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Departments of Obstetrics and Gynecology, Perinatal Research Laboratories (J.-Y.C., Y.S., R.R.M., J.Z.), Pediatrics (R.R.M.), and Animal Sciences (R.R.M.), University of Wisconsin, Madison, Wisconsin 53715; and Department of Obstetrics and Gynecology (Y.W.), Louisiana State University Medical Center, Shreveport, Louisiana 71130
Address all correspondence and requests for reprints to: Jing Zheng, Ph.D., Department of Obstetrics and Gynecology, University of Wisconsin-Madison, Perinatal Research Laboratories, 7E Meriter Hospital, 202 South Park Street, Madison, Wisconsin 53715. E-mail: jzheng{at}wisc.edu.
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Abstract |
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Introduction |
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Vascular endothelial growth factor (VEGF) is a critical regulator for vascular growth and function. Targeted disruption of a single allele of VEGF gene in mouse causes impaired cardiovascular development, leading to embryonic death (4, 5). Human VEGF has at least six isoforms of 121, 145, 165, 183, 189, and 206 amino acids. These VEGF isoforms differ in their bioavailability and biological activities (6). For example, VEGF121 does not bind heparin and is freely released from producing cells. VEGF165 is partially soluble with a significant fraction remaining bound to the cell surface and the extracellular matrix. Of these VEGF isoforms, VEGF121, 165, and 189 are widely expressed, whereas VEGF145, 183, and 206 expression is found in a few tissues (7, 8, 9). Moreover, VEGF165 is more potent than VEGF121 in stimulating endothelial cell proliferation (10). Thus, the expression of multiple VEGF isoforms in the same tissue and different biochemical properties of individual VEGF isoforms imply that they have distinct roles in regulating vascular development and function. This is supported by the fact that mouse embryos, which express only VEGF120, survive to term although they exhibit severe vascular defects (11). Additionally, expression of each VEGF isoform varies temporally and spatially in association with vascular formation, differentiation, and function in the developing mouse tissues (12, 13).
Endocrine gland-derived VEGF (EG-VEGF) is a newly identified angiogenic factor that is able to stimulate proliferation, migration, and fenestration in capillary endothelial cells selectively derived from endocrine glands (14). Expression of human EG-VEGF mRNA is restricted to the steroidogenic glands including placenta and is induced by hypoxia (14). However, to date, the data are lacking to compare EG-VEGF expression in human placentas from normal and preeclamptic (PE) pregnancies.
VEGF actions are mediated via binding to its receptors, which include VEGF receptor-1 (VEGFR-1/Flt1) and -2 (VEGFR-2/Flk1/KDR). VEGFR-2 is the major signal transducer of VEGF in endothelial cells and mediates most known VEGF bioactivities (i.e. cell proliferation, migration, and permeability) (15). In contrast, VEGFR-1 has been implicated in the inhibition of VEGF-dependent endothelial function (16, 17). Both these receptors, however, are critical for regulating vasculogenesis and angiogenesis, because null mutation of either of these receptors in the mouse results in abnormal vascular formation and development, leading to impaired vascular function and embryonic death (18, 19).
Neuropilin-1 (NP-1) and -2 (NP-2) are two VEGF receptors that bind to VEGF165, but not VEGF121 (20, 21, 22). Unlike VEGFR-1 and -2, neither NP-1 nor NP-2 has a tyrosine kinase domain and therefore cannot induce cellular responses by themselves alone. However, both NP-1 and NP-2 are required for normal vascular development because disruption of NP-1 or NP-2 gene in mouse results in abnormal vascular formation and development, leading to embryonic death (23). Moreover, a ternary complex of VEGF165, VEGFR-2, and NP-1 potentiates VEGF165 binding to VEGFR-2 and therefore enhances VEGF-induced angiogenesis (20, 24).
The data on VEGF expression in normal and PE pregnancies are controversial. The majority of these studies compared serum VEGF levels in the maternal circulation, reporting either decreased or increased levels in normal vs. PE pregnancy, largely depending on methodologies used (25). Similarly, placental VEGF mRNA levels have also been reported to be either decreased (26), increased (27), or unchanged (28) in normal vs. PE pregnancy. To date, little is known about protein expression of VEGF and VEGF receptors in placentas from normal and PE pregnancies. Thus, to determine whether expression of individual VEGF isoforms and VEGF receptors in placentas is associated with preeclampsia, in this study we have examined mRNA and protein expression of VEGF and its four receptors (VEGFR-1, VEGFR-2, NP-1, and NP-2) as well as EG-VEGF mRNA expression in placental tissues from normal and PE pregnancies.
