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Division of Human Development (P.B., I.P.C., C.P.S.), The Medical School, University of Manchester, Manchester M13 0JH, United Kingdom; Department of Obstetrics and Gynaecology (G.C.M.), Queens University Belfast, School of Medicine, Belfast BT7 1NN, Northern Ireland, United Kingdom; and The Rosie Maternity Hospital (J.C.B.), Addenbrooke’s Hospital, Cambridge University Hospitals National Health Service Foundation Trust, Cambridge DB2 2OO, United Kingdom
Address all correspondence and requests for reprints to: Mr. P. Brownbill, University Research Floor, St. Mary’s Hospital, Hathersage Road, Manchester M13 0JH, United Kingdom. E-mail: paul.brownbill{at}manchester.ac.uk.
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Introduction |
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A range of studies have shown VEGF production and secretion by the human placenta and fetal membranes. VEGF is expressed in the syncytiotrophoblast, chorionic and amniotic membranes and decidua (14), secreted by villous fibroblasts (15) and the villous trophoblast (16, 17). An RT-PCR study of human first-trimester placental villi revealed that isoforms 121, 165, and 189 are expressed in this tissue, with a suggested prominence of the VEGF-165 isoform (15). VEGF-165 is also the most abundant isoform in the ovine placenta (18).
Several receptors (VEGFRs) exist for VEGF: VEGFR-1, VEGFR-2, VEGFR-3, neuroplilin 1, neuroplilin-2, and soluble VEGFR (sVEGFR)-1 (19). In mammalian organs, VEGF elicits vasoactive effects predominantly through VEGFR-1 and VEGFR-2, with VEGFR-2 most widely implicated within the endothelium (10, 20). As regards the placenta and fetal membranes, VEGFR-1 expression is ubiquitous, occurring within the maternal decidua (14), fetal endothelial cells (21), human umbilical vein endothelial cells (22), villous stroma, syncytiotrophoblast (14), and extravillous trophoblast (11). In contrast, VEGFR-2 placental expression seems to be more limited to the fetoplacental endothelium (21) and the syncytiotrophoblast (23); it has also been found in human umbilical vein endothelial cells (22), in which it appears to mediate an enhanced permeability (24). VEGFR-3 is expressed in human placental villi but appears to be limited to the syncytiotrophoblast and therefore is not an obvious candidate receptor involved in the regulation of fetoplacental vascular tone (25).
sVEGFR-1 is a splice variant of the VEGFR-1 receptor, released by endothelial cells (26). There is good evidence that sVEGFR-1 is expressed and secreted by the placenta (27). Furthermore, there are increasing data to suggest that placental sVEGFR-1 secretion into the maternal circulation increases in the pregnancy complication of preeclampsia; in this condition it appears to competitively bind to circulating VEGF isomers, rendering them inactive and resulting in the systemic symptoms of this disease (7).
Despite this evidence of placental production of VEGF and of VEGF receptors in the cells of the placenta, there are no studies addressing whether the hormone has vasoactive or permeability effects in the organ itself. The human placenta has both a maternal and a fetoplacental circulation. Trophoblast invasion results in the maternal side circulation being devoid of endothelium, only bounded by the syncytiotrophoblast with limited potential for control of resistance. On the other hand, the fetoplacental circulation has a normal endothelium and appears to be a major site of vasomotor regulation (28). We and others have used the in vitro dually perfused human placental cotyledon to demonstrate vasomotor actions of several substances in the fetoplacental circulation (29, 30, 31, 32). In this study we used the technique to assess the relative release of total VEGF, sVEGFR-1, and free VEGF into the maternal and fetoplacental circulations of the placenta. We tested the hypothesis that VEGF has vasomotor and permeability effects in the fetoplacental circulation of the human placenta, and we examined the broad intracellular pathways involved in the vasodilator effect which we found.
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Materials and Methods |
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N-omega-nitro-L-arginine (L-NNA; Sigma-Aldrich) was prepared to 400 mM with 1 M hydrochloric acid. This was then serially diluted in PBS before experimentation by 1:40 and 1:20 to 10 and 20 mM, respectively, and stored on ice. Indomethacin (160 mM; Sigma-Aldrich) stock was prepared in ethyl acetate (Sigma-Aldrich) daily. This was serially diluted by 1:40 with PBS to 4 mM [2.5% (vol/vol) ethyl acetate] before experimentation and kept on ice. A 10-mM L-NNA and 4-mM indomethacin cocktail was also prepared in PBS from the above primary stocks and stored on ice. 0.85 mM placental protein-1, a selective inhibitor of src family tyrosine kinases (PP1; Biomol, c/o Affinity Research Products Ltd., Exeter, UK) was prepared in 50% ethyl acetate and 50% PBS and stored at –20 C. This was diluted to 17 µM with PBS daily and kept on ice before use.
Earle’s bicarbonate buffer (EBB) containing 402 µM L-arginine (VWR International Ltd., Lutterworth, UK), 5.6 mM glucose (VWR), 0.5 mM dextran 70 (average molecular mass 64–76 kDa; Sigma-Aldrich), 0.017 mM BSA (Sigma-Aldrich), and 5000 IU/liter heparin (sodium mucus; Multiparin, CP Pharmaceuticals, Wrexham, UK) was used as the perfusate, equilibrated with 95% O2-5% CO2 to a pH of 7.4. This perfusate was also prepared without heparin for the purpose of investigating heparin effects on VEGF-165 vasoactivity. During the permeability studies, the donor-side perfusate was supplemented with the following hydrophilic markers: 500 mg/liter creatinine (VWR), 200 mg/liter fluorescein isothiocyanate (FITC)-inulin (Sigma-Aldrich) and 100 mg/liter horseradish peroxidase (HRP; Sigma-Aldrich).
