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Angiogenic activity of human chorionic gonadotropin through LH receptor activation on endothelial and epithelial cells of the endometrium

Sarah Berndt^*, Sophie Perrier d’Hauterive^,, Silvia Blacher^*, Christel Péqueux^*, Sophie Lorquet^*,, Carine Munaut^*, Martine Applanat, Astrid Marie Hervé, Noël Lamandé^||, Pierre Corvol^||, Frédéric van den Brûle^*,, Françis Frankenne^*, Matti Poutanen^¶, Ilpo Huhtaniemi^¶,#, Vincent Geenen, Agnès Noël^* and Jean-Michel Foidart^*,

^* Laboratory of Tumor and Development Biology, Centre de Recherche en Cancérologie Expérimentale (CRCE), Université de Liège, Liège, Belgium;

Center of Immunology, Université de Liège, Liège, Belgium;

Department of Gynecology and Obstetrics, CHR-Citadelle, Liège, Belgium;

INSERM U135, Paris, France;

^|| Collège de France, INSERM U36, Paris, France;

^¶ Institute of Reproductive and Developmental Biology, Imperial College Faculty of Medicine, Hammersmith Campus, London, UK;

^# Department of Physiology, University of Turku, Turku, Finland

¹Correspondence: University of Liège, Laboratory of Tumor Biology and Development, Institute of Pathology CHU-B23, B-4000 Liège-Sart-Tilman, Belgium. E-mail: jmfoidart{at}ulg.ac.be

SPECIFIC AIMS

Although hCG is known to play a key role in materno-embryonic interactions occurring during embryo implantation, its involvement during endometrial angiogenesis is not well documented. The present study aims at 1) investigating the angiogenic activity of hCG in different in vitro and in vivo models; 2) determining the cellular targets of hCG (endothelial, stromal and/or epithelial cells); and 3) elucidating the cell surface receptor and signaling pathway involved in the angiogenic effects of hCG.

PRINCIPAL FINDINGS

1) hCG stimulates in vivo and in vitro angiogenesis
Incubation of rat aortic fragments with physiological concentrations of urinary hCG (uhCG) (8–800 IU/ml) or recombinant hCG (rhCG) resulted in a dose-dependent stimulation of vessel outgrowth (Fig. 1 ). The specificity of uhCG angiogenic effect was assessed by using two hormone preparations (rhCG and uhCG) (Fig. 1B) and neutralizing anti-hCG antibody (Ab) (Fig. 1C ). In the chicken chorioallantoic membrane (CAM) assay, uhCG also enhanced the complexity of vascular network. In addition, in vivo matrix invasion by functional blood vessels was enhanced (matrigel plug assay) by uhCG. Further in vitro experiments demonstrated the capacity of uhCG to stimulate human vascular endothelial cells (HUVEC) growth rate. This effect is specific to endothelial cells, since hCG did not affect endometrial epithelial cell proliferation.

View larger version (31K): [in this window] [in a new window]   Figure 1. Angiogenic effect of urinary hCG (uhCG) and recombinant hCG (rhCG) in aortic ring assay. Rat aortic rings were cultured in a collagen gel for 10 d with various hCG concentrations. A) Photomicrographs (original magnification 25x) of aortic rings after 10 d of incubation with uhCG (Pregnyl®, 100 IU/ml), with rhCG (100 IU/ml), or with vehicle (control). B) Quantification of microvessel outgrowth by computerized image analysis. Nv, vessel number; Nb, branching number; Lmax, maximal length (mm). n = 4, error bars = SE., *P < 0.05; **P < 0.01. C) Histogram of vessel number (Nv) outgrowth from rat aortic rings incubated during 10 d with uhCG (100 IU/ml) supplemented with various concentrations (1.2 and 2.4 µg/ml) of antihCG Ab. n = 4, error bars = SE., **P < 0.01. Experiments were repeated at least three times. Student’s t test was used to evaluate whether differences among groups were significant. Statistical significance was set at P < 0.05.

2) The angiogenic effect of hCG on endothelial cells is mediated via activation of LH/hCG receptor and cAMP/PKA pathway
The presence of LH/hCG receptor (LH/hCG R) at the surface of endothelial cells was evidenced by reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blotting analyses. Since interaction of hCG with its receptor could lead to the activation of Gs/adenyl cyclase (AC)/cAMP/protein kinase A (PKA) pathway, we tested the effect of PKA inhibitors and dibutyryl cAMP on endothelial cell proliferation in vitro and on endothelial cells spreading from aortic rings. Interestingly, hCG significantly induced cAMP production in HUVEC and dibutyryl cAMP (dbcAMP) stimulated microvessel outgrowth. In contrast, an adenylate cyclase inhibitor (MDL12) and a PKA inhibitor (H89) both abolished hCG effects. To further document the implication of LH/hCG R in hCG-driven angiogenesis, we evaluated the effect of uhCG on aortic rings issued from LH/hCG R knockout (LuRKO) mice. Although hCG induced an angiogenic response in aortic rings of wild-type (WT) mice, no stimulation was achieved when aortic rings of LuRKO mice were used (Fig. 2 ).

