Journal: Journal of Pain Research

Article Title: Effects and Mechanisms of Wrist-Ankle Acupuncture on Inflammatory Pain in the Rat Gastrocnemius Muscle

doi: 10.2147/JPR.S605568

Figure Lengend Snippet: Inflammatory cytokine levels in the gastrocnemius muscle of rats in each group. ( a ) IL-1β; ( b ) IL-6; ( c ) TNF-α; ( d ) TGF-β (n = 4). Data are presented as mean ± SEM. Differences between groups were compared using one-way ANOVA. * p < 0.05, ns p > 0.05.

Article Snippet: Complete freund’s adjuvant (CFA) (Sigma, USA, F5881), trichloroethanol (Aibei, Nanjing, CN, M2820), acupuncture needles (Jiajian Medical, CN), TENS-WAA equipment (Shanghai MicroPort Scientific Co, CN), Von Frey hairs mechanical stimulation needles (Yuyan Instrument, CN), Von Frey test cage (Yuyan Instrument, CN), Rat IL-6 ELISA Kit (Servicebio, CN, GER0001-96T), Rat IL-1β ELISA Kit (Servicebio, CN, GER0002-96T), Rat TNF-α ELISA Kit (Servicebio, CN, GER0004-96T), Rat TGF-β1 ELISA Kit (Servicebio, CN, GER0051-96T), BCA Protein Assay Kit (Servicebio, CN, G2026-1000T), TRIzol ® reagent (China, Servicebio, CN, G3013) SweScript All-in-One SuperMix for qPCR (Servicebio, CN, G3337), Blue SYBR Green qPCR Master Mix (Servicebio, CN, G3326).

Techniques:

Journal: BMC Cancer

Article Title: ARC (NSC 188491) has identical activity to Sangivamycin (NSC 65346) including inhibition of both P-TEFb and PKC

doi: 10.1186/1471-2407-9-63

Figure Lengend Snippet: ARC, sangivamycin and toyocamycin inhibit VEGF secretion . MCF7 cells were grown in 6 well dishes to 50% confluence, washed twice with PBS and 2 mL fresh RPMI-1640 media added. Media was then supplemented with the appropriate drug to a final concentration of 8 μM. After 24 h supernatant was removed and any cellular debris depleted by centrifugation. Adherent cells were trypsinized and cell numbers determined. An ELISA-based method was used to measure supernatant concentrations of VEGF. To avoid changes in cell number negatively influencing levels of VEGF secretion, results are expressed as picogram VEGF/mL per cell.

Article Snippet: The concentration of VEGF in cell-free culture supernatants was determined in triplicates using the Human VEGF Quantikine ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.

Techniques: Concentration Assay, Centrifugation, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Repulsive guidance molecule a (RGMa) expression increased in human umbilical artery endothelial cells (HUAECs), human umbilical vein endothelial cells (HUVECs), and rat brain microvascular endothelial cells (RBMECs) stimulated with VEGF. (A,B) Endothelial cell (EC) migration distance was evaluated by scratch assay. ECs were treated with a dose curve of RGMa (500–3000 ng/ml), and images were taken at the beginning and 12 h. (C,D) ECs were treated with RGMa (2 µg/ml), and lysates were collected over a time course of 50 min. The amount of phosphorylated focal adhesion kinase (p-FAK) protein was visualized with western blot analysis. (E,F) ECs were treated with VEGF (50 ng/ml), and lysates were collected over a course of 120 min. p-FAK protein levels were visualized with western blot analysis. (G–I) Quantitative real-time polymerase chain reaction showed RGMa mRNA level was upregulated in HUAECs, HUVECs, and RBMECs exposed to VEGF (50 ng/ml) for 30 min. (J–L) RGMa protein levels were visualized in HUAECs, HUVECs, and RBMECs exposed to VEGF at 30 min by western blot analysis. Data in bar graphs represent the means ± SD of ≥4 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, # VS 1500 ng/ml, ### P < 0.001.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: Expressing, Migration, Wound Healing Assay, Western Blot, Real-time Polymerase Chain Reaction

Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Repulsive guidance molecule a (RGMa) suppressed VEGF expression, phosphorylation of focal adhesion kinase (FAK), proliferation, migration, and tube formation in ECs. (A,B) Lysate was collected, and VEGF was detected by western blot in human umbilical artery endothelial cells (HUAECs) treated with RGMa (2 µg/ml); (C–E) ELISA kit assay showed VEGFA decreased in endothelial cell (EC)-culture supernatant exposed to RGMa compared with cell-culture supernatant from control group. (F–H) Cell proliferation was evaluated with cell-counting kit-8 and 5-ethynyl-2′-deoxyuridine (EdU) assays (I,J) . RGMa decreased proliferation of ECs stimulated and unstimulated with VEGF. (K,L) FAK (Tyr397) phosphorylation was measured with western blot in HUAECs treated with vehicle, RGMa (2 µg/ml), VEGF (50 ng/ml), or VEGF plus RGMa. (M–P) ECs were grown to 100% confluence, serum-starved overnight, wounded with a sterile pipette tip to remove cells, and treated with control, RGMa, VEGF, or VEGF plus RGMa. Photographs (40×) were taken at 12 h after injury. Wound closure of ≥3 wells was quantified and reported as mean ± SD. (Q–T) Migration activity of ECs treated with RGMa, VEGF, VEGF plus RGMa, or control was measured with transwell assay. Photographs (200×) were taken 18 h after treatment. (U–X) HUAECs were starved overnight, treated as indicated, and seeded into 96-well plates coated with Matrigel. Photographs (40×) were taken at 3 h after treatment. The number of tubes, tube area, and tube length were analyzed with Image J. Scale bar, 100 µm. Data shown are representative of experimental and quantitative results. N ≥ 4 independent experiments. Bars represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: Expressing, Phospho-proteomics, Migration, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture, Control, Cell Counting, Sterility, Transferring, Activity Assay, Transwell Assay

Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Repulsive guidance molecule a (RGMa) inhibited angiogenesis in vitro via neogenin. (A) Human umbilical artery endothelial cells (HUAECs) were transfected with CRISPR/Cas9 neogenin knockout kit and purified with puromycin, then the result of neogenin knockout was validated with western blot. (B,C) HUAECs transfected with SgRNA or neogenin gRNA were treated with vehicle, RGMa, VEGF, or VEGF plus RGMa. The focal adhesion kinase (FAK) (Tyr397) phosphorylation was measured with western blot. (D–G) Migration and (H–K) tube formation of HUAECs transfected with SgRNA or neogenin gRNA were determined by scratch, transwell, and Matrigel tube-formation assays. The relative number of tubes, tube area, and tube length were analyzed with Image J. Scale bar, 100 µm. Data shown are representative of experimental and quantitative results. N ≥ 4 independent experiments. Bars represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: In Vitro, Transfection, CRISPR, Knock-Out, Purification, Western Blot, Phospho-proteomics, Migration

Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Unc5b is involved in the effect of repulsive guidance molecule a (RGMa) on phosphorylation of focal adhesion kinase (FAK), migration, and tube formation in human umbilical artery endothelial cells (HUAECs). (A) HUAECs were transfected with CRISPR/Cas9 Unc5b knockout kits and purified with puromycin, then the effect of Unc5b knockout was validated with western blot. (B,C) HUAECs transfected with SgRNA or Unc5b gRNA were treated with control, RGMa, VEGF, or VEGF plus RGMa. FAK (Tyr397) phosphorylation was measured with western blot. (D–G) Migration and (H–K) tube formation of HUAECs transfected with SgRNA or Unc5b gRNA were determined with scratch, transwell, and Matrigel tube-formation assays. The relative number of tubes, tube area, and tube length was analyzed with Image J. Scale bar, 100 µm. Data shown are representative of experimental and quantitative results. N ≥ 4 independent experiments. Bars represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: Phospho-proteomics, Migration, Transfection, CRISPR, Knock-Out, Purification, Western Blot, Control

Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Repulsive guidance molecule a (RGMa) inhibited cytoskeleton reassembly, filopodia, and lamellipodia formation in human umbilical artery endothelial cells (HUAECs) via neogenin and Unc5b. Before the immunofluorescence experiment, HUAECs were treated with vehicle, RGMa, VEGF, or VEGF plus RGMa for 40 min. F-actin was stained with phalloidin conjugated with FITC and phosphorylated focal adhesion kinase (p-FAK) connected with primary antibody was labeled with Alexa Fluor 555 donkey anti-rabbit (H + L) secondary antibody. (A) Immunofluorescence showed the cytoskeleton (green) change and p-FAK (red) distribution. (B) Immunofluorescence showing the cytoskeleton change of HUAECs transfected with SgRNA or neogenin gRNA. (C) Immunofluorescence showed the cytoskeleton change of HUAECs transfected with SgRNA or Unc5b gRNA. The filopodia are indicated as sharp spikes (arrowhead), and lamellipodia (arrow) are indicated as flat intensive staining. The merged images are shown in the upper panels, and the amplified indicated areas are shown in the lower panel for different groups. Photographs were obtained with laser scanning confocal microscopy (Nikon, A1 + R, magnification 400×). Results shown are representative images of ≥4 independent experiments.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: Immunofluorescence, Staining, Labeling, Transfection, Amplification, Confocal Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Quercetin Mitigates Inflammatory Responses Induced by Vascular Endothelial Growth Factor in Mouse Retinal Photoreceptor Cells through Suppression of Nuclear Factor Kappa B