For the purpose of Western blotting, fetal and maternal venous perfusates, sampled at 120–1 min, were diluted 1:1 in a nonreducing sample buffer [1.5% (wt/vol) Tris hydroxymethyl methylamine, 4.1% (wt/vol) sodium dodecyl sulphate, 24% (wt/vol) urea, 20% (vol/vol) glycerol, 0.002% (wt/vol) bromophenol blue; reagents from Sigma-Aldrich and VWR]. Five percent nonfat dried milk in a 0.2% (wt/vol) Tween 20/PBS diluent was used as the membrane blocking solution. Mouse antihuman VEGF monoclonal antibody (5 µg/ml; R&D Systems, Abingdon, UK) was used as the primary antibody, capable of detecting free-form VEGF-121 and VEGF-165 VEGF-A isoforms. Goat antimouse HRP-conjugated polyclonal immunoglobulin (1:500) was used as the second antibody/detection system (Dako UK Ltd., Ely, UK). ECL Plus reagents (2.5 ml each of solution 1 and 2) were exposed to each membrane (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Kodak D-19 developer and Kodak fixative was obtained from Sigma-Aldrich as a powdered mixture and prepared following instructions.
Perfusion
The method of perfusion was that described by Schneider et al. (33), as adapted in our laboratory (31, 32, 34, 35). Term placentas were obtained within 30 min from normal delivery, or cesarean section. Informed, written consent of the mother was obtained by a clinician at an appropriate time before delivery, and this recruitment was approved by the Central Manchester Research Ethics Committee, the Clinical Research Director for Obstetrics and Gynaecology, and the hospital central delivery unit midwifery manager.
The fetal arterial cannula consisted of a 15-cm length of polythene tubing (Portex, Hythe, Kent, UK; code 800/100/300, inner diameter 1.0 mm, outer diameter 1.6 mm) attached to an 18G tubing adapter. The fetal venous cannula was a 15-cm length of polyvinyl chloride tubing (Portex, Hythe, Kent, UK; inner diameter 2 mm, outer diameter 3 mm). The maternal cannulae consisted of five 10-cm lengths of polythene tubing (Portex; Hythe; code 800/100200/100, inner diameter 0.58 mm, outer diameter 0.96 mm), cut to apices for insertion through the decidua and into the intervillous space, each attached at its opposite end to a modified 23G hypodermic needle, emanating in parallel from a bespoke perfusate distributor. The fetal and maternal arterial cannulae offered significant resistances to experimental flows, which were determined before experimentation, while held at their experimental height and inflow rates. Tubing resistance values were used to retrospectively correct all experimental hydrostatic pressure data. The intervillous space and the fetal circulation were perfused using roller pumps (Watson Marlow; Falmouth, UK) at 14 and 6 ml/min with EBB, respectively, in a humidified cabinet, maintained at 37 C. Fetal-side inflow hydrostatic pressure (FIHP) upstream of the fetoplacental circulation was continually recorded with a chart recorder (Multitrace 2; Lectromed, Letchworth, UK) via a pressure transducer (SensoNor, Horten, Norway) linked to the perfusate tubing. Perfusion rate was independent of inflow hydrostatic pressure in both circulations. When fetal venous outflow was less than 80% of fetal arterial inflow, at the commencement of experimentation, preparations were rejected from the study. When using an 80% threshold of venous outflow over arterial inflow, the rate of rejection of placentas for lower recoveries was approximately two thirds. Visible gross breakages of the villous structure were responsible for this fetomaternal leakage of fetal-side arterial perfusate. In the usable one third of placentas, there was frequently only one lobule per placenta suitable for perfusion when other broken lobules were avoided. This criterion was more stringent for the permeability investigations, during which any visible or measured fetomaternal leak resulted in rejection of the preparation. During the permeability investigation, a 100% fetal-side venous recovery criterion implied a rejection rate of approximately 75% of those placentas collected for study.
Only one placental lobule, perhaps comprising several associated cotyledons, was ever perfused from one placenta. There was a common channeling of fetal venous perfusate via a single fetal venous cannula, which was catheterized on the chorionic plate in which it drained several neighboring tributary villous stem veins from an equal number of cotyledons. Hence the variability seen in our data is true interplacental variability and the "n" numbers given relate directly to the number of placentas studied.
Venous perfusate sample collection
In six cotyledons, 1-min samples of fetal venous perfusate were taken from the fetal venous cannula and of maternal venous perfusate from a channeled drip point, commencing at 5, 10, 15, 30, 60, 120, and 180 min from the start of dual perfusion, into polypropylene (PP) tubes. Samples were centrifuged at 4 C at 1100 x g for 10 min in PP centrifuge tubes to remove blood cells. The supernatant was recovered, aliquoted into PP Eppendorphs, snap frozen in liquid nitrogen, and stored at –80 C until assayed. Perfused lobule wet weight was recorded for the purpose of correcting the reported endogenous levels of ligands and receptors for flow and tissue mass and extrapolating release rates to the whole placenta mass to allow comparisons with estimated in vivo release data, as outlined in Discussion. In doing so, we used data on in vivo term fetoplacental blood flow rates as 225 ml/min (36), maternoplacental blood flow as 600 ml/min (37) and an estimated total term placental mass to be around 560 g.
Arterial and venous cord sera collection
Twenty-five placentas were obtained within 15 min of delivery through labor or cesarean section. At least 3 ml each of paired arterial and venous cord bloods were taken from each cord. After 30 min of clotting in glass tubes at room temperature, the blood was centrifuged and the sera snap frozen, aliquoted, and stored, as above.