View larger version (27K): [in this window] [in a new window]   Figure 2. Comparison of uhCG-induced angiogenic effect between WT and LuRKO (KO) mice in the aortic ring assay. LuRKO mice aortic rings were cultured in a collagen gel during 9 d in presence of autologous sera (2%) and/or urinary hCG (100 IU/ml). A) Photomicrographs (original magnification 25x) of aortic rings from WT mice and LuRKO mice after incubation with autologous sera and supplementation with uhCG (100 IU/ml). B) Quantification of microvessel outgrowth (Nv) by computerized image analysis of aortic ring assay after 9 d of incubation with autologous sera supplemented or not with uhCG (100 IU/ml) in WT or KO mice aortics. sWT = WT mice serum. sKO = KO mice serum. Data are expressed as mean ± SD, n = 5, *P < 0.05. Experiments were repeated at least three times. Differences between the experimental conditions were evaluated using the ANOVA analysis (P values <0.05 were considered significant).

These data demonstrate that hCG exerts its angiogenic effect by interacting with hCG/LH receptor and through its main signaling pathway: adenylate cyclase/cAMP/PKA pathway.

3) hCG enhances vascular endothelial growth factor (VEGF) production through a paracrine effect on epithelial endometrial cells (EEC)
While hCG failed to modulate VEGF production by HUVEC, it significantly enhanced VEGF secretion by EEC. These results demonstrate that hCG can modulate endothelial cell proliferation and migration both directly and indirectly through a stimulation of VEGF production by EEC. In this context, of great interest is our finding that VEGF and uhCG displayed additive actions in the aortic ring assay. We therefore provide, for the first time, evidence for a functional link between hCG and VEGF in a paracrine loop involving trophoblasts, endothelial cells, and EEC.

CONCLUSION AND SIGNIFICANCE

In normal pregnancy, hCG expression is associated with an endometrial angiostimulation occurring early in gestation. hCG increases blood supply and alters the uterine vasculature via vasodilatation, increasing permeability, development, and maturation of new vessels. LH/hCG R expression has been documented in uterine endothelial cells by ligand binding of ¹²⁵I hCG and activity measurements.

Our study demonstrates clearly a direct dose-dependent angiogenic effect of hCG on endothelial cells in the aortic ring, chicken chorioallantoic membrane (CAM), matrigel plug, and endothelial cell proliferation assays. Urinary and recombinant hCG exerted similar angiogenic effects, excluding the putative involvement of contaminating growth factors in urinary preparation. In addition, the hCG angiogenic stimulation in the aortic ring assay was abrogated by anti-hCG Ab. The use of recombinant hCG, adenylcyclase activator/inhibitor (dbAMPc/MDL12), and PKA inhibitor (H89) demonstrate that hCG acts through interaction with LH/hCG receptor, a G protein-coupled receptor that activates mainly the cAMP/PKA pathway. In addition, the abrogation of hCG proangiogenic action in LuRKO aortic rings confirmed the authenticity of this effect. Altogether these data demonstrate that the hCG effect could not be attributed to a contaminant present in the urinary extracts of hCG, as documented by others for the anti-HIV activities. Nevertheless, others provided evidence for induction of apoptosis in Kaposi’s sarcoma spindle cells by the recombinant hCG ßbeta; subunit.

Of particular interest is our finding that treatment of EEC with hCG enhanced VEGF secretion, leading thereby to an indirect effect on angiogenesis. Accordingly, others have reported the presence of LH/hCG R in endothelial cells and smooth muscle cells of uterine blood vessels. Although hCG was also shown to elicit VEGF mRNA and/or protein production in ovarian hyperstimulation syndrome and in granulosa cells of various species, our study is the first one reporting an effect of hCG on VEGF production by EEC.

Collectively, our data suggest that the trophoblast may stimulate the maternal endometrial angiogenesis via at least two mechanisms: (i) a direct activation of endothelial cells mediated by hCG and (ii) a paracrine loop involving an enhanced secretion of VEGF by EEC. Finally, the additive effect of hCG and VEGF indicates that these growth factors operate through different but converging signaling pathways. In conclusion, hCG appears thus to play a key role in the angiogenic synchrony between blastocyst and maternal endometrium through interactions involving activation of LH/hCG R present at the surface of maternal endothelial and epithelial cells.

View larger version (48K): [in this window] [in a new window]   Figure 3. hCG plays a key role in the angiogenic synchrony between trophoblast, endothelial and EEC. Schematic diagram depicting the angiogenic activity of hCG during implantation regarding to our findings. 1) Direct angiogenic effect of hCG via interaction with LH/hCG R at the surface of endothelial cells resulting in an increase of proliferation and capillary outgrowth. 2) Indirect angiogenic effect via a paracrine loop involving an enhanced secretion of VEGF by EEC under the control of hCG. 3) Additive effect of hCG and VEGF in stimulating capillary outgrowth.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5885fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-5885fjev1 20/14/2630    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Berndt, S. Articles by Foidart, J.-M. Search for Related Content PubMed PubMed Citation Articles by Berndt, S. Articles by Foidart, J.-M.