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1Department of Obstetrics and Gynecology, University of Vermont College of Medicine, Burlington, Vermont; 2Department of Obstetrics and Gynecology, Brown University, Providence, Rhode Island; 3Department of Cell Biology, University of Calabria, Cosenza, Italy; and 4Department of Obstetrics and Gynecology, Karolinska Institutet, Clintec, Karolinska University Hospital-Huddinge, Stockholm, Sweden
Submitted 8 August 2007 ; accepted in final form 10 January 2008
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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vascular endothelial growth factor; nitric oxide; uterine circulation; rat; human resistance arteries; vascular endothelial growth factor receptor-1; fms-like tyrosine kinase-1
Placental growth factor (PlGF) is a member of the VEGF growth factor family that was discovered in 1991. Unlike some VEGF isoforms, which bind to VEGF receptor (VEGFR)-1 [fms-like tyrosine kinase (Flt)] and/or VEGFR-2 (kinase insert domain-containing receptor or fetal liver kinase), PlGF binds specifically to VEGFR-1. Thus far, four studies have examined the effects of PlGF on vascular reactivity, with reports of vasodilatory action in isolated renal (10), placental (14), pulmonary (7), and mammary vessels (17). The mechanism of dilation has not been determined. Vasodilation to VEGF has been more widely reported and is associated with signaling through VEGFR-2 via a mechanism that involves activation of phosphoinositide 3-kinase and Akt-dependent phosphorylation of endothelial nitric oxide (NO) synthase, resulting in increased NO production (e.g., Refs. 4, 15). In contrast, one report concluded that the vasodilator response may involve both types of receptor (7). VEGFR heterodimerization has also been reported and linked to prostanoid synthesis (11).
The physiological role of PlGF is not known; however, studies have shown that its circulating concentrations exceed those of VEGF by >40-fold during normal gestation, although its affinity for VEGFR-1 is only 1/10th of the affinity of VEGF (1). During preeclampsia, a disease associated with hypertension and uteroplacental underperfusion, free PlGF concentrations are significantly reduced due to elevations of a soluble form of VEGFR-1 (sFlt-1) (10, 16). Adenoviral overexpression of sFlt-1 in rats results in a preeclampsia-like syndrome characterized by hypertension, proteinuria, and renal endothelial glomerular endotheliosis (10). Recently, it has been suggested that a ratio of sFlt-1 to PlGF may be a useful predictive marker for the development of preeclampsia (16).
Based on these observations, we hypothesized that PlGF, like VEGF, may exert a vasodilatory effect on the peripheral vasculature and thus modulate regional blood flow and resistance. In view of previous results of enhanced uterine artery dilation to VEGF in pregnancy (2), we also measured uterine artery reactivity to PlGF as a function of concentration, as well as the expression of both the VEGFR-1 and VEGFR-2 message in uterine arteries from pregnant vs. nonpregnant animals to determine whether changes occur during pregnancy.
Pregnancy-induced, species-related, and regional differences in the vasodilatory actions of PlGF were evaluated by extending reactivity studies to mesenteric arteries in rats and to myometrial and subcutaneous vessels in humans, with and without inhibitors of endothelial dilators (NO, prostaglandins). We also examined its effect on the rat uterine veins that drain the placenta. Due to placental production and secretion of this growth factor, uterine veins are likely to contain the highest concentrations of PlGF before systemic dilution.
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METHODS |
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The uterus and a section of the gut 5–10 cm distal to the pylorus were removed and placed in separate petri dishes containing cold (4°C) HEPES-buffered physiological salt solution (PSS). HEPES-PSS was composed of (in mM) 141.8 NaCl, 4.7 KCl, 1.7 MgSO4, 0.5 EDTA, 2.8 CaCl2, 1.2 KH2PO4, 10.0 HEPES, and 5.0 glucose (pH = 7.4).
Segments (1–2 mm long) of uterine arcuate arteries, uterine arcuate veins, and third-order mesenteric arteries were dissected free from connective and adipose tissue and transferred to the chamber of a small-vessel arteriograph. One end of the vessel was tied onto a glass cannula and flushed of any luminal contents by increasing the pressure before securing the distal end onto a second cannula using a servo-null pressure system (Living Systems Instrumentation). All vessels were initially pressurized (arteries: 50 mmHg; veins: 10 mmHg) and equilibrated for 45–60 min at 37°C before the beginning of experimentation. Lumen diameter was measured by trans-illuminating each vessel segment and using a video dimension analyzer (Living Systems Instrumentation) in conjunction with data-acquisition software (Ionoptix) to continuously record lumen diameter.