Measurement of total combined VEGF-121 and -165
Total VEGF concentrations in all fetal and maternal venous perfusate samples (n = 6 each) and all arterial and venous cord sera samples (n = 25 each) were measured by RIA, using an adaptation of the method described by Anthony et al. (38). Recombinant human (rh) VEGF-165 (R&D Systems Europe, Ltd., Abingdon, Oxfordshire, UK) was used as the standard and 125I-labeled rh-VEGF (Amersham Pharmacia Biotech, Buckinghamshire, Little Chalfont, UK) as the tracer. The assay buffer was 0.2% BSA (Sigma Chemicals Co., St Louis, MO) in 0.04 M phosphate buffer (pH 7.4) containing 625 U/ml Trasylol (Bayer AG, Leverkusen, Germany). Aliquots of standard or unknown serum sample (100 µl) were diluted in the assay buffer with 0.14 M NaCl (VWR) and incubated with 100 µl of polyclonal goat antihuman VEGF antiserum (R&D Systems, Europe) at an initial dilution of 1:2000. 125I-VEGF (100 µl), diluted 1:300 and containing 3.4 IU heparin (CP Pharmaceuticals Ltd., Wrexham, UK) was added to each tube, and the tubes were incubated overnight at 4 C. The VEGF antibody was specific for both VEGF-165 and VEGF-121 on an equimolar basis and showed no cross-reactivity with other cytokines as specified in the R&D data sheet. A polyethylene glycol (Sigma-Aldrich) assisted double-antibody method was used to separate antibody bound 125I-VEGF from free. The antibody separation buffer was 0.1% BSA in 0.04 M phosphate buffer (pH 7.4) containing 0.1 M EDTA (VWR) and 0.5% Triton X-100 (Sigma). Normal goat serum carrier (100 µl) diluted 1:2000, 100 µl of 1:25 donkey antigoat (both IDS, Ltd., Bolden, UK) and 500 µl 4% polyethylene glycol were added to each tube before a 90-min incubation at 4 C. After centrifugation and removal of the supernatant, the radioactivity in the bound fraction was counted on a 
-radiation counter. Intra- and interassay coefficients of variation were 5.8 and 10.4%, respectively, at 4 ng/ml (105 pM, based on the molecular weight of the VEGF165 isoform), and the assay sensitivity limit was 1 ng/ml (26 pM, based on the molecular weight of the VEGF165 isoform). Results are presented as molarity calculated using the molecular weight of the recombinant VEGF-165 isoform because this was used as the standard.
Measurement of free-form VEGF-121, -145, and -165 and total sVEGFR-1
The combined free-form levels of VEGF-121, -145, and -165 were measured in the 180- to 181-min fetal and maternal venous perfusate samples (n = 6 each) and the arterial and venous cord sera samples (n = 9 each) using a sandwich ELISA kit (R&D Systems). This representative time point was in the steady-state release period for total VEGF. The expense of the ELISA kits prevented a full temporal release assay for free VEGF and sVEGFR-1. Separate standard curves were used for interpolating samples: using the serum/plasma assay kit for the sera (intra- and interassay coefficients of variation were 6.7 and 8.8% at 53.7 and 64.5 pg/ml, respectively) and the cell culture supernatant assay kit for the perfusate (intra- and interassay coefficients of variation were 6.5 and 8.5% at 29.1 and 32.8 pg/ml, respectively). The total sVEGFR-1 levels were measured in the 180- to 181-min fetal and maternal venous perfusate samples (n = 6 each) and the arterial and venous cord sera samples (n = 8 each) using a sandwich ELISA kit (R&D Systems; intra- and interassay coefficients of variation were 2.6 and 9.8% at 96.6 and 112 pg/ml, respectively). Results are presented as molarity, calculated using the molecular weight of the recombinant VEGF-165 isoform and the recombinant sVEGFR-1 structures because these molecules were used as the assay standards.
Investigation of released sVEGF isoforms
Western blotting was performed for the detection of soluble isoforms of VEGF-A in neat fetal and maternal venous perfusate (paired samples from n = 3 lobules), sampled at 120–121 min into perfusion during steady-state release. Thirty microliters of neat perfusate were run under nonreducing conditions in duplicate on 7.5% polyacrylamide resolving gels and a 3% polyacrylamide stacking gels, using the Mini-Protean II system (Bio-Rad, Hemel Hempstead, UK; 120 V, 70 min), following 1:1 dilution in nonreducing sample buffer, alongside Kaleidoscope molecular markers (Bio-Rad). Transfer occurred onto two nitrocellulose membranes using a semidry transfer cell (Bio-Rad; 10 V, 40 min).
Membranes were each transferred to the inside wall of a 50-ml seal-capped drum, blotted surface facing inward, and blocked with Tween 20/PBS diluent used as the membrane blocking solution for 1 h at ambient room temperature. After a multiple wash step with 0.2% (wt/vol) Tween 20/PBS solution, one membrane was incubated with 5 ml 5 µg/ml antihuman VEGF monoclonal antibody, and the other with 5 ml of the diluent alone to act as a negative control, for 1 h at ambient room temperature. Both membranes incurred another multiple wash step with Tween 20/PBS, and then both were incubated with polyclonal goat antimouse immunoglobulin-HRP conjugate for 2 h, followed by another multiple wash procedure, as above.
Detection of signal occurred within the dark room and involved the exposure to the chemiluminescence substrates for 1 min each. After this procedure, membranes were removed from their drums, placed inside a light-proof cassette, and covered with a transparent sheet of polythene and luminescent-sensitive photographic film for durations of 15 min each. Films were then developed for 1 min, washed for 1 min, and fixed for 2 min.
