Journal: bioRxiv

Article Title: Vascular endothelial growth factor-D improves lung vascular integrity during acute lung injury

doi: 10.1101/2024.12.16.628787

Figure Lengend Snippet: A) NICHES analysis of AFs, mesothelial cells, pericytes, gCap, and aCap in human microvascular niche yields a quantitative cell-cell signaling atlas of AF-aCap, AF-gCap, Mesothelial-aCap, Mesothelial-gCap, Pericyte-aCap, and Pericyte-gCap visualized by low-dimensional UMAP embedding. B) FeaturePlot of top differentially expressed L/R pairs ANGPT1 - TEK , SLIT2 - ROBO4 , VEGFD - KDR , BMP5-BMPR2 , ADM - RAMP2 , WNT5A - FZD6 , PTN - RACK1 , OXT - EDNRB , and ADCYAP1 - VIPR1 from NICHES analysis C) Top 10 differentially expressed L/R pairs between different cell-cell interaction signals processed through NICHES and plotted in DoHeatmap. D) Immunostaining of representative L/R pairs from AF to microvascular endothelial cells, including SLIT2 - ROBO4 , ADM - RAMP2 , BMP5 - BMPR2 , and VEGFD - VEGFR2 in human lung section. ITGA8 is a marker for AF; PRX is a marker for microvascular endothelial cells. Scale bar 20 μm.

Article Snippet: The protein levels of SLIT2, ADM, BMP5, and VEGFD in samples harvested from different timepoints were determined using corresponding ELISA kits: SLIT2 (# NB030384), ADM (# NBP2-78738), BMP5 (# NBP2-69996), VEGFD (# NBP2-78890), all from Novus Biologicals.

Techniques: Immunostaining, Marker

Journal: bioRxiv

Article Title: Vascular endothelial growth factor-D improves lung vascular integrity during acute lung injury

doi: 10.1101/2024.12.16.628787

Figure Lengend Snippet: A, B) The electrical resistance and permeability in HLMVECs after treatment with VEGF-D (1 μg/mL), adrenomedullin (ADM)(100 ng/mL), angiopoietin 1 (ANGPT1)(2 μg/mL), BMP-5 (1 μg/mL), SLIT2 (1 μg/mL), CXCL6 (1 μg/mL), Pleiotrophin (PTN)(1 μg/mL), SEMA6D (1 μg/mL), and TNF-α (1 ng/mL) were measured through electrical cell impendence sensing (ECIS) assay, and transwell assay, respectively. C) HLMVECs cultured on transwell treated with VEGF-D, followed by challenging with IL1β, TNF-α, or thrombin. The permeability was determined using a 20kDa FITC-dextran probe. D) Immunofluorescent images of F actin (red), and VE-Cadherin (green) in HLMVECs challenged with TNF-α or IL1β with or without VEGF-D. White boxes highlight VE-Cadherin junctions in all conditions. Scale bar 20 μm. E) Protein expression of phosphor-MLC in HLMVECs challenged with thrombin with different concentrations of VEGF-D was assessed through a western blot. Data presented as mean ± SEM. * and ** indicate p<0.05 and p<0.01, respectively.

Article Snippet: The protein levels of SLIT2, ADM, BMP5, and VEGFD in samples harvested from different timepoints were determined using corresponding ELISA kits: SLIT2 (# NB030384), ADM (# NBP2-78738), BMP5 (# NBP2-69996), VEGFD (# NBP2-78890), all from Novus Biologicals.

Techniques: Permeability, Transwell Assay, Cell Culture, Expressing, Western Blot

Journal: bioRxiv

Article Title: Vascular endothelial growth factor-D improves lung vascular integrity during acute lung injury

