand factor, VEGF-A, and bFGF respectively. (All original magnifications ×400 unless otherwise stated: scale bars = 100 μm.)

Techniques Used: Expressing

VEGF levels in (A) aqueous and (B) vitreous, showing high levels in most patients with NVI.
Figure Legend Snippet: VEGF levels in (A) aqueous and (B) vitreous, showing high levels in most patients with NVI.

Techniques Used:

3) Product Images from "hERG1 channels modulate integrin signaling to trigger angiogenesis and tumor progression in colorectal cancer"

Article Title: hERG1 channels modulate integrin signaling to trigger angiogenesis and tumor progression in colorectal cancer

Journal: Scientific Reports

doi: 10.1038/srep03308

Characterization of the β1/hERG1-dependent signaling pathway able to influence VEGF-a expression and secretion in HT116 cells. (A) Effect of β1 inhibition or activation on VEGF-A expression. (B) VEGF-A expression in HCT116 treated with E4031 or WAY (40 μM), in HCT116 and HCT8 transiently transfected with a mix of α- herg1 siRNAs 1 + 3 or in HCT116-Sh-hERG1. Controls are represented by cells cultured in standard conditions for the pharmacological treated samples, by HCT116-PLKO cells for HCT116-Sh-hERG1 and by HCT116 transfected with siRNA negative control for siRNAs 1 + 3, respectively. (C) Effect of hERG1 blocking on VEGF-A secretion in HCT116 and HCT8 cells. Cells were also used for RNA extraction and RT-qPCR assay shown in Table 3S . Data are means ± SEM of two-four separate experiments, each carried out in duplicate. (D) VEGF-A secretion in HEK-Mock and HEK-hERG1 cells and in HT29-Mock and HT29-hERG1 cells. For quantitative analysis of hERG1 expression, see Table 4S . (E) Effects of PI3K/Akt inhibitors LY294002 and perifosine on VEGF-A secretion (left panel) and of α-Akt1 and α-Akt2 siRNAs on VEGF-A expression (right panel). Data are reported as the percentage of control ± SEM of two experiments, each carried out in triplicate. (F) Effects of PI3K/Akt inhibitors LY294002 and perifosine, on HIF-1 transcriptional activity. Data are means ± SEM of three separate experiments. (G) Fold induction of HIF(s) target genes after hERG1, β1 inhibition and activation or after Akt1/2 silencing. Data are means ± SEM of three separate experiments, each carried out in duplicate. GLUT1 , glucose transporter 1; LDHA , lactate dehydrogenase A; ANGPTL-4 , angiopoietin-like 4.*: p
Figure Legend Snippet: Characterization of the β1/hERG1-dependent signaling pathway able to influence VEGF-a expression and secretion in HT116 cells. (A) Effect of β1 inhibition or activation on VEGF-A expression. (B) VEGF-A expression in HCT116 treated with E4031 or WAY (40 μM), in HCT116 and HCT8 transiently transfected with a mix of α- herg1 siRNAs 1 + 3 or in HCT116-Sh-hERG1. Controls are represented by cells cultured in standard conditions for the pharmacological treated samples, by HCT116-PLKO cells for HCT116-Sh-hERG1 and by HCT116 transfected with siRNA negative control for siRNAs 1 + 3, respectively. (C) Effect of hERG1 blocking on VEGF-A secretion in HCT116 and HCT8 cells. Cells were also used for RNA extraction and RT-qPCR assay shown in Table 3S . Data are means ± SEM of two-four separate experiments, each carried out in duplicate. (D) VEGF-A secretion in HEK-Mock and HEK-hERG1 cells and in HT29-Mock and HT29-hERG1 cells. For quantitative analysis of hERG1 expression, see Table 4S . (E) Effects of PI3K/Akt inhibitors LY294002 and perifosine on VEGF-A secretion (left panel) and of α-Akt1 and α-Akt2 siRNAs on VEGF-A expression (right panel). Data are reported as the percentage of control ± SEM of two experiments, each carried out in triplicate. (F) Effects of PI3K/Akt inhibitors LY294002 and perifosine, on HIF-1 transcriptional activity. Data are means ± SEM of three separate experiments. (G) Fold induction of HIF(s) target genes after hERG1, β1 inhibition and activation or after Akt1/2 silencing. Data are means ± SEM of three separate experiments, each carried out in duplicate. GLUT1 , glucose transporter 1; LDHA , lactate dehydrogenase A; ANGPTL-4 , angiopoietin-like 4.*: p

Techniques Used: Expressing, Inhibition, Activation Assay, Transfection, Cell Culture, Negative Control, Blocking Assay, RNA Extraction, Quantitative RT-PCR, Activity Assay

Role of hERG1 channels in the regulation of in vivo tumor growth and intratumoral angiogenesis in immunodeficient mice. (A) Volume of tumor masses obtained after injection of HEK-Mock and HEK-hERG1 cells. Data are reported as the mean ± SEM of three tumor masses. (B) The same experiments performed using HCT116-PLKO and HCT116-Sh-hERG1 cells. (C) Time course of tumor growth in control ( ), WAY- ( ) and E4031 ( )-treated mice, as well as untreated ( ) and E4031 ( )-treated mice injected with HCT116 p53-/- cells. (F) Histological analysis of VEGF-A, pAkt and Ki67 staining of tumor masses obtained in control and WAY-treated mice after injection of CRC cells. Bar: 100 μm for VEGF, 100 μm for Ki67, 100 μm for pAkt. (E) WB analysis of Akt phosphorylation and HIF expression in tumor masses obtained from the injection of HCT116 cells. For further details regarding densitometric analysis, see Fig. 6S DA . (F) CD34 staining of control and E4031-treated mice, as well as untreated and treated mice injected with p53 −/− . Further descriptions are reported in Supplementary Information . Full-length blots are reported in Supplementary Information section titled “Full-length blots relative to the cropped images showed in the main Figures”.
Figure Legend Snippet: Role of hERG1 channels in the regulation of in vivo tumor growth and intratumoral angiogenesis in immunodeficient mice. (A) Volume of tumor masses obtained after injection of HEK-Mock and HEK-hERG1 cells. Data are reported as the mean ± SEM of three tumor masses. (B) The same experiments performed using HCT116-PLKO and HCT116-Sh-hERG1 cells. (C) Time course of tumor growth in control ( ), WAY- ( ) and E4031 ( )-treated mice, as well as untreated ( ) and E4031 ( )-treated mice injected with HCT116 p53-/- cells. (F) Histological analysis of VEGF-A, pAkt and Ki67 staining of tumor masses obtained in control and WAY-treated mice after injection of CRC cells. Bar: 100 μm for VEGF, 100 μm for Ki67, 100 μm for pAkt. (E) WB analysis of Akt phosphorylation and HIF expression in tumor masses obtained from the injection of HCT116 cells. For further details regarding densitometric analysis, see Fig. 6S DA . (F) CD34 staining of control and E4031-treated mice, as well as untreated and treated mice injected with p53 −/− . Further descriptions are reported in Supplementary Information . Full-length blots are reported in Supplementary Information section titled “Full-length blots relative to the cropped images showed in the main Figures”.

Techniques Used: In Vivo, Mouse Assay, Injection, Staining, Western Blot, Expressing

Effects of hERG1 blockers in orthotopic and direct metastasis CRC mouse models. (A–B) Representative macroscopic images of the abdomen of control (A) and E4031-treated (B) mice (IP, 20 mg/Kg). Liver: gross images. Both hematoxylin/eosin (H E) and hMHCI staining (black arrows) of liver sections (A, Control mice, staining of liver metastases sections; B, E4031-treated mice, staining of liver sections) are reported. (C) IHC experiments on control mice (CONTROL) and E4031-treated (E4031) mice. Both hematoxylin/eosin (H E) and hMHCI staining of tumor masses and of a site of the coecum without macroscopic tumor masses (CONTROL), and of a site of the coecum without macroscopic tumor masses (E4031-treated)) are reported. Black arrows indicate the anti-hMHC staining. Bar: 100 μM for H E and for hMHC. (D) Representative pseudo-color BLI images tracking HCT116-luc cells emission in nude mice, 13 days after splenic HCT116-luc injection. Color bar represents light intensity levels reported as counts per minute (cpm). (E) Livers from animals sacrificed three weeks after cell injection and two weeks after beginning the pharmacological treatment. (F) H E and VEGF-A staining revealing larger necrotic areas and the presence of a higher amount of VEGF-A (brown staining) in Control compared to E4031-treated mice.
Figure Legend Snippet: Effects of hERG1 blockers in orthotopic and direct metastasis CRC mouse models. (A–B) Representative macroscopic images of the abdomen of control (A) and E4031-treated (B) mice (IP, 20 mg/Kg). Liver: gross images. Both hematoxylin/eosin (H E) and hMHCI staining (black arrows) of liver sections (A, Control mice, staining of liver metastases sections; B, E4031-treated mice, staining of liver sections) are reported. (C) IHC experiments on control mice (CONTROL) and E4031-treated (E4031) mice. Both hematoxylin/eosin (H E) and hMHCI staining of tumor masses and of a site of the coecum without macroscopic tumor masses (CONTROL), and of a site of the coecum without macroscopic tumor masses (E4031-treated)) are reported. Black arrows indicate the anti-hMHC staining. Bar: 100 μM for H E and for hMHC. (D) Representative pseudo-color BLI images tracking HCT116-luc cells emission in nude mice, 13 days after splenic HCT116-luc injection. Color bar represents light intensity levels reported as counts per minute (cpm). (E) Livers from animals sacrificed three weeks after cell injection and two weeks after beginning the pharmacological treatment. (F) H E and VEGF-A staining revealing larger necrotic areas and the presence of a higher amount of VEGF-A (brown staining) in Control compared to E4031-treated mice.

Techniques Used: Mouse Assay, Staining, Immunohistochemistry, Injection

4) Product Images from "Effect of Aplidin in acute lymphoblastic leukaemia cells"

Article Title: Effect of Aplidin in acute lymphoblastic leukaemia cells

Journal: British Journal of Cancer

doi: 10.1038/sj.bjc.6601130

Vascular endothelial growth factor-A concentration in the medium of ALL-PO cells treated for 1 h with 20 n M Aplidin and evaluated at 0, 6 and 24 h after drug-washout. The values express the % of VEGF concentration in the medium of treated cells with regard to control cells.
Figure Legend Snippet: Vascular endothelial growth factor-A concentration in the medium of ALL-PO cells treated for 1 h with 20 n M Aplidin and evaluated at 0, 6 and 24 h after drug-washout. The values express the % of VEGF concentration in the medium of treated cells with regard to control cells.

Techniques Used: Concentration Assay

Vascular endothelial growth factor mRNA levels in human leukaemic ALL-PO, ALL-MIK and Reh cell lines treated with 20 n M  Aplidin and performed at different time intervals after drug-washout. Data have been obtained by densitometric analysis and expressend as % of control untreated cells. Each column represents the mean of three independent replicates. The bars represent s.d.  * = P
Figure Legend Snippet: Vascular endothelial growth factor mRNA levels in human leukaemic ALL-PO, ALL-MIK and Reh cell lines treated with 20 n M Aplidin and performed at different time intervals after drug-washout. Data have been obtained by densitometric analysis and expressend as % of control untreated cells. Each column represents the mean of three independent replicates. The bars represent s.d. * = P

Techniques Used:

5) Product Images from "Basal and apical regulation of VEGF-A and placenta growth factor in the RPE/choroid and primary RPE"

Article Title: Basal and apical regulation of VEGF-A and placenta growth factor in the RPE/choroid and primary RPE

Journal: Molecular Vision

doi:

Influence of NF-κB on apical and basal VEGF-A secretion. In the RPE/choroid, the inhibition of nuclear factor-kappa B (NF-κB) significantly reduces vascular endothelial growth factor (VEGF)-A at 24 h and 48 h both on the apical ( A ) and basal sides ( B ). Similar results are obtained in primary RPE cell culture, with a significant reduction of VEGF-A secretion on the apical ( C ) and basal ( D ) sides, at 24 h and 48 h). Supernatants were collected for 24 h and were analyzed in enzyme-linked immunosorbent assay (ELISA). Significance was determined with the Student t test; + p
Figure Legend Snippet: Influence of NF-κB on apical and basal VEGF-A secretion. In the RPE/choroid, the inhibition of nuclear factor-kappa B (NF-κB) significantly reduces vascular endothelial growth factor (VEGF)-A at 24 h and 48 h both on the apical ( A ) and basal sides ( B ). Similar results are obtained in primary RPE cell culture, with a significant reduction of VEGF-A secretion on the apical ( C ) and basal ( D ) sides, at 24 h and 48 h). Supernatants were collected for 24 h and were analyzed in enzyme-linked immunosorbent assay (ELISA). Significance was determined with the Student t test; + p

Techniques Used: Inhibition, Cell Culture, Enzyme-linked Immunosorbent Assay

Influence of p38 on apical and basal VEGF-A secretion. A : In the RPE/choroid, inhibition of p38 significantly reduces the secretion of vascular endothelial growth factor (VEGF)-A on the apical side at 48 h. B : On the basal side, the inhibition of p38 reduces VEGF-A secretion at 24 h and at 48 h. In primary RPE cell culture, no influence of p38 on the secretion was found on the apical side ( C ), while p38 inhibition reduces VEGF-A secretion at the basal side significantly at 24 h and at 48 h ( D ). Supernatants were collected for 24 h and were analyzed in enzyme-linked immunosorbent assay (ELISA). Significance was determined with the Student t test; + p
Figure Legend Snippet: Influence of p38 on apical and basal VEGF-A secretion. A : In the RPE/choroid, inhibition of p38 significantly reduces the secretion of vascular endothelial growth factor (VEGF)-A on the apical side at 48 h. B : On the basal side, the inhibition of p38 reduces VEGF-A secretion at 24 h and at 48 h. In primary RPE cell culture, no influence of p38 on the secretion was found on the apical side ( C ), while p38 inhibition reduces VEGF-A secretion at the basal side significantly at 24 h and at 48 h ( D ). Supernatants were collected for 24 h and were analyzed in enzyme-linked immunosorbent assay (ELISA). Significance was determined with the Student t test; + p

Techniques Used: Inhibition, Cell Culture, Enzyme-linked Immunosorbent Assay

Influence of SP-1 on apical and basal VEGF-A secretion. In the RPE/choroid, the inhibition of SP-1 significantly reduces vascular endothelial growth factor (VEGF)-A at 24 h and 48 h on the apical ( A ) and basal sides ( B ). Similar results are obtained in primary RPE cell culture, with a significant decrease in VEGF-A secretion on the apical ( C ) and basal ( D ) sides, at 24 h and 48 h. Supernatants were collected for 24 h. Significance was determined with the Student t test; ++ p
Figure Legend Snippet: Influence of SP-1 on apical and basal VEGF-A secretion. In the RPE/choroid, the inhibition of SP-1 significantly reduces vascular endothelial growth factor (VEGF)-A at 24 h and 48 h on the apical ( A ) and basal sides ( B ). Similar results are obtained in primary RPE cell culture, with a significant decrease in VEGF-A secretion on the apical ( C ) and basal ( D ) sides, at 24 h and 48 h. Supernatants were collected for 24 h. Significance was determined with the Student t test; ++ p

Techniques Used: Inhibition, Cell Culture

Influence of VEGFR-2 on apical and basal VEGF-A secretion. In the RPE/choroid, the inhibition of vascular endothelial growth factor (VEGF)-2 reduces VEGF-A secretion on the apical side ( A ) but not on the basal side ( B ). In contrast, in primary RPE cell culture, the inhibition of VEGFR-2 did not influence the secretion of VEGFR-2, at the apical side ( C ) or at the basal side ( D ). Supernatants were collected for 24 h and were analyzed in enzyme-linked immunosorbent assay (ELISA). In the whole cell lysis of the primary RPE cells, analyzed with western blot, a significant reduction was found in VEGF-A expression after 48 h of VEGFR-2 inhibition. Representative blots of VEGF-A and β-actin control are shown, as well as a densitometric evaluation of VEGF-A normalized for β-actin ( E ). Significance was determined with the Student t test; + p
Figure Legend Snippet: Influence of VEGFR-2 on apical and basal VEGF-A secretion. In the RPE/choroid, the inhibition of vascular endothelial growth factor (VEGF)-2 reduces VEGF-A secretion on the apical side ( A ) but not on the basal side ( B ). In contrast, in primary RPE cell culture, the inhibition of VEGFR-2 did not influence the secretion of VEGFR-2, at the apical side ( C ) or at the basal side ( D ). Supernatants were collected for 24 h and were analyzed in enzyme-linked immunosorbent assay (ELISA). In the whole cell lysis of the primary RPE cells, analyzed with western blot, a significant reduction was found in VEGF-A expression after 48 h of VEGFR-2 inhibition. Representative blots of VEGF-A and β-actin control are shown, as well as a densitometric evaluation of VEGF-A normalized for β-actin ( E ). Significance was determined with the Student t test; + p

Techniques Used: Inhibition, Cell Culture, Enzyme-linked Immunosorbent Assay, Lysis, Western Blot, Expressing

Basal VEGF-A and PlGF secretion. A : In the RPE/choroid, vascular endothelial growth factor (VEGF)-A is constitutively secreted with significantly higher secretion on the basal side. B : Placental growth factor (PlGF) is also constitutively secreted with only minor secretion on the apical and significantly stronger secretion on the basal side. C : In the RPE cell culture, VEGF is basally and apically secreted, with a stronger secretion on the basal side. D : In RPE cell culture, no PlGF was found. E : The secretion of VEGF in the RPE cell culture is stable over time. Supernatants were collected for 24 h and were analyzed in enzyme-linked immunosorbent assay (ELISA). E : Supernatant was collected for 24 h, and the medium was changed and recollected after another 24 h. Significance was determined with the Student t test; +++ p
Figure Legend Snippet: Basal VEGF-A and PlGF secretion. A : In the RPE/choroid, vascular endothelial growth factor (VEGF)-A is constitutively secreted with significantly higher secretion on the basal side. B : Placental growth factor (PlGF) is also constitutively secreted with only minor secretion on the apical and significantly stronger secretion on the basal side. C : In the RPE cell culture, VEGF is basally and apically secreted, with a stronger secretion on the basal side. D : In RPE cell culture, no PlGF was found. E : The secretion of VEGF in the RPE cell culture is stable over time. Supernatants were collected for 24 h and were analyzed in enzyme-linked immunosorbent assay (ELISA). E : Supernatant was collected for 24 h, and the medium was changed and recollected after another 24 h. Significance was determined with the Student t test; +++ p

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

Regulation of PlGF secretion in the RPE/choroid. In general, placental growth factor (PlGF) secretion increased during cultivation. The inhibition of nuclear factor-kappa B (NF-κB) displayed a significant influence on PlGF secretion on the apical side ( A ) and on the basal side ( B ) at 24 h and at 48 h. The inhibition of SP-1 significantly reduced vascular endothelial growth factor (VEGF)-A secretion on the apical side after 48 h ( C ) and on the basal side at 24 h and at 48 h ( D ). The reduction of PlGF after p38 inhibition did not reach significance on the apical side ( E ) but did on the basal side after 48 h ( F ). The inhibition of VEGFR-2 did not significantly reduce PlGF at the apical side ( G ) but displayed significant effects on the basal side after 24 h and 48 h ( H ). Supernatants were collected for 24 h and were analyzed in enzyme-linked immunosorbent assay (ELISA). Significance was determined with the Student t test; + p
Figure Legend Snippet: Regulation of PlGF secretion in the RPE/choroid. In general, placental growth factor (PlGF) secretion increased during cultivation. The inhibition of nuclear factor-kappa B (NF-κB) displayed a significant influence on PlGF secretion on the apical side ( A ) and on the basal side ( B ) at 24 h and at 48 h. The inhibition of SP-1 significantly reduced vascular endothelial growth factor (VEGF)-A secretion on the apical side after 48 h ( C ) and on the basal side at 24 h and at 48 h ( D ). The reduction of PlGF after p38 inhibition did not reach significance on the apical side ( E ) but did on the basal side after 48 h ( F ). The inhibition of VEGFR-2 did not significantly reduce PlGF at the apical side ( G ) but displayed significant effects on the basal side after 24 h and 48 h ( H ). Supernatants were collected for 24 h and were analyzed in enzyme-linked immunosorbent assay (ELISA). Significance was determined with the Student t test; + p

Techniques Used: Inhibition, Enzyme-linked Immunosorbent Assay

6) Product Images from "Linking Power Doppler Ultrasound to the Presence of Th17 Cells in the Rheumatoid Arthritis Joint"

Article Title: Linking Power Doppler Ultrasound to the Presence of Th17 Cells in the Rheumatoid Arthritis Joint

Journal: PLoS ONE

doi: 10.1371/journal.pone.0012516

The presence of Th17 cells in SF is linked to increased VEGF in SF. A) VEGF-A levels in cell-free RA SF and matched paired serum were determined by ELISA (n = 15). B) Scatter plot of VEGF-A levels in SF vs. knee PDUS score, and the corresponding Spearman correlation coefficient and p-values. The regression line and 95% confidence intervals are shown. C) SF VEGF-A levels in RA patients stratified based on their frequency of total IL-17+ CD4+ T cells or total IFNγ+ CD4+ T cells (below or above the median level). Groups were compared using Mann Whitney U tests. (n = 12) D) RA synovial fibroblasts (n = 6) were cultured for 48 hours in the presence of 1 ng/ml rIL-17, 10 ng/ml rIL-17, 10 ng/ml rTNFα or 10 ng/ml rIFNγ. VEGF-A and IL-6 were measured in culture supernatants by ELISA. Comparison between groups was made using one-way ANOVA; * p
Figure Legend Snippet: The presence of Th17 cells in SF is linked to increased VEGF in SF. A) VEGF-A levels in cell-free RA SF and matched paired serum were determined by ELISA (n = 15). B) Scatter plot of VEGF-A levels in SF vs. knee PDUS score, and the corresponding Spearman correlation coefficient and p-values. The regression line and 95% confidence intervals are shown. C) SF VEGF-A levels in RA patients stratified based on their frequency of total IL-17+ CD4+ T cells or total IFNγ+ CD4+ T cells (below or above the median level). Groups were compared using Mann Whitney U tests. (n = 12) D) RA synovial fibroblasts (n = 6) were cultured for 48 hours in the presence of 1 ng/ml rIL-17, 10 ng/ml rIL-17, 10 ng/ml rTNFα or 10 ng/ml rIFNγ. VEGF-A and IL-6 were measured in culture supernatants by ELISA. Comparison between groups was made using one-way ANOVA; * p

Techniques Used: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Cell Culture

7) Product Images from "Continuous delivery of propranolol from liposomes-in-microspheres significantly inhibits infantile hemangioma growth"

