Supplementary Methods
Materials
Antibodies to the phosphorylated and total forms of AKT, GSK3, ICAM1 and KDR (Kinase domain
receptor, also known as vascular endothelial growth factor receptor 2 VEGFR2) were from Cell Signaling
Technology (Ipswich, UK). Anti-von Willebrand factor antibody was from DAKO (Ely, UK), antiendothelin from Abcam (Cambridge, UK), anti-VCAM from Insight Biotechnology (Wembley, UK).
Neutralising antibodies to FLT1 and KDR, and TNF- were from R&D Systems (Abingdon, UK) and
anti-VEGF-A neutralising antibody from Angio-Proteomie (Boston, MA, USA). Horseradish peroxidaseconjugated secondary antibodies were from Amersham Biosciences (Bucks., UK). Alexa-488 and Alexa568 fluorescently-labelled antibodies were from Invitrogen Molecular Probes (Leiden, The Netherlands)
and biotinylated secondary antibodies were from Vector Laboratories (Peterborough, UK). Soluble
VEGF-R1 was from Fitzgerald Industries International (cat. # 30R-AV013; Concord, MA, USA), this
comprises extracellular immunoglobulin-like domains 1-5
6
and importantly it is not an IgG fusion
protein. Recombinant proteins of that type readily form stable dimers in solution. siRNA transfection
was carried out using “smartpools” from Dharmacon Inc. (Loughborough, UK) and siFECTamine
transfection reagent (ICVEC, London, UK) according to the manufacturer’s instructions. siRNAs against
FLT1 smart pool (cat. #. M-003136-03-0005), KDR smart pool (M-003148-01-0005), individual FLT1
duplexes (cat. #s: D-003136-05-0005; D-003136-06-0005; D-003136-07-0005; D-003136-09-0005),
individual KDR duplexes (cat. #s: D-003148-05-0005; D-003148-06-0005; D-003148-07-0005; D003148-08-0005) and siControl (cat. # D-002050-01-20) were used at final concentrations of 100 nM.
SU5614 and LY294002 were from Calbiochem (Nottingham, UK) and PF573228 was from Tocris
Bioscience (Bristol, UK).
Culture and Treatment of HUVECs
Umbilical cords were collected from uncomplicated pregnancies with the informed written consent of the
patients and permission of the Local Research Ethics Committee. HUVECs were isolated as previously
described28. Cells were all used before passage 6 and, because of donor heterogeneity, cells from 3-7
individuals were pooled and used in each experiment. HUVECs were seeded into appropriate culture
wells in EGM-2 medium which contained 2% serum and growth factor supplements (Clonetics, Slough,
UK). The next day, cells were washed in PBS and pre-treated with sFLT1 (50-250 ng/mL) and/or
neutralising antibodies, i.e. anti-FLT1 (5 g/mL), anti-KDR (1 g/mL), anti-VEGF-A (5 g/mL), in
DMEM medium containing 2% FCS for 24 hr. On day 3, sFLT1 was re-applied into the medium and,
where applicable, cells were pre-incubated with SU5614 (5 mol/L), or LY294002 (0.1-10 mol/L) or
PF573228 (0.1-10 mol/L) for 30 min. A low dose of TNF- (0.5 ng/mL) was then added and cells were
incubated for a further 6 hr.
Cell adhesion assay
Pooled HUVECs were seeded into 96-well plates and treated with recombinant sFLT1 and TNF- in
quadruplicate, as described above.
In the meantime, differentiated HL60 cells (ECACC European
Collection of Cell Cultures) were spun down and resuspended at 1x106/ml in PBS. Cells were labelled by
incubation at 37°C for 20 min in cell tracker green CMFDA (5-chloromethylfluorescein diacetate) dye
(2.5 µM, Invitrogen Molecular Probes). Labelled cells and an aliquot of retained unlabelled HL60 cells
were washed twice in PBS and resuspended at 5x105/ml in DMEM. In the last hour of the HUVEC TNF incubation, 100 L of HL60 cell suspension was added to each well. The 96-well plate was incubated
for the remaining hour at 37°C, followed by aspiration of medium and three washes with 200 l PBS.
Finally, 100 l PBS was added to each well and fluorescence was measured (485/520 nm
excitation/emission wavelengths, respectively) using FLUOstar Optima Fluorimeter (BMG Labtech).