Following equilibration, all vessels were preconstricted with phenylephrine to produce a 50–70% reduction in baseline diameter. Once constriction was achieved, PlGF-2 (mouse, R&D Systems) was added in increasing concentrations (0.1 pM–10 nM), and the resulting dilation recorded. Separate vessel segments were preincubated with N
-nitro-L-arginine (L-NNA; 0.2 mM) to inhibit NO before the administration of PlGF.
To determine the receptor subtype responsible for PlGF-induced dilation in the uterine circulation, vessels were incubated with inhibitory antibodies to either VEGFR-1 (R&D Systems; 10 µg/ml) or VEGFR-2 (R&D Systems; 1 µg/ml) for 15 min before constriction with phenylephrine. These inhibiting antibodies were utilized at the maximal effective concentration specified by the manufacturer and confirmed experimentally in preliminary tests in our laboratory.
Once the concentration-response characteristics were defined, a separate group of vessels was prepared as described and incubated with VEGF-E, an isoform of VEGF that is selective for VEGFR-2 activation alone (1 nM arteries, 10 nM veins; Research Diagnostics), or in combination with PlGF (0.1 nM in arteries, 5 nM in veins).
Vascular reactivity of human subcutaneous and myometrial arteries. These studies were carried out in collaboration with the Karolinska Institute, Sweden, by two coauthors (K. Kublickiene and L. Luksha). The investigation was undertaken with the approval of the local research ethics committee. Biopsies of subcutaneous fat and myometrium were obtained from healthy pregnant women with a median age of 26 yr (range 24–34 yr) and at a median gestational age of 38 wk (range 37–39 wk) undergoing elective cesarean section due to breach presentation (n = 4) or psychological reasons (n = 3). Myometrial arterial segments were obtained from the upper edge of the transverse incision in the lower uterine segment during cesarean section. Biopsies were immediately placed in cold PSS (in mM: 119 NaCl, 4.7 KCl, 2.5 CaCl2, 1.17 MgSO4, 25 NaHCO3, 1.18 KH2PO4, 0.026 EDTA, 5.5 glucose) and utilized for isolation of small arteries, which were mounted in the organ baths of a four-channel wire myograph (Multimyograph, model 610, Danish Myo Technology, Aarhus, Denmark), as previously described (9). Each organ bath contained warmed (37°C) PSS that was continuously bubbled with 5% CO2–95% O2. Following a 30-min equilibration period, a passive circumference-tension curve was created for each segment to set optimum resting tension. Endothelium-dependent vasodilatation was assessed by the addition of a single concentration of bradykinin (1 µmol/l) to each chamber after preconstriction with norepinephrine (NE; 1 µmol/l). Arteries that did not achieve at least 70% vasorelaxation to bradykinin were excluded from the study.
Both myometrial and subcutaneous arteries were preconstricted with NE (3 µM), and concentration-response curves to PlGF (0.01–10 nM) were generated before and after a 30-min incubation with a solution containing a combination of N-nitro-L-arginine methyl ester (L-NAME; 0.3 mM) and indomethacin (Indo, 10 µM) to inhibit production of endothelial prostanoids and NO.
Measurement of message for VEGFR-1 and VEGFR-2 by RT-PCR. Main uterine arteries were isolated and flash-frozen in liquid nitrogen for subsequent extraction and purification of mRNA, followed by real-time RT-PCR Taqman assay to evaluate the mRNA expression of VEGFR-1 and VEGFR-2. Proprietary sequences were purchased from ABI. Total RNA was extracted using TRIzol (Invitrogen), as described by the manufacturer. RNA concentrations were determined by measuring the absorbance at a wavelength of 260 and 280 nm. RNA was then converted to cDNA using random hexamer primers and the Superscript First-strand Synthesis System and stored at –70°C until ready for use. Real-time quantification of target mRNA transcripts was performed using an ABI Prism 7000 Sequence Detection System. All samples were quantified in a multiplex reaction with 18s ribosomal RNA to control for differences in sample preparation. Control (ribosomal VIC RNA control; ABI) and targeted cDNA were amplified in a single reaction tube. The efficiency of amplification of each target transcript was evaluated by plotting cycle threshold to log (sample concentration) for each standard curve. Reactions were carried out using Universal Master Mix (ABI). Amplification was performed over 50 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 60 s. Cycle threshold values were determined for each reaction using real-time plots of fluorescence vs. cycle. Relative quantification was determined using the standard curve method. Target cDNA expression was normalized to 18s ribosomal RNA, and each sample was run in triplicate to obtain an average value.