Article Title: Continuous delivery of propranolol from liposomes-in-microspheres significantly inhibits infantile hemangioma growth

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S137634

The VEGF-A and bFGF expression level of HemSCs after treatment. Notes: After HemSCs were seeded on a fibronectin-coated 6-well plate, the cells were treated with various concentration of drugs for 96 hours. The VEGF-A and bFGF concentrations of the supernatant of the treated cells were measured by VEGF-A or bFGF ELISA kits, and the VEGF-A and bFGF mRNA level of the treated cells was measured by RT-PCR. The relative mRNA ( A and C ) or protein ( B and D ) level was expressed as the percentage of the mRNA or protein of treated group relative to the untreated group. Data are expressed as mean ± SD (n=3). * P
Figure Legend Snippet: The VEGF-A and bFGF expression level of HemSCs after treatment. Notes: After HemSCs were seeded on a fibronectin-coated 6-well plate, the cells were treated with various concentration of drugs for 96 hours. The VEGF-A and bFGF concentrations of the supernatant of the treated cells were measured by VEGF-A or bFGF ELISA kits, and the VEGF-A and bFGF mRNA level of the treated cells was measured by RT-PCR. The relative mRNA ( A and C ) or protein ( B and D ) level was expressed as the percentage of the mRNA or protein of treated group relative to the untreated group. Data are expressed as mean ± SD (n=3). * P

Techniques Used: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction

The VEGF-A and bFGF expression level of HemSCs after treatment. Notes: After HemSCs were seeded on a fibronectin-coated 6-well plate, the cells were treated with various concentration of drugs for 96 hours. The VEGF-A and bFGF concentrations of the supernatant of the treated cells were measured by VEGF-A or bFGF ELISA kits, and the VEGF-A and bFGF mRNA level of the treated cells was measured by RT-PCR. The relative mRNA ( A and C ) or protein ( B and D ) level was expressed as the percentage of the mRNA or protein of treated group relative to the untreated group. Data are expressed as mean ± SD (n=3). * P
Figure Legend Snippet: The VEGF-A and bFGF expression level of HemSCs after treatment. Notes: After HemSCs were seeded on a fibronectin-coated 6-well plate, the cells were treated with various concentration of drugs for 96 hours. The VEGF-A and bFGF concentrations of the supernatant of the treated cells were measured by VEGF-A or bFGF ELISA kits, and the VEGF-A and bFGF mRNA level of the treated cells was measured by RT-PCR. The relative mRNA ( A and C ) or protein ( B and D ) level was expressed as the percentage of the mRNA or protein of treated group relative to the untreated group. Data are expressed as mean ± SD (n=3). * P

Techniques Used: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction

8) Product Images from "Group V Secreted Phospholipase A2 Induces the Release of Proangiogenic and Antiangiogenic Factors by Human Neutrophils"

Article Title: Group V Secreted Phospholipase A2 Induces the Release of Proangiogenic and Antiangiogenic Factors by Human Neutrophils

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.00443

(A–D) Effect of Me-Indoxam or with Heparinase on human group V (hGV)-induced vascular endothelial growth factor (VEGF)-A, CXCL8/IL-8, Ang1, and VEGF-A 165b release from PMNs. hGV (3 µg/ml) was preincubated (37°C, 20 min) with Me-Indoxam (0.1 µM) or control medium. PMNs were then incubated (37°C, 1 h) with heparinase (0.4 U/ml) or control medium and then stimulated (37°C, 30 min) with hGV alone or with the combination of hGV with Me-Indoxam. (E,F) α V β 3 (P11) and α 4 β 1 (TCS2314) receptor antagonists inhibit GV-induced PMNs production of antiangiogenic factors. PMNs were preincubated (37°C, 30 min) with or without P11 and TCS 2314 (100 nM) and then stimulated (37°C, 30 min) with hGV (3 µg/ml). Data are the mean ± SD of eight different preparations of PMNs. * p
Figure Legend Snippet: (A–D) Effect of Me-Indoxam or with Heparinase on human group V (hGV)-induced vascular endothelial growth factor (VEGF)-A, CXCL8/IL-8, Ang1, and VEGF-A 165b release from PMNs. hGV (3 µg/ml) was preincubated (37°C, 20 min) with Me-Indoxam (0.1 µM) or control medium. PMNs were then incubated (37°C, 1 h) with heparinase (0.4 U/ml) or control medium and then stimulated (37°C, 30 min) with hGV alone or with the combination of hGV with Me-Indoxam. (E,F) α V β 3 (P11) and α 4 β 1 (TCS2314) receptor antagonists inhibit GV-induced PMNs production of antiangiogenic factors. PMNs were preincubated (37°C, 30 min) with or without P11 and TCS 2314 (100 nM) and then stimulated (37°C, 30 min) with hGV (3 µg/ml). Data are the mean ± SD of eight different preparations of PMNs. * p

Techniques Used: Incubation

Human secreted phospholipases A 2 (sPLA 2 s) induce the release of vascular endothelial growth factors (VEGFs), Ang1, and CXCL8/IL-8 from PMNs . PMNs were incubated (37°C, 3 h) with sPLA 2 [5 µg/ml, human group IB (hGIB), hGIIA, hGIIE, hGIIF, hGV, hGX, and hGXIIA] or control medium (A–D) . At the end of incubation, the supernatants were collected and centrifuged (1,000 × g , 4°C, 5 min). VEGF-A (A) , CXCL8/IL-8 (B) , Ang1 (C) , and VEGF-A 165b (D) were determined by ELISA. The values are expressed as picograms or nanograms of mediators per 10 6 cells. The results are the mean ± SD of eight different preparations of PMNs. * p
Figure Legend Snippet: Human secreted phospholipases A 2 (sPLA 2 s) induce the release of vascular endothelial growth factors (VEGFs), Ang1, and CXCL8/IL-8 from PMNs . PMNs were incubated (37°C, 3 h) with sPLA 2 [5 µg/ml, human group IB (hGIB), hGIIA, hGIIE, hGIIF, hGV, hGX, and hGXIIA] or control medium (A–D) . At the end of incubation, the supernatants were collected and centrifuged (1,000 × g , 4°C, 5 min). VEGF-A (A) , CXCL8/IL-8 (B) , Ang1 (C) , and VEGF-A 165b (D) were determined by ELISA. The values are expressed as picograms or nanograms of mediators per 10 6 cells. The results are the mean ± SD of eight different preparations of PMNs. * p

Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

Effect of increasing concentrations of human group V (hGV) and human group X (hGX) on vascular endothelial growth factor (VEGF)-A (A), CXCL8/IL-8 (B), angiopoietin 1 (Ang1) (C), and VEGF-A 165b (D) release from PMNs . PMNs were incubated (37°C, 3 h) with hGV and hGX (0.3–10 µg/ml) or control medium. (E–H) Kinetics of hGV-induced release of VEGFs, Ang1, and CXCL8/IL-8 from PMNs. The cells were incubated (37°C, 5–30 min) with hGV (3 µg/ml). At the end of incubations, the supernatants were collected and centrifuged (1,000 × g , 4°C, 5 min). Data are the mean ± SD of different eight preparations of PMNs *(for hGV) and § (for hGX) p
Figure Legend Snippet: Effect of increasing concentrations of human group V (hGV) and human group X (hGX) on vascular endothelial growth factor (VEGF)-A (A), CXCL8/IL-8 (B), angiopoietin 1 (Ang1) (C), and VEGF-A 165b (D) release from PMNs . PMNs were incubated (37°C, 3 h) with hGV and hGX (0.3–10 µg/ml) or control medium. (E–H) Kinetics of hGV-induced release of VEGFs, Ang1, and CXCL8/IL-8 from PMNs. The cells were incubated (37°C, 5–30 min) with hGV (3 µg/ml). At the end of incubations, the supernatants were collected and centrifuged (1,000 × g , 4°C, 5 min). Data are the mean ± SD of different eight preparations of PMNs *(for hGV) and § (for hGX) p

Techniques Used: Incubation

Effect of fMLF, LPS, and phorbolmyristate acetate (PMA) on vascular endothelial growth factors (VEGFs), angiopoietin 1 (Ang1), and CXCL8/IL-8 release from PMNs . PMNs were incubated (37°C, 3 h) with fMLF (50 nM), LPS (100 ng/ml), PMA (80 nM), or control medium (A–D) . At the end of incubation, the supernatants were collected and centrifuged (1,000 × g , 4°C, 5 min). VEGF-A (A) , CXCL8/IL-8 (B) , Ang1 (C) , and VEGF-A 165b (D) were determined by ELISA. The values are expressed as picograms or nanograms of mediators per 10 6 cells. The results are the mean ± SD of eight different preparations of PMNs. * p
Figure Legend Snippet: Effect of fMLF, LPS, and phorbolmyristate acetate (PMA) on vascular endothelial growth factors (VEGFs), angiopoietin 1 (Ang1), and CXCL8/IL-8 release from PMNs . PMNs were incubated (37°C, 3 h) with fMLF (50 nM), LPS (100 ng/ml), PMA (80 nM), or control medium (A–D) . At the end of incubation, the supernatants were collected and centrifuged (1,000 × g , 4°C, 5 min). VEGF-A (A) , CXCL8/IL-8 (B) , Ang1 (C) , and VEGF-A 165b (D) were determined by ELISA. The values are expressed as picograms or nanograms of mediators per 10 6 cells. The results are the mean ± SD of eight different preparations of PMNs. * p

Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

Effect of Me-Indoxam (A,B) and RO092906A (C,D) on fMLF-induced vascular endothelial growth factor (VEGF)-A and CXCL8/IL-8 release from PMNs . Cells were preincubated (37°C, 20 min) with or without Me-Indoxam and RO092906A (0.1 µM) and then stimulated (37°C, 1–6 h,) with fMLF (50 nM). VEGF-A (A–C) and CXCL8/IL-8 (B–D) release was determined by ELISA. Data are the mean ± SD of eight different preparations of PMNs. * p
Figure Legend Snippet: Effect of Me-Indoxam (A,B) and RO092906A (C,D) on fMLF-induced vascular endothelial growth factor (VEGF)-A and CXCL8/IL-8 release from PMNs . Cells were preincubated (37°C, 20 min) with or without Me-Indoxam and RO092906A (0.1 µM) and then stimulated (37°C, 1–6 h,) with fMLF (50 nM). VEGF-A (A–C) and CXCL8/IL-8 (B–D) release was determined by ELISA. Data are the mean ± SD of eight different preparations of PMNs. * p

Techniques Used: Enzyme-linked Immunosorbent Assay

Human neutrophils (PMNs) constitutively express different forms of vascular endothelial growth factors (VEGF) and angiopoietins . (A) VEGFA 165a , VEGFA 165b , VEGFB, VEGFC, VEGFD, Ang1 , and Ang2 mRNA expression in PMNs. The results are the mean ± SD of four different preparations of PMNs. RNA extraction from resting PMNs and RT-PCR was performed as described under Section “ Materials and Methods .” (B) Detection of VEGF and Ang proteins. Freshly isolated PMNs were lysed in Tryton 0.1%, and the concentrations of VEGFs and Angs were determined by ELISA. The results are the mean ± SD of six different preparations of PMNs.
Figure Legend Snippet: Human neutrophils (PMNs) constitutively express different forms of vascular endothelial growth factors (VEGF) and angiopoietins . (A) VEGFA 165a , VEGFA 165b , VEGFB, VEGFC, VEGFD, Ang1 , and Ang2 mRNA expression in PMNs. The results are the mean ± SD of four different preparations of PMNs. RNA extraction from resting PMNs and RT-PCR was performed as described under Section “ Materials and Methods .” (B) Detection of VEGF and Ang proteins. Freshly isolated PMNs were lysed in Tryton 0.1%, and the concentrations of VEGFs and Angs were determined by ELISA. The results are the mean ± SD of six different preparations of PMNs.

Techniques Used: Expressing, RNA Extraction, Reverse Transcription Polymerase Chain Reaction, Isolation, Enzyme-linked Immunosorbent Assay

9) Product Images from "Dietary oleuropein inhibits tumor angiogenesis and lymphangiogenesis in the B16F10 melanoma allograft model: a mechanism for the suppression of high-fat diet-induced solid tumor growth and lymph node metastasis"

Article Title: Dietary oleuropein inhibits tumor angiogenesis and lymphangiogenesis in the B16F10 melanoma allograft model: a mechanism for the suppression of high-fat diet-induced solid tumor growth and lymph node metastasis

Journal: Oncotarget

doi: 10.18632/oncotarget.16757

The cross-talk between tumor cells, M2-cells and adipocytes results in release of substantial amounts of VEGF-A and VEGF-D under hypoxic conditions stimulating angiogenesis and lymphangiogenesis, respectively B16F10s, M2-MΦs, and 3T3-L1 adipocytes were cultured separately or co-cultured under hypoxic conditions (1% O 2 , 5% CO 2 , and 94% N 2 ) with or without OL treatment (0 - 10 μmol/L). ( A ) The concentrations of VEGF-A in conditioned media (CM) were estimated by using ELISA. Each bar represents the mean ± SEM ( n = 4). ( B ) Cell proliferation was measured by using BrdU incorporation assay. HUVECs were treated for 6 h with CM collected under hypoxic conditions (hypoxic-CM). BrdU was then added, and the incubation was continued for a further 3 h to analyze BrdU incorporation into DNA ( n = 3). ( C ) For wound-healing assay, HUVECs were plated and grown to 100% confluence; subsequently, an injury line was created using a yellow pipette tip and the cells were treated with hypoxic-CM for 16 h. (Upper panel) Wound closure was visualized under phase-contrast microscopy. (Lower panel) Quantification of wound closure ( n = 3). ( D ) For tube formation assay, HUVECs were plated in Matrigel-coated plates. After 24 h, HUVECs were treated with hypoxic-CM in the absence or presence of an anti-VEGF-A antibody. (Upper panel) Tube formation was visualized under phase-contrast microscopy. (Lower panel) Quantification of HUVEC tube formation ( n = 3). ( E ) VEGF-D concentrations in CM were estimated using ELISA ( n = 4). ( F ) For tube formation assays, LECs were treated with hypoxic-CM in the absence or presence of an anti-VEGF-D antibody. (Upper panel) Tube formation was visualized under phase-contrast microscopy. (Lower panel) Quantification of LEC tube formation. Each bar represents the mean ± SEM ( n = 3). ♠ Significantly different from the DMEM group; *significantly different from the B16F10 group; $ significantly different from the M2-MΦ group; # significantly different from the 3T3-L1 group; and ψ significantly different from the B16F10/M2-MF/3T3-L1 co-culture group, P
Figure Legend Snippet: The cross-talk between tumor cells, M2-cells and adipocytes results in release of substantial amounts of VEGF-A and VEGF-D under hypoxic conditions stimulating angiogenesis and lymphangiogenesis, respectively B16F10s, M2-MΦs, and 3T3-L1 adipocytes were cultured separately or co-cultured under hypoxic conditions (1% O 2 , 5% CO 2 , and 94% N 2 ) with or without OL treatment (0 - 10 μmol/L). ( A ) The concentrations of VEGF-A in conditioned media (CM) were estimated by using ELISA. Each bar represents the mean ± SEM ( n = 4). ( B ) Cell proliferation was measured by using BrdU incorporation assay. HUVECs were treated for 6 h with CM collected under hypoxic conditions (hypoxic-CM). BrdU was then added, and the incubation was continued for a further 3 h to analyze BrdU incorporation into DNA ( n = 3). ( C ) For wound-healing assay, HUVECs were plated and grown to 100% confluence; subsequently, an injury line was created using a yellow pipette tip and the cells were treated with hypoxic-CM for 16 h. (Upper panel) Wound closure was visualized under phase-contrast microscopy. (Lower panel) Quantification of wound closure ( n = 3). ( D ) For tube formation assay, HUVECs were plated in Matrigel-coated plates. After 24 h, HUVECs were treated with hypoxic-CM in the absence or presence of an anti-VEGF-A antibody. (Upper panel) Tube formation was visualized under phase-contrast microscopy. (Lower panel) Quantification of HUVEC tube formation ( n = 3). ( E ) VEGF-D concentrations in CM were estimated using ELISA ( n = 4). ( F ) For tube formation assays, LECs were treated with hypoxic-CM in the absence or presence of an anti-VEGF-D antibody. (Upper panel) Tube formation was visualized under phase-contrast microscopy. (Lower panel) Quantification of LEC tube formation. Each bar represents the mean ± SEM ( n = 3). ♠ Significantly different from the DMEM group; *significantly different from the B16F10 group; $ significantly different from the M2-MΦ group; # significantly different from the 3T3-L1 group; and ψ significantly different from the B16F10/M2-MF/3T3-L1 co-culture group, P

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, BrdU Incorporation Assay, Incubation, Wound Healing Assay, Transferring, Microscopy, Tube Formation Assay, Co-Culture Assay

OL suppresses HFD-stimulated expression of proteins related to angiogenesis and lymphangiogenesis in the tumors and LNs ( A , B , G , H ) Tumor sections were stained with the indicated antibodies. (A, G) Representative immunofluorescence images. The staining intensity of (B) VEGF-A, VEGFR2, VEGF-C, VEGF-D, and VEGFR3 and (H) HIF-1α and GLUT-1 was quantified ( n = 5–9). ( C , D ) The concentrations of (C) VEGF-A and (D) VEGF-D in tumor tissues were measured using ELISA ( n = 10). ( E , F ) Sections of the LNs were stained with the indicated antibodies. Nuclei were counterstained with DAPI (blue). (E) Representative stained images are shown. (F) The staining intensity of VEGF-A, VEGF-C, and VEGF-D was quantified ( n = 4). Each bar represents the mean ± SEM ( n = 5–9). *Significantly different from the CD group, P
Figure Legend Snippet: OL suppresses HFD-stimulated expression of proteins related to angiogenesis and lymphangiogenesis in the tumors and LNs ( A , B , G , H ) Tumor sections were stained with the indicated antibodies. (A, G) Representative immunofluorescence images. The staining intensity of (B) VEGF-A, VEGFR2, VEGF-C, VEGF-D, and VEGFR3 and (H) HIF-1α and GLUT-1 was quantified ( n = 5–9). ( C , D ) The concentrations of (C) VEGF-A and (D) VEGF-D in tumor tissues were measured using ELISA ( n = 10). ( E , F ) Sections of the LNs were stained with the indicated antibodies. Nuclei were counterstained with DAPI (blue). (E) Representative stained images are shown. (F) The staining intensity of VEGF-A, VEGF-C, and VEGF-D was quantified ( n = 4). Each bar represents the mean ± SEM ( n = 5–9). *Significantly different from the CD group, P

Techniques Used: Expressing, Staining, Immunofluorescence, Enzyme-linked Immunosorbent Assay

10) Product Images from "Biocompatible, Purified VEGF-A mRNA Improves Cardiac Function after Intracardiac Injection 1 Week Post-myocardial Infarction in Swine"

Article Title: Biocompatible, Purified VEGF-A mRNA Improves Cardiac Function after Intracardiac Injection 1 Week Post-myocardial Infarction in Swine

Journal: Molecular Therapy. Methods & Clinical Development

doi: 10.1016/j.omtm.2018.04.003

VEGF-A Protein Pharmacokinetics and Effect on Cardiac Troponin I Release and Left Ventricular Function Post-myocardial Infarction following Intracardiac Injection of Purified VEGF mRNA in a Biologically Compatible Citrate-Saline Formulation (cs- VEGF mRNA) (A) VEGF-A protein pharmacokinetics after single cardiac injections of cs- VEGF mRNA versus RNAiMax (lipid nanoparticle [LNP]) in the mouse over 72 hr (n = 3 per time point and formulation). (B) Cross-species comparison of VEGF-A protein levels following increasing intracardiac dosing of cs- VEGF mRNA (n = 3–4 for each dose and species). (C) Pharmacokinetics of VEGF-A protein produced by increasing intracardiac doses, 15, 150, and 1,800 μg, of cs- VEGF mRNA (n = 3 per time point and dose) in the rat. (D) 100 μg of cs- VEGF mRNA expressed up to 192 hr (n = 3 per time point). (E) Circulating cardiac troponin I (cTnI) levels at day 1 following permanent occlusion of the left anterior descending coronary artery to induce MI in the rat. Citrate-saline vehicle (C/S, n = 8) or cs- VEGF mRNA (150 and 1,800 μg dose groups pooled; n = 19) was intracardially injected at the time of the induction of the MI. (F) Left ventricular ejection fraction (EF), assessed by cardiac magnetic resonance imaging 8 days after the occlusion of the coronary artery. Data are means ± SEM.
Figure Legend Snippet: VEGF-A Protein Pharmacokinetics and Effect on Cardiac Troponin I Release and Left Ventricular Function Post-myocardial Infarction following Intracardiac Injection of Purified VEGF mRNA in a Biologically Compatible Citrate-Saline Formulation (cs- VEGF mRNA) (A) VEGF-A protein pharmacokinetics after single cardiac injections of cs- VEGF mRNA versus RNAiMax (lipid nanoparticle [LNP]) in the mouse over 72 hr (n = 3 per time point and formulation). (B) Cross-species comparison of VEGF-A protein levels following increasing intracardiac dosing of cs- VEGF mRNA (n = 3–4 for each dose and species). (C) Pharmacokinetics of VEGF-A protein produced by increasing intracardiac doses, 15, 150, and 1,800 μg, of cs- VEGF mRNA (n = 3 per time point and dose) in the rat. (D) 100 μg of cs- VEGF mRNA expressed up to 192 hr (n = 3 per time point). (E) Circulating cardiac troponin I (cTnI) levels at day 1 following permanent occlusion of the left anterior descending coronary artery to induce MI in the rat. Citrate-saline vehicle (C/S, n = 8) or cs- VEGF mRNA (150 and 1,800 μg dose groups pooled; n = 19) was intracardially injected at the time of the induction of the MI. (F) Left ventricular ejection fraction (EF), assessed by cardiac magnetic resonance imaging 8 days after the occlusion of the coronary artery. Data are means ± SEM.