Transfection with siRNAs
Pooled HUVECs were seeded into the required number of T25 flasks at 5.0x105 cells per flask and
incubated overnight in EGM-2 medium. The T25 flasks were incubated with a mixture of siRNA (10 L
of 20 mol/L stock) in EBM-2 medium (906 L) and siFECTamine buffer (ICVEC, London, UK; 84 L)
for 3 hr at 37°C. The transfection medium was replaced with 5 ml EGM-2 and incubation continued for
another 3 hr at 37°C. Cells were trypsinised, counted and seeded into 96-wells or 12-wells at 1x104 and
5x104, respectively, and cultured for 24-48 hr. Subsequently, transfected HUVECs were treated with
sFLT1 and the HL60 adhesion assay performed. siRNA mediated knock-down efficiency was checked
by quantitative RT-PCR as previously described12 using pre-validated primers from Applied Biosystems
(Warrington, UK; FLT1, cat. # Hs01052961-m1, 18S, cat. # Hs99999901-s1).
Western Blotting
HUVECs were washed and lysed and scraped on ice in ice-cold lysis buffer containing 20 mM Tris, pH
7.4, 1 mM EGTA, 0.01 % Triton X100, 1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 10
mM β-glycerol phosphate, 50 mM sodium fluoride and a complete mini protease inhibitor cocktail
(Roche, Roche Diagnostics, East Sussex, UK). Cells homogenates were centrifuged at 15,000 x g, 4˚C
for 20 min and the supernatants removed. Protein concentrations were determined using a BCA protein
assay kit (Sigma, Poole, UK). Lysates were mixed with 3 x SDS PAGE sample buffer, boiled for 5 min
and allowed to return to room temperature. Equal amounts of protein (20-30 μg) were separated by
sodium dodecyl sulphate-polyacrylamide gel electrophoresis (7.5-15% polyacrylamide depending on the
molecular weight of the target protein), and transferred onto nitrocellulose membrane (Invitrogen,
Paisley, UK), and subjected to immunoblot analysis. Membranes were blocked for 1 h at 25˚C in 5%
milk diluted in Tris-buffered saline (TBS) and 0.1% Tween 20 and incubated with specific primary
antibodies overnight at 4˚C. After washing and incubating with secondary antibodies, immunoreactive
proteins were visualized by the ECL plus chemiluminescence system following the manufacturer’s
instructions (Amersham Biosciences). Protein bands were quantified using Image J software (National
Institutes of Health, http://rsb.info.nih.gov/ij/).
Protein loading was normalized against Ponceau S
staining or -actin staining. The values are expressed as a percentage of the control lysate (100%) for
each experiment.
Immunoprecipitation
Cells were lysed with lysis buffer (20 mM Tris, pH 7.4, 1 mM EGTA, 0.01 % Triton X100, 1 mM sodium
pyrophosphate, 1 mM sodium orthovanadate, 10 mM β-glycerol phosphate, 50 mM sodium fluoride and a
complete mini protease inhibitor cocktail (Roche, Roche Diagnostics, East Sussex, UK)) and the protein
concentration in the lysates determined using the Bicinchoninic acid method (Sigma-Aldrich). 250 g of
stimulated lysate was added to 20 L A/G PLUS-Agarose beads (Santa Cruz) and the mixture was
incubated at 4˚C for 30 min with gentle rocking. Beads were pelleted by centrifugation at 2,500
rpm for 5 min at 4°C and supernatant transferred to a fresh tube on ice. An appropriate
concentration of anti-KDR antibody (Cell Signaling) was added to each tube (usually 1-10 μl, i.e.
0.2-2 μg) that were incubated at 4°C overnight. 20 μl of resuspended volume of Protein A/G
PLUS-Agarose was added and tubes were incubated at 4°C for 6 hr (using rotor in the cold
room). Immunoprecipitates were collected by centrifugation at 2,500 rpm for 5 min at 4°C.
Supernatant was aspirated and stored. Pellets were washed 4 times with 1 ml lysis buffer, each
time repeating centrifugation step above. After final wash, pellets were resuspended in 15-20 μl
3x SDS-PAGE buffer. Samples were boiled for 3-5 min and analysed by electrophoresis.
Immunofluorescent Cytochemistry and Quantification
Cells were seeded onto gelatin-coated 13 mm glass cover-slips. At the end of the experiment, cells were
fixed in 2 % PFA at 4C for 30 min. Cover-slips were washed three times in PBS and if not stained
immediately, stored in PBS at 4C until required.
Prior to staining, cells were permeabilised by
incubating in TBS containing 1 % Triton X-100 and 1 % Tween 20 (TBS-TT) for approximately 30 min.
After blocking non-specific binding with 5 % goat serum and 2 % bovine serum albumin for 1 hr an
appropriate dilution of the primary antibody, diluted in TBS-TT was applied. Cover-slips were incubated
overnight at 4C in a humidified chamber. For double-labelling, antibodies raised in two different species
were used simultaneously. Cover-slips were washed three times in TBS and incubated with speciesspecific secondary antibody/ies (Alexa 568 or Alexa 488) at 1:100 for 1 hr at room temperature. Coverslips were washed in TBS as before and finally in water and mounted in Vectashield medium containing
DAPI (Vector Laboratories) as a nuclear stain. The edges of each cover-slip were sealed with nail varnish
and images captured using a Leica confocal microscope (LeicaTCS-NT, Leica Instruments GmbH,
Germany). All comparable images, i.e. controls and treated cells, were captured at the same laser and
pinhole setting. At least 6 fields per treatment were captured and quantified using the Leica software.
