Download PDF
Exp Clin Endocrinol Diabetes 2004; 112(4): 208-214
DOI: 10.1055/s-2004-817940
Article

J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York

Differential Regulation of ANG2 and VEGF-A in Human Granulosa Lutein Cells by Choriogonadotropin

D. Pietrowski1 , C. Keck1
  • 1Department of Obstetrics and Gynecology, University of Freiburg, Freiburg, Germany
Further Information

Publication History

Received: April 17, 2002 First decision: November 24, 2002

Accepted: October 6, 2003

Publication Date:
04 May 2004 (online)

Also available at
    Permissions and Reprints

Abstract

The growth and development of the corpus luteum after rupture of the follicle is a highly regulated process characterised by a rapid vascularization of the follicle surrounding granulosa cells. Vascularization is regulated by a large number of growth factors and cytokines whereas members of the angiopoietin family and VEGF-A are reported to play a principal role. The gonadotropic hormones luteinizing hormone and choriogonadotropin are reported to be essential for corpus luteum formation. In this study we investigated by RT PCR if the growth factors PGF, PDGF-A, PDGF-B, VEGF-A, VEGF-B, VEGF-C, VEGF-D, ANG1, ANG2, ANG3 and ANG4 are expressed in granulosa cells. We show the expression of VEGF-A, VEGF-B, PDGF-A, ANG1 and ANG2 in granulosa cells. Using RT-PCR and Real-Time PCR we demonstrate that angiopoietin 2 is downregulated in human granulosa cells in vitro after choriogonadotropin treatment whereas the expression of angiopoietin 1 is not significantly altered. The expression of VEGF on the RNA- and on the protein level was determined. It was shown that in granulosa cells VEGF is upregulated after choriogonadotropin treatment on the RNA level and that increasing concentrations of choriogonadotropin from 0 to 10 U/ml leads to an increasing amount of VEGF in the cell culture supernatants. The amount of VEGF in the supernatants reaches a plateau at 0.5 U/ml and is increased only slightly and not significantly after treatment of the cells with 10 U/ml choriogonadotropin compared to 0.5 U/ml.

In total these findings suggests that in granulosa cells the mRNA of various growth factors is detectable by RT-PCR and that VEGF-A and ANG2 is regulated by the gonadotropic hormone choriogonadotropin. These findings may add impact on the hypothesis of choriogonadotropin as a novel angiogenic factor.