doi: 10.1101/2024.12.16.628787

Figure Lengend Snippet: A) C57Bl/6 mice were injected with LPS for 18 hours, followed by a collection of bronchioalveolar lavage (BAL) fluid. The expression level of VEGF-D was measured in the BAL fluid in mice with or without LPS challenge. B) Mice were injected with LPS, followed by treatment with VEGF-D (225 μg/kg). The BAL fluid and histological samples were collected after 18 hours of treatment. C) The FITC-albumin probe was injected retro-orbitally 16 hours after the LPS challenge. The amount of probe in BAL fluid was quantified after another 2 hours. The FITC-albumin levels in BAL fluid in the control and experimental groups were determined. D) The BAL fluid was spun down at 5,000 g for 10 minutes and resuspended in 300 μL of 1XPBS. The number of cells in the BAL from different conditions was determined using a hemacytometer. n = 12 – 14 per group. E) LPS and LPS+VEGFD lungs were excised, fixed in 4% paraformaldehyde, embedded in paraffin, and used for histochemical analysis after haematoxylin and eosin staining. Images are representative of 6–9 lung specimens for each condition. Scale bar 150 μm. F) Lung pathological index was performed by a blinded reviewer from 1 to 4, where 4 was the worst pathology. The score was used for both peribronchial and alveolar inflammation. The average of two scores was taken to obtain a range between 1 (no inflammation or pathology) and 4 (maximum inflammation and pathology). The lung pathological index was calculated in LPS and LPS+VEGFD conditions. G-I) The expression level of TNF-α and IL-6 were measured in BAL fluid from LPS and LPS+VEGFD conditions using corresponding ELISA kits. Mice without LPS challenge were used as control. N = 12 - 14 per treatment group. Data presented as mean ± SEM. * and ** indicate p<0.05 and p<0.01, respectively.

Article Snippet: The protein levels of SLIT2, ADM, BMP5, and VEGFD in samples harvested from different timepoints were determined using corresponding ELISA kits: SLIT2 (# NB030384), ADM (# NBP2-78738), BMP5 (# NBP2-69996), VEGFD (# NBP2-78890), all from Novus Biologicals.

Techniques: Injection, Expressing, Control, Staining, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: ASK1/p38-mediated NLRP3 inflammasome signaling pathway contributed to aberrant retinal angiogenesis in diabetic retinopathy

doi: 10.1101/763102

Figure Lengend Snippet: (A) The inflammatory related cytokine IL-6, TNF-α and IL-1β mRNA expression levels were enhanced in DR group. (B) The expression of vascular marker CD31 was increased in DR group by confocal detection. Meanwhile, (C) the VEGF secretion levels in DR group was increased compared with control group. Values are expressed as the mean ± SD (n = 6, *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the control).

Article Snippet: The retinal tissue from control group, HG group and HG treated with inhibitor group were added in PBS and homogenized; the homogenate was centrifuged at 12000 g at 4°C for 10 min. Then, the supernatant was tested for VEGF by mouse VEGF ELISA kit (R&D Systems, MN, USA).

Techniques: Expressing, Marker, Control

Journal: bioRxiv

Article Title: ASK1/p38-mediated NLRP3 inflammasome signaling pathway contributed to aberrant retinal angiogenesis in diabetic retinopathy

doi: 10.1101/763102

Figure Lengend Snippet: (A) The protein expression level of NLRP3 was enhanced in DR groups; moreover, (B) blocking NLRP3, the mRNA expression of IL-6, TNF-α and IL-1β was down-regulated; meanwhile, (C) the vascular marker CD31 expression level and (D) VEGF secretion level were decreased through inhibiting NLRP3. In addition, in HRMEC cell model, (E) high glucose enhanced the protein expression of NLRP3, which was inhibited by NLPR3 inhibitor. (F) The inflammatory related cytokine IL-6, TNF-α and IL-1β was enhanced in HG-induced HRMEC cell model and was inhibited through blocking NLRP3. (G) The VEGF secretion level was enhanced, and was decreased after inhibiting NLRP3. Data are presented as the mean ± standard deviation from triplicate wells. *P < 0.05 and **P < 0.01 compared with the control. #P < 0.05 and ## P < 0.01 compared with the relative DR animal model group or HG-induced HRMEC cell group.

Article Snippet: The retinal tissue from control group, HG group and HG treated with inhibitor group were added in PBS and homogenized; the homogenate was centrifuged at 12000 g at 4°C for 10 min. Then, the supernatant was tested for VEGF by mouse VEGF ELISA kit (R&D Systems, MN, USA).

Techniques: Expressing, Blocking Assay, Marker, Standard Deviation, Control, Animal Model

Journal: bioRxiv

Article Title: ASK1/p38-mediated NLRP3 inflammasome signaling pathway contributed to aberrant retinal angiogenesis in diabetic retinopathy

doi: 10.1101/763102

Figure Lengend Snippet: (A) The protein expression levels of AKS1 and p38 were up-regulated in DR animal group. (B)The result of HG-induced HRMEC group was similar with DR animal group. Moreover, (C) the NLPR3, IL-6, TNF-α, IL-1β and VEGF protein expression levels were inhibited by (C) NLPR3 inhibitor and (D) p38 inhibitor.