Techniques Used: Injection, Purification, Produced, Magnetic Resonance Imaging

Effects of Intracardiac Purified cs- VEGF mRNA or Citrate-Saline Vehicle Injection 7 Days after Myocardial Infarction on Cardiac Function (A) Timeline of intracardiac injections of citrate-saline (vehicle), 1 mg cs- VEGF mRNA L, and 10 mg cs- VEGF mRNA H. (B) Cartoon representation of injection sites in green dots. (C) Left ventricular ejection fraction (LVEF) as measured by echocardiography before and after MI (day 0), 1 week after MI before injections (day 7), and 2 months after injections. Data are means ± SEM (n ≤ 5). *p
Figure Legend Snippet: Effects of Intracardiac Purified cs- VEGF mRNA or Citrate-Saline Vehicle Injection 7 Days after Myocardial Infarction on Cardiac Function (A) Timeline of intracardiac injections of citrate-saline (vehicle), 1 mg cs- VEGF mRNA L, and 10 mg cs- VEGF mRNA H. (B) Cartoon representation of injection sites in green dots. (C) Left ventricular ejection fraction (LVEF) as measured by echocardiography before and after MI (day 0), 1 week after MI before injections (day 7), and 2 months after injections. Data are means ± SEM (n ≤ 5). *p

Techniques Used: Purification, Injection

LacZ and VEGF mRNA Cardiac Transfection and Translation in Biologically Compatible Citrate-Saline Buffer (cs-mRNA) (A and B) 75 μg of lacZ mRNA was injected into the mouse heart and whole mount stained with X-gal after 48 hr. In situ (C and E) and immunohistochemistry (D and F) of 100 μg VEGF mRNA (C and D) intracardiac injection in rats and citrate-saline control (E and F). (G) Co-staining of troponin T and VEGF-A protein with immunohistochemistry showed that the injected VEGF mRNA was taken up and translated by cardiomyocytes. VEGF mRNA and protein expression were followed by in situ hybridization and immunohistochemistry 1 (H), 3 (I), 6 (J), 24 (K), and 48 (L) hr after intracardiac injection of 100 μg VEGF mRNA. (H) Arrow highlighting interstitial VEGF mRNA localization. (M) Cartoon representation of mRNA cardiac uptake.
Figure Legend Snippet: LacZ and VEGF mRNA Cardiac Transfection and Translation in Biologically Compatible Citrate-Saline Buffer (cs-mRNA) (A and B) 75 μg of lacZ mRNA was injected into the mouse heart and whole mount stained with X-gal after 48 hr. In situ (C and E) and immunohistochemistry (D and F) of 100 μg VEGF mRNA (C and D) intracardiac injection in rats and citrate-saline control (E and F). (G) Co-staining of troponin T and VEGF-A protein with immunohistochemistry showed that the injected VEGF mRNA was taken up and translated by cardiomyocytes. VEGF mRNA and protein expression were followed by in situ hybridization and immunohistochemistry 1 (H), 3 (I), 6 (J), 24 (K), and 48 (L) hr after intracardiac injection of 100 μg VEGF mRNA. (H) Arrow highlighting interstitial VEGF mRNA localization. (M) Cartoon representation of mRNA cardiac uptake.

Techniques Used: Transfection, Injection, Staining, In Situ, Immunohistochemistry, Expressing, In Situ Hybridization

Optimized VEGF-A mRNA Produces Functional Protein In Vitro (A) Purified mRNA with 1-methylpseudouridine (m1Ψ) produces higher levels of luciferase protein when compared to first generation (pseudouridine [Ψ]) in 20,000 HeLa cells with 200 ng of mRNA and (B) less innate immune reactivity measured as interferon-alpha secretion in 500,000 primary human PBMC cells after transfection with 500 ng of mRNA. (C) Time course of VEGF-A protein production in human aortic smooth muscle cells (hAoSMC) and in human iPS-derived cardiomyocytes (hiPS-CM) after transfection with purified VEGF mRNA, showing that relevant primary cardiac cell types can be transfected. VEGF protein transcribed from VEGF mRNA is biologically active and activates VEGFR2 and downstream signaling pathways, shown by phosphorylation of (D) VEGF-Receptor 2 in human endothelial cells (HUVECs), (E) endothelial nitric oxide synthase in HUVECs, and (F) AKT in mouse cardiac fibroblasts cells in culture (representative western blot images shown; n = 2). (G) VEGF-A protein produced from VEGF mRNA and recombinant VEGF-A (recVEGF-A) induce similar responses and increase endothelial cell proliferation (n = 3). (H) Endothelial cell migration (n = 2; VEGF-A dose: 10 ng/mL) and (I) angiogenic sprouting in endothelial cells (n = 3; VEGF-A dose: 10 ng/mL) compared to controls. (J) Representative images on angiogenic sprouting in control cells and cells stimulated with VEGF-A protein produced from VEGF mRNA. Data are presented as mean ± SEM. Control is medium only without VEGF-A protein. Asterisks represent: *p
Figure Legend Snippet: Optimized VEGF-A mRNA Produces Functional Protein In Vitro (A) Purified mRNA with 1-methylpseudouridine (m1Ψ) produces higher levels of luciferase protein when compared to first generation (pseudouridine [Ψ]) in 20,000 HeLa cells with 200 ng of mRNA and (B) less innate immune reactivity measured as interferon-alpha secretion in 500,000 primary human PBMC cells after transfection with 500 ng of mRNA. (C) Time course of VEGF-A protein production in human aortic smooth muscle cells (hAoSMC) and in human iPS-derived cardiomyocytes (hiPS-CM) after transfection with purified VEGF mRNA, showing that relevant primary cardiac cell types can be transfected. VEGF protein transcribed from VEGF mRNA is biologically active and activates VEGFR2 and downstream signaling pathways, shown by phosphorylation of (D) VEGF-Receptor 2 in human endothelial cells (HUVECs), (E) endothelial nitric oxide synthase in HUVECs, and (F) AKT in mouse cardiac fibroblasts cells in culture (representative western blot images shown; n = 2). (G) VEGF-A protein produced from VEGF mRNA and recombinant VEGF-A (recVEGF-A) induce similar responses and increase endothelial cell proliferation (n = 3). (H) Endothelial cell migration (n = 2; VEGF-A dose: 10 ng/mL) and (I) angiogenic sprouting in endothelial cells (n = 3; VEGF-A dose: 10 ng/mL) compared to controls. (J) Representative images on angiogenic sprouting in control cells and cells stimulated with VEGF-A protein produced from VEGF mRNA. Data are presented as mean ± SEM. Control is medium only without VEGF-A protein. Asterisks represent: *p

Techniques Used: Functional Assay, In Vitro, Purification, Luciferase, Transfection, Derivative Assay, Western Blot, Produced, Recombinant, Migration

Effects of Intracardiac Purified cs-VEGF mRNA or Citrate-Saline Vehicle Injection 7 Days after Myocardial Infarction on Infarct and Tissue Pathophysiology Assessed 2 Months after Injection (A) Representative slices encompassing VEGF mRNA injection sites processed immediately after harvesting the heart. (B) Schematic of tissue sampling regions used for downstream analyses. (C) Global infarct area. (D) Global infarct length. (E) Mid-ventricular infarct area. (F) Mid-ventricular infarct length. Quantification of (G) capillary growth, (H) arteriole growth, and (I) remote fibrosis. Representative images of each study arm for (J) isolectin-b4 (GFP)/Troponin (RFP) for angiogenesis assay, (K) SM-22α (GFP)/Troponin (RFP) staining for arteriogenesis assay, and (L) Masson Trichrome staining for remote fibrosis assay. Vehicle, cs- VEGF mRNA L, and cs- VEGF mRNA H represent citrate-saline, 1 mg cs- VEGF mRNA, and 10 mg cs- VEGF mRNA, respectively. Data are means ± SEM (n ≤ 5). *p
Figure Legend Snippet: Effects of Intracardiac Purified cs-VEGF mRNA or Citrate-Saline Vehicle Injection 7 Days after Myocardial Infarction on Infarct and Tissue Pathophysiology Assessed 2 Months after Injection (A) Representative slices encompassing VEGF mRNA injection sites processed immediately after harvesting the heart. (B) Schematic of tissue sampling regions used for downstream analyses. (C) Global infarct area. (D) Global infarct length. (E) Mid-ventricular infarct area. (F) Mid-ventricular infarct length. Quantification of (G) capillary growth, (H) arteriole growth, and (I) remote fibrosis. Representative images of each study arm for (J) isolectin-b4 (GFP)/Troponin (RFP) for angiogenesis assay, (K) SM-22α (GFP)/Troponin (RFP) staining for arteriogenesis assay, and (L) Masson Trichrome staining for remote fibrosis assay. Vehicle, cs- VEGF mRNA L, and cs- VEGF mRNA H represent citrate-saline, 1 mg cs- VEGF mRNA, and 10 mg cs- VEGF mRNA, respectively. Data are means ± SEM (n ≤ 5). *p

Techniques Used: Purification, Injection, Sampling, Angiogenesis Assay, Staining

11) Product Images from "Tie2 activation contributes to hemangiogenic regeneration after myelosuppression"

Article Title: Tie2 activation contributes to hemangiogenic regeneration after myelosuppression

Journal:

doi: 10.1182/blood-2004-11-4269

Role of VEGF-A. (A) Both 5-FU and Ang-1 induce VEGF-A expression in wild-type mice. Wild-type mice received a single intravenous injection of AdAng-1 (10 9 pfu), AdNull (10 9 pfu), or 5-FU (250 mg/kg). Introduction of Ang-1 and myelosuppression induced
Figure Legend Snippet: Role of VEGF-A. (A) Both 5-FU and Ang-1 induce VEGF-A expression in wild-type mice. Wild-type mice received a single intravenous injection of AdAng-1 (10 9 pfu), AdNull (10 9 pfu), or 5-FU (250 mg/kg). Introduction of Ang-1 and myelosuppression induced

Techniques Used: Expressing, Mouse Assay, Injection

12) Product Images from "Pancreatic cyst fluid VEGF-A and CEA: a highly accurate test for the diagnosis of serous cystic neoplasm"

Article Title: Pancreatic cyst fluid VEGF-A and CEA: a highly accurate test for the diagnosis of serous cystic neoplasm

Journal: Journal of the American College of Surgeons

doi: 10.1016/j.jamcollsurg.2017.05.003

ROC Curves ROC Curves for each biomarker alone and in combination. The AUC for CEA alone is 0.945, for VEGF-A alone is 0.983, and for VEGF-A/CEA Combination test is 0.993.
Figure Legend Snippet: ROC Curves ROC Curves for each biomarker alone and in combination. The AUC for CEA alone is 0.945, for VEGF-A alone is 0.983, and for VEGF-A/CEA Combination test is 0.993.

Techniques Used: Biomarker Assay

Pancreatic Cyst Fluid VEGF-A VEGF-A levels in pancreatic cyst fluid as determined by ELISA. Each VEGF-A value is shown on a log linear scale within the column consistent with pathologic diagnosis: SCN, pseudocyst, MCN, IPMN, or SPN. Horizontal black lines represent median values for each cyst type. The red dotted line is the cut-off (5,000 pg/mL) giving 100% sensitivity and 83.7% specificity.
Figure Legend Snippet: Pancreatic Cyst Fluid VEGF-A VEGF-A levels in pancreatic cyst fluid as determined by ELISA. Each VEGF-A value is shown on a log linear scale within the column consistent with pathologic diagnosis: SCN, pseudocyst, MCN, IPMN, or SPN. Horizontal black lines represent median values for each cyst type. The red dotted line is the cut-off (5,000 pg/mL) giving 100% sensitivity and 83.7% specificity.

Techniques Used: Enzyme-linked Immunosorbent Assay

CEA vs VEGF-A for SCN vs non-SCN CEA vs VEGF-A for SCN vs non-SCN. Each point displays the cyst fluid CEA and the VEGF-A level for a single pancreatic cyst on a log linear scale. Blue points are SCN, and red points are all other cysts. The horizontal dashed line is the CEA cut-off of 10 ng/mL; all cysts plotted below this line had a positive CEA test result. The vertical dotted line is the VEGF-A cut-off of 5,000 pg/mL; all cysts plotted to the right of this line had a positive VEGF-A result. The right lower quadrant formed by the two threshold lines contains cysts with a positive VEGF-A/CEA combination test. These are exclusively SCN, demonstrating 100% specificity and infinite positive likelihood ratio of the combination test.
Figure Legend Snippet: CEA vs VEGF-A for SCN vs non-SCN CEA vs VEGF-A for SCN vs non-SCN. Each point displays the cyst fluid CEA and the VEGF-A level for a single pancreatic cyst on a log linear scale. Blue points are SCN, and red points are all other cysts. The horizontal dashed line is the CEA cut-off of 10 ng/mL; all cysts plotted below this line had a positive CEA test result. The vertical dotted line is the VEGF-A cut-off of 5,000 pg/mL; all cysts plotted to the right of this line had a positive VEGF-A result. The right lower quadrant formed by the two threshold lines contains cysts with a positive VEGF-A/CEA combination test. These are exclusively SCN, demonstrating 100% specificity and infinite positive likelihood ratio of the combination test.

Techniques Used:

13) Product Images from "Semaphorin 6A regulates angiogenesis by modulating VEGF signaling"

Article Title: Semaphorin 6A regulates angiogenesis by modulating VEGF signaling

Journal: Blood

doi: 10.1182/blood-2012-02-410076

VEGF-A and FGF2 signaling in Sema6A -silenced endothelial cells. (A) Control and Sema6A -silenced HUVECs were incubated 15 minutes with VEGF-A (100 ng/mL). Cell lysates were tested for phosphorylated and total VEGFR2, AKT, and ERK1/2; β-actin staining
Figure Legend Snippet: VEGF-A and FGF2 signaling in Sema6A -silenced endothelial cells. (A) Control and Sema6A -silenced HUVECs were incubated 15 minutes with VEGF-A (100 ng/mL). Cell lysates were tested for phosphorylated and total VEGFR2, AKT, and ERK1/2; β-actin staining

Techniques Used: Incubation, Staining

Endothelial cells require VEGF signaling for survival. (A) VEGFA gene expression in Sema6A -silenced and control HUVECs was measured by quantitative PCR. The results reflect the mean (± SEM; n = 4) relative mRNA levels. (B) HUVECs were incubated
Figure Legend Snippet: Endothelial cells require VEGF signaling for survival. (A) VEGFA gene expression in Sema6A -silenced and control HUVECs was measured by quantitative PCR. The results reflect the mean (± SEM; n = 4) relative mRNA levels. (B) HUVECs were incubated

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Incubation

14) Product Images from "Hypoxic Conditioning Enhances the Angiogenic Paracrine Activity of Human Adipose-Derived Stem Cells"

Article Title: Hypoxic Conditioning Enhances the Angiogenic Paracrine Activity of Human Adipose-Derived Stem Cells

Journal: Stem Cells and Development

doi: 10.1089/scd.2012.0602

Degree of hypoxia differentially regulated the adipose derived stem cell (ASC) paracrine factor profile. Expression levels of the vascular endothelial growth factor-A (VEGF-A) (A) , VEGF-C (B) , angiogenin (ANG) (D) , and interleukin-8 (IL-8) (E) were upregulated
Figure Legend Snippet: Degree of hypoxia differentially regulated the adipose derived stem cell (ASC) paracrine factor profile. Expression levels of the vascular endothelial growth factor-A (VEGF-A) (A) , VEGF-C (B) , angiogenin (ANG) (D) , and interleukin-8 (IL-8) (E) were upregulated

Techniques Used: Derivative Assay, Expressing

The hypoxic ASC CM promoted angiogenesis in vivo in a VEGF-A- and ANG-dependent manner. (A) The hypoxic ASC CM increased CD31-positive vessels grown into the sponges at 2 weeks postimplantation. Removal of the VEGF-A and/or ANG from the hypoxic ASC CM significantly
Figure Legend Snippet: The hypoxic ASC CM promoted angiogenesis in vivo in a VEGF-A- and ANG-dependent manner. (A) The hypoxic ASC CM increased CD31-positive vessels grown into the sponges at 2 weeks postimplantation. Removal of the VEGF-A and/or ANG from the hypoxic ASC CM significantly

Techniques Used: In Vivo

Hypoxia increased VEGF-A, VEGF-C, and ANG secretion from ASCs. Treatment of ASCs with
Figure Legend Snippet: Hypoxia increased VEGF-A, VEGF-C, and ANG secretion from ASCs. Treatment of ASCs with

Techniques Used:

15) Product Images from "FcγRIIb inhibits immune complex-induced VEGF-A production and intranodal lymphangiogenesis"

Article Title: FcγRIIb inhibits immune complex-induced VEGF-A production and intranodal lymphangiogenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1413915111

IC-induced VEGF-A production abrogated by Erk inhibition. VEGF-A concentration in culture supernatants following incubation with media alone (−), ovalbumin (OVA), IgG-opsonized ovalbumin (OVA-IC), or OVA-IC with the addition of the MEK1/2 inhibitor
Figure Legend Snippet: IC-induced VEGF-A production abrogated by Erk inhibition. VEGF-A concentration in culture supernatants following incubation with media alone (−), ovalbumin (OVA), IgG-opsonized ovalbumin (OVA-IC), or OVA-IC with the addition of the MEK1/2 inhibitor

Techniques Used: Inhibition, Concentration Assay, Incubation

IgG immune complex-induced VEGF-A production by macrophages and DCs is inhibited by FcγRIIb. VEGF-A concentration in culture supernatants following 24-h incubation with media alone (−), ovalbumin (O), or IgG-opsonized ovalbumin (IC) using
Figure Legend Snippet: IgG immune complex-induced VEGF-A production by macrophages and DCs is inhibited by FcγRIIb. VEGF-A concentration in culture supernatants following 24-h incubation with media alone (−), ovalbumin (O), or IgG-opsonized ovalbumin (IC) using

Techniques Used: Concentration Assay, Incubation

Lymphangiogenesis and VEGF-A production occurs in autoimmune lymph nodes and immune complex-induced VEGF-A is increased in individuals with the lupus-associated FcγRIIB T/T232 polymorphism. ( A ) Representative confocal micrographs of inguinal lymph
Figure Legend Snippet: Lymphangiogenesis and VEGF-A production occurs in autoimmune lymph nodes and immune complex-induced VEGF-A is increased in individuals with the lupus-associated FcγRIIB T/T232 polymorphism. ( A ) Representative confocal micrographs of inguinal lymph

Techniques Used:

16) Product Images from "Active Immunotherapy Induces Antibody Responses that Target Tumor Angiogenesis"

Article Title: Active Immunotherapy Induces Antibody Responses that Target Tumor Angiogenesis

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-10-1852

Vaccinated melanoma patient K008 developed antibodies to multiple angiogenic cytokines. (A). Sera from K008, but not healthy donors (n=3) recognize VEGF-A, but not bFGF by immunoblotting (sera at 1:100). Control anti-cytokine antibodies are shown for comparison. (B). Longitudinal analysis of antibodies to VEGF family members in K008. Antigen specific IgG levels were determined with an ELISA (sera at 1:100). Arrows denote vaccinations. Day 0 is pre-vaccination. (C). Vaccination stimulated potent humoral reactions to angiopoietin-1 and -2 in K008. A subcutaneous nodule developed hemorrhagic necrosis after the fifth immunization. Day 0 is pre-vaccination.
Figure Legend Snippet: Vaccinated melanoma patient K008 developed antibodies to multiple angiogenic cytokines. (A). Sera from K008, but not healthy donors (n=3) recognize VEGF-A, but not bFGF by immunoblotting (sera at 1:100). Control anti-cytokine antibodies are shown for comparison. (B). Longitudinal analysis of antibodies to VEGF family members in K008. Antigen specific IgG levels were determined with an ELISA (sera at 1:100). Arrows denote vaccinations. Day 0 is pre-vaccination. (C). Vaccination stimulated potent humoral reactions to angiopoietin-1 and -2 in K008. A subcutaneous nodule developed hemorrhagic necrosis after the fifth immunization. Day 0 is pre-vaccination.

Techniques Used: Enzyme-linked Immunosorbent Assay

17) Product Images from "Nanoparticle-Mediated Delivery of shRNA.VEGF-A Plasmids Regresses Corneal Neovascularization"

Article Title: Nanoparticle-Mediated Delivery of shRNA.VEGF-A Plasmids Regresses Corneal Neovascularization

Journal: Investigative Ophthalmology & Visual Science

doi: 10.1167/iovs.11-9139

shRNA.VEGF-A NPs inhibit expression of VEGF-A protein. ELISA for total VEGF-A was performed on homogenates of vascularized corneas at 5 days after intrastromal injection with respective treatment or control ( n = 3–4/group; 6–8 corneas/group).
Figure Legend Snippet: shRNA.VEGF-A NPs inhibit expression of VEGF-A protein. ELISA for total VEGF-A was performed on homogenates of vascularized corneas at 5 days after intrastromal injection with respective treatment or control ( n = 3–4/group; 6–8 corneas/group).