doi: 10.3390/ijms18112497

Figure Lengend Snippet: Effect of quercetin on the expression of vascular epithelial growth factor (VEGF), VEGFR1, and VEGFR2 in VEGF-stimulated 661W cells. ( A ) Cells were treated with VEGF (20 ng/mL) in the absence or presence of quercetin (0.1 µM) for 16 h. The protein expression was detected by immunoblot. ( B – D ) Densitometry quantifications of VEGF ( B ), VEGFR1 ( C ), and VEGFR2 ( D ) protein expression were measured by ImageJ. Data are the means ± SDs of three independent experiments. * p < 0.05 and ** p < 0.01 indicate significant differences compared to the non-treated control group. # p < 0.05, ## p < 0.01 and ### p < 0.001 indicate significant differences compared to the VEGF-only treated group. ( E ) Cells were treated with VEGF (20 ng/mL) in the absence or presence of quercetin (0.1 µM) for 6 h. Gene expression was determined by RT-PCR. ( F – H ) Densitometry quantifications of vegf ( F ), vegfr1 ( G ), and vegfr2 ( H ) gene expression were measured by ImageJ. Data are the means ± SDs of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 indicate significant differences compared to the non-treated control group. # p < 0.05 and ## p < 0.01 indicate significant differences compared to the VEGF-only treated group.

Article Snippet: Mouse recombinant VEGF was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Control, Gene Expression, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Quercetin Mitigates Inflammatory Responses Induced by Vascular Endothelial Growth Factor in Mouse Retinal Photoreceptor Cells through Suppression of Nuclear Factor Kappa B

doi: 10.3390/ijms18112497

Figure Lengend Snippet: Effect of quercetin on the expression of ICAM1 and VCAM1 in VEGF-stimulated 661W cells. ( A ) Cells were treated with VEGF (20 ng/mL) in the absence or presence of quercetin (0.1 µM) for 16 h. The protein expression was detected by immunoblot. ( B , C ) Densitometry quantifications of ICAM1 ( B ) and VCAM1 ( C ) protein expression were measured by ImageJ. Data are the means ± SDs of three independent experiments. * p < 0.05 and ** p < 0.01 indicate significant differences compared to the non-treated control group. # p < 0.05 and ## p < 0.01 indicate significant differences compared to the VEGF-only treated group. ( D ) Cells were treated with VEGF (20 ng/mL) in the absence or presence of quercetin (0.1 µM) for 6 h. Gene expression was determined by RT-PCR. ( E , F ) Densitometry quantifications of icam1 ( E ) and vcam1 ( F ) gene expression were measured by ImageJ. Data are the means ± SDs of three independent experiments. ** p < 0.01 and *** p < 0.001 indicate significant differences compared to the non-treated control group. ## p < 0.01 indicates significant difference compared to the VEGF-only treated group.

Article Snippet: Mouse recombinant VEGF was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Control, Gene Expression, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Quercetin Mitigates Inflammatory Responses Induced by Vascular Endothelial Growth Factor in Mouse Retinal Photoreceptor Cells through Suppression of Nuclear Factor Kappa B

doi: 10.3390/ijms18112497

Figure Lengend Snippet: Effect of quercetin on the expression of MMP2 and MMP9 in VEGF-stimulated 661W cells. ( A ) Cells were treated with VEGF (20 ng/mL) in the absence or presence of quercetin (0.1 µM) for 16 h. The protein expression was detected by immunoblot. ( B , C ) Densitometry quantifications of MMP2 ( B ) and MMP9 ( C ) protein expression was measured by ImageJ. Data are the means ± SDs of three independent experiments. * p < 0.05 and *** p < 0.001 indicate significant differences compared to the non-treated control group. # p < 0.05 and ## p < 0.01 indicate significant differences compared to the VEGF-only treated group. ( D ) Cells were treated with VEGF (20 ng/mL) in the absence or presence of quercetin (0.1 µM) for 6 h. Gene expression was determined by RT-PCR. ( E , F ) Densitometry quantifications of mmp2 ( E ) and mmp9 ( F ) gene expression was measured by ImageJ. Data are the means ± SDs of three independent experiments. ** p < 0.01 and *** p < 0.001 indicate significant differences compared to the non-treated control group. ## p < 0.01 indicates significant difference compared to the VEGF-only treated group.