Investigation of potential constrictor effects of VEGF-165
In three preparations, successive 0.5-ml boluses of PBS alone and then incremental VEGF-165 doses (up to 20 nM) were administered to the fetal-side circulation via the fetal inflow perfusate tubing and FIHP was recorded. A 0.5-ml 250-nM angiotensin II bolus was added at the end of the experiment to confirm that the fetoplacental vasculature was potentially vasoconstrictive. After the administration of boli, 10 min were permitted for the PBS bolus and each of the four lowest VEGF dosage boli, before the next bolus was delivered, at least 30 min was permitted for a potential effect of the two highest VEGF dosage boli to be observed and a maximal effect of angiotensin II was observed at the end of the experiment, within 10 min of administering this bolus. In all cases, maximal effects were reported.
Investigation of potential dilator effects of VEGF-165
Seven lobules were dually perfused and the fetoplacental vasculature was preconstricted with an appropriate dose of the thromboxane mimetic U46619 to elevate baseline FIHP pressure, administered by constant infusion into the fetal perfusate line, using a syringe pump (Precidor model; Infors AG, Basel, Switzerland). U46619 infusion was maintained throughout the rest of the experiment. An elevated steady-state FIHP baseline was achieved and maintained throughout the experiment (interexperimental variation: U46619 perfusate concentration = 2.33–3.33 nM, elevated steady-state FIHP = 80–152 mm Hg). Preparations not achieving and maintaining a steady elevated baseline FIHP were rejected from the study. Successive 0.5-ml boluses of PBS alone and then six consecutive incremental VEGF-165 doses (1 pM to 20 nM) were administered to the fetal-side circulation. These boli became diluted 36.2-fold (as determined in preliminary experiments using bromophenol blue as a marker) in the fetal-side inflow perfusate tubing, before reaching the fetoplacental vasculature, so that the actual concentration range within the fetoplacental vascular lumen was 27.6 fM to 552 pM. FIHP was recorded throughout the experiment. FIHP in response to the PBS control bolus or each dose of VEGF was standardized against the respective previous steady-state value and expressed as a LOG of the percentage change in FIHP. The previous steady-state hydrostatic pressure value is unique to each bolus, being routinely read at the 1-min interval before injection of the bolus concerned and is the recovered hydrostatic pressure value from any previous intervention in the experiment.
Identification of receptors in the placenta mediating VEGF-165 evoked vasodilation
Placental-like growth factor (PlGF)-1 and VEGF-E are specific agonists for the VEGFR-1 and VEGFR-2 receptors, respectively (39, 40). Therefore, in the absence of other pharamacological tools or adequate specific antibodies, we used these agonists to address whether VEGFR-1 or VEGR-2 likely mediate vasodilatory effects of VEGF. Because PlGF-1 is known to have lower potency at VEGFR-1 receptor than VEGF (41), a dose-response investigation using higher maximum boli concentrations of PlGF-1, compared with that used for VEGF-165, was performed (1 pM to 100 nM, 0.5 ml boli, 
27.6 fM to 2.76 nM at the fetoplacental circulation, n = 7 perfused lobules). VEGF-E has been found to have a biological effect on VEGFR-2 at a similar potency to VEGF-(A)165 (42), so boli were prepared and administered at the same concentrations as described earlier for VEGF-165 (n = 3 perfused lobules). In each series of experiments, U46619 was used to preconstrict the fetal vasculature to elevated steady-state FIHP. Administration of PlGF-1 and VEGF-E and analysis of recorded FIHP were done as described for VEGF-165.
Investigation into the role of nitric oxide (NO) and prostacyclin as potential endothelial-derived second messengers of the VEGF-induced vasodilatory response
NO and prostacyclin are two potential downstream mediators of effects of VEGF (20, 43). To determine whether these were involved in the effects we observed, two further groups of lobules were perfused and constricted with U46619, as previously described. On achieving steady state, a 10-nM bolus of VEGF-165 was administered to the fetal-inflow perfusate line. A vasodilatory response was recorded as an internal control and recovery to the elevated FIHP baseline was permitted. Either 100 µM L-NNA (n = 6 lobules), an inhibitor of constitutive and inducible nitric oxide synthase (NOS), or 40 µM indomethacin, a nonspecific prostacyclin inhibitor with 100 µM L-NNA (n = 6 lobules), were administered to the fetal-side perfusate inflow line via a second syringe pump. Inhibition was maintained for the remaining course of the experiment. On achieving a new steady-state FIHP, a second 10-nM bolus of VEGF-165 was administered to the fetal circulation, and a vasodilatory response was recorded, as above. Changes in steady-state FIHP and VEGF-165 vasodilatory effects were compared in the absence and presence of L-NNA or indomethacin with L-NNA. To confirm that a maximum inhibition of the vasodilatory effect of NOS had occurred, a further series of experiments was performed with the administration of 200 µM L-NNA alone.
Investigation into the role of src-family tyrosine kinases in the upstream endothelial signaling of the VEGF-induced vasodilatory response
Because src-family tyrosine kinases are often involved in upstream VEGF signaling (44), we investigated their potential role in VEGF effects on placenta. The above protocol was repeated, but L-NNA/indomethacin was replaced with 170 nM PP1, a general src-family inhibitor. PP1 administration was maintained for the remaining course of the experiment. Changes in steady-state FIHP and VEGF-165 vasodilatory effects in the absence and presence of PP1 were compared. These experiments were repeated using 0.01% ethyl acetate alone (n = 3), as vehicle temporal control perfusions for the PP1 inhibitor investigation.