Techniques Used: shRNA, Expressing, Enzyme-linked Immunosorbent Assay, Injection

shRNA.VEGF-A NPs regress KNV . Immunostaining for CD31 (panendothelial marker) was performed on vascularized corneas harvested at four weeks post-intrastromal injection with either treatment or control. Images of immunostained corneal whole mounts were
Figure Legend Snippet: shRNA.VEGF-A NPs regress KNV . Immunostaining for CD31 (panendothelial marker) was performed on vascularized corneas harvested at four weeks post-intrastromal injection with either treatment or control. Images of immunostained corneal whole mounts were

Techniques Used: shRNA, Immunostaining, Marker, Injection

KNV regression on brightfield microscopy. Representative brightfield micrographs of mouse corneas. ( A ) Corneas treated with vehicle, pre-treatment. ( B ) Cornea treated with vehicle 4 weeks post-treatment. ( C ) Cornea treated with pSEC.shRNA.VEGF-A PLGA
Figure Legend Snippet: KNV regression on brightfield microscopy. Representative brightfield micrographs of mouse corneas. ( A ) Corneas treated with vehicle, pre-treatment. ( B ) Cornea treated with vehicle 4 weeks post-treatment. ( C ) Cornea treated with pSEC.shRNA.VEGF-A PLGA

Techniques Used: Microscopy, shRNA

shRNA.VEGF-A NPs inhibit expression of VEGF-A mRNA. RT-PCR was performed on vascularized corneas harvested 2 days post-intrastromal injection of treatment or control ( n = 4–5 corneas/group), to study gene expression profiles of VEGF-A (VEGF 164
Figure Legend Snippet: shRNA.VEGF-A NPs inhibit expression of VEGF-A mRNA. RT-PCR was performed on vascularized corneas harvested 2 days post-intrastromal injection of treatment or control ( n = 4–5 corneas/group), to study gene expression profiles of VEGF-A (VEGF 164

Techniques Used: shRNA, Expressing, Reverse Transcription Polymerase Chain Reaction, Injection

18) Product Images from "α-Mangostin inhibits hypoxia-driven ROS-induced PSC activation and pancreatic cancer cell invasion"

Article Title: α-Mangostin inhibits hypoxia-driven ROS-induced PSC activation and pancreatic cancer cell invasion

Journal: Cancer letters

doi: 10.1016/j.canlet.2014.02.003

α-mangostin abolishes secretion of IL-6, VEGF-A and SDF-1 in activated PSCs under hypoxia exposure. PSCs were treated with hypoxia for 24 h in the presence or absence of 20 mM NAC or 16 μM a-mangostin (ct-M); the cells were then serum-starved
Figure Legend Snippet: α-mangostin abolishes secretion of IL-6, VEGF-A and SDF-1 in activated PSCs under hypoxia exposure. PSCs were treated with hypoxia for 24 h in the presence or absence of 20 mM NAC or 16 μM a-mangostin (ct-M); the cells were then serum-starved

Techniques Used:

19) Product Images from "Secreted Phospholipases A2 in Hereditary Angioedema With C1-Inhibitor Deficiency"

Article Title: Secreted Phospholipases A2 in Hereditary Angioedema With C1-Inhibitor Deficiency

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01721

In vitro effects of plasma from healthy controls or patients with C1-INH-HAE on vascular permeability. (A) Bovine aortic endothelial cells (BAEC) were incubated (18 h, 37°C) with plasma from healthy controls or from symptom-free patients with C1-INH-HAE. The in vitro .” (B) Plasma of patients with C1-INH-HAE was incubated (20 min, 37°C) with anti-VEGF-A (1 µg/ml), anti-Ang2 (1 µg/ml), anti-Ang1 (1 µg/ml), Me-Indoxam (100 nM), RO032107A (100 nM), or control medium. BAEC were then pre-incubated (30 min, 37°C) with heparinase (0.4 U/ml) or control medium and stimulated (18 h, 37°C) with plasma of C1-INH-HAE patients alone or with the combination of C1-INH-HAE plasma with inhibitors and then we evaluated vascular permeability. Data are shown in relative fluorescence units. * p Value ≤0.05 and ** p value ≤0.01 vs untreated plasma.
Figure Legend Snippet: In vitro effects of plasma from healthy controls or patients with C1-INH-HAE on vascular permeability. (A) Bovine aortic endothelial cells (BAEC) were incubated (18 h, 37°C) with plasma from healthy controls or from symptom-free patients with C1-INH-HAE. The in vitro .” (B) Plasma of patients with C1-INH-HAE was incubated (20 min, 37°C) with anti-VEGF-A (1 µg/ml), anti-Ang2 (1 µg/ml), anti-Ang1 (1 µg/ml), Me-Indoxam (100 nM), RO032107A (100 nM), or control medium. BAEC were then pre-incubated (30 min, 37°C) with heparinase (0.4 U/ml) or control medium and stimulated (18 h, 37°C) with plasma of C1-INH-HAE patients alone or with the combination of C1-INH-HAE plasma with inhibitors and then we evaluated vascular permeability. Data are shown in relative fluorescence units. * p Value ≤0.05 and ** p value ≤0.01 vs untreated plasma.

Techniques Used: In Vitro, Permeability, Incubation, Fluorescence

Correlations between sPLA 2 activity and cleaved HK, VEGF-A, VEGF-C, and Angs concentrations. Correlations between two variables: sPLA 2 (U/ml) and cleaved HK (A) , sPLA 2 and VEGF-A (B) , sPLA 2 and VEGF-C (C) , sPLA 2 and Ang1 (D) , and sPLA 2 and Ang2 (E) , were assessed by Spearman’s correlation analysis and reported as coefficient of correlation ( r ).
Figure Legend Snippet: Correlations between sPLA 2 activity and cleaved HK, VEGF-A, VEGF-C, and Angs concentrations. Correlations between two variables: sPLA 2 (U/ml) and cleaved HK (A) , sPLA 2 and VEGF-A (B) , sPLA 2 and VEGF-C (C) , sPLA 2 and Ang1 (D) , and sPLA 2 and Ang2 (E) , were assessed by Spearman’s correlation analysis and reported as coefficient of correlation ( r ).

Techniques Used: Activity Assay

20) Product Images from "Decreases in GSH:GSSG activate vascular endothelial growth factor receptor 2 (VEGFR2) in human aortic endothelial cells"

Article Title: Decreases in GSH:GSSG activate vascular endothelial growth factor receptor 2 (VEGFR2) in human aortic endothelial cells

Journal: Redox Biology

doi: 10.1016/j.redox.2018.07.015

Hydrogen peroxide does not activate VEGFR2. (A and B) Western blot analysis for VEGFR2 phosphorylation at Y1175 after 15 min of H 2 O 2 treatment followed 15 min of VEGF-A treatment. [* = p
Figure Legend Snippet: Hydrogen peroxide does not activate VEGFR2. (A and B) Western blot analysis for VEGFR2 phosphorylation at Y1175 after 15 min of H 2 O 2 treatment followed 15 min of VEGF-A treatment. [* = p

Techniques Used: Western Blot

Diamide activates VEGFR2 and potentiates VEGF-A mediated receptor activation. (A) Western blot analysis for VEGFR2 phosphorylation at Y1175 after 15 min of diamide treatment followed by 15 min of VEGF-A treatment. [* = p
Figure Legend Snippet: Diamide activates VEGFR2 and potentiates VEGF-A mediated receptor activation. (A) Western blot analysis for VEGFR2 phosphorylation at Y1175 after 15 min of diamide treatment followed by 15 min of VEGF-A treatment. [* = p

Techniques Used: Activation Assay, Western Blot

Diamide does not stimulate VEGF release and inhibiting PTPs is insufficient to stimulate VEGFR2 activation. (A) VEGF-A was measured using ELISA following 30 min of diamide treatment. (B) Western blot analysis following 30 min of sodium orthovanadate (Na 3 VO 4 ) treatment (to inhibit PTP activity). (C) VEGFR2 phosphorylation at Y1175 following 30 min of 2-AAPA treatment (to inhibit GR activity) followed by 30 min of H 2 O 2 treatment. [* = p
Figure Legend Snippet: Diamide does not stimulate VEGF release and inhibiting PTPs is insufficient to stimulate VEGFR2 activation. (A) VEGF-A was measured using ELISA following 30 min of diamide treatment. (B) Western blot analysis following 30 min of sodium orthovanadate (Na 3 VO 4 ) treatment (to inhibit PTP activity). (C) VEGFR2 phosphorylation at Y1175 following 30 min of 2-AAPA treatment (to inhibit GR activity) followed by 30 min of H 2 O 2 treatment. [* = p

Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Activity Assay

21) Product Images from "Combinatorial conditioning of adipose derived‐mesenchymal stem cells enhances their neurovascular potential: Implications for intervertebral disc degeneration. Combinatorial conditioning of adipose derived‐mesenchymal stem cells enhances their neurovascular potential: Implications for intervertebral disc degeneration"

Article Title: Combinatorial conditioning of adipose derived‐mesenchymal stem cells enhances their neurovascular potential: Implications for intervertebral disc degeneration. Combinatorial conditioning of adipose derived‐mesenchymal stem cells enhances their neurovascular potential: Implications for intervertebral disc degeneration

Journal: JOR Spine

doi: 10.1002/jsp2.1072

Gene and protein regulation of pro‐angiogenic factors in human donor‐matched ASCs and MSCs exposed to cytokines IL‐1β or TNFα under 21% or 5% oxygen. Pro‐angiogenic factors Ang‐1 (A,B), Ang‐2 (C, D), FGF‐2 (E, F) and VEGF‐A (G, H) mRNA expression (A, C, E, G) and levels of secreted protein (B, D, F, H) in human ASCs and MSCs stimulated with either IL‐1β or TNFα under different oxygen concentrations for 48 hours. Gene expression was normalized to that of reference gene 18S. Values represent the mean ± SEM. Statistical significance determined using two‐way ANOVA with Tukey's multiple comparisons test, * = P ≤ .05 (* = significant compared to unstimulated control within the same group; # = significant compared to corresponding sample in 21% oxygen; + = significance between MSCs and ASCs of the same treatment; n = 3)
Figure Legend Snippet: Gene and protein regulation of pro‐angiogenic factors in human donor‐matched ASCs and MSCs exposed to cytokines IL‐1β or TNFα under 21% or 5% oxygen. Pro‐angiogenic factors Ang‐1 (A,B), Ang‐2 (C, D), FGF‐2 (E, F) and VEGF‐A (G, H) mRNA expression (A, C, E, G) and levels of secreted protein (B, D, F, H) in human ASCs and MSCs stimulated with either IL‐1β or TNFα under different oxygen concentrations for 48 hours. Gene expression was normalized to that of reference gene 18S. Values represent the mean ± SEM. Statistical significance determined using two‐way ANOVA with Tukey's multiple comparisons test, * = P ≤ .05 (* = significant compared to unstimulated control within the same group; # = significant compared to corresponding sample in 21% oxygen; + = significance between MSCs and ASCs of the same treatment; n = 3)

Techniques Used: Expressing

22) Product Images from "Angiopoietins, Vascular Endothelial Growth Factors and Secretory Phospholipase A2 in Ischemic and Non-Ischemic Heart Failure"

Article Title: Angiopoietins, Vascular Endothelial Growth Factors and Secretory Phospholipase A2 in Ischemic and Non-Ischemic Heart Failure

Journal: Journal of Clinical Medicine

doi: 10.3390/jcm9061928

( A ) Plasma concentrations of VEGF-A in IHF and NIHF patients and in healthy controls; ( B ) plasma concentrations of VEGF-C in IHF and NIHF patients and in healthy controls; ( C ) plasma concentrations of VEGF-D in IHF and NIHF patients and in healthy controls. Data are shown as the median (horizontal block line), the 25th and 75th percentiles (boxes), and the 5th and 95th percentiles (whiskers) (statistical analysis was performed by one-way ANOVA and Bonferroni’s multiple comparison test). * p
Figure Legend Snippet: ( A ) Plasma concentrations of VEGF-A in IHF and NIHF patients and in healthy controls; ( B ) plasma concentrations of VEGF-C in IHF and NIHF patients and in healthy controls; ( C ) plasma concentrations of VEGF-D in IHF and NIHF patients and in healthy controls. Data are shown as the median (horizontal block line), the 25th and 75th percentiles (boxes), and the 5th and 95th percentiles (whiskers) (statistical analysis was performed by one-way ANOVA and Bonferroni’s multiple comparison test). * p

Techniques Used: Immunohistofluorescence, Blocking Assay

( A ) Plasma concentrations ofvascular endothelial growth factor-A (VEGF-A) in heart failure (HF) patients and in healthy controls; ( B ) plasma concentrations of VEGF-C in HF patients and in healthy controls; ( C ) plasma concentrations of VEGF-D in HF patients and in healthy controls. Data are shown as the median (horizontal block line), the 25th and 75th percentiles (boxes), and the 5th and 95th percentiles (whiskers) (statistical analysis was performed by a Student’s t -test). *** p
Figure Legend Snippet: ( A ) Plasma concentrations ofvascular endothelial growth factor-A (VEGF-A) in heart failure (HF) patients and in healthy controls; ( B ) plasma concentrations of VEGF-C in HF patients and in healthy controls; ( C ) plasma concentrations of VEGF-D in HF patients and in healthy controls. Data are shown as the median (horizontal block line), the 25th and 75th percentiles (boxes), and the 5th and 95th percentiles (whiskers) (statistical analysis was performed by a Student’s t -test). *** p

Techniques Used: Blocking Assay

23) Product Images from "Vascular Endothelial Growth Factor Directly Inhibits Primitive Neural Stem Cell Survival But Promotes Definitive Neural Stem Cell Survival"

Article Title: Vascular Endothelial Growth Factor Directly Inhibits Primitive Neural Stem Cell Survival But Promotes Definitive Neural Stem Cell Survival

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.0526-06.2006

A , Adult brain tissue as well as d-NSC-derived clonal neurospheres from the adult brain express Flk1 mRNA. Clonal E14.5 brain neurospheres also expressed Flk1 mRNA. Nestin and control GAPDH mRNAs were expressed in adult brain tissue, clonal adult brain neurospheres, and clonal E14.5 brain neurospheres. B , Nestin and Flk1 mRNA expression were not changed by VEGF-A treatment of either p-NSCs or d-NSCs. Moreover, Brachyury mRNA expression was not induced by VEGF-A treatment of either p-NSCs or d-NSCs. C , Increasing concentrations of VEGF-A enhanced the formation of neurospheres from adult mouse d-NSCs. Moreover, this VEGF-A-induced increase in neurospheres was suppressed by the addition of SU1498, a VEGF-A signaling inhibitor. SU1498 also decreased the number of neurospheres in the absence of VEGF-A in the culture media. D , The enhanced numbers of E14.5 d-NSC spheres produced by VEGF-A were blocked by the addition of two VEGF-A inhibitors, VEGF-A antibodies and Flk1/Fc-soluble chimeric receptors. There was a significant interaction of VEGF-A dose and blocker ( F (3,20) = 36.86; p
Figure Legend Snippet: A , Adult brain tissue as well as d-NSC-derived clonal neurospheres from the adult brain express Flk1 mRNA. Clonal E14.5 brain neurospheres also expressed Flk1 mRNA. Nestin and control GAPDH mRNAs were expressed in adult brain tissue, clonal adult brain neurospheres, and clonal E14.5 brain neurospheres. B , Nestin and Flk1 mRNA expression were not changed by VEGF-A treatment of either p-NSCs or d-NSCs. Moreover, Brachyury mRNA expression was not induced by VEGF-A treatment of either p-NSCs or d-NSCs. C , Increasing concentrations of VEGF-A enhanced the formation of neurospheres from adult mouse d-NSCs. Moreover, this VEGF-A-induced increase in neurospheres was suppressed by the addition of SU1498, a VEGF-A signaling inhibitor. SU1498 also decreased the number of neurospheres in the absence of VEGF-A in the culture media. D , The enhanced numbers of E14.5 d-NSC spheres produced by VEGF-A were blocked by the addition of two VEGF-A inhibitors, VEGF-A antibodies and Flk1/Fc-soluble chimeric receptors. There was a significant interaction of VEGF-A dose and blocker ( F (3,20) = 36.86; p

Techniques Used: Derivative Assay, Expressing, Produced

A , The number of wild-type Flk1 +/+ p-NSC spheres is decreased by the addition of 100 ng/ml VEGF-A into the ES culture minimal medium ( t (4) = 2.6; p
Figure Legend Snippet: A , The number of wild-type Flk1 +/+ p-NSC spheres is decreased by the addition of 100 ng/ml VEGF-A into the ES culture minimal medium ( t (4) = 2.6; p

Techniques Used:

24) Product Images from "Circulating Angiopoietin-2 Is a Marker for Early Cardiovascular Disease in Children on Chronic Dialysis"

Article Title: Circulating Angiopoietin-2 Is a Marker for Early Cardiovascular Disease in Children on Chronic Dialysis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0056273

Circulating levels of VEGF-A and sFlt-1 in pre-dialysis CKD and dialysis patients. VEGF-A levels were significantly lower in individuals on dialysis compared with pre-dialysis CKD patients (A). In contrast, sFlt-1 were significantly higher in the dialysis patients (B)
Figure Legend Snippet: Circulating levels of VEGF-A and sFlt-1 in pre-dialysis CKD and dialysis patients. VEGF-A levels were significantly lower in individuals on dialysis compared with pre-dialysis CKD patients (A). In contrast, sFlt-1 were significantly higher in the dialysis patients (B)

Techniques Used:

Immunolocalisation of vascular growth factors in arteries. Ang-1 was detected in the media of vessels from both pre-dialysis CKD (A) and dialysis patients (B); no differences in staining intensity were observed between the two groups (C). Ang-2 was immunodetected in both the media and endothelia (arrows) in pre-dialysis CKD (D) and dialysis (E) vessels with similar intensity (F). The endothelial later was also positive for von Willebrand factor (arrows, G and H). VEGF-A immunostaining was prominent in the media of pre-dialysis CKD vessels (I), but was significantly decreased in dialysis patients (J and K). All fields taken with ×40 objective.
Figure Legend Snippet: Immunolocalisation of vascular growth factors in arteries. Ang-1 was detected in the media of vessels from both pre-dialysis CKD (A) and dialysis patients (B); no differences in staining intensity were observed between the two groups (C). Ang-2 was immunodetected in both the media and endothelia (arrows) in pre-dialysis CKD (D) and dialysis (E) vessels with similar intensity (F). The endothelial later was also positive for von Willebrand factor (arrows, G and H). VEGF-A immunostaining was prominent in the media of pre-dialysis CKD vessels (I), but was significantly decreased in dialysis patients (J and K). All fields taken with ×40 objective.

Techniques Used: Staining, Immunostaining

25) Product Images from "A phase I study of the combination of ro4929097 and cediranib in patients with advanced solid tumours (PJC-004/NCI 8503)"

Article Title: A phase I study of the combination of ro4929097 and cediranib in patients with advanced solid tumours (PJC-004/NCI 8503)

Journal: British Journal of Cancer

doi: 10.1038/bjc.2013.380

Concentration of circulating angiogenic factors measured in the sera of patients during cycle 1 of treatment. ( A ) VEGF-A, ( B ) VEGF-C and ( C ) SDF-1. Increases in these factors were detected after dosing with cediranib, but there was no suggestion of relationship with dose or time to treatment failure.
Figure Legend Snippet: Concentration of circulating angiogenic factors measured in the sera of patients during cycle 1 of treatment. ( A ) VEGF-A, ( B ) VEGF-C and ( C ) SDF-1. Increases in these factors were detected after dosing with cediranib, but there was no suggestion of relationship with dose or time to treatment failure.

Techniques Used: Concentration Assay

26) Product Images from "Argonaute‐2 Promotes miR‐18a Entry in Human Brain Endothelial Cells"

Article Title: Argonaute‐2 Promotes miR‐18a Entry in Human Brain Endothelial Cells

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

doi: 10.1161/JAHA.114.000968

Co‐treatment of miR‐18a and Ago‐2 in vivo “normalizes” TSP‐1 and VEGF‐A plasma levels. A, Athymic nude mice were implanted with glioma cells intracranially. After 3 days, animals were treated intravenously with vehicle, miR‐18a plus Ago‐2, miR‐18a alone or Ago‐2 alone every 48 hours for 3 cycles. Subsequently, plasma was tested for TSP‐1 (A) and VEGF‐A (B). miR‐18a and Ago‐2 combination treatment caused the most significant increase of TSP‐1 levels (n=5; * P
Figure Legend Snippet: Co‐treatment of miR‐18a and Ago‐2 in vivo “normalizes” TSP‐1 and VEGF‐A plasma levels. A, Athymic nude mice were implanted with glioma cells intracranially. After 3 days, animals were treated intravenously with vehicle, miR‐18a plus Ago‐2, miR‐18a alone or Ago‐2 alone every 48 hours for 3 cycles. Subsequently, plasma was tested for TSP‐1 (A) and VEGF‐A (B). miR‐18a and Ago‐2 combination treatment caused the most significant increase of TSP‐1 levels (n=5; * P

Techniques Used: In Vivo, Mouse Assay

27) Product Images from "Flexible heteroarotinoid (Flex-Het) SHetA2 inhibits angiogenesis in vitro and in vivo"

Article Title: Flexible heteroarotinoid (Flex-Het) SHetA2 inhibits angiogenesis in vitro and in vivo

Journal: Investigational New Drugs

doi: 10.1007/s10637-008-9175-7

Regulation of angiogenic cytokines in HUVEC cultures. HUVEC cultures were treated with 5 μM or the indicated doses of SHetA2 for 24 h. RNA isolated from the cultures was evaluated for expression of TP ( a ) and VEGF ( c ) mRNA using rt-PCR. Results represent the average and standard error of three independent experiments performed in triplicate. Protein extracts were evaluated by Western blot analysis for TP expression ( b ). Western blot results represent the average and standard error of two independent experiments. Data from one experiment was multiplied by a factor of 2 to normalize the results to the second experiment for presentation on a single graph. Conditioned media was evaluated by ELISA for VEGF expression ( d ). ELISA results represent the average and standard error of three independent experiments performed in triplicate
Figure Legend Snippet: Regulation of angiogenic cytokines in HUVEC cultures. HUVEC cultures were treated with 5 μM or the indicated doses of SHetA2 for 24 h. RNA isolated from the cultures was evaluated for expression of TP ( a ) and VEGF ( c ) mRNA using rt-PCR. Results represent the average and standard error of three independent experiments performed in triplicate. Protein extracts were evaluated by Western blot analysis for TP expression ( b ). Western blot results represent the average and standard error of two independent experiments. Data from one experiment was multiplied by a factor of 2 to normalize the results to the second experiment for presentation on a single graph. Conditioned media was evaluated by ELISA for VEGF expression ( d ). ELISA results represent the average and standard error of three independent experiments performed in triplicate

Techniques Used: Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

Regulation of bFGF and VEGF mRNA and protein expression. RNA isolated from A2780 cultures treated with 10 μM SHetA2 or solvent for the indicated time was analyzed by rt-PCR for bFGF ( a ) and VEGF ( b ) expression. Results or rtPCR experiments presented are representative of two independent experiments performed in triplicate. Media conditioned by incubation with A2780 cultures and other indicated cultures were evaluated for bFGF ( a ) or VEGF ( b ) protein secretion by ELISA. ELISA results are averages of three independent experiments performed in triplicate
Figure Legend Snippet: Regulation of bFGF and VEGF mRNA and protein expression. RNA isolated from A2780 cultures treated with 10 μM SHetA2 or solvent for the indicated time was analyzed by rt-PCR for bFGF ( a ) and VEGF ( b ) expression. Results or rtPCR experiments presented are representative of two independent experiments performed in triplicate. Media conditioned by incubation with A2780 cultures and other indicated cultures were evaluated for bFGF ( a ) or VEGF ( b ) protein secretion by ELISA. ELISA results are averages of three independent experiments performed in triplicate

Techniques Used: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Incubation, Enzyme-linked Immunosorbent Assay

28) Product Images from "VEGFR2 alteration in Alzheimer’s disease"

Article Title: VEGFR2 alteration in Alzheimer’s disease

Journal: Scientific Reports

doi: 10.1038/s41598-017-18042-1

Analysis of sVEGFR1 and sVEGFR2 concentrations in human plasma. Plasma sVEGFR1 and sVEGFR2 concentrations were measured by ELISA. The differences in the relative amounts of sVEGFR1 and sVEGFR2 were compared between dementia, MCI, and healthy controls by means of Mann-Whitney’s U-test within different groups. ( a ) Plasma sVEGFR1 levels in dementia subgroup were not closely correlated with VEGF-A ( r = −0.284, p = 0.013) ( b ) Plasma sVEGFR2 levels in dementia subgroup were significantly correlated with triglyceride ( r = 0.406, p = 0.0002).
Figure Legend Snippet: Analysis of sVEGFR1 and sVEGFR2 concentrations in human plasma. Plasma sVEGFR1 and sVEGFR2 concentrations were measured by ELISA. The differences in the relative amounts of sVEGFR1 and sVEGFR2 were compared between dementia, MCI, and healthy controls by means of Mann-Whitney’s U-test within different groups. ( a ) Plasma sVEGFR1 levels in dementia subgroup were not closely correlated with VEGF-A ( r = −0.284, p = 0.013) ( b ) Plasma sVEGFR2 levels in dementia subgroup were significantly correlated with triglyceride ( r = 0.406, p = 0.0002).