Article Snippet: Mouse recombinant VEGF was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Control, Gene Expression, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Quercetin Mitigates Inflammatory Responses Induced by Vascular Endothelial Growth Factor in Mouse Retinal Photoreceptor Cells through Suppression of Nuclear Factor Kappa B

doi: 10.3390/ijms18112497

Figure Lengend Snippet: Effect of quercetin on the expression of tight junction proteins in VEGF-stimulated 661W cells. Cells were treated with VEGF (20 ng/mL) in the absence or presence of quercetin (0.1 µM) for 16 h. ( A ) The immunostained cells with β-catenin or ZO-1 were prepared for fluorescence microscopy analysis. Scale bar means 25 µm. ( B ) The protein expression was detected by immunoblot. ( C , D ) Densitometry quantifications of β-catenin ( C ) and ZO-1 ( D ) protein expression were measured by ImageJ. Data are the means ± SDs of three independent experiments. ** p < 0.01 and *** p < 0.001 indicate significant differences compared to the non-treated control group. ### p < 0.001 indicates significant difference compared to the VEGF-only treated group (scale bar means 25 μm).

Article Snippet: Mouse recombinant VEGF was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Fluorescence, Microscopy, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Quercetin Mitigates Inflammatory Responses Induced by Vascular Endothelial Growth Factor in Mouse Retinal Photoreceptor Cells through Suppression of Nuclear Factor Kappa B

doi: 10.3390/ijms18112497

Figure Lengend Snippet: Effect of quercetin on activation of NF-κB in 661W cells. ( A ) Cells were treated with VEGF (20 ng/mL) in the absence or presence of quercetin (0.1 μM) for 1 h. Cells were prepared for fluorescence microscopy analysis. Scale bar means 25 µm; ( B ) cells were co-transfected with 1 µg of NF-κB promoter-containing GFP along with 20 ng of control pRL-TK DNA for 40 h. Transfected cells were treated with VEGF (20 ng/mL) in the absence or presence of quercetin (0.1 µM) for 6 h. Cells stained by DAPI were prepared for fluorescence microscopy analysis. Scale bar means 50 µm; ( C ) cells were treated with VEGF (20 ng/mL) in the absence or presence of quercetin (0.1 µM) for 1 h. After cell fractionation, nuclear translocation of NF-κB were determined by a immunoblot analysis; ( D ) Densitometry quantification of protein expression was measured by ImageJ. Data are the means ± SDs of three independent experiments. * p < 0.05 indicates significant difference compared to the non-treated control group. # p < 0.05 indicates significant difference compared to the VEGF-only treated group; ( E ) cells were treated with VEGF (20 ng/mL) in the absence or presence of quercetin (0.1 µM) for 1 h. Phosphorylation level of IKKαβ and IκBα were determined by a immunoblot analysis. ( F , G ) Densitometry quantifications of phosphorylation levels of IKKαβ (F) and IκBα (G) were measured by ImageJ. Data are the means ± SDs of three independent experiments. * p < 0.05 and *** p < 0.001 indicate significant differences compared to the non-treated control group. # p < 0.05 and ### p < 0.001 indicate significant differences compared to the VEGF-only treated group.

Article Snippet: Mouse recombinant VEGF was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Fluorescence, Microscopy, Transfection, Control, Staining, Cell Fractionation, Translocation Assay, Western Blot, Expressing, Phospho-proteomics

Journal: International Journal of Molecular Sciences

Article Title: Quercetin Mitigates Inflammatory Responses Induced by Vascular Endothelial Growth Factor in Mouse Retinal Photoreceptor Cells through Suppression of Nuclear Factor Kappa B

doi: 10.3390/ijms18112497

Figure Lengend Snippet: Effect of quercetin on the phosphorylation of MAPKs and Akt in VEGF-stimulated 661W cells. Cells were treated with VEGF (20 ng/mL) in the absence or presence of quercetin (0.1 µM) for 1 h. The phosphorylation levels of ERK ( A ), JNK ( B ), p38 ( C ), and Akt ( D ) were detected by immunoblot using corresponding antibodies. Densitometry quantifications of protein expression were measured by ImageJ. Data are the means ± SDs of three independent experiments. * p < 0.05 and ** p < 0.01 indicate significant differences compared to the non-treated control group. # p < 0.05 and ## p < 0.01 indicate significant differences compared to the VEGF-only treated group.