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Materials and Methods |
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Placentas were obtained immediately after delivery from women with normal and PE pregnancies. PE was defined according to standard criteria: onset of hypertension during late pregnancy with systolic and diastolic blood pressure more than 140/90 mm Hg on at least two occasions and urinary protein more than 2+ on dipstick or more than 0.3 g/24 h. Collection of placentas was approved by the Institutional Review Board, University of Wisconsin-Madison, and followed the recommended guidelines for using human subjects. Placental villi from beneath the chorionic and basal plates were quickly dissected (
10 g each), snap-frozen in liquid nitrogen, and stored at –80 C. Additional placental tissues were fixed overnight at 4 C in 4% paraformaldehyde in 10 mM PBS for immunohistochemical analysis.
Total RNA purification
Total RNA was extracted using an RNeasy Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The placental tissue (800 mg/placenta) was homogenized using a rotor-stator homogenizer (PawerGen700, Fisher Scientific, Hampton, NH) until the samples is uniformly homogeneous. The concentration of RNA was measured using a spectrophotometer (DU7400, Beckman, Fullerton, CA), and RNA quality was confirmed on agarose gels.
Real-time PCR analysis
A total RNA sample (2 µg/sample), 20 pmol oligo deoxythymidine primer, 2 µl deoxynucleotide triphosphate mix (5 mM each), and 1 µl reverse transcriptase (Omniscript Reverse Transcriptase, Qiagen) were used in a 20-µl scale to generate cDNA.
The primers (Table 1
) were designed for human VEGF, EG-VEGF, VEGFR-1, VEGFR-2, NP-1, and NP-2 using Primer Express 2.0 (Applied Biosystems, Foster City, CA). The primer set for VEGF was designed to detect all VEGF isoforms. The specificity of these primers was first analyzed by RT-PCR, and each PCR product was confirmed by sequencing.
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Semiquantitative RT-PCR for VEGF isoforms
Because real-time PCR cannot detect individual VEGF isoforms, mRNA expression of three VEGF isoforms was quantified by RT-PCR, using a specific Human VEGF 3 Isoforms and ß-Actin Genes Dual-PCR kit (Maxim Biotech, San Francisco, CA) according to the manufacturer’s instructions. The estimated sizes of RT-PCR products for ß-actin, VEGF189, VEGF165, and VEGF121 were 474, 306, 234, and 104 bp, respectively. Total RNA (2 µg/sample) was used and data were normalized to ß-actin.
Western blot analysis
Placental tissues were homogenized and lysed by sonication in buffer [50 mM HEPES, 0.1 M NaCl, 10 mM EDTA, 4 mM sodium pyrophosphate, 10 mM sodium fluoride, 2 mM sodium orthovanadate (pH 7.5), 1 mM phenylmethylsulfonylfluoride, 1% Triton X-100, 5 µg/ml leupeptin, 5 µg/ml aprotinin]. After centrifugation, protein concentrations of the supernatant were determined with BSA (fraction V; Sigma, St. Louis, MO) as standards.
Because the weak positive signal and intensive background were detected in the preliminary study, placental VEGF, NP-1, and NP-2 proteins were first enriched using immunoprecipitation, followed by Western blot analysis. Briefly, protein samples (2 mg for VEGF and 1 mg for NP-1 and NP-2) were precleaned by mixing the samples with 0.25 µg of rabbit control IgG (Vector Laboratories, Burlingame, CA) and 10 µl protein A-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at 4 C with end-over-end rotation. The supernatants were obtained and incubated with the polyclonal rabbit antibody (5 µg) against human VEGF (protein A-agarose conjugated), NP-1, or NP-2 (Santa Cruz Biotechnology) at 4 C for 2 h. For NP-1 and NP-2, the mixture of proteins and antibodies was subsequently incubated with 30 µl protein A- agarose at 4 C overnight. The immunoprecipitates were collected by centrifugation.
Western blot analysis was conducted as described (30, 31). The protein samples (for VEGFR-1 and VEGFR-2) or immunoprecipitates (for VEGF, NP-1, and NP-2) were heat-denatured (95 C, 5 min) in Laemmli buffer, separated on precasted SDS-PAGE gels (4–15% gradient, Bio-Rad, Hercules, CA), and electrically transferred to polyvinylidine difluoride membranes. The membranes were immunoblotted with a mouse monoclonal antibody against human VEGF (1:100), VEGFR-2 (1:500), NP-1 (1:100), or NP-2 (1:200) or with a rabbit antihuman VEGFR-1 antibody (1:500). All antibodies used were purchased from Santa Cruz Biotechnology. Proteins were visualized using enhanced chemiluminescence reagents, followed by exposure to chemiluminescence films. The immunoreactive signals were analyzed by densitometry. The membranes used for detecting VEGFR-1 and VEGFR-2 were reprobed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Research Diagnostics, Flanders, NJ). Recombinant human VEGF121 and 165 (R&D Systems, Inc., Minneapolis, MN) were used as positive controls for VEGF. Human umbilical vein endothelial cells (HUVEC; Cambrex, East Rutherford, NJ) were used as positive controls for the VEGF receptors.
