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Oncodevelopmental -Fetoprotein Acts as a Selective Proangiogenic Factor on Endothelial Cell from the Fetomaternal Unit

Departments of Obstetrics and Gynecology (O.D.L., J.E., J.R., N.B., F.H., M.Z.) and Biochemistry (O.D.L., K.T.P.), Justus-Liebig-University, D-35385 Giessen, Germany; and Department of Vascular Biology and Angiogenesis Research (T.K.), Tumor Biology Center at the University of Freiburg, D-79106 Freiburg, Germany

Address all correspondence and requests for reprints to: Marek Zygmunt, M.D., Ph.D., Department of Obsterics and Gynecology, University of Giessen, Klinikstr. 32, D-35385 Giessen, Germany. E-mail: marek.zygmunt{at}gyn.med.uni-giessen.de.

	Abstract

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The molecular coordination between angiogenesis and vascular remodeling is a critical step for the development of a functional vasculature in the placenta and the uterus during pregnancy. The oncodevelopmental albumin homolog -fetoprotein (AFP) is mainly synthesized in the developing fetus, and its expression has been found to be associated with highly vascularized tumors in the adult. In this study, we investigated the angiogenic activity of AFP and its possible role in the fetomaternal unit. Immunohistochemical studies revealed that the AFP-binding protein(s) is expressed in blood vessels of chorionic villi from placentae of the second and the third but not of the first trimester during pregnancy. At low concentrations, AFP directly stimulates or enhances, respectively, vascular endothelial growth factor-induced proliferation and sprout formation of endothelial cells isolated from the placenta and the uterus possibly by a MAPK-dependent pathway. Furthermore, AFP enhances blood vessel formation in a chick chorioallantoic membrane assay in vivo. Interestingly, AFP has no proliferative or migratory effects on endothelial cells isolated from the umbilical vein in the absence of vascular endothelial growth factor. These data indicate that AFP may act as a specific proangiogenic factor of endothelial cells within the fetomaternal unit during advanced stages in pregnancy.

	Introduction

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FOR AN UNDISTURBED pregnancy to occur, a close relationship must be established between the degree of embryonic development and the state of vascularization of the placental chorionic villi (1, 2). Whereas vascularization of the human embryo takes place very early in pregnancy (second week post conceptionem) and is initiated in the extraembryonic areas, subsequent adaptation of the uterine vasculature to the rising need of the fetus occurs through both vasodilation and angiogenesis. Transient, tightly regulated angiogenesis, the growth of blood vessels from preexisiting capillaries and postcapillary venules (3), is a characteristic of various developmental and physiological processes, although persistent, unregulated angiogenesis is associated with tumor growth, metastasis, or diabetic retinopathy. Angiogenesis is a morphogenetic process comprised of sequential steps, including activation of vascular endothelial cells by specific growth factors, penetration, and invasion of endothelial cell sprouts into extracellular matrix by combined migration and proliferation (4). The major angiogenic and permeability factor characterized is the vascular endothelial cell growth factor (VEGF) (5, 6), which is also the key growth factor during fetal development. It has been postulated that different organ-specific regulators of angiogenesis might be involved in this process in the fetomaternal unit, and we recently described human chorionic gonadotropin as such a factor (7).

-Fetoprotein (AFP) is known as an oncodevelopmental 70-kDa protein with a growth-regulative and immunosuppressive activity (8). AFP is secreted by the embryonic liver and yolk sac during perinatal development. After 12 wk of gestation, the yolk sac degenerates and the fetal liver becomes the main site of AFP synthesis. The protein expression level in the circulation of the fetus is very high (1–10 mg/ml), but it decreases abruptly soon after birth. By the end of second month postpartum, only trace amounts of AFP can be detected in the circulation, and it is almost completely substituted by human serum albumin (HSA) (8). At the molecular level, AFP shows strong structural homologies with HSA, i.e. 39% amino acid sequence identity and 66% homology (9). AFP from different species have a greater homology among each other than between albumin and AFP from the same species. The similarity in physical properties between AFP and HSA, and the fact that their expression occurs inversely indicates that AFP is the fetal counterpart of serum albumin (9). However, these analogies were not confirmed because many biological properties of AFP are different from those described for albumin (10). In adults, AFP is expressed by some types of malignant tumors, in which it has been associated with high microvessel density (8, 11, 12). In addition, AFP also appears to play a role in the suppression of the mother’s immune response to the developing embryo as well as in that of a patient to a developing tumor. AFP exerts various activities through binding to specific membrane binding-proteins (yet to be identified as receptors) expressed by different types of developing normal or tumor cells (13, 14). These diverse observations prompted us to investigate the proangiogenic activity of AFP in the fetomaternal unit and its possible role during pregnancy in the present study.