Effect of VEGF on paracellular permeability of the placenta to hydrophilic solutes
To investigate the effect of VEGF165 on maternofetal unidirectional paracellular transfer of three hydrophilic inert tracers, creatinine, inulin, and HRP (35, 45), three term healthy placental lobules were perfused with initial fetal venous outflow recoveries equaling 100% of fetal-side inflow. After an initial period of dual perfusion with EBB, the maternal inflow perfusate reservoir was switched to one containing hydrophilic markers (described above). This point represented experimental time, t = 0. At t = 30 min, VEGF165 was constantly administered into the fetal-side perfusate inflow line, from a syringe pump, through an injection port, before the peristaltic pump, giving a equilibrated fetoplacental luminal concentration of 50 pM. The experiment was terminated at t = 60 min. Fetal venous perfusate was sampled for 1 min durations every 5 min throughout the course of the perfusion, commencing at t = 4 min, and was centrifuged at 1100 x g for 10 min at 4 C. Supernatant was aliquoted and stored at –20 C, along with donor-side perfusate reservoir samples, until assays for the hydrophilic markers were conducted. The effect of VEGF-165 on unidirectional fetomaternal paracellular transfer was likewise investigated on a further three equally intact perfused lobules, with the fetal inflow perfusate reservoir switched to one containing the markers at t = 0, with VEGF-165 again administered to the fetal-side at t = 30 min and with maternal-side venous perfusate sampled throughout and processed as above.
Assay of paracellular markers
Creatinine was assayed using an Infinity Creatinine kit (Thermo Electron Corp., c/o Alpha Laboratories, Eastleigh, Hampshire, UK), which is a kinetic assay based on a modified Jaffe reaction (46). The peroxidase activity of HRP was measured using a kinetic assay measuring the rate of oxidation of HRP with the chromogen, 2'-2-azino-di-(3-ethyl-benzthiozoline-6-sulfonic acid) diammonium salt (Sigma-Aldrich), in the presence of hydrogen peroxide (VWR), producing an insoluble precipitate with a jade coloration (47). FITC-inulin was assayed using fluorescence spectrophotometry. In all assays, acceptor side venous perfusate and donor-side perfusate reservoir samples were measured for their hydrophilic marker concentrations against known standard concentrations. Unidirectional fetomaternal and maternofetal clearances were calculated and are proportional to the permeability surface area product at steady-state (48):
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Statistical analyses
All data were tested for normality of distribution using a Kolmogorov-Smirnov test to determine appropriate representation of the data and testing between groups. Data are expressed as mean ± SEM, or median with 25th and 75th percentiles, as appropriate, with n = the number of placentas studied. Arterial and venous cord sera levels of total VEGF were compared using a paired t test, except for a comparison of total VEGF levels between fetal venous serum and fetal-side venous perfusate, where a Mann-Whitney test was used. A two-way ANOVA was performed on the release of total VEGF into the fetal and maternal venous perfusate with time and side of release as variables. For the VEGF-165, PlGF-1 and VEGF-E dose response investigations, ANOVA was used to investigate the possibility of a relationship between FIHP and dosage, and a Dunnett post hoc test was used to assess whether individual doses might have significantly altered the FIHP differential from the effect of the PBS carrier. In L-NNA and indomethacin single and multiple administered studies and in the PP1 inhibitor study, paired t tests were used to compare the effects of the 10-nM VEGF-165 bolus with and without the inhibitor. In all cases, a significant effect was reported when P < 0.05. In the permeability studies, a mean value of unidirectional clearance data of the last two control samples (24–25 and 29–30 samples) was used as a reliable estimate of steady-state based on our previous transfer studies involving creatinine and 
-fetoprotein, a molecule of similar molecular radius to HRP (35). All clearance data in the presence of VEGF-165 was expressed as a percentage of the paired intraexperimental control steady-state value. One-way ANOVA with a Dunnett’s post hoc test was used to test for a significant change in the clearance trend and determine specific time points that were significantly different from the steady-state control period.
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Measurement of total VEGF, free VEGF, and sVEGFR-1
Total VEGF was detectable in both the fetal and maternal venous perfusates, with steady-state release occurring after 60 min of perfusion on both sides (Fig. 1
). The maternal-side venous perfusate level of total VEGF was much higher than the fetal-side venous level (50.3 ± 1.3 and 10.5 ± 7.9 pM, respectively, mean ± SE, sampling time: 180–181 min of perfusion; two-way ANOVA: effect of time: P < 0.0001, effect of side of release: P < 0.0001, interaction: P < 0.001). Figure 2 shows free and total levels of VEGF and total sVEGFR-1 in umbilical cord arterial (Fig. 2A) and venous (Fig. 2B) sera. In both sera, sVEGFR-1 levels were low (3.3, 1.5, and 3.9, and 2.4, 2.1, and 3.2 pM, median, 25th, and 75th percentiles, arterial and venous, respectively), whereas circulating total and free VEGF isomer levels were comparably high (total VEGF: 193.7, 170.2, and 233.0 and 204.2, 184.6, and 226.4 pM, median, 25th, and 75th percentiles; free VEGF: 43.8, 12.6, and 57.5 and 43.2, 16.4, and 57.6 pM, median, 25th, and 75th percentiles; arterial and venous, respectively). Total VEGF was 7% higher in cord venous serum than in cord arterial serum, but arterial and venous sera levels of both sVEGFR-1 and free VEGF were not different from each other. Figure 2 also provides a comparison of the combined free soluble VEGF-A isomer levels, the total VEGF and the total sVEGFR-1 levels in the fetal- (Fig. 2C) and maternal-side (Fig. 2D) venous perfusate after 180 min of perfusion. Fetal-side levels of total VEGF and sVEGFR-1 were much lower (2.6, 0.0, and 28.8 and 0.5, 0.1, and 0.6 pM, median, 25th, and 75th percentiles, respectively) than maternal-side levels (50.4, 47.1, and 53.0 and 34.0, 29.5, and 48.4 pM, respectively; median, 25th, and 75th percentiles). Free VEGF-A isomers were just detectable in the fetal venous perfusate (0.3, 0.1, and 0.5 pM, median, 25th, and 75th percentiles) but were undetectable in the maternal venous perfusate.
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). The rhVEGF-positive control exhibited a positive signal at 38 K (Fig. 3A), as expected from information provided by the supplier. Both of these bands were absent when the primary antibody was omitted (Fig. 3B).
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). In the same lobules, angiotensin II did cause a vasoconstrictive effect, proving that the vasculature was responsive to stimuli (Fig. 4A).
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, filled triangles). There was a lag time between administering bolus doses of VEGF-165 and the maximal vasodilatory response at all concentrations in which a response was observed; for the 10 nM bolus (276 pM at the fetoplacental circulation), this was between 5 and 11 min. Response recovery times between administering bolus doses of VEGF-165 varied according to the extent of the response but as a guide were 10–25 min at bolus concentrations less than 1 nM (27.6 pM at the fetoplacental circulation) and 18–52 min at bolus concentrations of 1–20 nM (27.6–552 pM at the fetoplacental circulation).
In a separate investigation, the repetitive administration of three boli of the 10 nM VEGF-165 bolus dose ( 276 pM at the fetoplacental circulation) to the fetoplacental circulation did not cause a desensitization of the vasodilatory response (68.6 ± 11.3, 68.6 ± 14.7 and 68.0 ± 8.4% of previous steady-state FIHP, consecutively, mean ± SE, n = 3 lobules).
In a further study, the effect of heparin on the VEGF-165 vasodilatory response was considered as this isoform has previously been shown to have heparin binding properties due to the inclusion of the exon-7 domain during its transcription (13). Heparin could potentially augment VEGF165 binding to the VEGFR-2 receptor (49) and inhibit binding to the VEGFR-1 receptor (50). The experimental time course was split into two periods, during which halfway through, the reservoir for dual perfusion was switched from one with to one without heparin and with sufficient washout time to equilibrate to the new conditions. The absence, or presence of heparin in the perfusate, had no effect on the vasodilatory response to the 10 nM VEGF-165 dose (276 pM at the fetoplacental circulation, vasodilatory responses: 73.4 ± 7.5 and 79.7 ± 3.2% of previous steady-state FIHP, respectively, mean ± SE, n = 3 lobules).
Identification of receptors in the placenta mediating VEGF-165 evoked vasodilation
PlGF-1 did not cause vasodilation of the fetoplacental vasculature (Fig. 4B, filled squares), whereas VEGF-E did cause vasodilation, with a similar effect to VEGF-165 at the maximum dose used (Fig. 4B, filled circles; 77.0 ± 7.7 and 79.3 ± 6.0% of previous steady-state FIHP, respectively, 20 nM boli dose 552 pM at the fetoplacental circulation).
Investigation into the role of NO and prostacyclin as potential endothelial-derived second messengers of the VEGF-induced vasodilatory response
A significant increase in steady-state FIHP occurred after the maintained administration of L-NNA (Fig. 5A), but this increase was not significant for the inhibitor cocktail of L-NNA and indomethacin (Fig. 5B). There was a significant decrease in the dilatory response to VEGF-165 in the presence of L-NNA (Fig. 5A) and in the presence of the indomethacin-L-NNA cocktail (Fig. 5B). NOS inhibition diminished the VEGF vasodilatory response by 57% and significantly reduced the duration of the vasodilatory response from 49 ± 3.5 to 28.4 ± 4.2 min (mean ± SE; P = 0.007, paired t test; n = 5). Inhibition of NOS with the higher 200-µM dose of L-NNA diminished the vasodilatory response of VEGF-165 in a comparable manner to the 100-µM L-NNA dose (86.5 ± 2.8 and 85.3 ± 2.9% of previous steady-state FIHP, respectively).
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) after the maintained administration of PP1, which was not observed in the presence of the inhibitor vehicle alone (data not shown). There was no significant decrease in the dilatory response to VEGF-165 in the presence of PP1 (Fig. 6).
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Discussion |
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Data from Western blotting, using a monoclonal antibody capable of recognizing isoforms 121 and 165 of the VEGF-A family, revealed a positive signal at 76 K, suggesting the detection of either a VEGF-165 homodimer, or a VEGF-PlGF heterodimer. It seems unlikely that this band is a VEGF-121 dimer (monomer = 28.4 K), but 76 K is an exact doubling of the molecular weights of either VEGF-165 or PlGF-1, both of which are 38 K. This 76 K-positive band was quite strong in the maternal venous perfusate from two of the three lobules investigated, compared with the 38 K recombinant human VEGF-165-positive control band. The 76 K band is too small to suggest the detection of VEGF sequestered by sVEGFR-1 or sVEGFR-2, and its strong native presence in maternal-side placenta venous perfusate was not detected by ELISA. Because the VEGF-PlGF-1 heterodimer is implicated in enhanced endothelial signaling as another bioactive molecular species of VEGF, our finding poses a further important question surrounding VEGF endocrinology of the maternal circulatory system, which is worthy of further investigation.
It has been suggested that the uteroplacental unit is an important source of sVEGFR-1 (51). Interestingly, the in vitro maternal-side venous perfusate levels of sVEGFR1 measured in our study (38.0 ± 4.4 pM, mean ± SE, n = 5 at 180 min of perfusion) very closely match term in vivo sera values from healthy pregnancy provided by Bujold et al. (51) (25.6 ± 8.8 pM, mean ± SE, n = 9). Allowing for a reduced maternal-side in vitro vs. in vivo perfusion rate and other possible environmental differences created by the in vitro model, such as soluble oxygen tension, our data are consistent with previous evidence that the normal placenta itself is a major source of sVEGFR-1 from the uteroplacental unit (52). Furthermore, the ratio of sVEGFR-1 to VEGF-121 and -165 in the maternal-side placental venous perfusate is so high that all monomeric VEGF-A is sequestered. If these data do reflect the in vivo situation, then they suggest that, in normal pregnancy, circulating maternal monomeric free VEGF levels must be maintained by VEGF secretion, or absorption and degradation of sVEGFR-1 by maternal tissues. The fine balance between placental and maternal production of this hormone and its soluble receptor emphasizes the significance of the increased sVEGFR-1 synthesis by the placenta in preeclampsia for the etiology of the symptoms of this disease (7).