Article Snippet: Mouse recombinant VEGF was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Phospho-proteomics, Western Blot, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Quercetin Mitigates Inflammatory Responses Induced by Vascular Endothelial Growth Factor in Mouse Retinal Photoreceptor Cells through Suppression of Nuclear Factor Kappa B

doi: 10.3390/ijms18112497

Figure Lengend Snippet: Primer information for PCR.

Article Snippet: Mouse recombinant VEGF was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques:

Journal: Bioactive materials

Article Title: Double hits with bioactive nanozyme based on cobalt-doped nanoglass for acute and diabetic wound therapies through anti-inflammatory and pro-angiogenic functions.

doi: 10.1016/j.bioactmat.2023.08.014

Figure Lengend Snippet: Fig. 5. Effects of CoNZ on pro-angiogenesis in vivo and in vitro. (A–C) In vivo experiments with STZ-induced diabetic skin wounds: (A) Immunohistochemical staining of tissue samples for CD31 and α-SMA (neovascularization) at weeks 1 and 3, with semi-quantification of signal intensity by ImageJ; both signals were expressed at significantly higher levels in the CoNZ group. (B) Western blot analysis of tissue samples for CD31 at week 1, showing significant differences between groups (defect control < BGn & CoNZ); the dotted line (intensity level ‘1’) represents the normal group. (C) ELISA analysis of tissue samples for VEGF at day 3. For the in vivo studies, nanoparticles of 10 μL from a concentration of 100 mg/1 mL were treated to each wound. (D–G) In vitro experiments with high glucose/H2O2-challenged inflammatory cells. (D) Schematic representation of the in vitro design; HUVECs challenged with high glucose and H2O2 were analyzed for angiogenic events. (E) Intracellular ROS level at 12 h; ROS levels substantially increased with high glucose/H2O2 challenge, which were significantly reduced by CoNZ treatment; ROS scavenging ability was observed in the order, CoNZ > CoNZ-ions ~ BGn > high glucose/H2O2 control. Nanoparticle concentration used for the in vitro study was 20 μg/mL. (F) Cellular migration was analyzed over 24 h by assessing the ability to fill a scratched multicellular gap; images of four representative groups at 12 h are presented; cell migration was recorded in the order, CoNZ ~ CoNZ-ions » BGn > high glucose/H2O2 control. (G) Tubular formation of cells was examined and quantified in terms of tubule and node number at 6 h and 24 h on a Matrigel substrate; cell images of four representative groups at 24 h are presented. Statistical significance was calculated between groups using one-way ANOVA with *p < 0.05, **p < 0.01, and ***p < 0.001 denoting significance compared to the defect group (in vivo) or HG (+) & H2O2 (+) group (in vitro); and +p < 0.05, ++p < 0.01, and +++p < 0.001 indicating significance compared to the BGn group (in vivo). All data are presented as mean ± one standard deviation and the sample size was n = 5.

Article Snippet: Quantikine murine VEGF ELISA kit (MMV00, R&D Systems) was used to detect the VEGF quantity released from HUVECs after culturing for 3 days, following the manufacturer’s protocols.

Techniques: In Vivo, In Vitro, Immunohistochemical staining, Staining, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Concentration Assay, Migration, Standard Deviation

Journal: Journal of leukocyte biology

Article Title: Advanced glycation end-products increase monocyte adhesion to retinal endothelial cells through vascular endothelial growth factor-induced ICAM-1 expression: inhibitory effect of antioxidants.

doi: 10.1189/jlb.0603265

Figure Lengend Snippet: Fig. 3. VEGF increases ICAM-1 expression on BRECs. (A) Confluent BRECs were treated with VEGF (20 ng/ml) for 12 h. Endothelial cell-surface expression of ICAM-1, VCAM-1, and E-selectin was determined by the cellular ELISA method as described in Materials and Methods. Data represent the mean SEM of four independent experiments. *, P 0.05, versus CTL. (B) Confluent BRECs were incubated with VEGF (20 ng/ml) for 12 h in the presence or absence of an antibody against ICAM-1 (10 g/ml). At the end of this incubation period, cells were washed twice with HBSS, and 230,000 freshly isolated human monocytes were added to each well. Monocyte adhesion to BRECs was determined by the MPO assay as described in Materials and Methods. Data represent the mean

