RICERCA ITALIANA     

Research program

The placenta as source of biophysical, molecular and biochemical markers in pathologies of the pregnancy

University Co-ordinator

Università Cattolica del Sacro Cuore - Clinica ostetrica e ginecologica - MILANO(MI)

Research Unit Leader

Antonio LANZONE

Description

The aim of the present study is to evaluate the hypothetical influence of ET-1, ET-3, CRF and CoCl2 (chemical hypoxia) on VEGF system in the main placental cellular compartments, that are trophoblast and endothelial cells. In particular, in these cells we will investigate the effect of the above-mentioned substances on the expression and production of VEGF-R1 and/or its soluble form, s-VEGFR1, that plays the role of potent functional antagonist of VEGF.
Moreover, in trophoblast cells we will study the effect of the above-mentioned stimuli on the expression of EG-VEGF, recently identified angiogenic factor that is potentially involved, as well as VEGF, in placental physiology and pathophysiology.
We will subsequently analyze the expression of ghrelin in trophoblast cells isolated from placentas at term of physiological or preeclamptic pregnancies.
Cultures of HUVEC will be also used to study the potential influence of ghrelin on endothelial function and proliferation. In particular, in HUVEC treated with ghrelin, PGF2alfa, PGE2 and PGI2 production will be evaluated by radioimmunologic assay (RIA), whereas NOS expression by reverse transcription-polymerase chain reaction (RT-PCR). In the same cells RT-PCR will also allow us to evaluate PA and PAI-1 expression. Finally, on VEGF synthesis the potential effect of ghrelin will be studied in both HUVEC and trophoblast cells. In the latter cells the effect of ghrelin on EG-VEGF expression will be also investigated.
Primary cultures of trophoblast cells
Placentas will be obtained from elective caesarean sections (breech delivery, previous caesarean section, previous myomectomy) at term of uncomplicated pregnancies. To study ghrelin expression, some experiments will be performed with placentas at term of preeclamptic pregnancies. Preeclampsia will be defined as a blood pressure of 140/90 mm Hg or higher on two separate readings with proteinuria (>300 mg/24 h). The villous tissue, removed excluding chorionic and basal plates, is washed repeatedly with 0.9% sodium chloride to remove blood from intervillous spaces. Subsequently, the villous tissue is subjected to enzymatic digestion by incubation with Dispase at 37 C for 15 min with constant shaking (15 g of tissue in 15 ml of enzymatic solution). The enzyme is inactivated by adding 0.5 M ethylendiamine tetraacetic acid (EDTA). The tissue suspension is centrifuged; the supernatant is discarded and the sediment suspended again in DMEM and incubated on ice for 20 min. In the meantime, the villous fragments settle to the bottom of the tube. The supernatant, containing trophoblast cells and the smaller tissue fragments, is filtered through a 100 micron pore size nylon mesh. This procedure is repeated twice. Further purification of trophoblast cell pellet is performed by Percoll density gradient centrifugation. The gradients are made from 45% to 15% Percoll (vol/vol) and cellular suspension is centrifuged for 30 min at 2000 rpm.
Subsequently, a negative immunoselection using the micro-beats technique (Miltenyi method) is used to obtain enriched trophoblast cell cultures. Finally, the cells are suspended again in appropriate medium (DMEM) containing 10% bovine fetal serum and plated (1000000 cells/ml). After 24h of pre-incubation, the medium is removed, and the cells are treated in serum-free medium with test substances. After 24h, the medium of incubation is harvested and stored at –20°C until sVEGFR-1 assay by Enzyme-Linked Immunosorbent Assay (ELISA), according to manufacturer's specifications. For RT-PCR, on the cells total RNA extraction is performed.
Primary cultures of purified HUVEC