Techniques Used: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

sVEGFR2 and sVEGFR1 mRNA expression in endothelial cells. ( a ) HUVEC were treated with 10 μM of Aβ 1–40 peptides for 8 h. Real-time PCR results showing relative mRNA expression levels of VEGF-A , sVEGFR2 , and sVEGFR1 (n = 3). ( b ) After HUVEC were treated with 10 μM of Aβ 1–40 peptides for 24 h, sVEGFR1 and sVEGFR2 mRNA expression levels were measured by real-time PCR (n = 3). ( c ) HBMEC were treated with 10 μM of Aβ 1–40 peptides for 24 h. sVEGFR2 and sVEGFR1 mRNA expression levels were measured by real-time PCR (n = 3). ( d ) Splicing variant form of VEGFR2 (sVEGFR2) protein levels were detected in HBMEC cell culture media. The cropped blot is displayed in the main figure, and its full-length blot is presented in Supplementary Figure 1 . Treatment of 10 μM of Aβ 1–40 peptides for 24 h decreased the sVEGFR2 levels (n = 3).
Figure Legend Snippet: sVEGFR2 and sVEGFR1 mRNA expression in endothelial cells. ( a ) HUVEC were treated with 10 μM of Aβ 1–40 peptides for 8 h. Real-time PCR results showing relative mRNA expression levels of VEGF-A , sVEGFR2 , and sVEGFR1 (n = 3). ( b ) After HUVEC were treated with 10 μM of Aβ 1–40 peptides for 24 h, sVEGFR1 and sVEGFR2 mRNA expression levels were measured by real-time PCR (n = 3). ( c ) HBMEC were treated with 10 μM of Aβ 1–40 peptides for 24 h. sVEGFR2 and sVEGFR1 mRNA expression levels were measured by real-time PCR (n = 3). ( d ) Splicing variant form of VEGFR2 (sVEGFR2) protein levels were detected in HBMEC cell culture media. The cropped blot is displayed in the main figure, and its full-length blot is presented in Supplementary Figure 1 . Treatment of 10 μM of Aβ 1–40 peptides for 24 h decreased the sVEGFR2 levels (n = 3).

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Variant Assay, Cell Culture

29) Product Images from "Tumour vasculature immaturity, oxidative damage and systemic inflammation stratify survival of colorectal cancer patients on bevacizumab treatment"

Article Title: Tumour vasculature immaturity, oxidative damage and systemic inflammation stratify survival of colorectal cancer patients on bevacizumab treatment

Journal: Oncotarget

doi: 10.18632/oncotarget.24276

Schematic depicting tumour and serum biomarkers that stratify survival in metastatic colorectal cancer patients following treatment with bevacizumab Up and down arrows indicate that altered levels (higher and lower respectively) of these biomarkers (red text) correlate with enhanced overall survival – a higher proportion of immature tumour vasculature, increased oxidative damage and lower levels of circulating inflammatory cytokines. For patients that have a lower survival, it is possible that these interconnected cancer-associated biological processes may have progressed beyond a point whereby subsequent treatment following surgery with anti-VEGF therapy is sufficient to enhance patient survival levels.
Figure Legend Snippet: Schematic depicting tumour and serum biomarkers that stratify survival in metastatic colorectal cancer patients following treatment with bevacizumab Up and down arrows indicate that altered levels (higher and lower respectively) of these biomarkers (red text) correlate with enhanced overall survival – a higher proportion of immature tumour vasculature, increased oxidative damage and lower levels of circulating inflammatory cytokines. For patients that have a lower survival, it is possible that these interconnected cancer-associated biological processes may have progressed beyond a point whereby subsequent treatment following surgery with anti-VEGF therapy is sufficient to enhance patient survival levels.

Techniques Used:

30) Product Images from "Cytokine Biomarkers Associated with Human Extra-Pulmonary Tuberculosis Clinical Strains and Symptoms"

Article Title: Cytokine Biomarkers Associated with Human Extra-Pulmonary Tuberculosis Clinical Strains and Symptoms

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.00275

Receiver operating curves (ROC) analysis of TNF-α and VEGF as markers to distinguish the differen clinical groups. VEGF ROC between EPTB and non-EPTB (A) , PTB (B) , Ctrl (C) , TNF-α ROC between PTB and non-EPTB (D) , EPTB (E) , Ctrl (F) . EPTB, bacteriologically confirmed extrapulmonary tuberculosis patients; non-EPTB, suspected-EPTB patients not bacteriologically confirmed; PTB, pulmonary tuberculosis patients; CTRL, healthy control group.
Figure Legend Snippet: Receiver operating curves (ROC) analysis of TNF-α and VEGF as markers to distinguish the differen clinical groups. VEGF ROC between EPTB and non-EPTB (A) , PTB (B) , Ctrl (C) , TNF-α ROC between PTB and non-EPTB (D) , EPTB (E) , Ctrl (F) . EPTB, bacteriologically confirmed extrapulmonary tuberculosis patients; non-EPTB, suspected-EPTB patients not bacteriologically confirmed; PTB, pulmonary tuberculosis patients; CTRL, healthy control group.

Techniques Used:

31) Product Images from "RSUME is implicated in tumorigenesis and metastasis of pancreatic neuroendocrine tumors"

Article Title: RSUME is implicated in tumorigenesis and metastasis of pancreatic neuroendocrine tumors

Journal: Oncotarget

doi: 10.18632/oncotarget.11081

Influence of RSUME on HIF-1α, VEGF-A and IL-8 production RSUME mRNA and protein level ( A ) were stimulated during hypoxia (1% O 2 ) for the indicated time points in BON1 cells. As expected RSUME mRNA and protein was down-regulated and less sensitive to hypoxia in BON1 cells with RSUME knock-down (BON1 RSUME-KD ) compared to scramble siRNA trasfected cells (BON1 Scramble ) ( B ). HIF-1a mRNA and in particular hypoxia-induced HIF-1a protein production was strongly impaired in BON1 RSUME-KD cells ( C ). Normoxic and hypoxic mRNA synthesis and secretion of VEGF-A was suppressed in BON1 RSUME-KD cells ( D ) whereas IL-8 mRNA and protein production was enhanced. ( E ). All experiments were performed three times and in B to E treatment time was 3 h for mRNA expression and 12 h for protein production studies, respectively. Results are expressed as mean ± SEM of triplicates for mRNA and quadruplicate for ELISA. * P
Figure Legend Snippet: Influence of RSUME on HIF-1α, VEGF-A and IL-8 production RSUME mRNA and protein level ( A ) were stimulated during hypoxia (1% O 2 ) for the indicated time points in BON1 cells. As expected RSUME mRNA and protein was down-regulated and less sensitive to hypoxia in BON1 cells with RSUME knock-down (BON1 RSUME-KD ) compared to scramble siRNA trasfected cells (BON1 Scramble ) ( B ). HIF-1a mRNA and in particular hypoxia-induced HIF-1a protein production was strongly impaired in BON1 RSUME-KD cells ( C ). Normoxic and hypoxic mRNA synthesis and secretion of VEGF-A was suppressed in BON1 RSUME-KD cells ( D ) whereas IL-8 mRNA and protein production was enhanced. ( E ). All experiments were performed three times and in B to E treatment time was 3 h for mRNA expression and 12 h for protein production studies, respectively. Results are expressed as mean ± SEM of triplicates for mRNA and quadruplicate for ELISA. * P

Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay

32) Product Images from "Circulating Fibroblast Growth Factor-2, HIV-Tat, and Vascular Endothelial Cell Growth Factor-A in HIV-Infected Children with Renal Disease Activate Rho-A and Src in Cultured Renal Endothelial Cells"

Article Title: Circulating Fibroblast Growth Factor-2, HIV-Tat, and Vascular Endothelial Cell Growth Factor-A in HIV-Infected Children with Renal Disease Activate Rho-A and Src in Cultured Renal Endothelial Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0153837

Characterization of the human glomerular endothelial cell line (HGEc-1). (A) Phase contrast microscopy picture of cultured HGEc-1, Magnification, X100. (B) Immunohistochemistry staining of cultured HGEc-1 expressing the endothelial cell marker Von Willebrand Factor (vWf), red color, Magnification, X100. Immunofluorescence staining of cultured HGEc-1 for (C) CD31, (D) VEGFR-2, (E) VE-cadherin, all in red color, and (F) human podocytes incubated with the anti- CD31 antibody (negative controls). Cell nuclei are visualized with DAPI in blue color. Scale bar = 20 μm. (G) Western blots show CD31, VEGFR2, VE-cadherin, and Beta actin expression in cultured HGEc-1, human umbilical vein endothelial cells (HUVEC), and human renal embryonic epithelial cells (HEK293). (H) Changes in trans-endothelial electrical resistance (TEER) induced by the cyclic AMP analogue 8-pCPT-cAMP (30 μm) in combination with the cAMP- phosphodiesterase inhibitor RO-20-1724 (20 μm); thrombin (100 units/ml), or VEGF-A (50 ng/ml). Results are expressed as changes relative to controls. Bar graphs show mean ± SEM corresponding to three different experiments. Values significantly different from control were marked with asterisk , *p
Figure Legend Snippet: Characterization of the human glomerular endothelial cell line (HGEc-1). (A) Phase contrast microscopy picture of cultured HGEc-1, Magnification, X100. (B) Immunohistochemistry staining of cultured HGEc-1 expressing the endothelial cell marker Von Willebrand Factor (vWf), red color, Magnification, X100. Immunofluorescence staining of cultured HGEc-1 for (C) CD31, (D) VEGFR-2, (E) VE-cadherin, all in red color, and (F) human podocytes incubated with the anti- CD31 antibody (negative controls). Cell nuclei are visualized with DAPI in blue color. Scale bar = 20 μm. (G) Western blots show CD31, VEGFR2, VE-cadherin, and Beta actin expression in cultured HGEc-1, human umbilical vein endothelial cells (HUVEC), and human renal embryonic epithelial cells (HEK293). (H) Changes in trans-endothelial electrical resistance (TEER) induced by the cyclic AMP analogue 8-pCPT-cAMP (30 μm) in combination with the cAMP- phosphodiesterase inhibitor RO-20-1724 (20 μm); thrombin (100 units/ml), or VEGF-A (50 ng/ml). Results are expressed as changes relative to controls. Bar graphs show mean ± SEM corresponding to three different experiments. Values significantly different from control were marked with asterisk , *p

Techniques Used: Microscopy, Cell Culture, Immunohistochemistry, Staining, Expressing, Marker, Immunofluorescence, Incubation, Western Blot

Rho-A activation increases the permeability of HGEc-1. (A) Cultured HGEc-1 cells were transfected with different plasmids, pCEFL-mock, constitutively active RhoAQL (pCEFL-AU5-RhoAQL), or dominant negative mutant RhoAN19 (pCEFL-AU5-RhoAN19). Twenty-four hours later, the cells were treated with VEGF-A (50 ng/ml) + Tat (100 ng/ml) + Heparin (50 units/ml), all together, and exposed to FITC-dextran as described in methods. (B) In other experiments HGEc-1 cells were treated for 5 min as described above and then harvested to assess the phosphorylation of Rho-A, MLC, Src as described in Methods. Panel B shows representative western blots corresponding to the phosphorylation changes. (C) The graphs show mean ± SEM values corresponding to three different Western blots that assessed the phosphorylation of Rho-A, MLC and Src in cultured HGEc-1. Results were expressed in arbitrary optical density units as a ratio of the total activity. In each group, mock, RhoAQL and RhoAN19 transfected cells were treated with either serum free media (Controls) (-), or VEGF-A + Tat + Heparin (+). Groups that were significantly different from controls (-) were labeled with asterisk , *p
Figure Legend Snippet: Rho-A activation increases the permeability of HGEc-1. (A) Cultured HGEc-1 cells were transfected with different plasmids, pCEFL-mock, constitutively active RhoAQL (pCEFL-AU5-RhoAQL), or dominant negative mutant RhoAN19 (pCEFL-AU5-RhoAN19). Twenty-four hours later, the cells were treated with VEGF-A (50 ng/ml) + Tat (100 ng/ml) + Heparin (50 units/ml), all together, and exposed to FITC-dextran as described in methods. (B) In other experiments HGEc-1 cells were treated for 5 min as described above and then harvested to assess the phosphorylation of Rho-A, MLC, Src as described in Methods. Panel B shows representative western blots corresponding to the phosphorylation changes. (C) The graphs show mean ± SEM values corresponding to three different Western blots that assessed the phosphorylation of Rho-A, MLC and Src in cultured HGEc-1. Results were expressed in arbitrary optical density units as a ratio of the total activity. In each group, mock, RhoAQL and RhoAN19 transfected cells were treated with either serum free media (Controls) (-), or VEGF-A + Tat + Heparin (+). Groups that were significantly different from controls (-) were labeled with asterisk , *p

Techniques Used: Activation Assay, Permeability, Cell Culture, Transfection, Dominant Negative Mutation, Western Blot, Activity Assay, Labeling

FGF-2 and VEGF-A in combination with HIV-Tat and heparin increase the permeability of cultured HGEc-1 through Rho-A and Src dependent pathways. (A) Monolayers of 5 hours-starved HGEc-1 were stimulated by thrombin (100 units/ml) as a positive control, Tat (100 ng/ml), FGF-2 (50 ng/ml), VEGF-A (50 ng/ml), and Heparin (50 units/ml) alone or in combination. The data represent FITC-dextran permeability changes expressed as fold increase. (B) Overnight-starved HGEc-1 monolayer were treated for 5 min as described above and then harvested to assess the phosphorylation of Rho-A, Rac1, MLC, and Src, as described in Methods. Panel B shows representative Western blots corresponding to the phosphorylation changes. (C) The graphs show mean ± SEM values corresponding to three different Western blots that assessed the phosphorylation of Rho-A, Rac1, MLC, and Src in cultured HGEc-1. Results were expressed in arbitrary optical density units as a ratio of the total activity. Values significantly different from control cells treated with serum free media were marked with asterisk , *p
Figure Legend Snippet: FGF-2 and VEGF-A in combination with HIV-Tat and heparin increase the permeability of cultured HGEc-1 through Rho-A and Src dependent pathways. (A) Monolayers of 5 hours-starved HGEc-1 were stimulated by thrombin (100 units/ml) as a positive control, Tat (100 ng/ml), FGF-2 (50 ng/ml), VEGF-A (50 ng/ml), and Heparin (50 units/ml) alone or in combination. The data represent FITC-dextran permeability changes expressed as fold increase. (B) Overnight-starved HGEc-1 monolayer were treated for 5 min as described above and then harvested to assess the phosphorylation of Rho-A, Rac1, MLC, and Src, as described in Methods. Panel B shows representative Western blots corresponding to the phosphorylation changes. (C) The graphs show mean ± SEM values corresponding to three different Western blots that assessed the phosphorylation of Rho-A, Rac1, MLC, and Src in cultured HGEc-1. Results were expressed in arbitrary optical density units as a ratio of the total activity. Values significantly different from control cells treated with serum free media were marked with asterisk , *p

Techniques Used: Permeability, Cell Culture, Positive Control, Western Blot, Activity Assay

FGF-2 and VEGF-A, in combination with HIV-Tat and heparin, induce the formation of stress fibers through Rho-A dependent pathways. (A) Panel A shows representative changes in the formation of stress fibers detected in cultured HGEc-1. Overnight-starved HGEc-1 monolayer were stimulated by thrombin (100 units/ml) as a positive control, Tat (100 ng/ml), FGF-2 (50 ng/ml), VEGF-A (50 ng/ml), and Heparin (50 units/ml) alone or in combination. The RhoA inhibitor: C3 transferase (20 ng/ml), was added 4 hrs. before stimulation and the inhibitor of Src family kinase SU6656 (1 μM) and ROCK inhibitor Y-27632 (10 μM) were added 1hr before stimulation and 20 min after treatment, F-actin fibers were visualized in cells by staining with 2 μg/ml of Alexa Fluor 488-labeled phalloidin. Cell nuclei were stained with Hoechst 33342. The scale bar is 10 μm. (B) The graphs show mean ± SEM values corresponding to the formation of stress fibers in three different experiments. Results were expressed as % changes in stress fibers formation relative to control cells. Values significantly different from controls were marked with an asterisk , *p
Figure Legend Snippet: FGF-2 and VEGF-A, in combination with HIV-Tat and heparin, induce the formation of stress fibers through Rho-A dependent pathways. (A) Panel A shows representative changes in the formation of stress fibers detected in cultured HGEc-1. Overnight-starved HGEc-1 monolayer were stimulated by thrombin (100 units/ml) as a positive control, Tat (100 ng/ml), FGF-2 (50 ng/ml), VEGF-A (50 ng/ml), and Heparin (50 units/ml) alone or in combination. The RhoA inhibitor: C3 transferase (20 ng/ml), was added 4 hrs. before stimulation and the inhibitor of Src family kinase SU6656 (1 μM) and ROCK inhibitor Y-27632 (10 μM) were added 1hr before stimulation and 20 min after treatment, F-actin fibers were visualized in cells by staining with 2 μg/ml of Alexa Fluor 488-labeled phalloidin. Cell nuclei were stained with Hoechst 33342. The scale bar is 10 μm. (B) The graphs show mean ± SEM values corresponding to the formation of stress fibers in three different experiments. Results were expressed as % changes in stress fibers formation relative to control cells. Values significantly different from controls were marked with an asterisk , *p

Techniques Used: Cell Culture, Positive Control, Staining, Labeling

VEGF-A in combination with HIV-Tat and heparin, increases the permeability of cultured HGEc-1 through Rho-A and Src dependent pathways. (A) Monolayers of 5 hours-starved HGEc-1 were stimulated by VEGF-A (50 ng/ml), Tat (100 ng/ml), and Heparin (50 units/ml) all combined. The Rho-A inhibitor: C3 transferase (20 ng/ml), was added 4 hours before stimulation and the inhibitor of Src family kinase SU6656 (1 μM) and ROCK inhibitor Y-27632 (10 μM) were added 1hr before stimulation. The data represent FITC-dextran permeability expresses as fold increase. (B) Overnight-starved HGEc-1 monolayers were treated for 5 min as described above and then harvested to assess the phosphorylation of Rho-A, Rac1, MLC and Src, as described in Methods. Panel B shows representative Western blots corresponding to the phosphorylation changes. (C) The graphs show mean ± SEM values corresponding to three different Western blots that assessed the phosphorylation of Rho-A, Rac1, MLC, and Src in cultured HGEc-1. Results were expressed in arbitrary optical density units expressed as a ratio of the total activity. Values significantly different from control cells treated with serum free media were marked with asterisks **p
Figure Legend Snippet: VEGF-A in combination with HIV-Tat and heparin, increases the permeability of cultured HGEc-1 through Rho-A and Src dependent pathways. (A) Monolayers of 5 hours-starved HGEc-1 were stimulated by VEGF-A (50 ng/ml), Tat (100 ng/ml), and Heparin (50 units/ml) all combined. The Rho-A inhibitor: C3 transferase (20 ng/ml), was added 4 hours before stimulation and the inhibitor of Src family kinase SU6656 (1 μM) and ROCK inhibitor Y-27632 (10 μM) were added 1hr before stimulation. The data represent FITC-dextran permeability expresses as fold increase. (B) Overnight-starved HGEc-1 monolayers were treated for 5 min as described above and then harvested to assess the phosphorylation of Rho-A, Rac1, MLC and Src, as described in Methods. Panel B shows representative Western blots corresponding to the phosphorylation changes. (C) The graphs show mean ± SEM values corresponding to three different Western blots that assessed the phosphorylation of Rho-A, Rac1, MLC, and Src in cultured HGEc-1. Results were expressed in arbitrary optical density units expressed as a ratio of the total activity. Values significantly different from control cells treated with serum free media were marked with asterisks **p

Techniques Used: Permeability, Cell Culture, Western Blot, Activity Assay

Rho-A activation increases the formation of stress fibers in HGEc-1. (A) HGEc-1 were transfected with the corresponding Rho-A constructs described above. Subsequently, 24 hours later, they were seeded on coverslips, treated with (VEGF-A + Tat + Heparin), and stained with F-actin to visualize the formation of stress fibers as described above. Scale bar = 20 μm. (B) The graphs show mean ± SEM values corresponding to three different experiments that assessed the formation of stress fibers in cultured HGEc-1. Results were expressed as % changes in stress fibers relative to controls. Control cells (-) were treated with serum free medium. Statistically significant differences between control cells (-) vs. VEGF-A + Tat + Heparin-treated cells (+), were highlighted with an asterisk , *p
Figure Legend Snippet: Rho-A activation increases the formation of stress fibers in HGEc-1. (A) HGEc-1 were transfected with the corresponding Rho-A constructs described above. Subsequently, 24 hours later, they were seeded on coverslips, treated with (VEGF-A + Tat + Heparin), and stained with F-actin to visualize the formation of stress fibers as described above. Scale bar = 20 μm. (B) The graphs show mean ± SEM values corresponding to three different experiments that assessed the formation of stress fibers in cultured HGEc-1. Results were expressed as % changes in stress fibers relative to controls. Control cells (-) were treated with serum free medium. Statistically significant differences between control cells (-) vs. VEGF-A + Tat + Heparin-treated cells (+), were highlighted with an asterisk , *p

Techniques Used: Activation Assay, Transfection, Construct, Staining, Cell Culture

33) Product Images from "Circulating Angiogenic Factors as Biomarkers of Disease Severity and Bacterial Burden in Pulmonary Tuberculosis"

Article Title: Circulating Angiogenic Factors as Biomarkers of Disease Severity and Bacterial Burden in Pulmonary Tuberculosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0146318

Positive relationship between systemic levels of angiogenic factors and smear grades in PTB individuals. The relationship between the plasma levels of VEGF-A, C, D, VEGF-R1, R2, R3 and smear grades as estimated by sputum smears was examined in all PTB individuals. The data are represented as scatter plots with each circle representing a single individual. P values were calculated using Linear trend post-test.
Figure Legend Snippet: Positive relationship between systemic levels of angiogenic factors and smear grades in PTB individuals. The relationship between the plasma levels of VEGF-A, C, D, VEGF-R1, R2, R3 and smear grades as estimated by sputum smears was examined in all PTB individuals. The data are represented as scatter plots with each circle representing a single individual. P values were calculated using Linear trend post-test.