Article Snippet: The retinal tissue from control group, HG group and HG treated with inhibitor group were added in PBS and homogenized; the homogenate was centrifuged at 12000 g at 4°C for 10 min. Then, the supernatant was tested for VEGF by mouse VEGF ELISA kit (R&D Systems, MN, USA).

Techniques: Expressing

Journal: Oncogene

Article Title: Oncogenes and tumor angiogenesis: the HPV-16 E6 oncoprotein activates the vascular endothelial growth factor (VEGF) gene promoter in a p53 independent manner.

doi: 10.1038/sj.onc.1203817

Figure Lengend Snippet: Figure 1 Comparison of VEGF mRNA and protein levels in HPV-16- positive and HPV-16- negative cells. (a) Northern blot analysis of VEGF expression in a panel of cell lines including human immortalized HPV-16- negative (HaCaT) and HPV-16- positive (HPK1A) keratinocytes, as well as cervical carcinoma derived HPV-16- negative (C33A) and HPV-16- positive (HeLa, CaSki) cells. VEGF mRNA levels were increased 4 ± 7-fold in HPV-16- positive cells as compared to HPV-16- negative immortalized keratinocytes, and 2 ± 3-fold compared to the HPV-16- negative cervical carcinoma cell line C33A. (b) Northern blot analysis of VEGF expression in HeLa cells as compared to HaCaT keratinocytes. VEGF mRNA levels were increased 7 ± 18-fold in HeLa, with the maximum dierence observed at low serum concentration (0.1%). The 28S and 18S mRNA bands show equal loading and RNA integrity (lower panel). Fold activation was calculated by densitometry with 28S serving as a normalization control. The value obtained for HaCaT keratinocytes in 0.1% FBS was taken as the basal level (1.0) in (b). (c) ELISA assay results showing the increase in VEGF concentration in conditioned medium collected from HeLa cells compared to that from HaCaT keratinocytes. Cells were incubated for 24 h after being seeded at dierent serum concentrations

Article Snippet: Measurement of human VEGF protein levels in conditioned medium (ELISA) A commercially available human VEGF ELISA kit (R&D Systems, Minneapolis, MN, USA) was used to quantitate the level of VEGF obtained from HaCaT, HeLa and A431 cells according to the manufacturer's instructions.

Techniques: Comparison, Northern Blot, Expressing, Derivative Assay, Concentration Assay, Activation Assay, Control, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Oncogene

Article Title: Oncogenes and tumor angiogenesis: the HPV-16 E6 oncoprotein activates the vascular endothelial growth factor (VEGF) gene promoter in a p53 independent manner.

doi: 10.1038/sj.onc.1203817

Figure Lengend Snippet: Figure 2 Eect of blockade of EGFR signaling on the production of VEGF protein by HeLa and A431 cells. (a) Eect of treatment with two anti-EGFR antibodies, C225 and hR3, on the secretion of VEGF by Hela cells. (b) Eect of equal treatments on the production of VEGF protein by A431 cells. No signi®cant dierences among the distinct treatment culture conditions in terms of VEGF secretion were detected in HeLa cells. In contrast, both antibodies down-regulated VEGF protein levels signi®cantly, up to a maximum of 50%, in A431 cells. (c) In agreement with the lack of eect of the EGFR neutralizing antibodies on HeLa, the anti-TGF-a antibody Ab-3 also failed to down-regulate VEGF protein production by HeLa cells. A one-way ANOVA test was used to compare the dierent treatment culture conditions for each experiment. A Dunnet post-test was used in order to compare each result with its respective control

Article Snippet: Measurement of human VEGF protein levels in conditioned medium (ELISA) A commercially available human VEGF ELISA kit (R&D Systems, Minneapolis, MN, USA) was used to quantitate the level of VEGF obtained from HaCaT, HeLa and A431 cells according to the manufacturer's instructions.

