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Department of Obstetrics and Gynecology (F.H., O.D.L., J.H., U.L., M.Z.), Justus-Liebig-University of Giessen, D-35385 Giessen, Germany; Department of Biochemistry (O.D.L., K.T.P.), Medical Faculty, Justus-Liebig-University of Giessen, D-35385 Giessen, Germany; and Medical Research Council Group in Fetal and Neonatal Health and Development (V.K.M.H.), Child Health Research Institute, University of Western Ontario, London N6A 4V2, Canada
Address all correspondence and requests for reprints to: Marek Zygmunt, M.D., Ph.D., Department of Obstetrics and Gynecology, University of Giessen, Klinikstr. 32, D-35385 Giessen, Germany. E-mail: marek.t.zygmunt{at}gyn.med.uni-giessen.de.
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Introduction |
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Another important fetal growth factor is IGF-II, a polypeptide of 6.7 kDa, which shares 70% amino acid sequence identity with IGF-I and 50% with insulin. The biological activities of these homologous proteins are mediated through respective cognate receptors: the IGF-I receptor (IGF-IR), the insulin receptor (IR) and the IGF-II/mannose-6 phosphate receptor (IGF-II/M6PR) (3). Although IGF-II biological actions are mediated mainly through IGF-IR, the IGF-II/M6PR participates in not only the clearance of extracellular IGF-II but also intracellular lysosomal enzyme transport. IGF-II is highly abundant in trophoblasts and fetal endothelial cells (4) and IGF-II/M6PR mediates IGF-II signals in migration of trophoblastic cell lines (5). The importance of IGF-II in embryonic and placental growth has been illustrated in experiments in which mice carrying a disrupted Igf2 gene had a growth-retarded phenotype, with a very early onset in embryonic development (6). Disruption of the Igf2/Mpr gene is generally lethal and leads to fetal overgrowth, indicating that the expression of Igf2/Mpr is essential for late embryonic development and growth regulation (7). Furthermore, increased expression of IGF-II is often associated with the growth and metastasis of tumors such as Wilms’ tumor (8), rhabdomyosarcoma (9), or neuroblastoma (10) as well as with the hypervascularized hepatocellular tumor (11).
The involvement of IGF-II and IGF-II/M6PR in the process of angiogenesis has been postulated in an earlier study. Volpert et al. (12) reported that the IGF-II/M6PR is necessary for the proliferin-induced angiogenesis in vitro and in vivo. Kim et al. (13) demonstrated that IGF-II secreted from human hepatocellular carcinoma has angiogenic activity both in vitro and in vivo. Moreover, IGF-II has been shown to induce the expression of hypoxia inducible factor-1
, which activates the transcription of VEGF to promote the adaptation to hypoxia, which may induce angiogenesis (14). In the present study, we identified IGF-II as an angiogenic factor in uterine vascular adaptation during pregnancy.
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Materials and Methods |
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Human uterine microvascular endothelial cells (UMVECs) were purchased from Bio Whittaker (Verviers, Belgium) and maintained in the selection medium as indicated by the provider (15). The cells were characterized by von Willebrand’s factor and CD 31-positive and -actin negative staining and grown in EGM-2 MV medium (BioWhittaker) containing 5% fetal calf serum (FCS). All cell lines were cultured in 5% CO2 atmosphere at 37 C.
In situ hybridization
The expression of IGF-II and IGF-IIR mRNAs in human uterine vessels was characterized using the in situ hybridization technique, which was performed as described previously (4, 16). Briefly, 5-µm-thick uterine tissue sections obtained from peripartal hysterectomy specimens were deparaffinized, rehydrated, and after prehybridization hybridized with 35S-labeled antisense cRNA or sense RNA probes at 55 C overnight, followed by washing at maximum stringency with 0.1 x standard saline citrate at 65 C for 30 min. 35S-radiolabeled antisense and sense RNA probes were generated from the human IGF-II cDNA or IGF-II/M6PR cDNA using appropriate RNA polymerases. Sections were dehydrated, coated with photoemulsion (NTB-3 nuclear track emulsion, Eastman Kodak, Rochester, NY) and exposed at 4 C for 2 wk. The photoemulsion was developed with a D-19 developer (Kodak), fixed, stained with Harris’s hematoxylin and eosin, and mounted with Permount (Fisher Scientific, Fair Lawn, NJ). The specificity of the in situ hybridization was demonstrated by the lack of specific hybridization signal when adjacent tissue sections were subjected to an identical in situ hybridization procedure with radiolabeled sense cRNA probes.
