ROLE OF VEGF RECEPTOR-1 (FLT-1) IN MEDIATING CALCIUM-DEPENDENT NITRIC-OXIDE RELEASE AND LIMITING DNA-SYNTHESIS IN HUMAN TROPHOBLAST CELLS

Vascular endothelial growth factor (VEGF) receptor KDR (kinase-insert- domain-containing receptor) is linked to endothelial cell proliferatio n, and VEGF receptor Flt-1 (fms-like tyrosine kinase) is essential for the organization of embryonic vasculature. Flt-1 is also known to be expressed on adult endothelial and trophoblast cells, although its fun ction has not yet been established. Herein we report that human tropho blast and endothelial cells contain functional Flt-1 receptors for VEG F that trigger the synthesis and release of nitric oxide (NO) by the a ctivation of constitutive NO synthase (cNOS). In first-trimester human trophoblast cells isolated by chorionic villous sampling, VEG(165) st imulated NO release in a concentration- and time-dependent manner, wit h a maximal increase of 60% (in comparison to basal release levels) oc curring within 30 minutes (basal: 1342 pmol/ml; VEGF (10 ng/ml): 2162 pmol/ml; p < 0.001), as measured by an NO chemiluminescence analyzer. VEGF(20), a peptide fragment that is composed of the first 20 amino ac ids at N-terminus, displayed properties of a partial agonist. VEGF(165 )- and VEGF(20)-mediated NO biosynthesis was attenuated by N-G-nitro-L -arginine in a concentration-dependent fashion, indicating NOS activat ion. VEGF-neutralizing anti-VEGF monoclonal antibody significantly inh ibited VEGF-mediated NO release (p < 0.001), and the addition of a neu tralizing anti-Flt-1 antibody inhibited the response by 79.6% +/- 7.59 %, an effect found to be reversible with higher concentrations of VEGF . In contrast, anti-KDR antibody had no significant inhibitory effect. RT-PCR confirmed the presence of mRNA encoding the Flt-1 and KDR rece ptors as well as the endothelial form of cNOS in trophoblast cells. VE GF(165)-stimulated NO release was inhibited by genistein (5 mu M; p < 0.001) as well as by the removal of calcium from the extracellular env ironment (p < 0.001), which suggests the contingency of this process o n tyrosine phosphorylation and extracellular calcium, respectively. Ad dition of sodium nitroprusside, an NO donor, inhibited trophoblast DNA synthesis in a concentration-dependent manner, as measured by [H-3]th ymidine incorporation, without affecting cell viability. VEGF under ma ximal NO production had no mitogenic activity, suggesting that trophob last-derived NO may limit trophoblast proliferation. Endogenous tropho blast DNA synthesis increased 3-fold in the presence of anti-Flt-1 ant ibody but not in the presence of anti-KDR antibody, suggesting that Fl t-1 functions as a growth suppressive receptor to counteract the proli ferative actions of KDR. Levels of immunoreactive endothelial cNOS wer e markedly increased in growth-restricted placentae (n = 4) in compari son to those of normal (n = 5) placentae, which may account for the re latively small-sized placentae associated with intrauterine growth res triction. VEGF(165) stimulated NO release via phosphorylation of the F lt-1 receptor, indicating that VEGF may be an autocrine regulator of N O biosynthesis by aiding trophoblast penetration into spinal arteriole s during the first trimester and preventing platelet aggregation withi n the placenta. Finally, the activation of Flt-1 receptor suppressed t rophoblast DNA synthesis within the placenta via NO.