Vegf r1 flt 1

Mouse VEGF R1/Flt-1 (R&D systems), PIGF-2 (R&D systems) and Endoglin/CD105 (R&D systems) were measured in plasma samples collected from pregnant mice on gd18.5. For collection of plasma, murine blood was collected in EDTA-coated tubes followed by centrifugation at 2000 × g for 15 min at room temperature. Supernatants (plasma) were collected, aliquoted and stored at –80 °C. Urine samples were also collected from pregnant female mice at gd18.5 (just prior to culling the mice for measurement of fetal weights), and aliquoted and stored at –80 °C. Albumin and creatinine concentrations in the urine were determined by an Albumin ELISA (Bethyl Laboratories) and a Creatinine colorimetric assay (Cayman Chemical), respectively. The ratio of urinary albumin to creatinine was calculated and considered as a measure of proteinuria.

Concentrations of the hormone human gonadotropin chorionic (βhCG) in culture supernatants of HPEs were determined by enzyme-linked immunosorbent assay (ELISA), using “DuoSet® ELISA kits” (R&D Systems) (Cat. Number: DY9034-05) according to the manufacturer’s instructions. Levels of the angiogenic factors: Placental Growth Factor (PlGF) (R&D Systems) (Cat. Number: DY264), Vascular Endothelial Growth Factor (VEGF) (R&D Systems) (Cat. Number: DY293B-05), VEGF receptor (VEGF R1/Flt-1) (R&D Systems) (Cat. Numbers: DY321B), and Endoglin/CD105 (END) (R&D Systems) (Cat. Number: DY1097) were also evaluated. Samples were added without further dilution. The absorbances were read at the Multiskan™ FC Microplate Photometer at 450 nm and the concentration of βhCG and angiogenic factors were determined in pg/mL by extrapolating the data from the absorbance against a standard curve, normalized for every 100 µg of tissue. The assay sensitivity limits of the kits are: 7.81 pg/mL to βhCG, 31.3 pg/mL to PIGF, 31.3 pg/mL to VEGF, 125 pg/mL to VEGF R1/Flt-1 and 46.27 pg/mL to END.

Bovine aortic endothelial cells (ECs) (passages 5–8) were obtained from Cell Applications, Inc. (San Diego, CA, USA). ECs were cultured in DMEM supplemented with 10% FBS (CellGro), 5.5 mM glucose, 4 mM L-glutamine, 1 mM sodium pyruvate and 100 U/ml penicillin/streptomycin at 37°C and 5% CO₂. Prior to experiments, ECs were cultured in DMEM containing 2% FBS for 24 h. Extravillous trophoblast cells (HTR-8/SVneo, passages 75–82), are a well characterized, authenticated and immortalized first-trimester extravillous trophoblast (EVT) cell line(Graham et al., 1993 (link)). HTR-8/SVneo were cultured in RPMI-1640 (Gibco, Grand Island, NY, USA) supplemented with 5% FBS and 100 U/ml penicillin/streptomycin at 37°C and 5% CO_2. To force mitochondrial metabolism, ECs and HTR-8/SVneo cells were grown in galactose supplemented media, matching the same glucose concentrations used previously, for two passages prior the analysis. Human recombinant sFlt-1 (VEGF-R1/Flt-1), and VEGF were obtained from R&D Systems, (Minneapolis, MN, USA). For in vitro correlation studies, cells were exposed to 2% serum from recruited women (Rodgers et al., 1988 (link); Maynard et al., 2003 (link)).