Drugs and solutions. All chemicals were purchased from Sigma Chemical (St. Louis, MO), including salts for buffer preparation, L-NNA, Indo, phenylephrine, diltiazem, and papaverine. PlGF-2 (mouse) and antibodies to VEGFRs were purchased from R&D Systems; VEGF-E was obtained from Research Diagnostics and used for both animal and human tissue studies.
Statistical analysis.
Data are expressed as means ± SE, where n is the number of arterial segments studied. The n values refer to both number of vessels and number of animals; wherever possible, two vessels were used from one animal to evaluate the effects of NO inhibition (i.e., one vessel with and one without preincubation in L-NNA). Hence, paired or unpaired Student's t-tests were used as appropriate to determine the significance of differences between sets of data, and differences were considered significant at P 
0.05. When more than two treatment groups were evaluated, as in the VEGF-E and antibody studies, differences in responses between groups were determined with ANOVA followed by a multiple-comparisons test (Tukey's) to evaluate the significance of differences between treatment means.
In wire-mounted human vessels, relaxation to PlGF was expressed as percent inhibition of the contraction induced by NE. Differences in responses between groups were determined by comparing concentration-response curves using a two-way repeated-measures ANOVA, using PlGF concentration as a within-subject factor and artery type or incubational conditions as a between-subject factors. All data are presented as means ± SE; n represents the number of vessels (one vessel per woman).
Calculations. Pharmacological sensitivity to vasodilation by PlGF (IC50) was calculated for each vessel by normalizing the fractional response to a particular concentration of PlGF to the maximal response obtained at the highest concentration of drug tested. Efficacy was defined as the percentage of maximal dilation, which was determined at the end of each experiment by the addition of diltiazem (10 µM) and papaverine (100 µM), a relaxing solution that induces complete vascular smooth muscle relaxation. In wire-mounted vessels, efficacy was defined as the percentage of maximal relaxation relative to preconstrictor level of tension.
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RESULTS |
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Vasodilatory effects of PlGF on rat uterine vessels; effects of gestation; expression of message for VEGFR-1 vs. VEGFR-2. Arcuate arteries and veins obtained from the uterine circulation dilated readily to PlGF. As previously observed with VEGF (12), arteries obtained from LP animals were significantly (1,000x) more sensitive than those from age-matched NP controls (IC50 values: NP = 2.7 ± 1.4 nM; LP = 0.003 ± 0.0015 nM; P < 0.01), although the extent of maximal dilation (efficacy) was comparable (NP = 86 ± 5.8%; LP = 78 ± 11%; P > 0.05).
Moreover, the sensitivity of LP uterine arteries to the vasodilator effects of PlGF was significantly (200x) greater than that of veins (IC50 value: 0.6 ± 0.17 nM in veins; Fig. 1B) and dilated to a greater extent than veins (efficacy averaged 78 and 39% of maximal dilation, respectively, at maximal concentrations; Fig. 1A).
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30% of the dilation remained (Fig. 2).
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50% of maximal dilation at the highest concentration, 10 nM) to a lesser extent than comparably sized uterine arteries (compare Figs. 6A vs. 1A); dilation was also considerably slower than that observed in uterine vessels, taking 15–45 min instead of 5–15 min to stabilize. Preincubation in L-NNA (0.2 mM) did not affect efficacy, but decreased sensitivity (IC50 values: control: 0.22 ± 0.04 nM; L-NNA: 1.20 ± 0.47 nM; P < 0.05; Fig. 6, A and B, respectively).
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50% of the relaxation response to PlGF in myometrial arteries (e.g., at 10 nM: 79 ± 7% PSS vs. 44 ± 14% L-NAME + Indo; n = 5, P < 0.05, ANOVA); however, it had no effect on efficacy in subcutaneous arteries (51 ± 7% PSS vs. 48 ± 9% in L-NAME + Indo; n = 6). The data shown in Fig. 7 indicate the extent of dilation under each condition relative to preconstriction level (efficacy). Calculation of pharmacological sensitivity requires the normalization of this parameter to the maximal effect observed in response to the compound in question (PlGF) for each vessel. Hence, the data in Fig. 7 were normalized in this manner to assess sensitivity and are replotted in Fig. 8. As indicated above, there was a difference in sensitivity to PlGF between subcutaneous and myometrial arteries (P < 0.05, ANOVA); however, after inhibition of NO synthase and cyclooxygenase products, the sensitivity of myometrial arteries was comparable to that obtained in subcutaneous arteries before and after L-NAME + Indo (P > 0.05, ANOVA). After preincubation with L-NAME + Indo, IC50 values were 2.2 ± 0.65 nM (myometrial) to 2.4 ± 0.5 nM (subcutaneous) and were no longer statistically different (P > 0.05).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In this study, we were interested in probing the role of tissue (nonsoluble) VEGFR-1 in uterine circulatory function during pregnancy. An in vitro approach using isolated vessels allowed us to quantitate the vasodilatory effects of PlGF, a selective agonist for VEGFR-1. As the results indicate, PlGF is an extremely potent vasodilator, with significant effects documented at subnanomolar concentrations, particularly in uterine arteries. The observation of a venodilatory effect is novel, and the fact that PlGF levels would be highest in the veins that drain the placenta suggests that it may play a role in regulating venous tone. Furthermore, uterine venorelaxation is associated with an increased permeability of the vascular wall (2), and, in view of the well-documented proximity of arteries to veins in the uterine circulation of several animal species, as well as in the human, its permeability-enhancing action may facilitate signal transfer from vein to artery (3) and thereby potentially alter uterine resistance artery tone, placental blood flow, and vascular structure.