Human umbilical cords will be obtained at the time of delivery from healthy, nonlaboring women with uncomplicated term pregnancies undergoing caesarean section because of breech delivery, previous caesarean section or previous myomectomy. After collection the umbilical cord is rapidly immersed in sterile saline solution and immediately processed for endothelial cell isolation. Under a laminar flow sterile hood, the umbilical vein is cannulated and thoroughly rinsed with sterile saline solution. After clamping the other extremity, the vein is filled with 0.03% type IA collagenase solution (in HBSS Ca and Mg free) prewarmed and incubated for 10 min at 37 C. The collagenase solution is then discarded, and the vein is gently washed with 40 ml HBSS that is collected in a 50 ml sterile polypropylene tube. The collected cells are pelleted by centrifugation at 4 C for 10 min. at 1300 rpm; the supernatant is discarded, and the cell pellet is gently resuspended in Medium 199 containing 10% heat-inactivated fetal bovine serum, antibiotics, endothelial cell growth factor and heparine. The cells are then plated on T25 culture flask. Once grown to confluence, the cells are plated again on T75 culture flask and grown up to the moment of the experiments when the cells are plated again on 48 well plates or T25 flask (75000 cells/well). After 24h of pre-incubation, the medium is removed, and cells are treated in serum-free medium with test substances. The medium of incubation is harvested and stored at –20°C until PGs assay by RIA and sVEGFR-1 assay by ELISA, according to manufacturer's specifications. For RT-PCR, on the cells total RNA extraction is performed.
Determination of cell proliferation
For the quantification of cell proliferation, HUVEC are plated in 96-well culture dishes and incubated with fresh serum-free medium alone (control) or containing ghrelin in different concentrations for 24 or 48h. Prolactin (PRL) 25 ng/ml is used as positive proliferating stimulus for HUVEC. Subsequently, the colorimetric immunoassay (ELISA) for the quantification of cell proliferation, based on the measurement of Bromo-deoxy-Uridine (BrdU) incorporation during DNA synthesis, are performed according to the instructions provided by the manufacturer. In particular, after cell incubation with the above-mentioned substances, BrdU is added to the wells and the cells are-incubated for 6h more. During this labelling period the pyrimidine analogue BrdU is incorporated in place of thymidine into the DNA of proliferating cells. After removing the culture medium, the cells have been fixed and the DNA denatured. A monoclonal antibody conjugated with peroxidase binds to the BrdU incorporated in newly synthesized cellular DNA; the immune complexes are detected by the substrate reaction and the reaction product is quantified by measuring the absorbance at the appropriate wavelength using a scanning multi-well spectrophotometer (ELISA reader). The developed color, and thereby the absorbance values, directly correlate to the amount of DNA synthesis and hereby to the number of proliferating cells in each well.
Prostaglandins assay
6-ketoPGF1alfa, stable prostacyclin metabolite, PGE2 and PGF2alfa will be assayed in colture media by RIA. Incubation mixtures of 1,5 ml will be prepared with 50 microl of incubation medium, 200 microl of 0.025 M phosphate buffer, tritiated 6-ketoPGF1alfa or PGE2 or PGF2alfa (2500-3500 cpm) and appropriately diluted antisera. A duplicate standard curve with variable range for each prostaglandin assay will be performed. All tubes will be incubated for 24 h. After separating antibody bound prostanoids with charcoal, tubes will be shacked, centrifuged and supernatants will be decanted into 10 ml of scintillation liquid for the measurement of radioactivity.
Total RNA extraction
HUVEC and trophoblast cells will be directly homogenized by TRIzol and total RNA extraction will be performed for RT-PCR analysis. The purity and concentration of the RNA will be checked spectroscopically.
Semiquantitative RT-PCR
Starting from 1 migrog of isolated total RNA, RT-PCR analysis will be carried out according with the instructions of the manufacturer where reagents of retrotranscription and amplification reactions will be purchased.
VEGFR-1, EG-VEGF, NOS, PA and PAI-1 expression will be evaluated using specific primers. For each substance the mRNA levels will be normalized against the expression of glyceraldehydes-3-phosphate dehydrogenase (GAPDH).
PCR products will be submitted to electrophoresis on agarose gel and stained with ethidium bromide to obtain their identification. Finally, the resulting bands will be quantified using an image densitometer.