	Materials and Methods

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Isolation of human placental microvascular endothelial cells (HPECs)

Human term placentae were collected aseptically after vaginal delivery or cesarean sections. HPECs were isolated from chorionic villi of placentae using mechanical and enzymatic methods. Briefly, cotyledons were excised from freshly delivered term placenta and sequentially digested with collagenase, dispase, and trypsin (15). The digestion mixture was placed onto a 90-µm sieve (Sigma, Taufkirchen, Germany) to remove the interstitial connective tissue. After several washes with PBS and centrifugation of the filtrate at 1500 rpm for 10 min, the cells in the pellet were cultured in EGM-2 MV complete medium (BioWhittaker, Verviers, Belgium) containing 20% fetal calf serum (FCS) in 5% CO₂ atmosphere at 37 C for 5 d. Immunomagnetic sorting was used to further purify the endothelial cells. Briefly, the cultures were treated with trypsin-EDTA (Sigma) and washed twice with EGM-2 MV containing 5% FCS. The recovered cells were incubated with magnetic microbeads coated with antibody against endothelial cell-specific CD105 following manufacturer’s instruction (Miltenyi, Bergisch Gladbach, Germany). The isolated cells were expanded for further 5 d in the EGM-2 MV complete medium containing 20% FCS before the immunomagnetic sorting was repeated once again. Purified HPECs were further cultivated in EGM-2 MV complete medium containing 5% FCS and used for experiments at passages 3–6. HPECs were analyzed by fluorescence- activated cell sorter and found to be more than 90% CD105 positive and contained von Willebrand factor, thrombomodulin (CD141), PECAM-1 (CD31), and VE-Cadherin (CD144). Acetylated Di-low-density lipoprotein uptake was positive, whereas cytokeratin and smooth muscle cell -actin were absent. Human uterine microvascular endothelial cells (UMVECs) were purchased from BioWhittaker and maintained in the selection medium. The cells were analyzed by fluorescence-activated cell sorter and found to be positive for von Willebrand factor and CD31, whereas and -actin was absent. They were grown in EGM-2 MV medium containing 5% FCS as described earlier (7). Human umbilical cord vein endothelial cells (HUVECs) were isolated and characterized based on established methods (16). The patients were informed about the objectives and procedures of the study. The study protocol was approved by the Ethics Committees of the Medical Faculty, Justus-Liebig-University, Giessen, Germany.

Immunohistochemistry

Immunohistochemical studies were performed using LSAB-kits (Dako, Hamburg, Germany). Briefly, serial sections (5 µm) of frozen placental tissue were submerged in Tris-buffered saline (TBS) for 5 min, followed by incubation for 30 min in methanol containing 0.6% H₂O₂. After three additional 5-min washings with TBS, nonspecific binding was blocked by 20% normal porcine serum in TBS for 10 min. Sections were then incubated overnight at 4 C in a humidified chamber with a mouse monoclonal antibody (mAb) directed against a putative human AFP-binding protein (AFPBP, Chemicon, Hofheim, Germany) 1:100 diluted in 20% normal porcine serum, a mAb against PECAM (CD31) and a mAb against cytokeratin (1:100, Dako) as positive control and normal mouse Ig (1:100, Dako) as negative control. After three washings with TBS, the sections were incubated with biotinylated antimouse Ig (Dako). The sections were then extensively washed with TBS and further incubated with streptavidin-horseradish peroxidase conjugate and diaminobenzidine substrate (Dako). The sections were then counterstained with hematoxylin for 30 sec and mounted with glycerin-gelatin solution.

