FMS-like tyrosine kinase 1 (FLT1) is a key regulator of fetoplacental endothelial cell migration and angiogenesis

Abstract

IntroductionFetoplacental angiogenesis plays a vital role in pregnancy outcome. Vascular endothelial growth factor A (VEGFA) is one major regulator of angiogenesis. It primarily binds to FMS-like tyrosine kinase (FLT1) and kinase insert domain receptor (KDR). In most vascular beds, KDR appears to be the main mediator of angiogenesis. However, the role of both receptors within the human placenta remains unknown. MethodsHuman fetoplacental ECs were isolated/cultured from placentas of full-term, uncomplicated pregnancies after scheduled Cesarean section. Cells were subjected to RNA interference of either FLT1 or KDR followed by MTT, wound scratch, and tube formation assays. ECs were serum-starved after RNA interference and treated with VEGFA (60 ng/ml), then subjected to western blot to investigate FLT1 or KDR-mediated signaling. All experiments were performed in triplicate utilizing ECs from at least three separate subjects. One-way ANOVA with Tukey post-hoc testing was utilized for statistical analysis. ResultsSignificant knock-down of FLT1 and KDR was confirmed by qPCR (p < 0.01) and WB (p < 0.0001). KDR knock-down decreased EC metabolic activity (p < 0.01), and FLT1 ablation unexpectedly increased EC proliferation (p < 0.01). There was no difference in apoptosis regardless of FLT-1 or KDR knock-down. FLT1 knock-down significantly impaired wound scratch closure (p < 0.0001) and tube formation (p < 0.001). Surprisingly, KDR effects on EC metabolism had no effect on migration, although KDR was important in VEGFA-stimulated Akt and ERK activation. In contrast, FLT1 effects on EC motility were Akt and ERK-independent. ConclusionHuman fetoplacental EC migration is primarily regulated by FLT1 but not KDR.