Immunolocalization
Immunolocalization of VEGF, VEGFR-1, VEGFR-2, NP-1, and NP-2 in the placental tissues from normal (n = 5) and PE (n = 5) pregnancies was visualized by indirect detection via the avidin:biotinylated-peroxidase complex method with 3-amino-9-ethylcarbazole as the chromogen (30, 31) (Vector Laboratories). Briefly, tissue sections were heated in sodium citrate buffer (0.1 M; pH 6.0) in a microwave for 5 min. Endogenous peroxidase in tissue sections was quenched with 3% H2O2 in methanol for 15 min. The sections were stained briefly with Harris’ hematoxylin. After washing with PBS containing 0.3% Triton X-100, nonspecific binding was blocked with 1% normal goat serum for 20 min. The tissue sections were incubated with a rabbit anti-VEGF as we described (31), VEGFR-1, VEGFR-2, NP-1, or NP-2 antibody (4 µg/ml; Santa Cruz Biotechnology) for 1 h. Controls consisted of replacing the primary antibody with preimmune rabbit IgG (Vector Laboratories) at the same concentration as the primary antibody.
Statistical analysis
The data were analyzed by the Student’s t test. Results were considered significant at P < 0.05.
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Results |
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). One band for each mRNA studied was observed at the estimated size as shown in Table 1
, and these PCR products were confirmed by sequencing, indicating the specificity of each primer set. The mRNA levels of total VEGF, EG-VEGF, and the four VEGF receptors quantified using real-time PCR are shown in Fig. 2. The mRNA levels of total VEGF and VEGFR-1 were increased 2.8- and 2.7-fold (P < 0.05), respectively, in PE vs. normal placentas. No significant difference in mRNA levels of EG-VEGF and the other three VEGF receptors was observed between PE and normal placentas. Among these genes studied, VEGFR-1 was the most abundant (1/8 of ß-actin), followed by VEGFR-2 (1/19), NP-1 (1/66), VEGF (1/186), EG-VEGF (1/473), and NP-2 (1/631) in normal placentas.
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). The overall mRNA levels of these three VEGF isoforms were increased (P < 0.05) 1.8-fold in PE vs. normal placentas. Compared with normal pregnancy, the placental mRNA levels of three VEGF isoforms in PE were elevated (P < 0.05) 1.8-, 1.9-, and 1.7-fold, respectively, for VEGF189, 165, and 121.
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). The VEGF antibody detected two major bands at approximately 20 and 25 kDa (Fig. 4A). The former is corresponding to the molecular mass of recombinant human VEGF165, whereas the latter is similar to the reported molecular mass of VEGF189 (32). There was no significant difference in protein expression of both VEGF isoforms between normal and PE placentas (Fig. 4B). For VEGFR-1, VEGFR-2, NP-1, and NP-2, one major band, corresponding to its respective control, was also identified at approximately 180, 235, 130, and 130 kDa, respectively (Fig. 4A). VEGFR-1, but not the other three receptors (VEGFR-2, NP-1, and NP-2), protein levels were increased (P < 0.05) 1.7-fold in PE vs. normal placentas (Fig. 4B).
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). No apparent difference in the staining intensity for all five antigens examined was noted between PE and normal placentas. No staining was observed in the IgG controls (Fig. 5).