Proliferation assays

Endothelial cells were seeded in quadruplicates on gelatin-coated 24-well plates (HPEC: approximately 2500 cells/well, UMVECs and HUVECs: approximately 5000 cells/well), reached 50% confluence after approximately 3 d in EGM-2 MV containing 5% FCS, and they were washed and further cultured in serum-free medium for 24 h before use. AFP (ICN Biochemicals, Eschwege, Germany), whose purity was confirmed by electrospray ionization analysis and SDS-PAGE, was diluted in basal medium containing 1% FCS, and cells were incubated with 10 ng/ml, 50 ng/ml, 100 ng/ml, 150 ng/ml, 1 µg/ml, or 10 µg/ml of AFP for 2 d. Total cell number was measured with a Casy cell analyzer (Schärfe System, Reutlingen, Germany). Possible synergetic effects of AFP with VEGF were tested using VEGF (2.5 ng/ml) and AFP at various concentrations indicated above. As positive control 20% FCS was applied to the cells, whereas basal medium containing 1% FCS was used as negative control. HSA (Sigma) at different concentrations was also tested in parallel. For specificity testing, antibodies against AFP (Santa Cruz Biotechnology, Temecula, CA) or the mAb against AFPBP was added to the assay. Mouse normal Ig was used in parallel experiments as negative control. Proliferation index was calculated considering basal proliferation as 1.

Microcarrier beads sprouting in vitro angiogenesis assay

The three-dimensional in vitro angiogenesis assay was described previously by Nehls and Drenckhahn (17). Briefly, UMVECs were seeded on MEM (Life Technologies, Inc., Karlsruhe, Germany)-washed microcarriers (Sigma) and allowed to grow to confluence (approximately 30 cells/microcarrier) for 24 h in endothelial cell growth medium (EGM-2 MV, Bio Whittaker). Fibrinogen (Sigma) was dissolved to 3 mg/ml in PBS (PBS Dulbecco, Life Technologies, Inc.) and filled into a 12-well plate (0.6 ml/well) together with the cell-covered microcarriers. AFP at 10–150 ng/ml final concentrations were applied to the wells before fibrin polymerization was initiated by thrombin (0.65 U/ml, Sigma). After 3 d of incubation, the number of sprouts was quantified in eight separate microscope fields per well. All assays were repeated at least three times and four wells were used for each treatment. Sprouting index was calculated considering basal sprouting as 1.

Spheroid sprouting in vitro angiogenesis assay

The spheroid sprouting in vitro angiogenesis assay using HUVECs was performed as described earlier (18). The angiogenic activity of AFP was tested at concentrations of 10 ng/ml, 50 ng/ml, 100 ng/ml, and 150 ng/ml alone or together with VEGF (2.5 ng/ml). Cumulative sprout length was calculated as described previously (18).

Chick chorioallantoic membrane (CAM) in vitro angiogenesis assay

The CAM assay was performed similarly to that described earlier (19) to determine the angiogenic effect of AFP in vivo. Fertilized chicken eggs were prepared by creating a window in the shell on d 3 of incubation at 37 C in a humidified incubator. On d 10, methylcellulose disks saturated with basic fibroblast growth factor (bFGF) (50 ng/pellet) as a positive control, AFP (1 or 2 µg/pellet) in the absence or presence of anti-AFP antibodies (0.2 µg/pellet), anti-AFP alone (0.2 µg/ml·pellet), HSA (2 µg/pellet), and H₂O as a negative control were laid onto the chorioallantoic membrane in separate eggs. Blood vessel density around (magnification: x10) or within the disks (magnification x16) was photographed and evaluated using a stereomicroscope (Leica, Wetzlar, Germany) on d 13. Five CAMs were performed for each test group and the experiments were repeated at least three times. Relative microvessel density was determined with software program Quantity one (Bio-Rad Laboratories, Hercules, CA).