Key words

Angiopoietin - corpus luteum - gonadotropin - granulosa cell - VEGF-A

References

  • 1 Agrawal R, Jacobs H, Payne N, Conway G. Concentration of vascular endothelial growth factor released by cultured human luteinized granulosa cells is higher in women with polycystic ovaries than in women with normal ovaries.  Fertil Steril. 2002;  78 1164-1169
    CrossrefPubMedGoogle Scholar
  • 2 Antczak M, Van Blerkom J. The vascular character of ovarian follicular granulosa cells: phenotypic and functional evidence for an endothelial-like cell population.  Hum Reprod. 2000;  15 2306-2318
    CrossrefPubMedGoogle Scholar
  • 3 Asahara T, Chen D, Takahashi T, Fujikawa K, Kearney M, Magner M, Yancopoulos G D, Isner J M. Tie2 receptor ligands, angiopoietin-1 and angiopoietin-2, modulate VEGF-induced postnatal neovascularization.  Circ Res. 1998;  83 233-240
    PubMedGoogle Scholar
  • 4 Carmeliet P, Moons L, Luttun A, Vincenti V, Compernolle V, De Mol M, Wu Y, Bono F, Devy L, Beck H, Scholz D, Acker T, DiPalma T, Dewerchin M, Noel A, Stalmans I, Barra A, Blacher S, Vandendriessche T, Ponten A, Eriksson U, Plate K H, Foidart J M, Schaper W, Charnock-Jones D S, Hicklin D J, Herbert J M, Collen D, Persico M G. Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions.  Nat Med. 2001;  7 575-583
    CrossrefPubMedGoogle Scholar
  • 5 Flamme I, Frolich T, Risau W. Molecular mechanisms of vasculogenesis and embryonic angiogenesis.  J Cell Physiol. 1997;  173 206-210
    CrossrefPubMedGoogle Scholar
  • 6 Fraser H M, Dickson S E, Lunn S F, Wulff C, Morris K D, Carroll V A, Bicknell R. Suppression of luteal angiogenesis in the primate after neutralization of vascular endothelial growth factor.  Endocrinology. 2000;  141 995-1000
    CrossrefPubMedGoogle Scholar
  • 7 Fraser H M, Lunn S F. Angiogenesis and its control in the female reproductive system.  Br Med Bull. 2000;  56 787-797
    CrossrefPubMedGoogle Scholar
  • 8 Fraser H M, Wulff C. Angiogenesis in the primate ovary.  Reprod Fertil Dev. 2001;  13 557-566
    CrossrefPubMedGoogle Scholar
  • 9 Geva E, Jaffe R B. Role of angiopoietins in reproductive tract angiogenesis.  Obstet Gynecol Surv. 2000;  55 511-519
    CrossrefPubMedGoogle Scholar
  • 10 Goede V, Schmidt T, Kimmina S, Kozian D, Augustin H G. Analysis of blood vessel maturation processes during cyclic ovarian angiogenesis.  Lab Invest. 1998;  78 1385-1394
    PubMedGoogle Scholar
  • 11 Grutzkau A, Kruger-Krasagakes S, Baumeister H, Schwarz C, Kogel H, Welker P, Lippert U, Henz B M, Moller A. Synthesis, storage, and release of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) by human mast cells: implications for the biological significance of VEGF206.  Mol Biol Cell. 1998;  9 875-884
    PubMedGoogle Scholar
  • 12 Hazzard T M, Christenson L K, Stouffer R L. Changes in expression of vascular endothelial growth factor and angiopoietin-1 and -2 in the macaque corpus luteum during the menstrual cycle.  Mol Hum Reprod. 2000;  6 993-998
    CrossrefPubMedGoogle Scholar
  • 13 Hazzard T M, Molskness T A, Chaffin C L, Stouffer R L. Vascular endothelial growth factor (VEGF) and angiopoietin regulation by gonadotrophin and steroids in macaque granulosa cells during the peri-ovulatory interval.  Mol Hum Reprod. 1999;  5 1115-1121
    CrossrefPubMedGoogle Scholar
  • 14 Heid C A, Stevens J, Livak K J, Williams P M. Real time quantitative PCR.  Genome Res. 1996;  6 986-994
    PubMedGoogle Scholar
  • 15 Higuchi R, Dollinger G, Walsh P S, Griffith R. Simultaneous amplification and detection of specific DNA sequences.  Biotechnology (NY). 1992;  10 413-417