Techniques: Control

Journal: Oncogene

Article Title: Oncogenes and tumor angiogenesis: the HPV-16 E6 oncoprotein activates the vascular endothelial growth factor (VEGF) gene promoter in a p53 independent manner.

doi: 10.1038/sj.onc.1203817

Figure Lengend Snippet: Figure 3 Transcriptional activation of VEGF promoter by a plasmid expressing the HPV-16 E6 oncoprotein. (a) Schematic representation of the activator and the reporter construct. The plasmid pJ4O16E6 expresses the E6 gene of HPV-16, driven by MoMu LV LTR promoter (Crook et al., 1991). The reporter 2.6 VEGF is a VEGF promoter (72361 to +298)-Luciferase construct described previously (Mukhopadhyay et al., 1997). (b and c) Eect of HPV-16 E6 gene product on VEGF promoter activity in either HaCaT keratinocytes or NIH3T3 cells, respectively. Signi®cant dierences (P50.01) were detected for both cell lines by applying unpaired t-test. Dose-dependence of VEGF-promoter induction by E6 expression is seen in (b). Plasmid 2.6 VEGF (1.5 mg) was co-transfected into HaCaT cells along with increasing concentrations of either HPV-16 E6 (0 ± 3 mg) expressing plasmid or control plasmid pJ4O (3 mg). Values are presented as averages of three independent experiments in terms of normalized luciferase activity with the standard error (s.e.) of the mean noted. Fold induction is indicated at the top of the bars

Article Snippet: Measurement of human VEGF protein levels in conditioned medium (ELISA) A commercially available human VEGF ELISA kit (R&D Systems, Minneapolis, MN, USA) was used to quantitate the level of VEGF obtained from HaCaT, HeLa and A431 cells according to the manufacturer's instructions.

Techniques: Activation Assay, Plasmid Preparation, Expressing, Construct, Luciferase, Activity Assay, Transfection, Control

Journal: Oncogene

Article Title: Oncogenes and tumor angiogenesis: the HPV-16 E6 oncoprotein activates the vascular endothelial growth factor (VEGF) gene promoter in a p53 independent manner.

doi: 10.1038/sj.onc.1203817

Figure Lengend Snippet: Figure 4 Identi®cation of the E6-responsive region in the human VEGF promoter. Dierent deletion constructs were co- transfected into HaCaT cells with either E6 expression plasmid (&) or control plasmid (&). Left panel presents a schematic representation of the VEGF promoter deletions including consensus binding sites of dierent transcription factors. The E6- responsive region is localized between bp 7194 and 750 of the VEGF gene promoter. Values are presented as averages of three independent experiments in function of normalized luciferase activity with the s.e. of the mean noted. Fold induction in luciferase activity is depicted to the right side of the bars

Article Snippet: Measurement of human VEGF protein levels in conditioned medium (ELISA) A commercially available human VEGF ELISA kit (R&D Systems, Minneapolis, MN, USA) was used to quantitate the level of VEGF obtained from HaCaT, HeLa and A431 cells according to the manufacturer's instructions.

Techniques: Construct, Transfection, Expressing, Plasmid Preparation, Control, Binding Assay, Luciferase, Activity Assay

Journal: Oncogene

Article Title: Oncogenes and tumor angiogenesis: the HPV-16 E6 oncoprotein activates the vascular endothelial growth factor (VEGF) gene promoter in a p53 independent manner.

doi: 10.1038/sj.onc.1203817

Figure Lengend Snippet: Figure 5 Activation of VEGF promotor by E6 in p53 null cells. The plasmid 2.6 VEGF (1.5 mg) was co-transfected into MEF p53+/+ or MEF p537/7 cells together with 3 mg of the HPV- 16 E6 expression plasmid (pJ4O16E6) or 3 mg of the control plasmid (pJ4O). The HPV-16 E6 appears to induce transcription from VEGF promoter even in the absence of a functional p53 gene. Values are presented as averages of three independent experiments in terms of normalized luciferase activity with the s.e. of the mean noted. Fold induction is indicated at the top of the bars

Article Snippet: Measurement of human VEGF protein levels in conditioned medium (ELISA) A commercially available human VEGF ELISA kit (R&D Systems, Minneapolis, MN, USA) was used to quantitate the level of VEGF obtained from HaCaT, HeLa and A431 cells according to the manufacturer's instructions.

