Endothelial Sprouting Assay
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Endothelial sprouting assay was performed as described 4 ESC were seeded in 35 mm dishes in 1.2 mg/ml collagen, type I neutralized with 0.1 N NaOH.
- Collagen media contained 15% FBS, 450 µm MTG, 10 µg/ml insulin , 50 ng/ml human vascular endothelial growth factor (VEGF, , ), 100 ng/ml human basic fibroblast growth factor (FGF-2, , ) in IMDM.
- Cells were incubated at 37°C, 5% CO2.
- At day 6, 200 µl media without collagen was added and EB were analyzed on day 11.
- Image analysis was performed using AxioVision LE Software .
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Endothelial differentiation and immunocytochemical analysis
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Endothelial differentiation was induced as described previously with some modifications [2) in each well.
- 1 × 104 hADSCs were seeded in plates and incubated for up to 15 days in medium'>medium'>medium'>endothelial medium'>medium'>differentiation medium'>medium containing medium'>medium'>medium'>endothelial medium'>growth medium'>medium (EGM2-MV) supplemented with 50 ng/mL vascular medium'>medium'>medium'>endothelial medium'>growth factor-165 (VEGF165) , 100 U/mL penicillin, and 100 μg/mL streptomycin.
- 15 days after endothelial differentiation started, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature, and rinsed with PBS.
- The fixed cells were then incubated for 1 hour at 37°C with mouse antibodies against human CD31 or CD34 , KDR at 1:500 dilution.
- After incubation in a blocking solution containing 1% normal goat serum, they were…
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Endothelial differentiation was induced as described previously with some modifications [2) in each well.
- 1 × 104 hADSCs were seeded in plates and incubated for up to 15 days in medium'>medium'>medium'>endothelial medium'>medium'>differentiation medium'>medium containing medium'>medium'>medium'>endothelial medium'>growth medium'>medium (EGM2-MV) supplemented with 50 ng/mL vascular medium'>medium'>medium'>endothelial medium'>growth factor-165 (VEGF165) , 100 U/mL penicillin, and 100 μg/mL streptomycin.
- 15 days after endothelial differentiation started, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature, and rinsed with PBS.
- The fixed cells were then incubated for 1 hour at 37°C with mouse antibodies against human CD31 or CD34 , KDR at 1:500 dilution.
- After incubation in a blocking solution containing 1% normal goat serum, they were incubated with secondary antibodies.
- A streptavidin-biotin peroxidase detection system was used to detect antibody binding.
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Effects of VEGF and TNFá on Tie1 and Tie2 Cleavage.
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HUVEC were incubated with VEGF A or TNFα B for times up to 24 h as indicated before cell lysis and detection of cellular full-length Tie1 by immunoblotting.
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2.3. Differentiation of hESC
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H1 hESCs were plated at 3 × 106 per well of 6-well ultralow attachment (ULA) plates in a total volume of 4 mL of medium'>X-VIVO-15 medium , supplemented with 1 mM sodium pyruvate, nonessential amino acids, 2 mM L-glutamine (all PAA Laboratories GmbH) and 5 μM 2-mercaptoethanol .
- The following growth factors were added: 50 ng/mL recombinant human bone morphogenetic protein-4 (BMP-4, & ), 50 ng/mL recombinant human vascular endothelial growth factor , 20 ng/mL recombinant human stem cell factor , and 50 ng/mL recombinant human granulocyte macrophage-colony stimulating factor (GM-CSF, & ).
- After 2-3 days, the medium was topped up with 2 mL of fresh supplemented X-VIVO-15 medium to produce a total volume of 6 mL.
- Subsequent feeding was performed every 2-3 days by replacing 2-3 mL of old medium with new supplemented X-VIVO-15 medium from which every 5 days a growth factor was removed starting with BMP-4 at day 5, followed by VEGF at…
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H1 hESCs were plated at 3 × 106 per well of 6-well ultralow attachment (ULA) plates in a total volume of 4 mL of medium'>X-VIVO-15 medium , supplemented with 1 mM sodium pyruvate, nonessential amino acids, 2 mM L-glutamine (all PAA Laboratories GmbH) and 5 μM 2-mercaptoethanol .
- The following growth factors were added: 50 ng/mL recombinant human bone morphogenetic protein-4 (BMP-4, & ), 50 ng/mL recombinant human vascular endothelial growth factor , 20 ng/mL recombinant human stem cell factor , and 50 ng/mL recombinant human granulocyte macrophage-colony stimulating factor (GM-CSF, & ).
- After 2-3 days, the medium was topped up with 2 mL of fresh supplemented X-VIVO-15 medium to produce a total volume of 6 mL.