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Discussion |
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We demonstrate that mRNA of total VEGF and all three VEGF isoforms (VEGF121, 165, and 189) tested increase in PE placentas as compared with normal placentas. We also note that the ratio of the individual isoforms in the total three VEGF isoforms combined is similar between PE and normal placentas (40, 34, and 26% of the total three VEGF isoforms for VEGF121, 165, and 189, respectively). These data suggest that the increase in total VEGF mRNA levels in PE placentas is attributed to proportional elevations in each individual VEGF isoform. In the current study, two isoforms of VEGF proteins (VEGF165 and 189) were detected in human placentas, indicating that they are the two major VEGF isoforms expressed in human placentas. We further provided evidence showing that protein levels of VEGF165 and 189 did not increase as their mRNA expression did in PE placentas. It is not clear from these data alone whether the lack of increase in protein levels of these VEGF isoforms is due to decreased translation or increased protein degradation. However, PE pregnancy is associated with increases in VEGF levels in maternal circulation and VEGF mRNA expression in placentas (27, 35, 36). Moreover, in parallel with these increases, elevations in soluble VEGFR-1 (sVEGFR-1) protein, which has a high affinity to VEGF, in the amniotic fluid and maternal circulation (25, 37, 38) and in its mRNA expressions in placentas (25) have also been reported. Therefore, it is more likely that translation of these VEGF isoforms in PE placentas may keep the same pace as their increased mRNA levels. After being released from its cells of origin or from extracellular matrix by plasmin cleavage, these VEGF could speculatively bind to sVEGFR-1 and be rapidly transported into the circulation, so contributing to the higher VEGF levels in the maternal circulation and maintaining local VEGF protein levels in PE placentas at the same levels as normal placentas.
Our findings that VEGFR-1 mRNA levels are increased in PE placentas are consistent with a previous report (27). VEGFR-1 mRNA detected herein was a full-length VEGFR-1 using the specific primers, which cannot detect sVEGFR-1. More importantly, we demonstrated, for the first time, an elevated VEGFR-1 protein expression in PE placental tissues. It is noteworthy that only VEGFR-1, of the five proteins studied, exhibits an increase in PE vs. normal placentas. These observations indicate that this increase in VEGFR-1 protein levels is specific in PE placentas and is not due to any potential change in placental tissue or cell architecture. Moreover, we also found in the current study that in addition to endothelial cells, VEGFR-1 is also localized in trophoblast cells, confirming the previous studies (33, 39, 40, 41, 42). Because the majority of placental cellular mass consists of trophoblast cells, the increased VEGFR-1 expression observed could be attributed largely to trophoblast cells. An important question that follows is whether and how this increased VEGFR-1 expression in trophoblast affects placental functions associated with preeclampsia. Because VEGF is expressed in placental trophoblast cells as shown in the current study and by other investigators (33, 39, 40, 41, 42), VEGF is able to affect multiple trophoblast functions (i.e. invasion, differentiation, proliferation, and releasing vasorelaxants) via an autocrine loop (33, 39, 40, 41, 42). Moreover, abnormal trophoblast invasion and differentiation are believed to be associated with preeclampsia (3, 42). Thus, it is plausible that the increased VEGFR-1 levels observed may substantially alter normal trophoblast function in PE placentas.
In addition to affecting their own functions, the trophoblast could also influence adjacent placental endothelial cells by producing and releasing vasoactive factors such as sVEGFR-1, nitric oxide, endothelin, and prostacyclin, possibly leading to endothelial dysfunction (3, 33, 42, 43). Endothelial dysfunction has been postulated to be responsible for initiating and/or associating with many of the changes in preeclampsia, which is characterized by the increased endothelial-mediated vasoconstriction, increased vascular permeability, and increased endothelial-mediated platelet aggregation leading to maternal hypertension, proteinuria, and thrombocytopenia (3). Moreover, although the increased VEGFR-1 expression observed could be attributed largely to trophoblast as discussed above, it is possible that a portion of this increase is derived from endothelial cells because VEGFR-1 is also expressed in endothelial cells. In this case, the increased VEGFR-1 in endothelial cells of PE placentas may interfere with VEGF-mediated actions on these endothelial cells. This concept is supported by the observations that VEGF, acting through VEGFR-1, is able to inhibit VEGFR-2-mediated endothelial proliferation and migration (16, 17), and to reduce endothelial-dependent uterine blood vessel relaxation in pregnancy (34).
Although the role of EG-VEGF in placental endothelial cells remained unsolved, EG-VEGF is expressed in placentas and has similar biological activity to VEGF in endocrine glands (14). Our observation that EG-VEGF mRNA is expressed in human placentas is in agreement with a previous report (14). We also have, for the first time, provided evidence showing that there is no difference in EG-VEGF mRNA levels between normal and PE placentas. Because of the lack of a commercially available EG-VEGF antibody, we were unable, at this time, to measure EG-VEGF protein expression. However, because EG-VEGF regulates angiogenesis and endothelial permeability, both of which are key endothelial functions associated with preeclampsia, future studies are needed to investigate EG-VEGF protein expression as well as expression of its receptors in placentas.
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Acknowledgments |
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Footnotes |
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Abbreviations: EG-VEGF, Endocrine gland-derived VEGF; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HUVEC, human umbilical vein endothelial cells; NP, neuropilin; PE, preeclamptic; sVEGFR-1, soluble VEGFR-1; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.
Received September 10, 2003.
Accepted January 27, 2004.
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