Techniques: Activation Assay, Plasmid Preparation, Transfection, Expressing, Control, Functional Assay, Luciferase, Activity Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Vascular endothelial growth factor production by fibroblasts in response to factor VIIa binding to tissue factor involves thrombin and factor Xa.

doi: 10.1161/01.atv.20.5.1374

Figure Lengend Snippet: Figure 1. A, Effect of hirudin (Hir) and FXa inhibitors on ACSET- induced VEGF secretion. Confluent fibroblasts were incubated with 10 U/mL Hir, 10 mmol/L DX9065a (DX), 50 mg/mL TAP, or Hir in combination with either DX or TAP for 30 minutes at 37°C before a 24-hour incubation at 37°C with 100 nmol/L ACSET. Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction of VEGF in treated fibroblasts com- pared with unstimulated fibroblasts (NS). Each point represents the mean6SD of at least 3 different determinations, each per- formed in triplicate. *P,0.0001 vs unstimulated fibroblasts. lP,0.05 vs ACSET-stimulated fibroblasts. B, Kinetics of thrombin and FXa generation in the culture medium of confluent human fibroblasts incubated with 100 nmol/L ACSET. Thrombin and FXa generation were assessed by hydrolysis of the thrombin-sensitive chromogenic substrate S-2238 and by hydrolysis of the FXa-sensitive chromogenic substrate S-2765, respectively, in culture supernatants. Results of a typical experi- ment are shown.

Article Snippet: Human VEGF Immunoassay Human VEGF concentrations in fibroblast culture supernatants were determined by using the Quantikine human VEGF kit (R&D Systems Europe).11

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Vascular endothelial growth factor production by fibroblasts in response to factor VIIa binding to tissue factor involves thrombin and factor Xa.

doi: 10.1161/01.atv.20.5.1374

Figure Lengend Snippet: Figure 2. A, Concentration effect of thrombin on VEGF produc- tion. Confluent fibroblasts were incubated for 24 hours with or without 0.5, 1, and 10 U/mL thrombin at 37°C. Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction of VEGF in treated fibroblasts compared with unstimulated fibroblasts (basal VEGF level 124673 pg/mL). Each point represents the mean6SD of 4 different determina- tions, each performed in triplicate. *P,0.05 vs unstimulated fibroblasts. B, Kinetics of thrombin-induced VEGF secretion. Confluent fibroblasts were incubated with 10 U/mL thrombin for 2, 4, 6, 12, and 24 hours at 37°C. Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction of VEGF in thrombin-treated fibroblasts compared with unstimu- lated fibroblasts at the same time. Results of a typical experi- ment are shown. C, Concentration effect of TRAP on VEGF secretion. Confluent fibroblasts were incubated for 24 hours with or without 10, 50, and 100 mmol/L of TRAP at 37°C. Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction in VEGF secretion compared with unstimulated fibroblasts. Each point represents the mean6SD of 3 different determinations, each performed in triplicate. *P,0.05 vs unstimulated fibroblasts.

Article Snippet: Human VEGF Immunoassay Human VEGF concentrations in fibroblast culture supernatants were determined by using the Quantikine human VEGF kit (R&D Systems Europe).11

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

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Vascular endothelial growth factor production by fibroblasts in response to factor VIIa binding to tissue factor involves thrombin and factor Xa.

doi: 10.1161/01.atv.20.5.1374

Figure Lengend Snippet: Figure 3. A, Concentration of FXa on VEGF secretion. Conflu- ent fibroblasts were incubated for 24 hours with or without 22.8, 57, 114, and 228 nmol/L of FXa at 37°C. Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction of VEGF secretion compared with unstimulated fibro- blasts. Each point represents the mean6SD of 3 different deter- minations, each performed in triplicate. *P,0.05 vs unstimulated fibroblasts. Insert, Additive effect of thrombin (IIa) and FXa on VEGF secretion. Confluent fibroblasts were incubated at 37°C for 24 hours with 1 U/mL of IIa or 100 nmol/L of FXa or a com- bination of both. Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction of VEGF secre- tion compared with unstimulated fibroblasts (NS). Each point represents the mean6SD of 3 different determinations, each performed in triplicate. lP,0.01 vs NS. B, Effect of FXa inhibi- tors, Hir, and anti-TF antibodies on FXa-induced VEGF produc- tion. Confluent fibroblasts were incubated either with 10 mg/mL anti-TF antibodies (TFab) for 30 minutes at 4°C or with 10 mmol/L DX, 50 mg/mL TAP, or 10 U/mL Hir for 30 minutes at 37°C before 24-hour incubation at 37°C with 114 nmol/L FXa. Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction of VEGF secretion compared with NS. Each point represents the mean6SD of at least 3 different determinations, each performed in triplicate. *P,0.0001 vs NS. lP,0.01 vs FXa-stimulated fibroblasts.

