The choriocapillaris endothelial cell culture as a model of angiogenesis in vitro
A. Zubilewicz*, J. C. Jeanny**, G. Soubrane ***, F. Mascarelli**
The Aim:The purpose of this study was to establish a pure CEC culture and to investigate the mitogenic and survival effects of various growth factors, especially FGF2 and VEGF, the two main angiogenic factors.
Material and Methods: Bovine CEC were obtained by the method described by Hoffman et al.,1998,using the polystyrene superparamagnetic beads coated with lectine Lycopersicon esculentum which selectively binds to fucose residues on the surface of choriocapillaris endothelial cells. The endothelial characteristic of the cultured cells was confirmed by immunocytochemistry using anti-factor von Willebrand and anti-CD31 antibodies. Proliferation and survival of CEC in the presence of FGF2 anf VEGF were analysed using haemacytometer of Mallasez. The production of FGF2 and activation of the intracellular signaling mediated by FGF2 was analysed by Western blotting using specific antibodies.
Results: The method of isolation of the microvascular endothelial cells used in that study provide a pure CEC culture with confirmation of cellular type by positive immunocytochemical staining with antibodies against anti-vWillebrand factor and anti-CD31. The CEC proliferation and survival are optimate in Endothelial Growth Medium enriched with 5% fetal calf serum, human epidermal growth factor (1m g/ml), bovine brain extract (12 m g/ml) and hydrocortisone (1m g/ml). However, in the presence of exogenous FGF2 (15ng/ml) or VEGF (15ng/ml), the CEC metabolism was maintained even in serum-deprived medium. The CEC in culture produce FGF2 in form monomeric and dimeric. FGF2-induced CEC proliferation was mediated by ERK 1 and 2 activation (phosphorylation). Inhibition of ERK pathway by the MEK1 inhibitor PD 98059 blocks FGF2-mediated cell proliferation.
Conclusion: CEC culture provide a very good in vitro model to study the effects of various growth factors and angiogenic molecules. In further study anti-angiogenic molecules will be analysed. The better understanding of mechanisms, implicated in choroidal neovascularisation can be of value in looking for the therapeutic possibilities in AMD.
*Tadeusz Krwawicz Chair of Ophthalmology and 1st Eye Hospital, Medical University School,
** Unite 450, INSERM, Paris, France
*** Clinique Ophtalmologique de Creteil, Creteil, France