MAPK assay

The MAPK assay approximated the procedure of the manufacturer (New England Biolabs, Frankfurt, Germany) to test for activation of this signal transduction pathway, whereby HPEC monolayers in 24-well cluster plates, after 48 h at 37 C in 5% CO₂ in serum-free EGM, were incubated with various concentrations of AFP or positive control for a stimulation period of 20 min. The resultant samples were processed using SDS-PAGE and Western blotting (Bio-Rad Laboratories) to identify activated MAPK-positive bands by a monoclonal antiactive phospho-p42/p44 antibody (New England Biolabs) and visualized employing a horseradish peroxidase-conjugated rabbit antimouse antibody (Dako) with enhanced chemiluminescence-signal amplification (Amersham Pharmacia Biotech, Freiburg, Germany).

Statistics

Statistic significance (two-tailed P value) of the experimental results were evaluated using a software program (GraphPad, San Diego, CA).

	Results

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Expression of putative AFPBP during early pregnancy

Using immunohistochemical techniques, we could demonstrate the localization of the putative AFPBP in the blood vessel wall of the chorionic villi in placenta tissue of the second trimester (23 wk, Fig. 1F) and third trimester (39 wk, Fig. 1I), whereas the AFPBP was not detectable in the chorionic villi from placentae of the first trimester (9 wk, Fig. 1C). As positive control, chorionic villi vessels were stained with anti-PECAM (CD31) (Fig 1, B, E, and H) and as negative control mouse Ig was applied in parallel (Fig. 1, A, D, and G).

View larger version (139K): [in this window] [in a new window]   FIG. 1. Localization of the putative AFPBP (arrows) in placental villi. Cross-sections of placental tissue from the first (A–C), second (D–F), and third (G–I) trimesters were stained with normal mouse Ig, mAb against PECAM (CD31), or mAb against AFPBP. The AFPBP is expressed in blood vessels of chorionic villi from placentae of the second and the third but not of the first trimester during pregnancy. Original magnification, x200.

AFP-stimulated proliferation of HPECs and UMVECs in a dose-dependent manner, showing the highest response at a concentration of 100 ng/ml for both endothelial cell types (Fig. 2, data for UMVECs not shown). As shown in lower panel, AFP at 100 ng/ml induced phosphorylation of MAPK (p42/p44), which coincided with the optimal concentration stimulating HPEC proliferation. Both anti-AFP and anti-AFPBP antibodies could reverse AFP-induced proliferation (Fig. 3), whereas normal mouse Ig had no effect (data not shown). Higher concentrations (1 or 10 µg/ml) of AFP or HSA within the concentration range of 5–200 ng/ml had no stimulatory effect on the cells (data not shown).

View larger version (18K): [in this window] [in a new window]   FIG. 2. Induction of fetomaternal endothelial cell proliferation by AFP (upper panel with error bars indicating SDs) and activation of MAPK (p42/p44) in HPECs by 100 ng/ml AFP (lower panel), which coincided with the optimal concentration stimulating HPEC proliferation. *, P < 0.01.

View larger version (13K): [in this window] [in a new window]   FIG. 3. Specificity of AFP-induced HPEC proliferation. Antibodies against AFP or AFPBP were coincubated with the cells during the proliferation assay. Both anti-AFP and anti-AFPBP antibodies could reverse AFP-induced proliferation, whereas normal mouse Ig had no effect. Error bars indicate SDs. *, P < 0.01; **, P < 0.05; ns, not significant.

VEGF-induced proliferation of both HPECs and UMVECs was enhanced by AFP in a dose-dependent manner with optimal concentration at 100 ng/ml (Fig. 4A). This enhancing effect of AFP was blocked by specific antibodies against AFP or the putative AFPBP (Fig. 4B), in which antibodies alone had no effect on VEGF-induced proliferation (data not shown). Whereas AFP alone at concentrations 10–150 ng/ml had no stimulatory effect on HUVEC proliferation, AFP enhanced VEGF-induced HUVEC proliferation (data not shown).