Article Snippet: Human VEGF Immunoassay Human VEGF concentrations in fibroblast culture supernatants were determined by using the Quantikine human VEGF kit (R&D Systems Europe).11

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

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Vascular endothelial growth factor production by fibroblasts in response to factor VIIa binding to tissue factor involves thrombin and factor Xa.

doi: 10.1161/01.atv.20.5.1374

Figure Lengend Snippet: Figure 5. Effect of FXa inhibitors and TFab on FVIIa1FX- induced VEGF secretion. Confluent fibroblasts were incubated with FXa inhibitors (TAP, NAP5, and NAPc2 at 50 mg/mL and DX at 10 mmol/L) and with TFab for 30 minutes at either 37°C or 4°C, respectively, before 24-hour incubation with 100 nmol/L FVIIa and 90 nmol/L FX. Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction of VEGF secretion compared with NS. Each point represents the mean6SD of at least 3 different determinations, each performed in triplicate. *P,0.0001 vs NS. lP,0.0001 vs FXa-stimulated fibroblasts.

Article Snippet: Human VEGF Immunoassay Human VEGF concentrations in fibroblast culture supernatants were determined by using the Quantikine human VEGF kit (R&D Systems Europe).11

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Vascular endothelial growth factor production by fibroblasts in response to factor VIIa binding to tissue factor involves thrombin and factor Xa.

doi: 10.1161/01.atv.20.5.1374

Figure Lengend Snippet: Figure 6. Effect of activated clotting factors on VEGF mRNA induction. Confluent fibroblasts were incubated for 24 hours at 37°C with 100 nmol/L FVIIa in combination with 90 nmol/L FX, 114 nmol/L FXa, or 1 U/mL thrombin. Cells were preincubated for 30 minutes at 37°C either with 50 mg/mL TAP before incuba- tion with FVIIa-FX and FXa or with 10 U/mL Hir before incuba- tion with thrombin. Five micrograms of total RNA was analyzed by RT-PCR. A, RNA was quantified by scanning the Polaroid negative by laser densitometry. The density of the 180-bp band was normalized to the density of the mimic, and the fold induc- tion of VEGF mRNA, induced in the different conditions of stim- ulation compared with NS, was plotted. Each point represents the mean6SD of 4 experiments. *P,0.05 vs NS. lP,0.05 vs FVIIa-FX, FXa, or thrombin-stimulated fibroblasts. B, Photograph from a representative experiment is shown. Three VEGF mRNA transcripts are seen (180, 312, and 384 bp). The internal stan- dard, mimic (M), is also visible. L indicates 100-bp DNA ladder.

Article Snippet: Human VEGF Immunoassay Human VEGF concentrations in fibroblast culture supernatants were determined by using the Quantikine human VEGF kit (R&D Systems Europe).11

Techniques: Coagulation, Incubation, Reverse Transcription Polymerase Chain Reaction

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Vascular endothelial growth factor production by fibroblasts in response to factor VIIa binding to tissue factor involves thrombin and factor Xa.

doi: 10.1161/01.atv.20.5.1374

Figure Lengend Snippet: Figure 7. A, Effect of FVIIa and FVIIai on p44/42 MAP kinase activation. Fibroblasts were incubated with either 100 nmol/L FVIIa or 100 nmol/L FVIIai for 5, 10, and 15 minutes. Phosphor- ylated p44/42 MAP kinases (p44ERK1 and p42ERK2) and total p44/42 MAP kinases (ERK1 and ERK2) were studied by Western blotting. B, Western blot analysis of thrombin and FXa-induced phosphorylation of p44/42 MAP kinases. Fibroblasts were incu- bated with 1 U/mL of thrombin for 2, 5, 7, and 10 minutes or with 100 nmol/L of FXa for 2, 5, 10, and 15 minutes. p44ERK1, p42ERK2, ERK1, and ERK2 are shown. C, Effect of MAP kinase inhibitors on FXa- and thrombin-induced VEGF secretion. Con- fluent fibroblasts were incubated with MAP kinase inhibitors (50 mmol/L PD 98059 [PD] and 10 mmol/L SB 203580 [SB]) for 30 minutes at 37°C before 24-hour incubation with either FXa (114 nmol/L) or thrombin (1 U/mL). Secreted VEGF was assessed by a specific ELISA. Results are expressed as fold induction of VEGF secretion compared with unstimulated fibro- blasts. Each point represents the mean6SD of 4 different deter- minations, each performed in triplicate. *P,0.0005 vs NS. lP,0.05 vs FXa- or thrombin- stimulated fibroblasts.