    PubMedGoogle Scholar
  • 16 Higuchi R, Fockler C, Dollinger G, Watson R. Kinetic PCR analysis: real-time monitoring of DNA amplification reactions.  Biotechnology (NY). 1993;  11 1026-1030
    PubMedGoogle Scholar
  • 17 Holash J, Maisonpierre P C, Compton D, Boland P, Alexander C R, Zagzag D, Yancopoulos G D, Wiegand S J. Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF.  Science. 1999;  284 1994-1998
    CrossrefPubMedGoogle Scholar
  • 18 Kamat B R, Brown L F, Manseau E J, Senger D R, Dvorak H F. Expression of vascular permeability factor/vascular endothelial growth factor by human granulosa and theca lutein cells. Role in corpus luteum development.  Am J Pathol. 1995;  146 157-165
    PubMedGoogle Scholar
  • 19 Keck C, Felberbaum R. Application of Cetrorelix - a GnRH-antagonist in reproductive medicine.  Geburtsh Frauenheilk. 2000;  60 212-217
    PubMedGoogle Scholar
  • 20 Kimura H, Weisz A, Ogura T, Hitomi Y, Kurashima Y, Hashimoto K, D'Acquisto F, Makuuchi M, Esumi H. Identification of hypoxia-inducible factor 1 ancillary sequence and its function in vascular endothelial growth factor gene induction by hypoxia and nitric oxide.  J Biol Chem. 2001;  19 (276) 2292-2298
    PubMedGoogle Scholar
  • 21 Korpelainen E I, Alitalo K. Signaling angiogenesis and lymphangiogenesis.  Curr Opin Cell Biol. 1998;  10 159-164
    CrossrefPubMedGoogle Scholar
  • 22 Laitinen M, Ristimaki A, Honkasalo M, Narko K, Paavonen K, Ritvos O. Differential hormonal regulation of vascular endothelial growth factors VEGF, VEGF-B, and VEGF-C messenger ribonucleic acid levels in cultured human granulosa-luteal cells.  Endocrinology. 1997;  138 4748-4756
    CrossrefPubMedGoogle Scholar
  • 23 Levy A P, Levy N S, Goldberg M A. Hypoxia-inducible protein binding to vascular endothelial growth factor mRNA and its modulation by the von Hippel-Lindau protein.  J Biol Chem. 1996;  271 25492-25497
    CrossrefPubMedGoogle Scholar
  • 24 Licht P, Neuwinger J, Fischer O, Siebzehnrubl E, Wildt L. Peripheral levels of vascular endothelial growth factor (VEGF) are higher in gonadotropin stimulated as compared to natural ovarian cycles.  Exp Clin Endocrinol Diabetes. 2001;  109 345-349
    Article in Thieme ConnectPubMedGoogle Scholar
  • 25 Licht P, Neuwinger J, Fischer O, Siebzehnrubl E, Wildt L. VEGF plasma pattern in ovulation induction: evidence for an episodic secretion and lack of immediate effect of hCG.  Exp Clin Endocrinol Diabetes. 2002;  110 130-133
    Article in Thieme ConnectPubMedGoogle Scholar
  • 26 Livak K J, Flood S J, Marmaro J, Giusti W, Deetz K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization.  PCR Methods Appl. 1995;  4 357-362
    PubMedGoogle Scholar
  • 27 Machens H G, Morgan J R, Berthiaume F, Stefanovich P, Siemers F, Krapohl B, Berger A, Mailander P. Platelet-derived growth factor-AA-mediated functional angiogenesis in the rat epigastric island flap after genetic modification of fibroblasts is ischemia dependent.  Surgery. 2002;  131 393-400
    CrossrefPubMedGoogle Scholar
  • 28 Maisonpierre P C, Suri C, Jones P F, Bartunkova S, Wiegand S J, Radziejewski C, Compton D, McClain J, Aldrich T H, Papadopoulos N, Daly T J, Davis S, Sato T N, Yancopoulos G D. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis.  Science. 1997;  277 55-60
    CrossrefPubMedGoogle Scholar
  • 29 Mochizuki Y, Nakamura T, Kanetake H, Kanda S. Angiopoietin 2 stimulates migration and tube-like structure formation of murine brain capillary endothelial cells through c-Fes and c-Fyn.  J Cell Sci. 2002;  115 175-183