Our data demonstrate VEGF release into the fetoplacental circulation. There is a significantly higher concentration of VEGF in cord venous serum vs. paired arterial serum. Furthermore, there is steady production of VEGF for at least 3 h into both maternal and fetal circulations of the dually perfused placenta, albeit greater into the maternal circulation. Correcting fetal- and maternal-side venous perfusate levels of total VEGF for respective in vitro perfusate flow rates and a mean perfused lobule fresh weight of 31.5 g, fetal- and maternal-side release rates were calculated to be 2.1 and 22.3 pM/g per circuit of the placental circulations, respectively, in the in vitro-perfused lobule. Correcting the arteriovenous cord serum differential in total VEGF (15.7 pM, difference in means) for an estimated term placental tissue mass of 560 g and an in vivo term 50th centile fetoplacental blood flow of 225 ml/min (36), in vivo release rate is calculated to be 6.3 pM/g per circuit of the fetoplacental circulation, i.e. approximately 3 times higher than that found for in vitro release. This reduced in vitro release might be explained by the superoxic nature of the perfusate currently used or perhaps the deficiency of amino acids in the perfusate, limiting peptide synthesis with perfusion time. The requirement of oxygen delivery has prevented the use of more physiological oxygen tensions in perfusion experiments such as performed by ourselves (as here) and others (53, 54). However, the known effect of oxygen tension on VEGF production, through the stabilization of its mRNA (55) and enhancement of translation (56), suggests that it remains an important goal to develop a normoxic but viable perfusion system for further investigations of VEGF production by placenta.
By contrast with the maternal side, our data suggest a fetal-side release ratio of VEGF and soluble receptor, which enables a small, but measurable level of free VEGF-A in the fetal venous perfusate, detectable by ELISA but not Western blotting. This observation of free VEGF fetal serum levels exceeding maternal serum levels is in keeping with the work of others (57), although the levels of free cord serum VEGF levels in our study exceed those of others (57, 58). Given the relatively low level of release of sVEGFR-1 into the fetal circulation and its ratio to total VEGF, it might be expected that the free form VEGF level would be higher than that actually found. One possible explanation for this could be the significant presence of sVEGFR-2 in the fetal circulation, which can have a similar sequestering role to sVEGFR-1 (59) and might occur at levels in excess of that found in the peripheral maternal circulation. The presence of free VEGF in the fetoplacental circulation suggests that an autocrine role in this compartment is a physiological possibility.
Here we have demonstrated that VEGF-165 mediates a dose-dependent vasodilation of the fetoplacental vasculature. Importantly, the free VEGF ex vivo serum level of around 39 pM (mean), which we have measured, very closely approximates to the vasodilatory EC50 of 71 pM for recombinant human VEGF165 in the in vitro dual perfusion model. This gives physiological relevance to our finding that VEGF is a potent vasodilator of the fetoplacental vasculature. Consistent with our results, Szukiewicz et al. (60) found that supraphysiological levels of VEGF evoked a 100% maximal vasodilatory response in isolated human chorionic plate arteries preconstricted with phenylephrine but showed a similar vasodilatory response to our data at the physiological level of fetal serum. In contrast to our findings, Szukiewicz et al. (60) reported a significant vasodilatory response to PlGF. However, there are differences between the two models, in that perfusion additionally incurs luminal flow and sheer stress, the chorionic plate artery ring preparations record changes in resistance appropriate to one vessel type and therefore reflect only part of the potential resistance within the fetoplacental vascular network, and pharmacological preconstriction was different between the two studies.
The positive dose-dependent vasodilatory effect of VEGF-E and the absence of an effect of PlGF-1 provide strong evidence for the role of VEGFR-2 but not VEGFR-1 in the mediation of VEGF-induced vasodilation within this tissue (19). However, an EC50 of 71 pM for the VEGF-165 vasodilatory response is very low for a VEGFR-2 mediated response (20). Also, there is immunocytochemical evidence that VEGFR-1 receptors are expressed in the locality of fetoplacental vessels (23) and that such receptors are capable of eliciting vasodilatory responses in piglet pulmonary vessels (39). Therefore, our conclusion that VEGFR-2 receptors are involved in the vasodilatory response to VEGF in the fetoplacental circulation should be regarded as preliminary. It is plausible that the simplified nature of the perfusate has masked possible complex heterodimeric VEGF-PlFG1 ligand interactions found in serum, which has the capacity to engage VEGFR-1 with VEGFR-2 in a synergized endothelial response (40).
The experiments with the NOS antagonist L-NNA suggest that NO release, assumed to be endothelial derived, explains half of the VEGF-induced vasodilatory response. This agonist-induced NO production is additional to any underlying sheer stress-induced NO production caused by perfusion per se in the U46619 constricted preparation as well as in the basally perfused lobule (data not shown). The remaining 50% of this agonist-mediated vasodilation is not through prostacyclin because a cocktail of indomethacin and L-NNA did not inhibit vasodilation beyond the L-NNA inhibitory effect alone.