View larger version (26K): [in this window] [in a new window]   FIG. 4. A, AFP potentiation of VEGF-induced endothelial cell proliferation of HPECs (filled bars) and UMVECs (empty bars). B, This enhancement by AFP was blocked by specific antibodies against AFP or AFPBP. Error bars indicate SDs. *, P < 0.01; **, P < 0.05.

Incubation of UMVECs with AFP at concentrations of 5–150 ng/ml resulted in a significant dose-dependent increase of endothelial sprout formation (Fig. 5). As for proliferation, the highest response was observed at a concentration of 100 ng/ml. Furthermore, AFP was also found to significantly increase VEGF-induced HUVEC sprouting in the spheroid in vitro angiogenesis assay, whereas AFP alone had no angiogenic effect on HUVECs (Fig. 6). This potentiation of VEGF was again blocked on addition of specific antibody against AFP at 1 µg/ml or antibody against the putative AFPBP at 3 µg/ml (Fig. 6).

View larger version (13K): [in this window] [in a new window]   FIG. 5. Microcarrier beads in vitro angiogenesis assay. Induction of capillary sprout formation by AFP in microcarrier bead in vitro angiogenesis using UMVECs. Error bars indicate SDs. *, P < 0.01, compared with unstimulated control.

View larger version (47K): [in this window] [in a new window]   FIG. 6. Spheroid in vitro angiogenesis assay using HUVECs. AFP potentiated VEGF-induced sprouting (A–C) and specificity of this effect was confirmed using specific antibodies to AFP (D) or AFPBP (E). Error bars indicate SDs. *, P < 0.01; **, P < 0.05.

AFP caused undulating vessel formation increasing vascular density and tortuosity within the methylcellulose membrane disk at a concentration of 1 µg/pellet, compared with the regular straight vessel formation in the adjacent area of the pellet or to the regular straight vessel formation within the disk containing 2 µg/pellet HSA (data not shown). Methylcellulose disk containing 50 ng/pellet bFGF was used as positive control (Fig. 7A), whereas disks containing only H₂O (Fig. 7B), 2 µg/pellet HSA (Fig. 7C) or 0.2 µg/pellet anti-AFP antibodies (Fig. 7D) were used as negative controls. AFP at 2 µg/pellet induced strong angiogenesis (Fig. 7E), which could be attenuated by simultaneous addition of 0.2 µg/pellet anti-AFP antibodies (Fig. 7F). Relative microvessel density surrounding the disk is also presented (Fig. 7B).

View larger version (98K): [in this window] [in a new window]   FIG. 7. AFP-induced in vivo angiogenesis in CAM assay. Whereas a methylcellulose disk containing 50 ng/pellet bFGF was used as a positive control (A) and disk containing only H2O (B), 2 µg/pellet HSA (C) or 0.2 µg/pellet anti-AFP antibodies (D) was used as negative control. Disk containing 2 µg/pellet AFP induced strong angiogenesis (E), which could be attenuated by simultaneous addition of 0.2 µg/pellet anti-AFP antibodies (F). Arrows indicate areas of capillary budding and new blood vessel formation. Relative microvessel density surrounding the disk is presented in G with error bars indicating SDs. Original magnification, x10. *, P < 0.01.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

As a large serum glycoprotein belonging to the class of oncodevelopmental proteins, the expresssion of AFP can be detected very early in the pregnancy and its concentration in maternal circulation correlates positively with the vascular adaptation during pregnancy. AFP has attracted considerable attention because the change in its serum level during pregnancy is associated with the development of numerous embryonic disorders, and the increase in its content in the plasma of adults correlates with the appearance of, for example, different types of tumors presented with high microvessel density (8). However, little is known about the potential role of AFP as an angioregulatory factor within the fetomaternal unit during pregnancy.