125I-IGF-II cross-linking
For cross-linking, UMVECs were subplated at a cell density of 1.0 x 106 cells/well in six-well multiwell dishes in RPMI 1640 medium containing 10% FCS. HTR-8/SV neo cells were used as a control. The cells were grown to 80–90% confluence (48–72 h), and the medium was changed to serum-free RPMI 1640 containing 0–100 ng/ml IGF-II for another 12 h. The cells were washed twice with 0.1 M HEPES buffer containing BSA 10 mg/ml (pH 8.0) and incubated with 125I-IGF-II (1 x 105 cpm/dish) in the same HEPES buffer at 4 C for 16–18 h. The incubation solution was then aspirated, and the cultures were washed three times with cold HEPES buffer (pH 7.4) without BSA to remove the unbound radiolabel. The cell surface-associated 125I-IGF-II was cross-linked with 0.01 mM disuccinimidyl suberate for 30 min at 22 C. The reaction was then stopped by adding 1 ml of 10 mM Tris HCl. The cells were scraped from the bottom of the dish and solubilized in 2% sodium dodecyl sulfate containing 12.5 mM Tris, 0.002% bromphenol blue, 8% glycerol, and 100 mM dithiothreitol. Samples were boiled for 5 min before electrophoresis on a 3–14% polyacrylamide linear gradient gel. After separation the gel was fixed, dried, and exposed to x-ray film (XAR, Kodak) (17).
In vitro angiogenesis assay
The three-dimensional in vitro angiogenesis assay was described previously (18). UMVECs were seeded in MEM-washed plastic microcarriers (MCs; Sigma, Deisenhofen, Germany) and allowed to grow to confluence (approximately 30 cells per MC) for 24 h in endothelial cell growth medium. Fibrinogen (Sigma) was dissolved in PBS (PBS Dulbecco, pH 7.4, 3 mg/ml) and filled into a 12-well plate (0.6 ml/well). Test substances (final concentrations) [r-IGF-II (1–100 ng/ml, ICN, Eschwege, Germany), VEGF (10–100 ng/ml, R&D, Wiesbaden, Germany), the specific inhibitor of IGF-II/M6PR, ß galactosidase (19) (1–2 mU/ml), dbcAMP (0.1–1 µmol/liter), forskolin (1–10 µmol/liter), phorbol,12,13-dibutyrate (PDBu; 1–10 nmol/liter, Calbiochem, La Jolla, CA), GRGDTP peptide (0–10 µg/ml), and pertussis toxin (0.1–100 ng/ml) (all purchased from Sigma)], protein kinase A (PKA), and protein kinase C (PKC) inhibitory peptides (0–200 nM, Calbiochem) or their combinations were each added to the system. In parallel, neutralizing anti-IGF-II antibodies (1:25 to 1:500, R&D, Minneapolis, MN), heat-inactivated recombinant human IGF-II, polyclonal rabbit antihuman IGF-II/M6PR IgG, or monoclonal antibody (mAb) against IR or IGF-IR was applied to the cells grown on MCs 30 min before the assay was started by addition of thrombin (0.65 U/ml, Sigma) to initiate fibrin polymerization. After 3 d of incubation, the number of sprouts (capillary-like outgrowth from the MC) was quantified in eight separate microscope fields. All assays were repeated at least three times, and four wells were used for each treatment.
Chicken chorioallantoic membrane (CAM) assay
To determine the angiogenic effect of IGF-II in vivo, a modified CAM assay was performed as described earlier (18). Fertilized chicken eggs were prepared by cutting a window into the shell on d 3 of incubation at 37 C in a humidified egg incubator. On d 10, methylcellulose disks saturated with bovine fibroblast growth factor (bFGF) (25 µg/ml; positive control) or IGF-II (5–25 µg/ml) were laid onto the chorioallantoic membrane of the eggs. On d 13 blood vessel density around or within the disks were evaluated and photographed using a stereomicroscope (magnification x23–30). Five CAMs were evaluated for each test group, and the experiments were repeated at least three times. Quantification was performed using Meta Morph Imaging and Analysis System (Universal Imaging Corp., Downington, PA). Equal-sized quarters (four per pellet) were subjected to color recognition and integral analysis. The proportion of areas occupied by blood vessels (red) were assessed, and the vascularity index (VI; defined as B-A/A, in which A is vascular density distant from the stimulated area and B is vascular density within the stimulated area after 3 d of incubation with test substances) was compared between treatment groups.