This lack of involvement of prostacyclin contrasts with its known signaling role in ovine uterine artery endothelial cells stimulated by VEGF, in which both NO and prostacyclin are thought to play an important role in enhancing blood flow during pregnancy (61). NO and prostacyclin can be synthesized in endothelia via a common constitutive pathway after VEGFR-2/c-src phosphorylation, phospholipase C activation, and inositol 1,4,5-triphosphate formation, which evokes elevations in intracellular calcium (43, 62, 63, 64). In addition to this calcium-dependent pathway leading to dual second-messenger synthesis, NO synthesis is possible via a calcium-independent endothelial signaling pathway (61, 65). Our data, including the lack of an effect of the Src-family inhibitor in our system (see above), are consistent with the idea of such an alternative pathway leading to NO generation, which is independent of Src-family phosphorylation and the associated release of prostacyclin, perhaps using the phosphatidylinositol-3-OH-kinase/Akt associated endothelial NOS pathway seen in the activation of NO generation by insulin (65, 66).
The acute administration of a physiologically relevant level of VEGF165 did not evoke enhanced permeability of the placenta to hydrophilic inert solutes. In general, Src activation appears to be an important upstream process for NO generation to enhance the permeability of endothelial cells (43). The absence of a permeability effect of VEGF in our study is therefore consistent with our inability to observe src-dependent vasodilation. It is a possibility, but not a certainty, that the important src-activated signaling pathway, necessary for altered permeability, has been silenced in our model, possibly due to an unfavorable superoxic in vitro environment. We can eliminate an environmental effect on VEGFR-2 inhibition, the receptor commonly associated with enhanced permeability of endothelial cells, because we have proven its active role in vasodilation to VEGF-E. As noted above, ideally this investigation would have been performed using placental lobules in a normoxic environment, but delivery of blood-free perfusate with soluble oxygen concentration similar to that found in vivo (67, 68) would cause certain hypoxia for most of the tissue mass due to metabolism. In considering the lack of effect of VEGF here on placental permeability, it should be remembered that we are measuring transfer across the entire maternofetal exchange barrier, both syncytiotrophoblast and fetal capillary endothelium. It is therefore possible that an effect on endothelium could be masked by opposite changes in the syncytiotrophoblast permeability or indeed that endothelial permeability contributes only a small fraction of total placental permeability so that any effect of VEGF on this cell layer is not apparent in our experimental system.
In summary, the placenta is an important source of VEGF and sVEGFR-1, particularly for the maternal circulation. Free VEGF is present in the fetoplacental circulation. We have demonstrated that VEGF is a potent vasodilator of the fetoplacental circulation at physiological concentrations. This dilation of fetoplacental vasculature is probably mediated by the VEGFR-2 receptor and an uncharacterized endothelial signaling pathway, resulting in NO generation as a second messenger. VEGF must act through at least one other myocyte-communicating pathway to explain the residual NO-independent vasodilation. Our data provide no evidence for any effect of VEGF on placental permeability.
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Disclosure Statement: The authors have nothing to declare.
First Published Online July 19, 2007
Abbreviations: EBB, Earle’s bicarbonate buffer; FIHP, fetal-side inflow hydrostatic pressure; FITC, fluorescein isothiocyanate; HRP, horseradish peroxidase; L-NNA, N-omega-nitro-L-arginine; NO, nitric oxide; NOS, nitric oxide synthase; PlGF, placental-like growth factor; PP, polypropylene; PP1, placental protein-1; rh, recombinant human; sVEGFR, soluble VEGFR; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.
Received February 9, 2007.
Accepted for publication July 6, 2007.
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References |
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-fetoprotein transfer in the perfused human placental cotyledon from uncomplicated pregnancy. J Clin Invest 96:2220–2226[Medline](v)ß5 in vascular endothelial growth factor signaling. J Cell Biol 157:149–160-fetoprotein and creatinine across the human syncytiotrophoblast. Am J Physiol Regul Integr Comp Physiol 278:R677–R683
B pathways. J Biol Chem 281:11792–11804
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) and maternal venous (
) perfusate of the in vitro dually perfused human placental lobule (mean ± SE, n = 6 lobules). SE was small in maternal side samples after 60 min and falls within the symbols on the figure.
) VEGF-165 (
) on fetal-side inflow hydrostatic pressure in the in vitro dually perfused human placenta lobule with basal fetoplacental tone (Kruskal-Wallis: P = 0.7883, Dunn’s multiple comparison test comparing all VEGF effects with PBS: NS, all comparisons; mean ± SE for PBS, lowest and highest VEGF dose and angiotensin II are: 3.77 ± 0.44 kPa, 3.72 ± 0.48 kPa, 3.18 ± 0.30 kPa and 15.37 ± 4.61 kPa, respectively, n = 3). B, Dose-dependent effect of PBS (
, 
), VEGF-165 (
, n = 3) in the in vitro dually perfused human placenta lobule during a maintained constriction of the fetoplacental vasculature with U46619. The x-axis is shown as a log scale, representing the agonist concentration in the fetoplacental vascular lumen. Pressure data are expressed as absolute pressure (kPa, A) and as LOG percentage change from respective prebolus steady-state values (B). The significance of individual agonist doses tested against the effect of the carrier (PBS) alone was analyzed using an ANOVA followed by a Dunnett’s post hoc test comparing the effect of each dose against the associated PBS control data (VEGF-165: one-way ANOVA: P = 0.0002, EC50 = 71 pM, maximal effect = 77% of previous steady-state FIHP, *, P < 0.05, **, P < 0.01; PlGF-1: one-way ANOVA: P = 0.2922; VEGF-E: one-way ANOVA: P = 0.0034, ##, P < 0.01). The range of maintained doses of U46619 used and the resulting elevated steady-state FIHP values achieved for the PlGF-1 and VEGF-E groups were not significantly different from the VEGF-165 investigation or from each other (one-way ANOVA with Dunn’s post hoc test, P > 0.05 all comparisons).