Earlier, our laboratory (12) reported a significant effect of pregnancy on uterine artery sensitivity to VEGF and, in a subsequent study, determined this to be secondary to elevated circulating levels of estrogen by studying oophorectomized animals in combination with selective hormone replacement (13). These findings did not distinguish the receptor subtype involved in VEGF vasodilation, since VEGF activates both VEGFR-1 and VEGFR-2. In this study, we demonstrate a similar change in reactivity to PlGF, a selective agonist for the VEGFR-1 receptor subtype, and implicate a transcriptional mechanism as the underlying cause since: 1) VEGFR-1 but not VEGFR-2 message was significantly upregulated in pregnancy, and, as shown by the experiments that utilized receptor blocking antibodies, 2) only VEGFR-1 appears to be involved in the vasodilatory process, at least in the vessels studied herein. This finding does not preclude altered postreceptor signaling as an additional, or complementary, mechanism for enhanced vasodilator sensitivity.
The important question from a physiological standpoint is whether PlGF signaling contributes to the profound increase in uterine blood flow that characteristically occurs in the pregnant state. In this regard, it was recently noted (8) that fetal and placental weights were significantly reduced in a rat model in which PlGF/VEGF signaling was attenuated by adenoviral overexpression of sFlt-1. Although uterine blood flow was not measured in that study, there is a longstanding association between reduced uteroplacental perfusion and reduced fetal and placental weights in studies that utilize a surgical approach to impose a state of reduced uteroplacental perfusion in rats (the reduction of uterine perfusion pressure model; e.g., Refs. 6, 18). More directly pertinent to this study, it was recently shown that, in addition to the hypertension that is associated with reduction of uterine perfusion pressure, there is also a significant (800%) increase in plasma sFlt-1a concentration. As might be predicted, this excess of soluble receptor was associated with significant reductions in the circulating free concentrations of both PlGF and VEGF (5).
The actions of PlGF extend to the human, as it was a potent vasodilator of human myometrial resistance arteries, with IC50 values in the subnanomolar range. As in rat uterine arteries, in which NO accounted for >90% of the dilation to PlGF, NO contributed significantly to human uterine artery vasodilation, since its inhibition eliminated >40% of the vasodilation. In contrast, endothelial NO synthase inhibition was of little consequence in PlGF-induced dilation of rat mesenteric and human subcutaneous arteries, demonstrating regional differences in its mechanism of action.
The experiments with anti-VEGFR-1 and anti-VEGFR-2 antibodies and the complete absence of a vasodilatory effect of VEGF-E, at least in rat uterine vessels, confirm that dilation is mediated entirely through VEGFR-1. Although this is not surprising in view of the specificity of PlGF for VEGFR-1 signaling, it is noteworthy in that other reports have associated vasodilation and the production of NO most closely with VEGFR-2 signaling through mechanisms involving phospholipase C and Akt-1 (8, 15).
It is also interesting to note that only VEGFR-1, and not VEGFR-2, message was upregulated in gestation, pointing to an unrecognized role for this receptor and of signal transduction pathways subsequent to its activation in the adaptation of the maternal uterine circulation to pregnancy. Combined with two earlier reports that showed an increased sensitivity to VEGF dilation in rat uterine arteries from pregnant rats (12), and of its linkage to estrogen (13), the present findings support the hypothesis that enhanced vasodilation may result from an estrogen-induced increase in VEGFR-1 in the vascular wall. Changes in other signaling pathways downstream to the receptor (e.g., production of NO or other vasodilators) cannot be ruled out and needs to be evaluated in future studies.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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) and veins (10 mmHg; n = 3; 
) to placental growth factor (PlGF). Efficacy (maximal dilation) of arteries was twice that of veins (78 vs. 39%; P < 0.01). B: same data shown as sensitivity (normalized to maximal dilation obtained in each vessel to PlGF). Note 