Proliferation assays
A colorimetric nonradioactive assay was used for the quantification of cell proliferation and viability, based on the cleavage of tetrazolium salt WST-1 by mitochondrial dehydrogenases (Roche Molecular Biochemicals, Mannheim, Germany). After starvation of cultured UMVECs for 4 h, they were incubated with various concentrations of IGF-II (1–100 ng/ml), insulin (1 µg/ml), or VEGF (10–100 ng/ml) for 24 h followed by addition of cell proliferation reagent WST-1 (10 µl/well) and incubation for 2 h. Absorbance at wavelengths of 450 and 650 nm was measured using a microtiter plate reader and compared with a standard curve (20).
In addition, a flow cytometric proliferation assay was performed I which UMVECs were seeded in a gelatin-coated 24-well plate (5000 cells/well) and cultured for 24 h. After washing the cells were cultured in supplement-free medium containing 0.2% FCS for another 24 h, followed by stimulation with IGF-II (10–250 ng/ml) for 3 d. Total cell numbers were measured in a Casy counter (Schärfe System, Reutlingen, Germany). As a positive control, 20% FCS was applied, compared with unstimulated cells, with 0.2% FCS (21).
Migration assay
For analysis of cell migration, uncoated 8-µm porous filters in a Boyden chamber (BD, Heidelberg, Germany) were used as previously described (20). Briefly, cells were starved for 4 h and a total number of 5 x 104 cells in endothelial cell medium without any supplements were added to the upper chamber of the chamber inserts. Various IGF-II concentrations (1–100 ng/ml) as well as VEGF (10 ng/ml) as a positive control were then added to the lower chamber, and the system was incubated at 37 C for 24 h in 5% CO2. After incubation and fixation, nonmigrating cells were removed with a cotton swab, and the remaining were stained with hematoxylin and eosin. Filters were examined microscopically for cells on the lower side of the membrane. The number of cells in eight microscopic fields was counted and the median of three wells was determined. As a further evaluation method, slides were digitally scanned, and the cell number was determined electronically via measuring the amount of pixels of all cells of the whole slide divided by the amount of pixels of one cell (http://www.scioncorp.com/).
Statistics
The data were analyzed for statistical significance with Graph Pad Instruments (GraphPad Software Inc., San Diego, CA). One-way ANOVA (for single treatments) and the Kolmogorov-Smirnov test (testing for normality) were used as primary tests, and the post test was processed by Dunnet (parametric) where appropriate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As revealed by in situ hybridization, both IGF-II and IGF-II/M6PR mRNA were highly expressed in endothelial cells of uterine tissue sections, whereas only very low levels of IGF-IR mRNA were found (Fig. 1
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Chemical cross-linking of 125I-IGF-II to UMVECs resulted in labeled complexes with multiple sizes. As shown in Fig. 2, the 230-kDa moiety relates to the IGF-II/M6PR because this band also showed positive immunoreactivity with the anti-IGF-II/M6PR antibody. The 130-kDa band relates to the -subunit of the IGF-IR detected with a specific mAb as shown previously (22), and the 20- and 60-kDa bands represent cell surface-associated IGF-binding protein (IGFBP), whose identity was not further characterized (Fig. 2, lane a). Cross-linking to all moieties was specific because the indicated radiolabeled bands disappeared when 200 ng/ml of unlabeled IGF-II was present during the reaction (Fig. 2, lane b), but they remained in the presence of insulin (Fig. 2, lane c).