Article Snippet: Recombinant human VEGF and antibodies to ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), and E-selectin were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Incubation, Isolation, MPO Assay

Journal: Journal of leukocyte biology

Article Title: Advanced glycation end-products increase monocyte adhesion to retinal endothelial cells through vascular endothelial growth factor-induced ICAM-1 expression: inhibitory effect of antioxidants.

doi: 10.1189/jlb.0603265

Figure Lengend Snippet: Fig. 2. Effect of anti-ICAM-1 or anti-VEGF antibodies on AGE-induced monocyte adhesion. Confluent BRECs were incubated for 12 h with AGEs (100 g/ml) in the presence or absence of an antibody against ICAM-1 (10 g/ml) or VEGF (2 g/ml). At the end of this incubation period, cells were washed twice with HBSS, and 230,000 freshly isolated human monocytes were added to each well. Monocyte adhesion to BRECs was determined by the MPO assay as described in Materials and Methods. Data represent the mean SEM of five independent experiments. **, P 0.01, versus CTL; ##, P 0.01, versus AGE.

Article Snippet: Recombinant human VEGF and antibodies to ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), and E-selectin were obtained from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Isolation, MPO Assay

Journal: Journal of leukocyte biology

Article Title: Advanced glycation end-products increase monocyte adhesion to retinal endothelial cells through vascular endothelial growth factor-induced ICAM-1 expression: inhibitory effect of antioxidants.

doi: 10.1189/jlb.0603265

Figure Lengend Snippet: Fig. 5. PKC and NF-B activation is involved in AGE- and VEGF-induced monocyte adhesion to BRECs. Confluent BRECs were treated for 12 h with AGEs (100 g/ml; A) or VEGF (20 ng/ml; B) in the presence or absence of inhibitors of PKC (GF10923X, 20 nM) or NF-B (BAY117086, 40 M). At the end of this incubation period, cells were washed, and monocyte adhesion to BRECs was measured by the MPO assay as described in Materials and Methods. Data represent the mean SEM of four independent experiments. **, P 0.01, versus CTL; ***, P 0.001, versus CTL; ##, P 0.05, versus AGE ; ###, P 0.001, versus VEGF.

Article Snippet: Recombinant human VEGF and antibodies to ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), and E-selectin were obtained from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Incubation, MPO Assay

Journal: Journal of leukocyte biology

Article Title: Advanced glycation end-products increase monocyte adhesion to retinal endothelial cells through vascular endothelial growth factor-induced ICAM-1 expression: inhibitory effect of antioxidants.

doi: 10.1189/jlb.0603265

Figure Lengend Snippet: Fig. 4. Effect of gliclazide and vitamin E on VEGF-induced monocyte adhesion and ICAM-1 expression on BRECs. Confluent BRECs were incubated with AGEs (100 g/ml) in the presence or absence of gliclazide (2.5 g/ml) or vitamin E (50 M) for 12 h. Monocyte adhesion to BRECs (A) and endothelial cell-surface expression of ICAM-1 (B) were measured by the MPO assay and the cellular ELISA method, respectively. Data represent the mean SEM of four independent experiments. *, P 0.05, versus CTL; **, P 0.01, versus CTL; #, P 0.05, versus VEGF; ##, P 0.01, versus VEGF.

Article Snippet: Recombinant human VEGF and antibodies to ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), and E-selectin were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Incubation, MPO Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of leukocyte biology

Article Title: Advanced glycation end-products increase monocyte adhesion to retinal endothelial cells through vascular endothelial growth factor-induced ICAM-1 expression: inhibitory effect of antioxidants.

doi: 10.1189/jlb.0603265

Figure Lengend Snippet: Fig. 6. AGEs and VEGF induce ICAM-1 expression on BRECs through PKC and NF-B activation. Confluent BRECs were treated for 6 h with AGEs (100 g/ml; A) or VEGF (20 ng/ml; B) in the presence or absence of inhibitors of PKC (GF10923X, 20 nM) or NF-B (BAY117086, 40 M). Endothelial cell-surface expression of ICAM-1 was determined by the cellular ELISA method as described in Materials and Methods. Data represent the mean SEM of four independent experiments. **, P 0.01, versus CTL; ***, P 0.001, versus CTL; ##, P 0.05, versus AGE or VEGF; ###, P 0.001, versus AGE.

Article Snippet: Recombinant human VEGF and antibodies to ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), and E-selectin were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay