Recombinant growth factors were purchased from RELIATech (Brauschweig, Germany). Rabbit polyclonal antibodies against phospho- endothelial nitric oxide synthase (eNOS) at serine-1177 (p-eNOSSer1177), phospho-ERK-1/-2 MAPK and phospho-VEGF receptor-2 (VEGFR-2) tyrosine-951 antibodies were purchased from Calbiochem (Nottingham, UK). Small inhibitory RNAs (siRNA) and oligonucleotide primers were purchased from Eurogentec (Southampton, UK). Luciferase reporter assay and cDNA synthesis kits were from Promega (Southampton, UK). All other cell culture reagents and chemicals were obtained from Sigma Aldrich (Poole, UK).
Human placental tissue was obtained from normal pregnancies and gestationally-matched pregnancies complicated by preeclampsia. Preeclampsia was defined as blood pressure > 140/90 mm Hg on at least two consecutive measurements and proteinuria of at least 300 mg per 24 hours. Informed consent was obtained from the patients and the study had the approval of the South Birmingham Ethical Committee (Birmingham, UK).
Primary human umbilical vein endothelial cells (HUVEC) were isolated and cultured as described . Cells were used at passage two or three for experiments and serum-starved in endothelial cell serum-free medium (Gibco-BRL, UK) supplemented with 0.2% bovine serum albumin for 24 hours prior to stimulation.
Adenoviral gene transfer
The recombinant, replication-deficient adenoviruses encoding sFlt-1 (Ad-sFlt-1) VEGF (Ad-VEGF) and PTEN (Ad-PTEN) were used as described previously [27–29].
Quantitative Real-Time PCR
Sample preparation and real-time PCR was performed as described previously . Briefly, mRNA was prepared using TRIzol and DNase-1 digestion/purification on RNAeasy columns (Qiagen), and reverse transcribed with the cDNA Synthesis Kit (Promega). Triplicate cDNA samples and standards were amplified in SensiMix containing SYBR green (Quantace) with primers specific for sFlt-1 . The mean threshold cycle (CT) was normalized to β-actin and expressed relative to control.
siRNA knock-down of sFlt-1
Two siRNA sequences to the unique 3' sequence of sFlt-1 (sFlt-1 A sense: 5'-TAACAGUUGUCUCAUAUCAtt-3' and antisense: 5'-UGAUAUGAGACAACUGUUAtt-3'; sFlt-1 B sense: 5'-UCUCGGAUCUCCAAAUUUAtt-3' and antisense 5'-UAAAUUUGGAGAUCCGAGAtt-3') were designed using the Dharmacon siDESIGN tool . HUVEC (~ 1 × 106 cells) were electroporated with ~ 3 μg of sFlt-1, or a universal control siRNA (Dharmacon) using the HUVEC kit II and Amaxa nucleofector (Amaxa GmbH, Cologne, Germany) as described .
Transduction of chimeric VEGF Receptors in HUVEC
A chimeric VEGF/epidermal growth factor (EGF) receptor comprising the intracellular and transmembrane domains of VEGFR-2 fused to the extracellular domain of the human EGF receptor . EGF does not bind to VEGF receptors, therefore, it does not activate the endogenous VEGF receptors. EGDR and its tyrosine-to-phenylalanine mutants (EGDR-Y951F) were generated and cloned into the pMMP retroviral vector, and retrovirus-containing cell supernatant was harvested and used immediately to infect HUVEC . Following 16 hours of incubation, the medium was replaced with fresh growth medium and the HUVEC were used 48 hours after infection.
Nitric oxide (NO) Release
Total NO in conditioned media was assayed as nitrite, the stable breakdown product of NO, using a Sievers NO chemiluminescence analyzer (Analytix, Sunderland, UK) as described previously .
Tube Formation Assay
The formation of capillary-like structures was examined on growth factor-reduced Matrigel in 24-well plates as described previously . Tube formation was quantified by measuring the total tube length in five random x200 power fields per well using a Nikon phase-contrast inverted microscope with Image ProPlus image analysis software (Media Cybernetics, Silver Spring, USA). Mean total tube length was calculated from three independent experiments performed in duplicate.
flt-1gene promoter activity assay
A 1.3 Kb fragment of the human flt-1 gene corresponding to -1214 to +155 bp relative to the first exon in the pGL2 luciferase vector (Promega) was used to determine flt-1 promoter activity . Briefly, porcine aortic endothelial cells (PAEC) were transfected with the flt-1 promoter-reporter construct using Exgen 500 (Fermentas, UK) and the cell lysates assayed as described previously .
Cells lysates were immunoblotted as described previously . Membranes were probed with rabbit polyclonal antibodies against phospho-eNOS-Ser1177, anti-ERK-1/-2 or anti-VEGFR-2 phosphotyrosine-951 at 4°C overnight. Proteins were visualised using the ECL detection kit (Amersham-Pharmacia, UK).
Soluble Flt-1 (sFlt-1) levels in culture supernatants were measured as previously described .
Formalin-fixed, paraffin-embedded tissues were used for immunohistochemistry as previously described .
All data are expressed as mean ± SEM. Statistical comparisons were performed using one-way ANOVA followed by the Student-Newman-Keuls test as appropriate. Statistical significance was set at a value of p < 0.05.