Techniques Used:

Elevated systemic levels of VEGF-A, VEGF-C and VEGF-R2 in bilateral and cavitary disease in PTB individuals. (A) The plasma levels of VEGF-A, C and D, VEGF-R1, R2 and R3 were measured in PTB individuals with bilateral versus unilateral disease. (B) The plasma levels of VEGF-A, C and D, VEGF-R1, R2 and R3 were measured in PTB individuals with cavitary versus non-cavitary disease. The data are represented as scatter plots with each circle representing a single individual. P values were calculated using the Mann-Whitney test.
Figure Legend Snippet: Elevated systemic levels of VEGF-A, VEGF-C and VEGF-R2 in bilateral and cavitary disease in PTB individuals. (A) The plasma levels of VEGF-A, C and D, VEGF-R1, R2 and R3 were measured in PTB individuals with bilateral versus unilateral disease. (B) The plasma levels of VEGF-A, C and D, VEGF-R1, R2 and R3 were measured in PTB individuals with cavitary versus non-cavitary disease. The data are represented as scatter plots with each circle representing a single individual. P values were calculated using the Mann-Whitney test.

Techniques Used: MANN-WHITNEY

Diminished systemic levels of angiogenic factors following anti-tuberculous treatment in PTB individuals. The plasma levels of VEGF-A, C and D, VEGF-R1, R2 and R3 were measured in PTB individuals before (pre-T) and after (post-T) standard anti-tuberculous chemotherapy. The data are represented as line graphs with each line representing a single individual. P values were calculated using the Wilcoxon signed rank test.
Figure Legend Snippet: Diminished systemic levels of angiogenic factors following anti-tuberculous treatment in PTB individuals. The plasma levels of VEGF-A, C and D, VEGF-R1, R2 and R3 were measured in PTB individuals before (pre-T) and after (post-T) standard anti-tuberculous chemotherapy. The data are represented as line graphs with each line representing a single individual. P values were calculated using the Wilcoxon signed rank test.

Techniques Used:

ROC analysis to estimate the discriminatory power of systemic angiogenic factors in PTB individuals. (A) ROC analysis to estimate the sensitivity, specificity and area under the curve was performed using all the systemic angiogenic factors (VEGF-A, C, VEGF-R1, R2 and R3) to estimate the capacity of these factors to distinguish PTB versus LTB individuals. (B) ROC analysis to estimate the sensitivity, specificity and area under the curve was performed using all the systemic angiogenic factors (VEGF-A, C and D, VEGF-R1, R2 and R3) to estimate the capacity of these factors to distinguish PTB versus NTB individuals.
Figure Legend Snippet: ROC analysis to estimate the discriminatory power of systemic angiogenic factors in PTB individuals. (A) ROC analysis to estimate the sensitivity, specificity and area under the curve was performed using all the systemic angiogenic factors (VEGF-A, C, VEGF-R1, R2 and R3) to estimate the capacity of these factors to distinguish PTB versus LTB individuals. (B) ROC analysis to estimate the sensitivity, specificity and area under the curve was performed using all the systemic angiogenic factors (VEGF-A, C and D, VEGF-R1, R2 and R3) to estimate the capacity of these factors to distinguish PTB versus NTB individuals.

Techniques Used:

Elevated systemic levels of circulating angiogenic factors in PTB individuals. The plasma levels of (A) vascular endothelial growth factors (VEGF-A, C and D) and (B) VEGF-receptors (VEGF-R1, R2 and R3) were measured in PTB (n = 44), LTB (n = 44) and NTB (n = 44) individuals. The data are represented as scatter plots with each circle representing a single individual. P values were calculated using the Kruskal-Wallis test with Dunn's post hoc comparison.
Figure Legend Snippet: Elevated systemic levels of circulating angiogenic factors in PTB individuals. The plasma levels of (A) vascular endothelial growth factors (VEGF-A, C and D) and (B) VEGF-receptors (VEGF-R1, R2 and R3) were measured in PTB (n = 44), LTB (n = 44) and NTB (n = 44) individuals. The data are represented as scatter plots with each circle representing a single individual. P values were calculated using the Kruskal-Wallis test with Dunn's post hoc comparison.

Techniques Used:

34) Product Images from "C-reactive protein can upregulate VEGF expression to promote ADSC-induced angiogenesis by activating HIF-1α via CD64/PI3k/Akt and MAPK/ERK signaling pathways"

Article Title: C-reactive protein can upregulate VEGF expression to promote ADSC-induced angiogenesis by activating HIF-1α via CD64/PI3k/Akt and MAPK/ERK signaling pathways

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-016-0377-1

CRP induces phosphorylation of ERK and Akt, and inhibiting both pathways abrogated the increasing of VEGF production. a, b CRP induced phosphorylation of ERK, Akt, and NF-kB; the effect peaked at 120 min. Pharmacological inhibitors of MAPK (PD98059), PI3K/AKT (LY294002), and NF-ĸB (BAY-11-7082) inhibited the CRP-mediated increase of phosphorylated kinases. LY249002 (10 μM) for PI3K-specific inhibitor, PD98059 (10 μM) for MAPK inhibitor, BAY-11-7082 (10 μM) for inhibitor for NF-ĸB. Inhibitor concentrations were chosen based on the manufacturer’s recommendations and our preliminary experimental findings. c Effects of kinase inhibitors on CRP-induced VEGF production examined by ELISA. Inhibition of the MAPK and PI3K/AKT signaling pathways but not NF-ĸB/IkBα and cycloheximide partly abrogated the increased CRP-induced VEGF production. Columns , mean; bars , SE. * p
Figure Legend Snippet: CRP induces phosphorylation of ERK and Akt, and inhibiting both pathways abrogated the increasing of VEGF production. a, b CRP induced phosphorylation of ERK, Akt, and NF-kB; the effect peaked at 120 min. Pharmacological inhibitors of MAPK (PD98059), PI3K/AKT (LY294002), and NF-ĸB (BAY-11-7082) inhibited the CRP-mediated increase of phosphorylated kinases. LY249002 (10 μM) for PI3K-specific inhibitor, PD98059 (10 μM) for MAPK inhibitor, BAY-11-7082 (10 μM) for inhibitor for NF-ĸB. Inhibitor concentrations were chosen based on the manufacturer’s recommendations and our preliminary experimental findings. c Effects of kinase inhibitors on CRP-induced VEGF production examined by ELISA. Inhibition of the MAPK and PI3K/AKT signaling pathways but not NF-ĸB/IkBα and cycloheximide partly abrogated the increased CRP-induced VEGF production. Columns , mean; bars , SE. * p

Techniques Used: Enzyme-linked Immunosorbent Assay, Inhibition

Schematic representation of the molecular mechanism for CRP regulation of VEGF in ADSCs. CRP binds to the membrane surface antigen FCγRI (CD64) on ADSCs and then induces ERK1/2 and Akt phosphorylation, which activates HIF-1α to enter the nucleus and bind to hypoxia-response element sites in the VEGF promoter, stimulating VEGF gene and protein expression. CRP C-reactive protein, VEGF vascular endothelial growth factor, HIF-1α , hypoxia inducible factor-1α, HRE hypoxia-response element
Figure Legend Snippet: Schematic representation of the molecular mechanism for CRP regulation of VEGF in ADSCs. CRP binds to the membrane surface antigen FCγRI (CD64) on ADSCs and then induces ERK1/2 and Akt phosphorylation, which activates HIF-1α to enter the nucleus and bind to hypoxia-response element sites in the VEGF promoter, stimulating VEGF gene and protein expression. CRP C-reactive protein, VEGF vascular endothelial growth factor, HIF-1α , hypoxia inducible factor-1α, HRE hypoxia-response element

Techniques Used: Expressing

CRP stimulated VEGF expression through HIF-1α, which was linked to activation of the PI3K/AKT1 and MAPK/ERK1/2 pathways. a CRP increased HIF-1α production as assessed by western blotting. b HIF-1α inhibitor (2-methoxyestradiol, 10 μM) prevented CRP-induced HIF-1α and VEGF protein expression. c Effects of kinase inhibitors on CRP-induced HIF-1α and VEGF expression examined by western blotting. MAPK and PI3K signaling pathway inhibition suppressed CRP-induced HIF-1α and VEGF expression. Columns , mean; bars , SE. * p
Figure Legend Snippet: CRP stimulated VEGF expression through HIF-1α, which was linked to activation of the PI3K/AKT1 and MAPK/ERK1/2 pathways. a CRP increased HIF-1α production as assessed by western blotting. b HIF-1α inhibitor (2-methoxyestradiol, 10 μM) prevented CRP-induced HIF-1α and VEGF protein expression. c Effects of kinase inhibitors on CRP-induced HIF-1α and VEGF expression examined by western blotting. MAPK and PI3K signaling pathway inhibition suppressed CRP-induced HIF-1α and VEGF expression. Columns , mean; bars , SE. * p

Techniques Used: Expressing, Activation Assay, Western Blot, Inhibition

CD64 mediated CRP-induced VEGF expression regulation in ADSCs. a Immunofluorescence staining of ADSCs for FcγRIII (CD16), FcγRII (CD32), and FcγRI (CD64) expression in ADSCs. b mRNA expression of CD16, CD32, and CD64 detected by PCR amplification and agarose gel electrophoresis. c CRP stimulation significantly increased CD64 mRNA expression in ADSCs, whereas no significant difference was detected in CD16 and CD32, * p
Figure Legend Snippet: CD64 mediated CRP-induced VEGF expression regulation in ADSCs. a Immunofluorescence staining of ADSCs for FcγRIII (CD16), FcγRII (CD32), and FcγRI (CD64) expression in ADSCs. b mRNA expression of CD16, CD32, and CD64 detected by PCR amplification and agarose gel electrophoresis. c CRP stimulation significantly increased CD64 mRNA expression in ADSCs, whereas no significant difference was detected in CD16 and CD32, * p

Techniques Used: Expressing, Immunofluorescence, Staining, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

CRP treatment upregulates VEGF protein and production levels and promotes angiogenesis in ADSCs. a CRP increased VEGF but not HGF production as assessed by western blotting. Values were normalized to β-tubulin as a control, * p
Figure Legend Snippet: CRP treatment upregulates VEGF protein and production levels and promotes angiogenesis in ADSCs. a CRP increased VEGF but not HGF production as assessed by western blotting. Values were normalized to β-tubulin as a control, * p

Techniques Used: Western Blot

CM with or without added CRP analyzed by antibody-based protein arrays. ADSCs were cultured with 10 % FBS low-glucose DMEM until 80–90 % confluence was reached and then incubated in DMEM for 24 hours. The CM was then collected for protein assays; increased proteins after CRP treatment are indicated with letters. CRP C-reactive protein, VEGF vascular endothelial growth factor
Figure Legend Snippet: CM with or without added CRP analyzed by antibody-based protein arrays. ADSCs were cultured with 10 % FBS low-glucose DMEM until 80–90 % confluence was reached and then incubated in DMEM for 24 hours. The CM was then collected for protein assays; increased proteins after CRP treatment are indicated with letters. CRP C-reactive protein, VEGF vascular endothelial growth factor

Techniques Used: Cell Culture, Incubation

35) Product Images from "Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity"

Article Title: Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity

Journal: Scientific Reports

doi: 10.1038/srep07518

Characterization of VEGFR2-binding Affibody molecules from phage display selection. (a) Sensorgrams from SPR analysis of Z VEGFR2_1 and Z VEGFR2_2 binding to immobilized human VEGFR2. Affibody molecules were injected at concentrations ranging from 50 nM to 500 nM. Data is double referenced by subtraction of simultaneous responses from reference surface and a buffer injection. The experiment was performed in duplicates. (b) Representative results from SPR-based competition assay. Sensorgrams were obtained from a double injection, where a first injection of 1 μM of Z VEGFR2_1 (I) was immediately followed by a second injection (II) of either a combination of 1 μM of Z VEGFR2_1 and 1 μM of Z VEGFR2_2 , or 2 μM of Z VEGFR2_1 , over immobilized human VEGFR2. The experiment was performed in duplicates. (c) Representative results from SPR-based analysis of human VEGF blocking. 40 nM of human VEGFR2, which had been pre-incubated for 40 min with a 25 × molar excess of Z VEGFR2_1 , Z VEGFR2_2 or PBS (control), was injected over a surface of immobilized human VEGF-A. The experiment was performed in duplicates.
Figure Legend Snippet: Characterization of VEGFR2-binding Affibody molecules from phage display selection. (a) Sensorgrams from SPR analysis of Z VEGFR2_1 and Z VEGFR2_2 binding to immobilized human VEGFR2. Affibody molecules were injected at concentrations ranging from 50 nM to 500 nM. Data is double referenced by subtraction of simultaneous responses from reference surface and a buffer injection. The experiment was performed in duplicates. (b) Representative results from SPR-based competition assay. Sensorgrams were obtained from a double injection, where a first injection of 1 μM of Z VEGFR2_1 (I) was immediately followed by a second injection (II) of either a combination of 1 μM of Z VEGFR2_1 and 1 μM of Z VEGFR2_2 , or 2 μM of Z VEGFR2_1 , over immobilized human VEGFR2. The experiment was performed in duplicates. (c) Representative results from SPR-based analysis of human VEGF blocking. 40 nM of human VEGFR2, which had been pre-incubated for 40 min with a 25 × molar excess of Z VEGFR2_1 , Z VEGFR2_2 or PBS (control), was injected over a surface of immobilized human VEGF-A. The experiment was performed in duplicates.

Techniques Used: Binding Assay, Selection, SPR Assay, Injection, Competitive Binding Assay, Blocking Assay, Incubation

Characterization of affinity matured VEGFR2-binding Affibody molecules selected by staphylococcal display. (a) Representative sensorgrams from SPR analysis of affinity-matured Affibody molecules (Z VEGFR2_22 and Z VEGFR2_40 ) binding to immobilized VEGFR2, showing the response signal. Affibody molecules were injected at concentrations of 5, 10 and 20 nM. Data is referenced by subtraction of simultaneous responses from reference surface. The experiment was performed in duplicates. (b) Representative results from SPR-based competition assay. Sensorgrams were obtained from a double injection, where 1 μM of Z VEGFR2_22 was injected (1), immediately followed by a second injection (2) of either a combination of 1 μM of Z VEGFR2_22 and 1 μM of Z VEGFR2_40 , or 2 μM of Z VEGFR2_22 , over immobilized human or murine VEGFR2. The experiment was performed in duplicates. (c) Representative results from SPR-based assay of VEGF blocking. 40 nM of human VEGFR2, which had been pre-incubated for 40 min with a 25 × molar excess of Z VEGFR2_22 , was injected over a surface of immobilized human VEGF-A. The experiment was performed in duplicates.
Figure Legend Snippet: Characterization of affinity matured VEGFR2-binding Affibody molecules selected by staphylococcal display. (a) Representative sensorgrams from SPR analysis of affinity-matured Affibody molecules (Z VEGFR2_22 and Z VEGFR2_40 ) binding to immobilized VEGFR2, showing the response signal. Affibody molecules were injected at concentrations of 5, 10 and 20 nM. Data is referenced by subtraction of simultaneous responses from reference surface. The experiment was performed in duplicates. (b) Representative results from SPR-based competition assay. Sensorgrams were obtained from a double injection, where 1 μM of Z VEGFR2_22 was injected (1), immediately followed by a second injection (2) of either a combination of 1 μM of Z VEGFR2_22 and 1 μM of Z VEGFR2_40 , or 2 μM of Z VEGFR2_22 , over immobilized human or murine VEGFR2. The experiment was performed in duplicates. (c) Representative results from SPR-based assay of VEGF blocking. 40 nM of human VEGFR2, which had been pre-incubated for 40 min with a 25 × molar excess of Z VEGFR2_22 , was injected over a surface of immobilized human VEGF-A. The experiment was performed in duplicates.

Techniques Used: Binding Assay, SPR Assay, Injection, Competitive Binding Assay, Blocking Assay, Incubation

Characterization of biparatopic Affibody constructs. (a) Schematic overview of the design of the dimeric constructs. (b) Schematic overview of SPR-based off rate analysis assay. HSA was immobilized on the chip surface. A first injection of dimeric Affibody constructs resulted in a negligible off rate due to the femtomolar affinity of ABD for HSA. VEGFR2 binding was analyzed by subsequent injection of monomeric VEGFR2. The experiments were performed in duplicates. (c) Representative sensorgrams obtained from the SPR-based off-rate analysis assay, showing the injection of 40 nM monomeric human VEGFR2 over each of the four dimeric Affibody molecules. Data is double referenced by subtraction of simultaneous responses from reference surface and a buffer injection. (d) Flow-cytometric analysis of binding of dimeric Affibody constructs to VEGFR2-expressing 293/KDR cells. Binding of the Affibody constructs is monitored by the binding of fluorescently labeled HSA to the ABD tag. A higher shift in mean log fluorescence intensity compared to the negative control construct Z Taq -ABD-Z Taq or cells labeled with secondary reagent only was observed for the heterodimeric constructs than for the homodimeric constructs upon binding to VEGFR2-expressing cells. The experiment was performed in duplicates. (e) Inhibition of VEGF-A induced phosphorylation of VEGFR2 on 293/KDR cells. Cells were pre-treated with the biparatopic binder Z VEGFR2_22 -(S 4 G) 4 -ABD 035 -(S 4 G) 4 -Z VEGFR2_40 , a combination of 30 nM or 3 nM of each of the monomeric binders Z VEGFR2_22 and Z VEGFR2_40 , 30 nM or 3 nM of the negative control construct Z Taq -ABD 035 -Z Taq , or PBS, followed by stimulation with VEGF-A. VEGFR2 phosphorylation was determined by ELISA. The biparatopic binder and the combination of monomers both resulted in a decrease in phosphorylation level compared to the controls, and a more potent inhibition was observed for the biparatopic binder. The data is presented as the OD450 for each sample normalized against the OD450 of untreated cells. The experiment was performed in duplicates.
Figure Legend Snippet: Characterization of biparatopic Affibody constructs. (a) Schematic overview of the design of the dimeric constructs. (b) Schematic overview of SPR-based off rate analysis assay. HSA was immobilized on the chip surface. A first injection of dimeric Affibody constructs resulted in a negligible off rate due to the femtomolar affinity of ABD for HSA. VEGFR2 binding was analyzed by subsequent injection of monomeric VEGFR2. The experiments were performed in duplicates. (c) Representative sensorgrams obtained from the SPR-based off-rate analysis assay, showing the injection of 40 nM monomeric human VEGFR2 over each of the four dimeric Affibody molecules. Data is double referenced by subtraction of simultaneous responses from reference surface and a buffer injection. (d) Flow-cytometric analysis of binding of dimeric Affibody constructs to VEGFR2-expressing 293/KDR cells. Binding of the Affibody constructs is monitored by the binding of fluorescently labeled HSA to the ABD tag. A higher shift in mean log fluorescence intensity compared to the negative control construct Z Taq -ABD-Z Taq or cells labeled with secondary reagent only was observed for the heterodimeric constructs than for the homodimeric constructs upon binding to VEGFR2-expressing cells. The experiment was performed in duplicates. (e) Inhibition of VEGF-A induced phosphorylation of VEGFR2 on 293/KDR cells. Cells were pre-treated with the biparatopic binder Z VEGFR2_22 -(S 4 G) 4 -ABD 035 -(S 4 G) 4 -Z VEGFR2_40 , a combination of 30 nM or 3 nM of each of the monomeric binders Z VEGFR2_22 and Z VEGFR2_40 , 30 nM or 3 nM of the negative control construct Z Taq -ABD 035 -Z Taq , or PBS, followed by stimulation with VEGF-A. VEGFR2 phosphorylation was determined by ELISA. The biparatopic binder and the combination of monomers both resulted in a decrease in phosphorylation level compared to the controls, and a more potent inhibition was observed for the biparatopic binder. The data is presented as the OD450 for each sample normalized against the OD450 of untreated cells. The experiment was performed in duplicates.