The fact that expression of a putative AFPBP was found to be restricted to the chorionic villi vessel wall indicates that AFP in the fetal circulation can be bound to the vascular endothelium and subsequent cellular activities of AFP are initiated. The lack of immunohistochemically detectable expression of AFPBP in the chorionic villi of placenta from the first trimester of pregnancy is likely due to its low abundance, in which further investigation at mRNA expression level is required. However, to date genetic information of the AFPBP is not yet clarified. Molecular cloning of a putative 67-kDa AFPBP recognized by the anti-AFPBP mAb is currently being carried out in our laboratory.

Although AFP stimulated proliferation of different carcinoma cell types has been reported (11, 12), in the present work, we demonstrate that AFP induces proliferation of two endothelial cell types isolated from the fetomaternal unit. It is noteworthy that in our assays placental endothelial cells were more sensitive than uterine endothelial cells on AFP stimulation, which is in accordance with the different growth rates of placenta and uterus during pregnancy. However, the difference in the expression levels of AFPBP and other AFP-binding sites from the two cell types has yet to be investigated in detail. For the first time, we showed that VEGF-induced proliferation of different endothelial cell types could be significantly enhanced by low concentrations of AFP. In support of our observation, it was reported that AFP could augment the mitogenic activity of epidermal growth factor and may modulate the growth factor-mediated cell proliferation of porcine granulosa cells (20) or mammary carcinoma cells in vitro (21). In the spheroid in vitro angiogenesis assay, AFP was shown to potentiate VEGF-induced sprouting in a similar manner. The specificity of AFP-related angiogenic effects was confirmed when antibodies against AFP or AFPBP abolished AFP-induced synergism with VEGF. Notably, inhibitory effects caused by the mAb against the AFPBP were less pronounced than that of antibodies against AFP in both proliferation as well as in spheroid in vitro angiogenesis assays. Conceivably, the mAb might not have blocked other putative cellular AFP-binding sites (22). AFP alone did not induce sprout formation of HUVECs, suggesting that the proangiogenic activity of AFP might be restricted to certain cell types, similarly to the organ-specific regulation of angiogenesis, which was reported recently (7, 23).

AFP can apparently regulate growth of different types of tumors as well as normal cells by several mechanisms, which include apoptotic regulation and cytoplasmic signaling modulation. Because AFP was reported to protect HL-60 cells and HepG2 cells against apoptosis caused by various factors (24, 25), it is tempting to speculate that fine-tuning of AFP concentrations could result in switching its mode of action, also depending on the relative concentration of exogenous or endogenous cytokines, hormones, and growth factors. Whereas experiments in our laboratory indicated that AFP alone could induce MAPK (p42/p44) phosphorylation, it still remains to be explored to what extent the mitogenic activity of AFP per se, the synergetic effect with endogenous growth factors such as VEGF, or the suppression of apoptosis have contributed to the overall increased angiogenic activities. Taken together, the development of a functioning fetomaternal vascular network requires a remarkable degree of coordination between different vascular endothelial cell-specific growth factors and various other regulators of angiogenesis, and we have defined new functions of AFP in this regard.

	Acknowledgments

 
We thank Bettina Gill and Anna Hoffmann for skillful assistance in immunohistochemistry and Carolin Szardening-Kirchner and Susanne Vogelsberger for help in MAPK experiments.

	Footnotes

 
Results from this work were presented at the Annual Meeting of the Society of Gynecologic Investigation, Los Angeles, CA, 2002.

This work was supported by a grant from the DFG-German Research Council (Zy 19/3-1, Bonn, Germany).

Abbreviations: AFP, -Fetoprotein; AFPBP, AFP-binding protein; bFGF, basic fibroblast growth factor; CAM, chick chorioallantoic membrane; FCS, fetal calf serum; HPEC, human placental microvascular endothelial cell; HSA, human serum albumin; HUVEC, human umbilical cord vein endothelial cell; mAb, monoclonal antibody; TBS, Tris-buffered saline; UMVEC, uterine microvascular endothelial cell; VEGF, vascular endothelial cell growth factor.

Received October 6, 2003.

Accepted December 2, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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