Regulatory effects of soluble growth factors on choriocapillaris endothelial growth and survival.

The effects of soluble growth factors on regulating the survival and growth of choriocapillaris endothelial (CCE) cells were investigated in vitro. CCE cells were cultured in a serum-free medium in the presence or absence of various soluble growth factors. Cell growth and cell viability were assessed by counting viable cells. Results showed that acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) significantly stimulated CCE growth in a dose-dependent manner. Platelet-derived growth factor BB (PDGF, 0.4-10 ng/ml), insulin-like growth factor (IGF, 0.4-10 ng/ml) or insulin (0.4-10 micrograms/ml) alone did not affect the growth of CCE cells. In the presence of insulin (10 micrograms/ml), however, PDGF stimulated CCE growth in a dose-dependent manner. By contrast, transforming growth factor beta 1 (TGF-beta 1) induced CCE death in the absence of other growth factors and inhibited the CCE growth induced by aFGF, bFGF or VEGF. When CCE cells had been cultured in media containing aFGF, bFGF, VEGF or the combination of PDGF and insulin for 48 h, withdrawal of aFGF, bFGF or VEGF, but not PDGF, from the media resulted in CCE death. The CCE death induced by either an introduction of TGF-beta 1 or withdrawal of aFGF, bFGF or VEGF was defined as apoptosis based on morphologic characteristics (condensation and fragmentation of nuclei, shrinkage of cells in size) and DNA fragmentation in multiples of approximately 180 base pairs. Phorbol 12-myristate 13-acetate (PMA, 2 or 5 nM) protected CCE cells against apoptosis induced by the introduction of TGF-beta 1 and withdrawal of aFGF, bFGF or VEGF, while H7 (50 microM), but not HA1004 (50 microM), abrogated the protective effect of PMA on CCE apoptosis. However, cycloheximide (0.1 microM), a protein synthesis inhibitor, was only effective to protect CCE cells from apoptosis induced by aFGF, bFGF or VEGF withdrawal, but not that induced by the introduction of TGF-beta 1. Moreover, cycloheximide and a low concentration of PMA (2 nM) showed an additive effect on protecting CCE cells from apoptosis as the result of a growth factor withdrawal. These data may suggest that some growth factors, such as aFGF, bFGF, VEGF and TGF-beta 1, play a more critical role than the others, such as PDGF and IGF, in the regulation of CCE growth and survival. CCE apoptosis as a result of withdrawal of a survival factor or of receiving a death factor such as TGF-beta 1 may involve different mechanisms, which merits further study.