Pre-eclampsia is a serious complication of pregnancy. Soluble Fms-like tyrosine kinase-1 (sFlt1) is central to the pathophysiology of pre-eclampsia. Released from the placenta into the maternal circulation, it antagonises vascular endothelial growth factor (VEGF) and causes severe endothelial dysfunction and maternal organ injury. Excitingly, in 2010 three groups reported the predominant placental-derived sFlt-1 isoform is a newly identified splice variant, named sFlt1-e15a. Given this is the main sFlt-1 isoform released from placenta, it may be the key pathophysiological factor of pre-eclampsia. To date, the reports of e15a have only been descriptive. Notably, there have been no functional studies of sFlt1-e15a. We therefore set out to examine whether sFlt1-e15a protein is biologically active.
To test the functional capacity of sFlt1-e15a to neutralise VEGF, we used a novel bioassay where BAF3 cells have been engineered so that they require the presence of VEGF to survive. Recombinant sFlt-e15a dose dependently reduced BaF3 cell viability in the presence of VEGF, suggesting it directly antagonises VEGF and is biologically active. Heat inactivated sFlt1-e15a induced no significant reduction in BaF3 viability. We next assessed whether sFlt1-e15a blocks VEGF induced migration and invasion using the xCELLigence assay (measures experiments in real time). In the presence of VEGF, sFlt1-e15a potently blocked primary HUVEC migration (n=3) and invasion (n=3). We next assessed whether sFlt1-e15a blocked VEGF induced signalling of Akt by exposing primary HUVECs to VEGF ± sFlt-1-e15a for 30 minutes. Densitometric analysis of western blots showed VEGF induced phosphorylation of Akt (normalised to total Akt), but this was potently blocked by sFLt1-e15a.
In conclusion, the novel primate and placental specific sFlt1-e15a is biologically active and potentially neutralises VEGF. Importantly this BaF3 assay provides a valuable screening tool to screen candidate sFlt1-e15a neutralising antibodies as potential therapeutics for preeclampsia (work that is currently under progress).