Techniques Used: Construct, SPR Assay, Chromatin Immunoprecipitation, Injection, Binding Assay, Flow Cytometry, Expressing, Labeling, Fluorescence, Negative Control, Inhibition, Enzyme-linked Immunosorbent Assay

36) Product Images from "Serum VEGF-A and Tumor Vessel VEGFR-2 Levels Predict Survival in Caucasian but Not Asian Patients Undergoing Resection for Gastric Adenocarcinoma"

Article Title: Serum VEGF-A and Tumor Vessel VEGFR-2 Levels Predict Survival in Caucasian but Not Asian Patients Undergoing Resection for Gastric Adenocarcinoma

Journal: Annals of surgical oncology

doi: 10.1245/s10434-015-4790-y

Scatter plot of serum VEGF-A levels in gastric and gastroesophageal junction cancers in Caucasian and Asian patients
Figure Legend Snippet: Scatter plot of serum VEGF-A levels in gastric and gastroesophageal junction cancers in Caucasian and Asian patients

Techniques Used:

37) Product Images from "Induction of Podocyte VEGF164 Overexpression at Different Stages of Development Causes Congenital Nephrosis or Steroid-Resistant Nephrotic Syndrome"

Article Title: Induction of Podocyte VEGF164 Overexpression at Different Stages of Development Causes Congenital Nephrosis or Steroid-Resistant Nephrotic Syndrome

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2010.091146

Podocyte VEGF 164 overexpression during organogenesis. A–C: VEGF-A immunohistochemistry showing increased immunoreactive VEGF-A (brown) in glomeruli from pod-rtTA:tet-O-VEGF 164 mice on doxycycline ( A , + dox) versus single transgenic littermates ( B , + dox) and uninduced pod-rtTA:tet-O-VEGF 164 mice ( C , − dox). D: VEGF measured by enzyme-linked immunosorbent assay in whole kidney lysate, showing a twofold increase in mice overexpressing podocyte VEGF 164 (TOPO) versus controls. * P
Figure Legend Snippet: Podocyte VEGF 164 overexpression during organogenesis. A–C: VEGF-A immunohistochemistry showing increased immunoreactive VEGF-A (brown) in glomeruli from pod-rtTA:tet-O-VEGF 164 mice on doxycycline ( A , + dox) versus single transgenic littermates ( B , + dox) and uninduced pod-rtTA:tet-O-VEGF 164 mice ( C , − dox). D: VEGF measured by enzyme-linked immunosorbent assay in whole kidney lysate, showing a twofold increase in mice overexpressing podocyte VEGF 164 (TOPO) versus controls. * P

Techniques Used: Over Expression, Immunohistochemistry, Mouse Assay, Transgenic Assay, Enzyme-linked Immunosorbent Assay

VEGF-induced MCD is steroid-resistant. A: Western blot showing albuminuria from induced pod-rtTA:tet-O-VEGF 164 (+ dox), which does not improve with methylprednisolone, whereas uninduced mice (− dox) receiving methylprednisolone do not develop proteinuria. B: TEM images showing extensive podocyte effacement in induced kidneys (+ dox, top panels ), which did not improve with methylprednisolone ( top right panel ), and normal ultrastructure in uninduced kidneys (− dox, bottom panels ), unchanged by methylprednisolone ( bottom right panel ). efp, effaced foot process; cap, capillary; P, podocyte; EC, endothelial cell. C: Western blots showing VEGF 164 -induced decreased nephrin levels, further decreased by methylprednisolone; expression level changes are shown as fold mean ± SEM change from uninduced baseline ( n = 6 blots). Podocin and WT1 immunoblots showed no protein level changes ( n = 4 blots each). * P
Figure Legend Snippet: VEGF-induced MCD is steroid-resistant. A: Western blot showing albuminuria from induced pod-rtTA:tet-O-VEGF 164 (+ dox), which does not improve with methylprednisolone, whereas uninduced mice (− dox) receiving methylprednisolone do not develop proteinuria. B: TEM images showing extensive podocyte effacement in induced kidneys (+ dox, top panels ), which did not improve with methylprednisolone ( top right panel ), and normal ultrastructure in uninduced kidneys (− dox, bottom panels ), unchanged by methylprednisolone ( bottom right panel ). efp, effaced foot process; cap, capillary; P, podocyte; EC, endothelial cell. C: Western blots showing VEGF 164 -induced decreased nephrin levels, further decreased by methylprednisolone; expression level changes are shown as fold mean ± SEM change from uninduced baseline ( n = 6 blots). Podocin and WT1 immunoblots showed no protein level changes ( n = 4 blots each). * P

Techniques Used: Western Blot, Mouse Assay, Transmission Electron Microscopy, Expressing

38) Product Images from "Co-suppression of VEGF-A and VEGF-C inhibits development of experimental hemangioma"

Article Title: Co-suppression of VEGF-A and VEGF-C inhibits development of experimental hemangioma

Journal: American Journal of Translational Research

doi:

Proliferating-phase of the hemangiomas expresses significantly higher VEGF-A and VEGF-C than involuting-phase. A. RT-qPCR for VEGF family members in hemangiomas at involuting-phase and proliferating-phase. B. ELISA for VEGF family members in hemangiomas at involuting-phase and proliferating-phase. *P
Figure Legend Snippet: Proliferating-phase of the hemangiomas expresses significantly higher VEGF-A and VEGF-C than involuting-phase. A. RT-qPCR for VEGF family members in hemangiomas at involuting-phase and proliferating-phase. B. ELISA for VEGF family members in hemangiomas at involuting-phase and proliferating-phase. *P

Techniques Used: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Co-suppression of both VEGF-A and VEGF-C has a more pronounced effect on HemECs cell proliferation than suppression of either VEGF-A or VEGF-C alone. (A, B) BrdU assay on control (scrambled), or shVEGF-A or shVEGF-C or combined shVEGF-A and shVEGF-C (shVEGF-A+C) cells, shown by representative images (A), and by quantification (B). BrdU in red and DNA in blue. Co-suppression of both VEGF-A and VEGF-C has a more pronounced effect on HemECs cell proliferation than suppression of either VEGF-A or VEGF-C alone. (C, D) Western blotting for cell-cycle regulators in transduced HemECs, shown by representative blots (C), and by quantification (D). shVEGF-A significantly enhanced cell-cycle suppressor p21, significantly reduced cell-cycle activators CyclinD1 and CDK4, without affecting cell-cycle activator CyclinB2 and cell-cycle suppressor p27. On the other hand, shVEGF-C significantly enhanced cell-cycle suppressor p27, significantly reduced cell-cycle activator CyclinB2, without affecting cell-cycle activators CyclinD1 and CDK4 and cell-cycle suppressor p21. Co-suppression of both VEGF-A and VEGF-C significantly enhanced both p21 and p27, and significantly reduced CyclinD1, CDK4 and CyclinB2. *P
Figure Legend Snippet: Co-suppression of both VEGF-A and VEGF-C has a more pronounced effect on HemECs cell proliferation than suppression of either VEGF-A or VEGF-C alone. (A, B) BrdU assay on control (scrambled), or shVEGF-A or shVEGF-C or combined shVEGF-A and shVEGF-C (shVEGF-A+C) cells, shown by representative images (A), and by quantification (B). BrdU in red and DNA in blue. Co-suppression of both VEGF-A and VEGF-C has a more pronounced effect on HemECs cell proliferation than suppression of either VEGF-A or VEGF-C alone. (C, D) Western blotting for cell-cycle regulators in transduced HemECs, shown by representative blots (C), and by quantification (D). shVEGF-A significantly enhanced cell-cycle suppressor p21, significantly reduced cell-cycle activators CyclinD1 and CDK4, without affecting cell-cycle activator CyclinB2 and cell-cycle suppressor p27. On the other hand, shVEGF-C significantly enhanced cell-cycle suppressor p27, significantly reduced cell-cycle activator CyclinB2, without affecting cell-cycle activators CyclinD1 and CDK4 and cell-cycle suppressor p21. Co-suppression of both VEGF-A and VEGF-C significantly enhanced both p21 and p27, and significantly reduced CyclinD1, CDK4 and CyclinB2. *P

Techniques Used: BrdU Staining, Western Blot

Co-suppression of both VEGF-A and VEGF-C has a more pronounced effect on HemECs growth than suppression of either VEGF-A or VEGF-C alone. Cultured hemangioma-derived endothelial cells (HemECs) were transduced with AAV carrying either control (scrambled), or shVEGF-A or shVEGF-C or combined shVEGF-A and shVEGF-C (shVEGF-A+C). A. RT-qPCR for VEGF-A and VEGF-C on the transduced cells, which confirmed the effects of shVEGF-A and shVEGF-C. B. An MTT assay for the transduced cells. Co-suppression of both VEGF-A and VEGF-C has a more pronounced effect on HemECs growth than suppression of either VEGF-A or VEGF-C alone. *P
Figure Legend Snippet: Co-suppression of both VEGF-A and VEGF-C has a more pronounced effect on HemECs growth than suppression of either VEGF-A or VEGF-C alone. Cultured hemangioma-derived endothelial cells (HemECs) were transduced with AAV carrying either control (scrambled), or shVEGF-A or shVEGF-C or combined shVEGF-A and shVEGF-C (shVEGF-A+C). A. RT-qPCR for VEGF-A and VEGF-C on the transduced cells, which confirmed the effects of shVEGF-A and shVEGF-C. B. An MTT assay for the transduced cells. Co-suppression of both VEGF-A and VEGF-C has a more pronounced effect on HemECs growth than suppression of either VEGF-A or VEGF-C alone. *P

Techniques Used: Cell Culture, Derivative Assay, Transduction, Quantitative RT-PCR, MTT Assay

Suppression of both VEGF-A and VEGF-C has more pronounced effects on the growth of the implanted hemangiomas than suppression of either VEGF-A or VEGF-C HemECs transduced with control or shVEGF-A or shVEGF-C or shVEGF-A+C were implanted into nude mice with HUVECs and the tumor growth was assessed after 4 weeks. (A, B) Bioluminescence assay, shown by representative images (A), and by quantification (B). (C, D) Tumor size, shown by gross view (C), and by quantification (D). *P
Figure Legend Snippet: Suppression of both VEGF-A and VEGF-C has more pronounced effects on the growth of the implanted hemangiomas than suppression of either VEGF-A or VEGF-C HemECs transduced with control or shVEGF-A or shVEGF-C or shVEGF-A+C were implanted into nude mice with HUVECs and the tumor growth was assessed after 4 weeks. (A, B) Bioluminescence assay, shown by representative images (A), and by quantification (B). (C, D) Tumor size, shown by gross view (C), and by quantification (D). *P

Techniques Used: Transduction, Mouse Assay, ATP Bioluminescent Assay

Co-suppression of both VEGF-A and VEGF-C has a more pronounced effect on HemECs cell apoptosis than suppression of either VEGF-A or VEGF-C. (A, B) Annexin V assay on control (scrambled), or shVEGF-A or shVEGF-C or combined shVEGF-A and shVEGF-C (shVEGF-A+C) cells, shown by representative flow charts (A), and by quantification (B). (C, D) Western blotting for apoptosis-associated proteins in transduced HemECs, shown by representative blots (C), and by quantification (D). shVEGF-A significantly enhanced pro-apoptosis protein CYTC and caspase3, significantly reduced anti-apoptosis protein Bcl-2, without affecting pro-apoptosis protein caspase9. On the other hand, shVEGF-C significantly enhanced pro-apoptosis protein caspase9, significantly reduced anti-apoptosis protein Bcl-2, without affecting pro-apoptosis protein CYTC and caspase3. Co-suppression of both VEGF-A and VEGF-C significantly enhanced CYTC, caspase3 and caspase9, and more pronouncedly reduced Bcl-2. *P
Figure Legend Snippet: Co-suppression of both VEGF-A and VEGF-C has a more pronounced effect on HemECs cell apoptosis than suppression of either VEGF-A or VEGF-C. (A, B) Annexin V assay on control (scrambled), or shVEGF-A or shVEGF-C or combined shVEGF-A and shVEGF-C (shVEGF-A+C) cells, shown by representative flow charts (A), and by quantification (B). (C, D) Western blotting for apoptosis-associated proteins in transduced HemECs, shown by representative blots (C), and by quantification (D). shVEGF-A significantly enhanced pro-apoptosis protein CYTC and caspase3, significantly reduced anti-apoptosis protein Bcl-2, without affecting pro-apoptosis protein caspase9. On the other hand, shVEGF-C significantly enhanced pro-apoptosis protein caspase9, significantly reduced anti-apoptosis protein Bcl-2, without affecting pro-apoptosis protein CYTC and caspase3. Co-suppression of both VEGF-A and VEGF-C significantly enhanced CYTC, caspase3 and caspase9, and more pronouncedly reduced Bcl-2. *P

Techniques Used: Annexin V Assay, Flow Cytometry, Western Blot

39) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus Induces Sustained Levels of Vascular Endothelial Growth Factors A and C Early during In Vitro Infection of Human Microvascular Dermal Endothelial Cells: Biological Implications ▿"

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Induces Sustained Levels of Vascular Endothelial Growth Factors A and C Early during In Vitro Infection of Human Microvascular Dermal Endothelial Cells: Biological Implications ▿

Journal:

doi: 10.1128/JVI.00873-07

Exogenous addition of VEGF-A or VEGF-C activates KSHV ORF50 promoter and gene expression. 293 cells were transfected with control p-Luc or ORF50 promoter-Luciferase constructs and after 24 h, cells were either uninfected or infected with KSHV at an MOI
Figure Legend Snippet: Exogenous addition of VEGF-A or VEGF-C activates KSHV ORF50 promoter and gene expression. 293 cells were transfected with control p-Luc or ORF50 promoter-Luciferase constructs and after 24 h, cells were either uninfected or infected with KSHV at an MOI

Techniques Used: Expressing, Transfection, Luciferase, Construct, Infection

Exogenous addition of VEGF-A or VEGF-C activates KSHV ORF73 promoter and gene expression. 293 cells were transfected with control pGL3-Luc or ORF73 promoter-luciferase construct and after 24 h cells were either uninfected or infected with KSHV at an MOI
Figure Legend Snippet: Exogenous addition of VEGF-A or VEGF-C activates KSHV ORF73 promoter and gene expression. 293 cells were transfected with control pGL3-Luc or ORF73 promoter-luciferase construct and after 24 h cells were either uninfected or infected with KSHV at an MOI

Techniques Used: Expressing, Transfection, Luciferase, Construct, Infection

Blocking KSHV binding by heparin inhibits VEGF-A expression. (A) KSHV was incubated at 37°C for 1 h with DMEM containing 100 μg/ml of soluble heparin. This mixture was then added to a serum-starved (8 h) HMVEC-d cell monolayer and incubated
Figure Legend Snippet: Blocking KSHV binding by heparin inhibits VEGF-A expression. (A) KSHV was incubated at 37°C for 1 h with DMEM containing 100 μg/ml of soluble heparin. This mixture was then added to a serum-starved (8 h) HMVEC-d cell monolayer and incubated

Techniques Used: Blocking Assay, Binding Assay, Expressing, Incubation

Schematic model depicting the potential implications of VEGF-A and VEGF-C induction during in vitro KSHV infection of endothelial cells. KSHV has been shown to be reactivated under immunosuppression conditions, resulting in increased circulating virus.
Figure Legend Snippet: Schematic model depicting the potential implications of VEGF-A and VEGF-C induction during in vitro KSHV infection of endothelial cells. KSHV has been shown to be reactivated under immunosuppression conditions, resulting in increased circulating virus.

Techniques Used: In Vitro, Infection

Detection of VEGF-A and VEGF-C mRNA and protein in KSHV-infected HMVEC-d cells. HMVEC-d cells grown to 80 to 90% confluence were serum starved for 8 h and infected with KSHV at an MOI of 10. (A and C) Infected and uninfected cells were washed
Figure Legend Snippet: Detection of VEGF-A and VEGF-C mRNA and protein in KSHV-infected HMVEC-d cells. HMVEC-d cells grown to 80 to 90% confluence were serum starved for 8 h and infected with KSHV at an MOI of 10. (A and C) Infected and uninfected cells were washed

Techniques Used: Infection

Immunofluorescent detection of KSHV infection-induced VEGF-A and -C protein expression in HMVEC-d cells. (A and B) Uninfected HMVEC-d cells (panels a, b, and c) or cells infected with KSHV (MOI, 10) for 72 h (panels d, e, and f) were permeabilized and
Figure Legend Snippet: Immunofluorescent detection of KSHV infection-induced VEGF-A and -C protein expression in HMVEC-d cells. (A and B) Uninfected HMVEC-d cells (panels a, b, and c) or cells infected with KSHV (MOI, 10) for 72 h (panels d, e, and f) were permeabilized and

Techniques Used: Infection, Expressing

Induction of VEGF-A by UV-inactivated KSHV and viral glycoproteins gB and gpK8.1A. (A) HMVEC-d cells (80 to 90% confluence) serum starved for 8 h were uninfected or infected with either live KSHV or UV-irradiated KSHV for the indicated times at
Figure Legend Snippet: Induction of VEGF-A by UV-inactivated KSHV and viral glycoproteins gB and gpK8.1A. (A) HMVEC-d cells (80 to 90% confluence) serum starved for 8 h were uninfected or infected with either live KSHV or UV-irradiated KSHV for the indicated times at

Techniques Used: Infection, Irradiation

40) Product Images from "Pathological neovascularization is reduced by inactivation of ADAM17 in endothelial cells, but not in pericytes"

Article Title: Pathological neovascularization is reduced by inactivation of ADAM17 in endothelial cells, but not in pericytes

Journal: Circulation research

doi: 10.1161/CIRCRESAHA.109.207415

Endothelial cell proliferation and tube formation assays (A) Proliferation assay and (B) tube formation assay with endothelial cells isolated from Adam17flox/flox/Tie2-Cre mice or Adam17flox/flox controls (see materials and methods). There was no significant difference in proliferation at day 4 (d4) of the culture plated a comparable density on day 0 (d0) (A), but a clear decrease in tube formation was seen in endothelial cells from Adam17flox/flox/Tie2-Cre mice compared to controls (B), which could be largely rescued by treatment with 5ng/ml HB-EGF, but not with 5ng/ml VEGF-A.
Figure Legend Snippet: Endothelial cell proliferation and tube formation assays (A) Proliferation assay and (B) tube formation assay with endothelial cells isolated from Adam17flox/flox/Tie2-Cre mice or Adam17flox/flox controls (see materials and methods). There was no significant difference in proliferation at day 4 (d4) of the culture plated a comparable density on day 0 (d0) (A), but a clear decrease in tube formation was seen in endothelial cells from Adam17flox/flox/Tie2-Cre mice compared to controls (B), which could be largely rescued by treatment with 5ng/ml HB-EGF, but not with 5ng/ml VEGF-A.

Techniques Used: Proliferation Assay, Tube Formation Assay, Isolation, Mouse Assay

Related Articles

BIA-KA:

Article Title: Hypoxia-Inducible Factor-1 Activation by (-)-Epicatechin Gallate: Potential Adverse Effects of Cancer Chemoprevention with High-Dose Green Tea Extracts
Article Snippet: .. The levels of VEGF proteins in the cell lysate were determined using an ELISA assay for human VEGF proteins (R & D Systems) as described and the protein concentration in the cell lysate were determined using a micro BCA assay kit (Pierce). .. Data comparisons were performed using ANOVA and Fisher’s PLSD post hoc analyses (StatView® Software Version 5.01, SAS Institute Inc).

Cell Culture:

Article Title: Fluorescence and Bioluminescence Imaging of Angiogenesis in Flk1-Nano-lantern Transgenic Mice
Article Snippet: .. Individual rings were embedded in type I collagen gel (Nitta Gelatin, Osaka, Japan) on glass-bottom dishes and cultured in DMEM containing 50 ng/ml human recombinant VEGF-A (R & D systems). ..

Enzyme-linked Immunosorbent Assay:

Article Title: Vascular endothelial growth factor/vascular permeability factor is an autocrine growth factor for AIDS-Kaposi sarcoma
Article Snippet: .. Recombinant human VEGF (rhVEGF) and VEGF ELISA kits were purchased from R & D Systems. .. BALB/c Nu+/NU+ athymic mice were purchased from Simonsen Laboratories (Gilroy, CA).

Article Title: The Terpenoid Tetrahydroisoquinoline Alkaloids Emetine, Klugine, and Isocephaeline Inhibit the Activation of Hypoxia-Inducible Factor-1 (HIF-1) in Breast Tumor Cells
Article Snippet: .. The level of secreted VEGF proteins in the conditioned media was determined using a modified ELISA assay for human VEGF proteins (R & D Systems, Minneapolis, MN). .. Exponentially grown T47D cells were plated at the density of 30,000 cells per well in a volume of 100 µL DMEM/F12 medium supplemented with 10% FCS (v/v) and 0.5% penicillin/streptomycin into 96-well tissue culture plate (Corning Inc., Corning, NY).

Article Title: Hypoxia-Inducible Factor-1 Activation by (-)-Epicatechin Gallate: Potential Adverse Effects of Cancer Chemoprevention with High-Dose Green Tea Extracts
Article Snippet: .. The levels of VEGF proteins in the cell lysate were determined using an ELISA assay for human VEGF proteins (R & D Systems) as described and the protein concentration in the cell lysate were determined using a micro BCA assay kit (Pierce). .. Data comparisons were performed using ANOVA and Fisher’s PLSD post hoc analyses (StatView® Software Version 5.01, SAS Institute Inc).

Concentration Assay:

Article Title: von Willebrand factor fibers promote cancer-associated platelet aggregation in malignant melanoma of mice and humans
Article Snippet: .. To measure VEGF-tinzaparin interaction, we added tinzaparin (concentration range, 5 µM to 80 µM) to 0.5 µM human recombinant VEGF-A (R & D Systems) in PBS. .. The reaction mixtures were incubated at room temperature for 20 minutes and measured in a fluorescence spectrometer (excitation, 290 nm; emission, 320-450 nm; Tecan infinite M200, Tecan Group Ltd.).

Article Title: ETS-1 Protein Regulates Vascular Endothelial Growth Factor-induced Matrix Metalloproteinase-9 and Matrix Metalloproteinase-13 Expression in Human Ovarian Carcinoma Cell Line SKOV-3 *
Article Snippet: .. The human recombinant VEGF-A (hereafter referred to as VEGF) was purchased from R & D Systems, Minneapolis, MN and used at a concentration of 20 ng/ml unless otherwise specified. .. To study the effect of VEGF receptor tyrosine kinase inhibitor-II (Calbiochem), the cells were pretreated with a 20 n m concentration of this inhibitor for 30 min followed by measurement of VEGF-induced cell invasion, scattering, and gene expression.

Blocking Assay:

Article Title: VEGF-C sustains VEGFR2 activation under bevacizumab therapy and promotes glioblastoma maintenance
Article Snippet: .. Inhibitors, Growth Factors, and Blocking Antibodies We purchased SU1498 (Millipore), bevacizumab (Avastin, 25 mg/mL, Roche), recombinant VEGF-A-165aa (50 µg/mL, Miltenyi Biotec), and recombinant human mature VEGF-C (Thr103-Arg227~21 kDa; 10 µg/mL, R & D Systems). .. SiRNA Transfection/shRNA Transduction Cells were transfected using 75 pmol of either VEGF-C–small interfering (si)RNA (siVEGF-C-1: esiRNA pool/EHU013781, Sigma-Aldrich; siVEGF-C-2: single siRNA/HSS111277, ThermoFisher Scientific) or scrambled control siRNA (siCtrl: Stealth RNAi Negative Control Duplex Med CG, ThermoFisher Scientific).

Protein Concentration:

Article Title: Hypoxia-Inducible Factor-1 Activation by (-)-Epicatechin Gallate: Potential Adverse Effects of Cancer Chemoprevention with High-Dose Green Tea Extracts
Article Snippet: .. The levels of VEGF proteins in the cell lysate were determined using an ELISA assay for human VEGF proteins (R & D Systems) as described and the protein concentration in the cell lysate were determined using a micro BCA assay kit (Pierce). .. Data comparisons were performed using ANOVA and Fisher’s PLSD post hoc analyses (StatView® Software Version 5.01, SAS Institute Inc).

Modification:

Article Title: The Terpenoid Tetrahydroisoquinoline Alkaloids Emetine, Klugine, and Isocephaeline Inhibit the Activation of Hypoxia-Inducible Factor-1 (HIF-1) in Breast Tumor Cells
Article Snippet: .. The level of secreted VEGF proteins in the conditioned media was determined using a modified ELISA assay for human VEGF proteins (R & D Systems, Minneapolis, MN). .. Exponentially grown T47D cells were plated at the density of 30,000 cells per well in a volume of 100 µL DMEM/F12 medium supplemented with 10% FCS (v/v) and 0.5% penicillin/streptomycin into 96-well tissue culture plate (Corning Inc., Corning, NY).

Recombinant:

Article Title: von Willebrand factor fibers promote cancer-associated platelet aggregation in malignant melanoma of mice and humans
Article Snippet: .. To measure VEGF-tinzaparin interaction, we added tinzaparin (concentration range, 5 µM to 80 µM) to 0.5 µM human recombinant VEGF-A (R & D Systems) in PBS. .. The reaction mixtures were incubated at room temperature for 20 minutes and measured in a fluorescence spectrometer (excitation, 290 nm; emission, 320-450 nm; Tecan infinite M200, Tecan Group Ltd.).

