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: Advanced Science

Article Title: CRIP1 Reshapes the Gastric Cancer Microenvironment to Facilitate Development of Lymphatic Metastasis

doi: 10.1002/advs.202303246

Figure Lengend Snippet: VEGFC is upregulated by CRIP1 in GC cells and involved in CRIP1‐mediated lymphangiogenesis and LM. A,B) ELISA showing the effect of CRIP1 overexpression on VEGFC A) and VEGFD B) secretion. C,D) ELISA showing the effect of CRIP1 knockdown on VEGFC C) and VEGFD D) secretion. E,F) RT‐qPCR showing the effect of CRIP1 overexpression E) and knockdown F) on VEGFC mRNA expression. G) Western blot showing the effect of CRIP1 overexpression and knockdown on VEGFC protein expression. H) Western blot showing the expression of CRIP1 and VEGFC in human GC samples. NT, nontumorous tissues; GC, gastric cancer tissues. I) Representative images (left panel) and quantification (right panel) of the Matrigel tube formation assay with human lymphatic endothelial cells (HLECs). HLECs were cultured with conditioned medium derived from GC cells treated as indicated. Scale bars = 200 µm. J) Representative images of enucleated popliteal lymph nodes across groups. K) Histogram analysis of lymph node volumes across groups. L) Ratios of metastatic to total dissected popliteal lymph nodes from mice inoculated with the indicated cells. χ ‐square test was performed to assess the statistical significance in (L). Error bars represent the mean±SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cell supernatants were collected to quantify the secretion of VEGFC (DVEC00, R&D systems, MN), VEGFD (DVED00, R&D systems, MN), CCL5 (mlbio, Shanghai, China), and TNF‐ α (mlbio, Shanghai, China) via ELISA assay.

Techniques: Enzyme-linked Immunosorbent Assay, Over Expression, Knockdown, Quantitative RT-PCR, Expressing, Western Blot, Tube Formation Assay, Cell Culture, Derivative Assay