    PubMedGoogle Scholar
  • 30 Neulen J, Yan Z, Raczek S, Weindel K, Keck C, Weich H A, Marme D, Breckwoldt M. Human chorionic gonadotropin-dependent expression of vascular endothelial growth factor/vascular permeability factor in human granulosa cells: importance in ovarian hyperstimulation syndrome.  J Clin Endocrinol Metab. 1995;  80 1967-1971
    CrossrefPubMedGoogle Scholar
  • 31 Nicosia R F, Villaschi S. Autoregulation of angiogenesis by cells of the vessel wall.  Int Rev Cytol. 1999;  185 1-43
    PubMedGoogle Scholar
  • 32 Olofsson B, Jeltsch M, Eriksson U, Alitalo K. Current biology of VEGF-B and VEGF-C.  Curr Opin Biotechnol. 1999;  10 528-535
    CrossrefPubMedGoogle Scholar
  • 33 Papapetropoulos A, Garcia-Cardena G, Dengler T J, Maisonpierre P C, Yancopoulos G D, Sessa W C. Direct actions of angiopoietin-1 on human endothelium: evidence for network stabilization, cell survival, and interaction with other angiogenic growth factors.  Lab Invest. 1999;  79 213-223
    CrossrefPubMedGoogle Scholar
  • 34 Risau W. Mechanisms of angiogenesis.  Nature. 1997;  386 671-674
    CrossrefPubMedGoogle Scholar
  • 35 Stratmann A, Risau W, Plate K H. Cell type-specific expression of angiopoietin-1 and angiopoietin-2 suggests a role in glioblastoma angiogenesis.  Am J Pathol. 1998;  153 1459-1466
    PubMedGoogle Scholar
  • 36 Teichert-Kuliszewska K, Maisonpierre P C, Jones N, Campbell A I, Master Z, Bendeck M P, Alitalo K, Dumont D J, Yancopoulos G D, Stewart D J. Biological action of angiopoietin-2 in a fibrin matrix model of angiogenesis is associated with activation of Tie2.  Cardiovasc Res. 2001;  49 659-670
    CrossrefPubMedGoogle Scholar
  • 37 Thurston G, Suri C, Smith K, McClain J, Sato T N, Yancopoulos G D, McDonald D M. Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1.  Science. 1999;  286 2511-2514
    CrossrefPubMedGoogle Scholar
  • 38 Wang T H, Horng S G, Chang C L, Wu H M, Tsai Y J, Wang H S, Soong Y K. Human chorionic gonadotropin-induced ovarian hyperstimulation syndrome is associated with up-regulation of vascular endothelial growth factor.  J Clin Endocrinol Metab. 2002;  87 3300-3308
    CrossrefPubMedGoogle Scholar
  • 39 Wulff C, Dickson S E, Duncan W C, Fraser H M. Angiogenesis in the human corpus luteum: simulated early pregnancy by HCG treatment is associated with both angiogenesis and vessel stabilization.  Hum Reprod. 2001;  16 2515-2524
    CrossrefPubMedGoogle Scholar
  • 40 Wulff C, Wilson H, Largue P, Duncan W C, Armstrong D G, Fraser H M. Angiogenesis in the human corpus luteum: localization and changes in angiopoietins, tie-2, and vascular endothelial growth factor messenger ribonucleic acid.  J Clin Endocrinol Metab. 2000;  85 4302-4309
    CrossrefPubMedGoogle Scholar
  • 41 Yan Z, Weich H A, Bernart W, Breckwoldt M, Neulen J. Vascular endothelial growth factor (VEGF) messenger ribonucleic acid (mRNA) expression in luteinized human granulosa cells in vitro.  J Clin Endocrinol Metab. 1993;  77 1723-1725
    CrossrefPubMedGoogle Scholar
  • 42 Zygmunt M, Herr F, Keller-Schoenwetter S, Kunzi-Rapp K, Munstedt K, Rao C V, Lang U, Preissner K T. Characterization of human chorionic gonadotropin as a novel angiogenic factor.  J Clin Endocrinol Metab. 2002;  87 5290-5296
    CrossrefPubMedGoogle Scholar

Dr. Detlef Pietrowski

Universitäts Frauenklinik Freiburg

Hugstetter Straße 55

79106 Freiburg

Germany

Phone: + 49(0)7612703188

Fax: + 49 (0) 76 12 70 30 37

Email: pietrowski@frk.ukl.uni-freiburg.de

      >