Article Snippet: Human VEGF Immunoassay Human VEGF concentrations in fibroblast culture supernatants were determined by using the Quantikine human VEGF kit (R&D Systems Europe).11

Techniques: Activation Assay, Incubation, Western Blot, Phospho-proteomics, Enzyme-linked Immunosorbent Assay

Journal: Vascular Cell

Article Title: EGCG, a major green tea catechin suppresses breast tumor angiogenesis and growth via inhibiting the activation of HIF-1α and NFκB, and VEGF expression

doi: 10.1186/2045-824X-5-9

Figure Lengend Snippet: Oral EGCG at 50–100 mg/kg/d in drinking water significantly reduced intratumoral microvessel density (Panel A: 109 ± 20 vs. 156 ± 12 microvessel #/mm^2; P < 0.01), plasma VEGF levels (Panel B; 26.48 ± 3.76 vs. 40.79 ± 3.5 pg/ml; P < 0.01), and tumor VEGF expression (Panel B; 45.72 ± 1.4 vs. 59.03 ± 3.8 pg/mg; P < 0.01) over the control, respectively in mice (n = 8). The digital images show CD31 immunohistochemistry staining in OCT-embedded cryosections of mouse breast cancer tumors obtained from a control (Figure A) or EGCG-treated (Figure A) mouse.

Article Snippet: Protein levels of VEGF in plasma, breast tumor, the heart, the limb muscle, and the medium cultured with E0771 cells were determined using mouse VEGF ELISA kits (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions.

Techniques: Clinical Proteomics, Expressing, Control, Immunohistochemistry, Staining

Journal: Vascular Cell

Article Title: EGCG, a major green tea catechin suppresses breast tumor angiogenesis and growth via inhibiting the activation of HIF-1α and NFκB, and VEGF expression

doi: 10.1186/2045-824X-5-9

Figure Lengend Snippet: EGCG at 50 μg/ml significantly inhibited VEGF expression (Panel A, 1752 ± 49 vs. 2254 ± 91 pg/mg, n = 6, P < 0.01), the activation of HIF-1α (Panel B, 0.11 ± 0.02 vs. 0.24 ± 0.02, n = 6, P < 0.01) and NFκB (Panel C, 1.15 ± 0.21 vs. 1.61 ± 0.32; n = 6, P < 0.01) in cultured E0771 cells, compared to the control, respectively.

Article Snippet: Protein levels of VEGF in plasma, breast tumor, the heart, the limb muscle, and the medium cultured with E0771 cells were determined using mouse VEGF ELISA kits (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions.

Techniques: Expressing, Activation Assay, Cell Culture, Control

Journal: Vascular Cell

Article Title: EGCG, a major green tea catechin suppresses breast tumor angiogenesis and growth via inhibiting the activation of HIF-1α and NFκB, and VEGF expression

doi: 10.1186/2045-824X-5-9

Figure Lengend Snippet: EGCG treatment did not affect the capillary density (3270 ± 162 vs. 3103 ± 226 #/mm^2; n = 8; P = 0.5215), and VEGF expression (261 ± 22 vs. 245 ± 19 pg/mg; n = 8; P = 0.4517) in the mouse heart, compared to the control group (Panel A), respectively. There was no significant difference in the capillary density (370 ± 55 vs. 381 ± 44 #/mm^2; n = 8; P = 0.5401), and VEGF expression (225 ± 16 vs. 214 ± 20 pg/mg; n = 8; P = 0.7825) in the limb skeletal muscles between the EGCG-treated mice and the control mice (Panel B ), respectively. The digital images show CD31 immunohistochemistry staining in OCT-embedded cryosections of the heart (Panel A ) and the limb muscle (Panel B ) of control mouse and EGCG-treated mouse, respectively.

Article Snippet: Protein levels of VEGF in plasma, breast tumor, the heart, the limb muscle, and the medium cultured with E0771 cells were determined using mouse VEGF ELISA kits (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions.

Techniques: Expressing, Control, Muscles, Immunohistochemistry, Staining