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Incubation of UMVECs with physiological concentrations of IGF-II (1–100 ng/ml) resulted in a significant dose- dependent increase of endothelial sprout formation (P < 0.05), in which addition of 100 ng/ml caused a 2.5-fold increase in the number of outgrown sprouts, compared with nontreated cells (Fig. 3, A and B). This increase in tube formation was similar to the effect of 10 ng/ml VEGF. The angiogenic activity of IGF-II was inhibited by a mAb against IGF-II or an IgG fraction of rabbit-antihuman IGF-II/M6PR, whereas a control mAb against CD 45 had no effect (Fig. 3, C and D). Although mAb against IGF-IR or IR could inhibit IGF-I or insulin-induced endothelial cell sprouting (data not shown), respectively, they had no effect on IGF-II-induced tube formation. Both pertussis toxin (0.1–10 ng/ml) and GRGDTP peptide (0–10 µg/ml) could abolish IGF-II-induced capillary formation (data not shown). Treatment of UMVECs with 10 nM PDBu (a PKC activator) but not with 1–10 mM dbcAMP (a PKA inducer) mimicked the effects of IGF-II on capillary formation. In addition, a PKC inhibitory peptide (PKCI, 0–200 nM) but not a PKA inhibitory peptide (PKAI, 0–200 nM) could abolish IGF-II-induced sprout formation (Fig. 4).
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Microscopic inspection of the CAMs analyzed in the described angiogenesis assay indicated that the methylcellulose disks adsorbed with IGF-II (Fig. 5B) or bFGF (Fig. 5C) were surrounded by allantoic vessels that developed toward the implant, whereas no vascular reaction was detectable around the methylcellulose implants containing purified water (Fig. 5A) only. Semiquantitation of the angiogenic response demonstrated that the microvessel density in the methylcellulose containing IGF-II was less than the response to bFGF but significantly more than in controls. Quantitative evaluation of the assays revealed a significant increase (P < 0.05) in the VI after incubation with IGF-II (VI = 0.66; SD = 0.02) or bFGF (VI = 0.63; SD = 0.04), compared with unstimulated controls (VI = 0.20: SD = 0.03) (Fig. 5B).
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Although different concentrations of IGF-II (10–250 ng/ml) showed no significant proliferative effect on UMVECs, in neither the colorimetric (results not shown) nor flow cytometric proliferation assay, compared with the negative control (Fig. 6A), increased UMVEC migration (1.5-fold, P < 0.05) in vitro was induced by physiological concentrations of IGF-II (50–250 ng/ml) to a similar degree as by VEGF (Fig. 6, B and C). In parallel experiments, IGF-I (data not shown) and FCS (20%) increased the proliferation of UMVECs 3.3-fold and migration 2-fold, respectively, during the same incubation period, compared with unstimulated controls.
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Discussion |
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Using in situ hybridization, we observed that uterine vasculature cells and UMVECs expressed both IGF-II and IGF-II/M6PR mRNAs, suggesting a regulatory role of IGF-II at the fetomaternal interface. Physiological concentrations of IGF-II significantly increased capillary-like outgrowth formation in vitro in the three-dimensional sprouting assay and blood vessel formation in vivo in the CAM assay. The biological activities of IGF-II can be modulated by the IGF-IR; the IGF-II/M6PR; and, to a much lesser extent, the IR (3). Despite the overlapping activities, the observed angiogenic response in our study was IGF-II-specific and likely mediated via the IGF-II/M6PR because addition of antibodies against IGF-II or IGF-II/M6PR but not against insulin or IGF-IR prevented or blocked IGF-II-induced sprout formation, although an IGF-IR involvement in angiogenesis is known (23).
With regard to the complex mechanisms of endothelial cell interactions in angiogenesis, IGF-II was shown to promote migration of UMVECs, correlating with its ability to stimulate extravillous trophoblast invasion (5). The lack of a proliferative effect of IGF-II on UMVECs may be due to the low cellular abundance of IGF-IR mRNA, which is the main mediator for its mitogenic activity (24), as shown by in situ hybridization. In addition, although IGF-II is a potent mitogen for many types of cultured cells and tissues and this biological effect is mediated mainly through IGF-IR, it was shown previously that not all IGF-IR expressing cells do respond with an increased proliferation to IGF-II stimulation (25). On the other hand, the proliferative response to IGFs or insulin can be mediated by all three receptors including IGF-IR, IGF-IIR, and IR (26), and different expression patterns of the respective receptors on the cell surface can determine their biological effect under different growth conditions (27).
Interestingly, a recent study (28) suggested that IGF-II could promote tumor growth through suppression of apoptosis. It remains to be clarified to which extent the mitogenic activity of IGF-II per se and the suppression of apoptosis can contribute to the angiogenic effect of IGF-II. Because IGF-II has been shown to induce the expression of both VEGF mRNA and protein in human hepatoma cells (HepG2) and the induction of VEGF by IGF-II was additively increased by hypoxia (13), it is possible that IGF-II in addition to its direct IGF-II/M6PR-mediated effect may have an indirect VEGF-mediated angiogenic effect.