Article Title: Vascular endothelial growth factor/vascular permeability factor is an autocrine growth factor for AIDS-Kaposi sarcoma
Article Snippet: .. Recombinant human VEGF (rhVEGF) and VEGF ELISA kits were purchased from R & D Systems. .. BALB/c Nu+/NU+ athymic mice were purchased from Simonsen Laboratories (Gilroy, CA).

Article Title: ETS-1 Protein Regulates Vascular Endothelial Growth Factor-induced Matrix Metalloproteinase-9 and Matrix Metalloproteinase-13 Expression in Human Ovarian Carcinoma Cell Line SKOV-3 *
Article Snippet: .. The human recombinant VEGF-A (hereafter referred to as VEGF) was purchased from R & D Systems, Minneapolis, MN and used at a concentration of 20 ng/ml unless otherwise specified. .. To study the effect of VEGF receptor tyrosine kinase inhibitor-II (Calbiochem), the cells were pretreated with a 20 n m concentration of this inhibitor for 30 min followed by measurement of VEGF-induced cell invasion, scattering, and gene expression.

Article Title: Fluorescence and Bioluminescence Imaging of Angiogenesis in Flk1-Nano-lantern Transgenic Mice
Article Snippet: .. Individual rings were embedded in type I collagen gel (Nitta Gelatin, Osaka, Japan) on glass-bottom dishes and cultured in DMEM containing 50 ng/ml human recombinant VEGF-A (R & D systems). ..

Article Title: VEGF-C sustains VEGFR2 activation under bevacizumab therapy and promotes glioblastoma maintenance
Article Snippet: .. Inhibitors, Growth Factors, and Blocking Antibodies We purchased SU1498 (Millipore), bevacizumab (Avastin, 25 mg/mL, Roche), recombinant VEGF-A-165aa (50 µg/mL, Miltenyi Biotec), and recombinant human mature VEGF-C (Thr103-Arg227~21 kDa; 10 µg/mL, R & D Systems). .. SiRNA Transfection/shRNA Transduction Cells were transfected using 75 pmol of either VEGF-C–small interfering (si)RNA (siVEGF-C-1: esiRNA pool/EHU013781, Sigma-Aldrich; siVEGF-C-2: single siRNA/HSS111277, ThermoFisher Scientific) or scrambled control siRNA (siCtrl: Stealth RNAi Negative Control Duplex Med CG, ThermoFisher Scientific).

Article Title: Potent anti-angiogenesis and anti-tumor activity of a novel human anti-VEGF antibody, MIL60
Article Snippet: .. Human recombinant VEGF-A was purchased from R & D Systems (Minneapolis, MN, USA). .. Human colon carcinoma HT-29 cells and the human ovarian cancer cell line SKOV3 were obtained from the American Type Culture Collection.

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    R&D Systems vegf a
    Chemotactic Migration of EPCs toward mRNA-Engineered EPCs 1 × 10 5 murine EPCs were cultivated overnight, and then they were transfected with 1.6 μg <t>ANG-1,</t> 0.8 μg <t>VEGF-A,</t> or 0.5 μg SDF-1α mRNA or with an mRNA cocktail containing 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α mRNA. Migration behavior of untreated EPCs (5 × 10 4 ) seeded on transwell inserts toward mRNA-transfected EPCs was analyzed using a chemotactic migration assay. As a control, EPCs incubated with medium or medium containing transfection reagent (TR) were used. After 6 hr, migrated EPCs through 8-μm transwell inserts were stained with DAPI, and cell numbers were calculated using ImageJ software. Scale bars represent 100 μm. Results are shown as mean + SEM (n = 4). Statistical differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test (***p
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    Chemotactic Migration of EPCs toward mRNA-Engineered EPCs 1 × 10 5 murine EPCs were cultivated overnight, and then they were transfected with 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with an mRNA cocktail containing 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α mRNA. Migration behavior of untreated EPCs (5 × 10 4 ) seeded on transwell inserts toward mRNA-transfected EPCs was analyzed using a chemotactic migration assay. As a control, EPCs incubated with medium or medium containing transfection reagent (TR) were used. After 6 hr, migrated EPCs through 8-μm transwell inserts were stained with DAPI, and cell numbers were calculated using ImageJ software. Scale bars represent 100 μm. Results are shown as mean + SEM (n = 4). Statistical differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test (***p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Improving the Angiogenic Potential of EPCs via Engineering with Synthetic Modified mRNAs

    doi: 10.1016/j.omtn.2018.09.005

    Figure Lengend Snippet: Chemotactic Migration of EPCs toward mRNA-Engineered EPCs 1 × 10 5 murine EPCs were cultivated overnight, and then they were transfected with 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with an mRNA cocktail containing 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α mRNA. Migration behavior of untreated EPCs (5 × 10 4 ) seeded on transwell inserts toward mRNA-transfected EPCs was analyzed using a chemotactic migration assay. As a control, EPCs incubated with medium or medium containing transfection reagent (TR) were used. After 6 hr, migrated EPCs through 8-μm transwell inserts were stained with DAPI, and cell numbers were calculated using ImageJ software. Scale bars represent 100 μm. Results are shown as mean + SEM (n = 4). Statistical differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test (***p

    Article Snippet: The concentrations of ANG-1, VEGF-A, and SDF-1α were determined as duplicate in 100 μL using human ANG-1, CXCL-12/SDF-1α, and VEGF-A DuoSet ELISA (R & D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

    Techniques: Migration, Transfection, Incubation, Staining, Software

    Analysis of Wound-Healing Capacity of mRNA-Engineered EPCs via Wound Scratch Migration Assay 1 × 10 5 murine EPCs were cultivated overnight, and then they were transfected with 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with an mRNA cocktail containing 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α mRNA. The next day, cells were detached, and 28,000 EPCs with or without mRNA transfection were seeded in each chamber of Culture-Insert 3 wells in μ-dishes. After 5 hr, when the cells completely attached and covered the surface, an open wound field was generated. Immediately after the generation of wound areas (0 hr) and after 12, 24, and 36 hr, phase-contrast images were taken and closed wound areas were calculated using Tscratch software. Scale bar represents 500 μm. Results are shown as mean + SD (n = 8). Statistical differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test (*p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Improving the Angiogenic Potential of EPCs via Engineering with Synthetic Modified mRNAs

    doi: 10.1016/j.omtn.2018.09.005

    Figure Lengend Snippet: Analysis of Wound-Healing Capacity of mRNA-Engineered EPCs via Wound Scratch Migration Assay 1 × 10 5 murine EPCs were cultivated overnight, and then they were transfected with 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with an mRNA cocktail containing 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α mRNA. The next day, cells were detached, and 28,000 EPCs with or without mRNA transfection were seeded in each chamber of Culture-Insert 3 wells in μ-dishes. After 5 hr, when the cells completely attached and covered the surface, an open wound field was generated. Immediately after the generation of wound areas (0 hr) and after 12, 24, and 36 hr, phase-contrast images were taken and closed wound areas were calculated using Tscratch software. Scale bar represents 500 μm. Results are shown as mean + SD (n = 8). Statistical differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test (*p

    Article Snippet: The concentrations of ANG-1, VEGF-A, and SDF-1α were determined as duplicate in 100 μL using human ANG-1, CXCL-12/SDF-1α, and VEGF-A DuoSet ELISA (R & D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

    Techniques: Migration, Transfection, Generated, Software

    Expression of ANG-1, VEGF-A, and SDF-1α after the Transfection of Synthetic mRNA into Murine EPCs 1 × 10 5 EPCs were seeded and transfected the next day with (A) 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with (B) an mRNA cocktail containing 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α mRNA. The protein expression was analyzed in supernatants 24 hr after the transfection using ELISA. Cells treated with only medium or medium and transfection reagent (TR) served as negative controls. Results are shown as mean + SEM (n = 4). Statistical differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test (****p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Improving the Angiogenic Potential of EPCs via Engineering with Synthetic Modified mRNAs

    doi: 10.1016/j.omtn.2018.09.005

    Figure Lengend Snippet: Expression of ANG-1, VEGF-A, and SDF-1α after the Transfection of Synthetic mRNA into Murine EPCs 1 × 10 5 EPCs were seeded and transfected the next day with (A) 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with (B) an mRNA cocktail containing 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α mRNA. The protein expression was analyzed in supernatants 24 hr after the transfection using ELISA. Cells treated with only medium or medium and transfection reagent (TR) served as negative controls. Results are shown as mean + SEM (n = 4). Statistical differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test (****p

    Article Snippet: The concentrations of ANG-1, VEGF-A, and SDF-1α were determined as duplicate in 100 μL using human ANG-1, CXCL-12/SDF-1α, and VEGF-A DuoSet ELISA (R & D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

    Techniques: Expressing, Transfection, Enzyme-linked Immunosorbent Assay

    Analysis of In Vivo Angiogenetic Potential of mRNA-Engineered EPCs in Chick Embryo Chorioallantoic Membrane Assay (A) Schematic representation of the chorioallantoic membrane (CAM) assay. ANG-1, SDF-1α, and VEGF-A mRNA-transfected and untreated cells (medium or medium containing transfection reagent [TR]) were applied in silicone rings (8-mm inner diameter) onto the CAMs at the ninth day of incubation at 37°C and 60% humidity. At the fourth day of incubation, the CAMs were fixed and excised. (B) Analysis of the region of the inner ring circle with Wimasis WimCAM image software to quantify angiogenesis. Scale bar of photographs (upper) and analyzed pictures (lower) represents 2.7 mm. (C) Quantification of angiogenesis using Wimasis WimCAM web-based service. The vessel density, total branching points, total vessel network length, and total segments were quantified and compared to the medium control. Results are shown as mean + SD (n = 3). Statistical differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test (**p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Improving the Angiogenic Potential of EPCs via Engineering with Synthetic Modified mRNAs

    doi: 10.1016/j.omtn.2018.09.005

    Figure Lengend Snippet: Analysis of In Vivo Angiogenetic Potential of mRNA-Engineered EPCs in Chick Embryo Chorioallantoic Membrane Assay (A) Schematic representation of the chorioallantoic membrane (CAM) assay. ANG-1, SDF-1α, and VEGF-A mRNA-transfected and untreated cells (medium or medium containing transfection reagent [TR]) were applied in silicone rings (8-mm inner diameter) onto the CAMs at the ninth day of incubation at 37°C and 60% humidity. At the fourth day of incubation, the CAMs were fixed and excised. (B) Analysis of the region of the inner ring circle with Wimasis WimCAM image software to quantify angiogenesis. Scale bar of photographs (upper) and analyzed pictures (lower) represents 2.7 mm. (C) Quantification of angiogenesis using Wimasis WimCAM web-based service. The vessel density, total branching points, total vessel network length, and total segments were quantified and compared to the medium control. Results are shown as mean + SD (n = 3). Statistical differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test (**p

    Article Snippet: The concentrations of ANG-1, VEGF-A, and SDF-1α were determined as duplicate in 100 μL using human ANG-1, CXCL-12/SDF-1α, and VEGF-A DuoSet ELISA (R & D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

    Techniques: In Vivo, Chick Chorioallantoic Membrane Assay, Transfection, Incubation, Software

    Influence of mRNA Transfection on the Viability of Murine EPCs 1 × 10 5 EPCs were transfected with 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with an mRNA cocktail containing 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α mRNA. Viability was determined 24 hr post-transfection using PrestoBlue assay. The viability of cells incubated with Opti-MEM (medium) was set to 100%, and the viability of samples was expressed relative to these cells. The data are shown as mean + SEM (n = 3). No statistically significant differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Improving the Angiogenic Potential of EPCs via Engineering with Synthetic Modified mRNAs

    doi: 10.1016/j.omtn.2018.09.005

    Figure Lengend Snippet: Influence of mRNA Transfection on the Viability of Murine EPCs 1 × 10 5 EPCs were transfected with 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with an mRNA cocktail containing 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α mRNA. Viability was determined 24 hr post-transfection using PrestoBlue assay. The viability of cells incubated with Opti-MEM (medium) was set to 100%, and the viability of samples was expressed relative to these cells. The data are shown as mean + SEM (n = 3). No statistically significant differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test.

    Article Snippet: The concentrations of ANG-1, VEGF-A, and SDF-1α were determined as duplicate in 100 μL using human ANG-1, CXCL-12/SDF-1α, and VEGF-A DuoSet ELISA (R & D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

    Techniques: Transfection, Prestoblue Assay, Incubation

    Quality Control of the Generated PCR Products and In Vitro -Transcribed mRNAs ANG-1-, SDF-1α-, and VEGF-A-encoding mRNAs were synthesized and modified with 3′-PolyA 120 tail, 5′-ARCA, 100% m5CTP, 100% Ψ-UTP, and post-transcriptional phosphatase treatment. The specific lengths of the amplified DNA and the synthetic mRNA were detected using 1% agarose gel electrophoresis and GelRed staining at approximately 1.8 kb for ANG-1, 0.6 kb for SDF-1α, and 0.9 kb for VEGF-A. The 0.08- to 10-kb range mix DNA ladder and the 0.5- to 10-kb RNA ladder were used as length markers (Ms).

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Improving the Angiogenic Potential of EPCs via Engineering with Synthetic Modified mRNAs

    doi: 10.1016/j.omtn.2018.09.005

    Figure Lengend Snippet: Quality Control of the Generated PCR Products and In Vitro -Transcribed mRNAs ANG-1-, SDF-1α-, and VEGF-A-encoding mRNAs were synthesized and modified with 3′-PolyA 120 tail, 5′-ARCA, 100% m5CTP, 100% Ψ-UTP, and post-transcriptional phosphatase treatment. The specific lengths of the amplified DNA and the synthetic mRNA were detected using 1% agarose gel electrophoresis and GelRed staining at approximately 1.8 kb for ANG-1, 0.6 kb for SDF-1α, and 0.9 kb for VEGF-A. The 0.08- to 10-kb range mix DNA ladder and the 0.5- to 10-kb RNA ladder were used as length markers (Ms).

    Article Snippet: The concentrations of ANG-1, VEGF-A, and SDF-1α were determined as duplicate in 100 μL using human ANG-1, CXCL-12/SDF-1α, and VEGF-A DuoSet ELISA (R & D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

    Techniques: Generated, Polymerase Chain Reaction, In Vitro, Synthesized, Modification, Amplification, Agarose Gel Electrophoresis, Staining, Mass Spectrometry

    Analysis of Angiogenic Potential of mRNA-Engineered EPCs by Tube Formation Assay 1 × 10 5 murine EPCs were cultivated overnight, and then they were transfected with 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with an mRNA cocktail containing 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α mRNA. After 24 hr, EPCs were detached and 1 × 10 4 EPCs were seeded on Matrigel-coated angiogenesis slides. After 4 hr of incubation at 37°C, the formation of tubes was examined by phase-contrast microscopy. Microscopic images were analyzed using NIH ImageJ software with Angiogenesis Analyzer plugin, and segments are shown in magenta, master segments in orange, branches in green, and meshes in blue. The numbers (Nb) of nodes, segments, and master segments and the total (Tot.) segment length, total mesh area, and branching interval were quantified and compared to the medium control. The unit of area and length is pixel (px). Scale bars represent 100 μm. Results are shown as mean + SD (n = 3). Statistical differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test (*p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Improving the Angiogenic Potential of EPCs via Engineering with Synthetic Modified mRNAs

    doi: 10.1016/j.omtn.2018.09.005

    Figure Lengend Snippet: Analysis of Angiogenic Potential of mRNA-Engineered EPCs by Tube Formation Assay 1 × 10 5 murine EPCs were cultivated overnight, and then they were transfected with 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with an mRNA cocktail containing 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α mRNA. After 24 hr, EPCs were detached and 1 × 10 4 EPCs were seeded on Matrigel-coated angiogenesis slides. After 4 hr of incubation at 37°C, the formation of tubes was examined by phase-contrast microscopy. Microscopic images were analyzed using NIH ImageJ software with Angiogenesis Analyzer plugin, and segments are shown in magenta, master segments in orange, branches in green, and meshes in blue. The numbers (Nb) of nodes, segments, and master segments and the total (Tot.) segment length, total mesh area, and branching interval were quantified and compared to the medium control. The unit of area and length is pixel (px). Scale bars represent 100 μm. Results are shown as mean + SD (n = 3). Statistical differences were determined using one-way ANOVA followed by Bonferroni multiple comparison test (*p

    Article Snippet: The concentrations of ANG-1, VEGF-A, and SDF-1α were determined as duplicate in 100 μL using human ANG-1, CXCL-12/SDF-1α, and VEGF-A DuoSet ELISA (R & D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

    Techniques: Tube Formation Assay, Transfection, Incubation, Microscopy, Software

    ELISA analysis of the secretion of the angiogenic factor secreted by the rat adipose-derived stem cells (rADSCs) on the modified scaffolds. a Vascular endothelial growth factor (VEGF), b hepatocyte growth factor (HGF) and c basic fibroblast growth factor (bFGF) secretion by the rADSCs after 7 and 14 days ( n = 6). Note that rADSCs on the PRP+PM and PM scaffolds secreted significantly more angiogenic factors including VEGF, HGF and bFGF than those on the PU and PRP scaffolds ( p

    Journal: Stem Cell Research & Therapy

    Article Title: Argon plasma surface modification promotes the therapeutic angiogenesis and tissue formation of tissue-engineered scaffolds in vivo by adipose-derived stem cells

    doi: 10.1186/s13287-019-1195-z

    Figure Lengend Snippet: ELISA analysis of the secretion of the angiogenic factor secreted by the rat adipose-derived stem cells (rADSCs) on the modified scaffolds. a Vascular endothelial growth factor (VEGF), b hepatocyte growth factor (HGF) and c basic fibroblast growth factor (bFGF) secretion by the rADSCs after 7 and 14 days ( n = 6). Note that rADSCs on the PRP+PM and PM scaffolds secreted significantly more angiogenic factors including VEGF, HGF and bFGF than those on the PU and PRP scaffolds ( p

    Article Snippet: Quantification of the secretion of the angiogenic growth factors using ELISA Firstly, the angiogenic response of the rADSCs was assessed by quantification of angiogenic growth factors, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF), using sandwich enzyme-linked immunosorbent (ELISA; Quantikine, R & D System, Abingdon, UK) on days 7 and 14 of culture (n = 6).

    Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Modification

    (A) New vessels (arrows) close to the anterior surface of the pupillary margin of the iris in an eye with NVI (von Willebrand factor), and (B) the same eye showing bFGF expression in some of the stromal cells (arrows). (C) Positivity of the ciliary body epithelium (arrowheads) and smooth muscle (arrow) for VEGF-A. (D) Attached retina adjacent to uveal melanoma showing mild positivity for VEGF-A, with strong positivity in ganglion cells. (E) Strong VEGF-A positivity in detached retina with considerable atrophy. (F) bFGF expression in atrophic retina. Artefactual detachment of the retina is easily determined histologically by the presence of pigment granules in retinal pigment epithelium processes stripped with the retina forming a line along the base of the photoreceptor outer segments: the detachments shown in both (E) and (F) were tumour related. (G–I) Negative controls for von Willebrand factor, VEGF-A, and bFGF respectively. (All original magnifications ×400 unless otherwise stated: scale bars = 100 μm.)

    Journal: The British Journal of Ophthalmology

    Article Title: Vascular endothelial growth factor is elevated in ocular fluids of eyes harbouring uveal melanoma: identification of a potential therapeutic window

    doi:

    Figure Lengend Snippet: (A) New vessels (arrows) close to the anterior surface of the pupillary margin of the iris in an eye with NVI (von Willebrand factor), and (B) the same eye showing bFGF expression in some of the stromal cells (arrows). (C) Positivity of the ciliary body epithelium (arrowheads) and smooth muscle (arrow) for VEGF-A. (D) Attached retina adjacent to uveal melanoma showing mild positivity for VEGF-A, with strong positivity in ganglion cells. (E) Strong VEGF-A positivity in detached retina with considerable atrophy. (F) bFGF expression in atrophic retina. Artefactual detachment of the retina is easily determined histologically by the presence of pigment granules in retinal pigment epithelium processes stripped with the retina forming a line along the base of the photoreceptor outer segments: the detachments shown in both (E) and (F) were tumour related. (G–I) Negative controls for von Willebrand factor, VEGF-A, and bFGF respectively. (All original magnifications ×400 unless otherwise stated: scale bars = 100 μm.)

    Article Snippet: ELISAs for VEGF-A and bFGF were performed using commercially available kits (R & D Systems, Abingdon, Oxford, UK) according to the manufacturer's instructions.

    Techniques: Expressing

    VEGF levels in (A) aqueous and (B) vitreous, showing high levels in most patients with NVI.

    Journal: The British Journal of Ophthalmology

    Article Title: Vascular endothelial growth factor is elevated in ocular fluids of eyes harbouring uveal melanoma: identification of a potential therapeutic window

    doi:

    Figure Lengend Snippet: VEGF levels in (A) aqueous and (B) vitreous, showing high levels in most patients with NVI.

    Article Snippet: ELISAs for VEGF-A and bFGF were performed using commercially available kits (R & D Systems, Abingdon, Oxford, UK) according to the manufacturer's instructions.

    Techniques:

    Intracellular Ca 2+ measurements, VEGF-A release, and proliferation in response to ANG II in angiomyolipoma cells. A : representative tracings of intracellular Ca 2+ concentration ([Ca 2+ ] i ) responses to ANG II (1 μM) in TRI102 and TRI103 cells. B : measurements of changes in [Ca 2+ ] i (calculated as the maximum fura-2 ratio value after treatment minus the average baseline ratio value) in the presence or absence of the ARB valsartan (Val; 1 μM). Values are expressed as means ± SE. *** P

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Evidence for pericyte origin of TSC-associated renal angiomyolipomas and implications for angiotensin receptor inhibition therapy

    doi: 10.1152/ajprenal.00569.2013

    Figure Lengend Snippet: Intracellular Ca 2+ measurements, VEGF-A release, and proliferation in response to ANG II in angiomyolipoma cells. A : representative tracings of intracellular Ca 2+ concentration ([Ca 2+ ] i ) responses to ANG II (1 μM) in TRI102 and TRI103 cells. B : measurements of changes in [Ca 2+ ] i (calculated as the maximum fura-2 ratio value after treatment minus the average baseline ratio value) in the presence or absence of the ARB valsartan (Val; 1 μM). Values are expressed as means ± SE. *** P

    Article Snippet: After 24 h of treatment, conditioned media were collected, and VEGF-A levels were determined by ELISA according to the manufacturer's specifications (R & D Systems).

    Techniques: Concentration Assay