- Subsequent feeding was performed every 2-3 days by replacing 2-3 mL of old medium with new supplemented X-VIVO-15 medium from which every 5 days a growth factor was removed starting with BMP-4 at day 5, followed by VEGF at day 10 and SCF at day 15 of differentiation [
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Met knockdown alters secretion of angiogenic factors.
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VEGF ELISA shows decreased VEGF levels in BxPC-3 and ASPC-1 MetKD xenografts relative to NT control tumors (***p<0.001; ANOVA).
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Specificity of the VEGF-A (a), Flt-1 (b) and KDR (c) antibodies in western blot analyses of ovine choroid plexus (CP) homogenates.
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d Representative blots of recombinant human (rh)VEGF-A121 (line 1), ovine CPs (lines 2 and 3) and rhVEGF-A165 (lines 4 and 5) resolved by SDS-PAGE and immunoblotted with VEGF-A antibodies used in (a).
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SDS-PAGE and immunoblotting
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The second part of CP was cut into small pieces, placed frozen into lysing Matrix D tubes with 500 μl of ice-cold lysis buffer consisting in 100 mM NaCl, 1 % Triton X-100, 2 mM EDTA, 0.2 % SDS, 0.5 % sodium deoxycholate and 1 % protease inhibitor cocktail and homogenized in the FastPrep instrument at an oscillation speed of 6.5 for 30 s. Disruption was repeated 3 times and between the cycles, samples were placed on ice.
- After the last cycle, tubes were briefly centrifuged at 5,000g to remove any undisrupted tissues.
- Homogenates were then transferred into new tubes and centrifuged at 13,000g for 30 min at 4 °C.
- The obtained supernatants were used for protein quantification using a Bradford kit (kit, , ).
- Aliquots of 100 μg of protein were stored at −20 °C until being loaded on SDS-polyacrylamide gradient gels (6–15 % for Flt-1 and KDR and 6–12 % for VEGF-A) and then transferred to a 0.45-μm PVDF membrane…
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The second part of CP was cut into small pieces, placed frozen into lysing Matrix D tubes with 500 μl of ice-cold lysis buffer consisting in 100 mM NaCl, 1 % Triton X-100, 2 mM EDTA, 0.2 % SDS, 0.5 % sodium deoxycholate and 1 % protease inhibitor cocktail and homogenized in the FastPrep instrument at an oscillation speed of 6.5 for 30 s. Disruption was repeated 3 times and between the cycles, samples were placed on ice.
- After the last cycle, tubes were briefly centrifuged at 5,000g to remove any undisrupted tissues.
- Homogenates were then transferred into new tubes and centrifuged at 13,000g for 30 min at 4 °C.
- The obtained supernatants were used for protein quantification using a Bradford kit (kit, , ).
- Aliquots of 100 μg of protein were stored at −20 °C until being loaded on SDS-polyacrylamide gradient gels (6–15 % for Flt-1 and KDR and 6–12 % for VEGF-A) and then transferred to a 0.45-μm PVDF membrane using wet (VEGF-A) or semi-dry (Flt-1, KDR) techniques.
- Molecular weight standards were included for each immunoblot.
- The membranes were then blocked with 5 % non-fat milk in TBST (Tris-buffered saline with 0.5 % Tween-20) buffer for 1.5 h at room temperature, extensively washed in TBST and incubated overnight at 4 °C with the appropriate primary antibody solution.
- The following primary antibodies were used: rabbit polyclonal anti Flt-1 (C-17; 1:40), rabbit polyclonal anti-Flk-1 (C-20; 1:40), rabbit polyclonal anti-VEGF-A (A-20; 1:200) (all from ).
- VEGF-A immunoblots were washed in TBST 3 times and then incubated for 1.5 h at room temperature with goat anti-rabbit alkaline phosphatase-conjugated polyclonal antibodies at a dilution of 1: 20,000.
- Binding of the secondary antibody was visualized with NBT/BCIP solution .
- Next, the blots were examined under white light .
- The Flt-1 and KDR immunoblots were incubated for 1.5 h at room temperature with goat anti-rabbit biotin-conjugated antibodies included in the WesternDot™ kit ( by , , , ) and visualized with Qdot 625 streptavidin conjugate ( by ) according to the manufacturer’s instructions.
- Then, the blots were examined under UV light .
- The blots were stripped and re-probed with rabbit polyclonal anti-GAPDH antibody conjugated to horseradish peroxidase ( Cruz ) as the protein loading control, which was then detected with the enhanced chemiluminescence SuperSignal® Dura Kit ( , Palm , , ) and imaged with G BOX iChemi XT .
- Additionally, some blots were incubated with antibodies pre-adsorbed with excess amounts of their respective peptides (Flt-1 (C-17), , VEGF-A (A-20) Cruz ).