The intracellular signal transduction cascade of the IGF-II/M6PR is not completely understood. Unlike the classical G protein-coupled receptors, which contain a seven-transmembrane region, the IGF-II/M6PR represents a single-chain membrane-spanning glycoprotein, with a major role for the intracellular transport of lysosomal enzymes (3). Yet the inhibitory effect of pertussis toxin during IGF-II-induced angiogenesis indicated that a G protein-coupled receptor might be involved in the signal transduction, which is in accordance with earlier studies in which G protein coupling to the IGF-II/M6PR was seen (5, 29). Moreover, a PKCI abrogated sprouting induced by IGF-II or PDBu (a PKC activator), whereas a PKAI had no effect, indicating that the PKC signaling pathway might be involved. In agreement with earlier studies, the intracellular signal molecules PKC and adenylate cyclase were shown to be counterparts in regulation of angiogenesis because PKC leads to an activation of angiogenesis, which is abolished by an increased intracellular cAMP level (30).
Taken together, we demonstrated a defined angiogenic activity of IGF-II on UMVECs, which could be attributed mostly to a migratory but not proliferative function of IGF-II toward these cells. Because IGFBP-1 has been shown to be expressed in the decidua (4) and some endothelial cells (31), one could speculate that IGFBP-1 might play an important role in the regulation of IGF-II bioavailability in the fetomaternal unit (32) and indirectly influence uterine angiogenesis. On the other hand, because IGFBP-1 and -2 express RGD (Arg-Gly-Asp) sequence (33) and blocking peptides against RGD sequence are potent inhibitors of angiogenesis (18, 34), it appears possible that IGFBPs might be directly involved in the regulation of angiogenesis.
Another recently characterized link between hCG and IGFBP-1 also indicates an additional possible interaction in the regulation of angiogenesis through the IGF system in the fetomaternal unit (35). Unpublished data from our laboratory (Zygmunt, M., T. McKinnon, P. K. Lala, and U. K. M. Han, unpublished observations) revealed a further interaction between hCG and the IGF system: Because hCG increased the IGF-IIR number at the plasma membrane by influencing its externalization rate, another important aspect in the complex interaction during the establishment of pregnancy would be added.
These findings have important implications for the successful implantation of the conceptus during pregnancy. In addition, failure of a successful implantation process has been associated with pregnancy diseases such as preeclampsia and intrauterine growth retardation. The role of IGF-II in preeclampsia has been suggested by studies from our laboratory that showed an increased expression of IGF-II in preeclamptic placentae, particularly close to the infarcts (36). Although physiological neovascularization is tightly regulated both temporally and spatially, pathophysiological angiogenesis (e.g. during tumor growth) is characterized by uncontrolled growth of vessels (37). Understanding the underlying mechanism of angiogenesis during pregnancy can not only improve the chance of a successful outcome but also open up new therapeutic avenues.
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Acknowledgments |
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Footnotes |
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Abbreviations: bFGF, Bovine fibroblast growth factor; CAM, chicken chorioallantoic membrane; FCS, fetal calf serum; hCG, human chorionic gonadotropin; IGFBP, IGF-binding protein; IGF-IR, IGF-I receptor; IR, insulin receptor; mAb, monoclonal antibody; MC, microcarrier; M6PR, mannose-6 phosphate receptor; PDBu, phorbol,12,13-dibutyrate; PKA, protein kinase A; PKAI, PKA inhibitory peptide; PKC, protein kinase C; PKCI, PKC inhibitory peptide; UMVEC, uterine microvascular endothelial cell; VEGF, vascular endothelial growth factor; VI, vascularity index.
Received February 13, 2003.
Accepted July 8, 2003.
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References |
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and insulin-like growth factor II in chronic active hepatitis B, cirrhosis and hepatocellular carcinoma. J Hepatol 22:286–294[CrossRef][Medline]
and insulin-like growth factor 2. Cancer Res 59:3915–39185 ß1 integrin by means of its Arg-Gly-Asp sequence. Proc Natl Acad Sci USA 15;90:10553–10557
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