In-vitro evidence of autocrine secretion of vascular endothelial growth factor by endothelial cells from human placental blood vessels

Vascular endothelial growth factor (VEGF), a highly specific mitogen for va scular endothelial cells, is involved in placental vascular growth and remo delling. The aim of this study was to investigate whether placental endothe lial cells secrete VEGF in an autocrine manner and if this secretion is cor related with endothelial cell growth. Blood vessels, excised from the apica l surface of three human placentae, were sectioned into 40 fragments per pl acenta and cultured in fibrin gel matrix for 27 days. Immunohistochemical d etection of placental endothelial cells was performed by positive staining with anti-human factor VIII-associated antigen and negative staining with a ntihuman a-actin and desmin. To investigate the production and autocrine ac tion of VEGF, VEGF concentrations in culture media were measured and the ef fect of an anti-VEGF neutralizing antibody on endothelial cell growth was o bserved. The results demonstrate that soluble VEGF is secreted by placental endothelial cells reaching a plateau from day 24 (68.74 +/- 7.52 pg/ml) to day 27 (67.20 +/- 6.28 pg/ml). Furthermore, VEGF concentrations in media c ollected on days 6, 12, 18, 21 and 27 of culture were found to be directly correlated to the sprouting parameter of endothelial cells, as calculated b y image analysis on the same day (P < 0.001, r(2) = 0.95). The use of 10 an d 100 ng/ml of a neutralizing antibody against human VEGF suppressed cell p roliferation, compared to that observed in the untreated controls, by 74.8 +/- 7.3 and 89.4 +/- 3.9% respectively. In conclusion, this study reports t he first evidence of autocrine secretion of VEGF by human placental endothe lial cells and demonstrates the involvement of VEGF in endothelial cell gro wth within a fibrin gel culture.