Values for each treatment were expressed as cell fluorescence per m. All experiments were repeated at
least 3 times.
Colorimetric immunohistochemistry (IHC)
Paraformaldehyde-fixed tissues embedded in paraffin wax were sectioned at 7 µm. After dewaxing and
blocking of endogenous peroxidases by incubation with 3 % H2O2 for 30 min, the sections were incubated
with non-immune serum for 20 min. Primary antibodies were applied overnight at 4C, and binding was
detected using Vectastain Elite ABC kits (Vector Laboratories) and SigmaFast DAB (Sigma), according
to the manufacturers’ instructions. Sections were lightly counterstained with haematoxylin. When
necessary, antigen retrieval was performed prior to blocking using 0.01 mol/L sodium citrate buffer at pH
6.0 in a pressure cooker for 2 min. Negative controls were performed by replacement with equal
concentrations of non-immune or isotype matched irrelevant control.
Flow cytometry
Adherent cultured HUVECs were dissociated using 0·2% trypsin. An initial incubation with human IgG
(Sigma) was performed to block Fcγ receptor-mediated mAb binding. Unlabelled primary ICAM1
antibody was then added and detected with Alexa488-conjugated anti-rabbit secondary antibody
(Invitrogen). Cells were analysed using a FACscan flow cytometer and CELLQUEST software (Becton
Dickinson, Oxford, UK).
Supplementary figure legends
Supplementary Figure 1. Flow cytometric analysis of treated HUVECs to validate immunofluorescent
quantification. A) HUVECs were pre-treated with sFLT1, or anti-KDR, or anti-VEGF for 24 hr and
subsequently treated with TNF-α for 6 hr. Cells were trypsinised, stained with anti-ICAM and then with a
fluorescent secondary antibody.
CELLQUEST
Analysis was performed using a FACscan flow cytometer and
software. Pre-treatment with sFLT1, anti-KDR or anti-VEGF increased the surface ICAM
staining in TNF-α-treated cells, compared to control HUVECs treated with TNF-α alone. B) Mean
fluorescence intensity readings from the FACS experiments were plotted for each treatment to compare
them to the values of immunofluorescence quantification (C) extracted from Fig. 3A. This confirms our
data obtained by immunofluorescent quantification.
Supplementary Figure 2. The simultaneous treatment of HUVECs with sFLT1 and TNF- induces
changes in the expression of ICAM1, VCAM1, vWF and ET-1. HUVECs were pre-treated with/without
sFLT1 for 24 hr, followed by TNF- incubation for 6 hr. Fixed HUVECs were immunostained for
ICAM1 (A), VCAM1 (B), vWF (C) and ET-1 (D) and immunofluorescence was quantified (see Fig. 1BE).
Supplementary Figure 3. HUVECs were transfected with individual duplexes of siFLT1 (A) or siKDR (B)
as well as with siFLT1pool (A) or siKDRpool (B) for 24 hr, pre-treated with/without sFLT for 24 hr and then
treated with TNF-α for 6 hr. Fluorescently labelled HL60 leukocytes were added in the final hour of
TNF-α incubation and fluorescence of adherent cells was quantified. Different letters indicate groups that
are significantly different using the PLSD test with P<0.05.
Supplementary Figure 4. HUVECs were transfected with individual duplexes targetting siFLT1 as well as
with siFLT1pool for 24 hr and then treated with TNF- for 6 hr. RNA was isolated and relative levels of
FLT1 mRNA were detected using quantitative real-time RT-PCR. FLT1 mRNA levels were normalised
to the 18S RNA levels.
Supplementary Figure 5. Transfection of HUVECs with siFLTpool and siKDRpool alone or in combination.
HUVECs were transfected with individual pools of FLT1 or KDR siRNAs used at 100 nM (siFLT or
siKDR) and also with combined pools of both FLT1 and KDR siRNAs, used at 100 nM each (siF/K100)
or 50 nM (siF/K50) each. We found that the dual transfection with FLT1 and KDR siRNAs did not
increase the level of leukocyte adhesion, compared to individual transfections with 100 nM of either
FLT1 or KDR siRNAs.
Supplementary Figure 6. HUVECs were pre-treated with/without sFLT1 (200 ng/ml) for 24 hr. Cell
lysates were immunoprecipitated with immobilised anti-KDR antibody and the bead eluent and protein
lysate were immunoblotted with antibodies against FLT1 and KDR.