- To identify VEGF-A isoforms, western blot analyses were performed using recombinant human (rh) VEGF-A121 and rhVEGF-A165 isoforms .
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Generation and administration of UC-MSCs
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UCs (n = 10, clinically normal pregnancies, approved by the Qilu hospital’s human research ethics committee) were excised and washed in a 0.1 mol/l phosphate buffer (pH 7.4) to remove excess blood.
- The cords were dissected and the blood vessels were removed.
- The remaining tissues were cut into small pieces (1–2 mm3) and placed in plates with low-glucose medium'>ulbecco-modified medium'>Eagle medium (L-MEM) , supplemented with 10% fetal bovine serum (FBS), 2 ng/mL vascular endothelial growth factor (VEGF& , , ), 2 ng/mL epidermal growth factor (EGF& ), 2 ng/mL fibroblast growth factor (FGF& ), 100 U/ml penicillin, and 100 μg/ml streptomycin .
- Cultures were maintained at 37°C in a humidified atmosphere with 5% CO2.
- The media were changed every 3–4 d. Adherent cells proliferated from individual explanted tissues 7–12 d after initiating incubation.
- At this time, the small tissue pieces were removed from the culture and the adherent fibroblast-like…
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UCs (n = 10, clinically normal pregnancies, approved by the Qilu hospital’s human research ethics committee) were excised and washed in a 0.1 mol/l phosphate buffer (pH 7.4) to remove excess blood.
- The cords were dissected and the blood vessels were removed.
- The remaining tissues were cut into small pieces (1–2 mm3) and placed in plates with low-glucose medium'>ulbecco-modified medium'>Eagle medium (L-MEM) , supplemented with 10% fetal bovine serum (FBS), 2 ng/mL vascular endothelial growth factor (VEGF& , , ), 2 ng/mL epidermal growth factor (EGF& ), 2 ng/mL fibroblast growth factor (FGF& ), 100 U/ml penicillin, and 100 μg/ml streptomycin .
- Cultures were maintained at 37°C in a humidified atmosphere with 5% CO2.
- The media were changed every 3–4 d. Adherent cells proliferated from individual explanted tissues 7–12 d after initiating incubation.
- At this time, the small tissue pieces were removed from the culture and the adherent fibroblast-like cells were cultured to confluence, which subsequently took 2–3 weeks in culture.
- The cells were then trypsinized using 0.25% trypsin (Gibco-BRL) and passaged at 1 × 104 cells/cm2 in the medium described above.
- The cells were used after five or more passages.
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Isolation of primary human LSECs
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Human liver endothelial cells were isolated from liver tissue from patients undergoing surgical liver resection.
- The tissue was collected with informed consent and under local ethics committee approval of the University of Bern.
- Approximately 30 g of liver was cut into 2 mm3 pieces and digested in 50 ml PBS containing 10 mg/ml collagenase 1A for 90 min at 37 °C.
- The resultant solution was then filtered through a sterile 0.5 mm steel mesh, the supernatant centrifuged at 300 × g for 8 min, and the pellet then resuspended on 10 ml PBS.
- This digest was then layered on a 33%/77% Percoll density gradient, centrifuged for 30 min at 800 × g 4 °C and the non-parenchymal layer removed, pelleted and resuspended in 500 μl of PBS.
- Endothelial cells were isolated from this fraction by immunomagnetic selection using a mouse anti-CD31 antibody , followed by an anti-mouse IgG Dynabead, 30 min incubation with each and magnetic separation was performed on MACS columns.
- Isolated LSECs were…
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Human liver endothelial cells were isolated from liver tissue from patients undergoing surgical liver resection.
- The tissue was collected with informed consent and under local ethics committee approval of the University of Bern.
- Approximately 30 g of liver was cut into 2 mm3 pieces and digested in 50 ml PBS containing 10 mg/ml collagenase 1A for 90 min at 37 °C.
- The resultant solution was then filtered through a sterile 0.5 mm steel mesh, the supernatant centrifuged at 300 × g for 8 min, and the pellet then resuspended on 10 ml PBS.
- This digest was then layered on a 33%/77% Percoll density gradient, centrifuged for 30 min at 800 × g 4 °C and the non-parenchymal layer removed, pelleted and resuspended in 500 μl of PBS.
- Endothelial cells were isolated from this fraction by immunomagnetic selection using a mouse anti-CD31 antibody , followed by an anti-mouse IgG Dynabead, 30 min incubation with each and magnetic separation was performed on MACS columns.
- Isolated LSECs were cultured on collagen-coated plates.
- Human LSECs were cultured in SFM endothelial basal media with 10% human serum, 0.1% gentamycin, 5 ng/ml hepatocyte growth factor and 10 ng/ml vascular endothelial growth factor (VEGF, ).
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