82. Disulfiram inhibits placental sFLT-1 secretion

Abstract

Introduction Preeclampsia is associated with elevated placental secretion of the anti-angiogenic factor sFLT-1. Surprisingly, little is understood about its regulation, identifying such mechanisms may uncover therapeutic leads. Objectives Our objective was to determine the effects of disulfiram, a 20S subunit proteasome inhibitor, on placental secretion of sFLT-1 and endothelial dysfunction. Methods Primary trophoblast were isolated from normotensive term placenta, treated with disulfiram and mRNA expression of sFLT-1 variants and protein secretion assessed. To assess the effects of disulfiram on the 20S subunit of the proteasome, primary trophoblast were treated with disulfiram in the presence of Suc-LLVY-AMC fluorogenic substrate. Next, we investigated the effect of disulfiram on endothelial dysfunction using isolated primary human umbilical vein endothelial cells (HUVECs) treated with 5% preeclamptic serum +/− disulfiram. The effect of disulfiram on markers of endothelial dysfunction were measured via qRT-PCR for vascular cell adhesion molecule-1 (VCAM-1) and adhesion of fluorescently labelled peripheral blood mononuclear cells (PBMCs) to HUVECs. Results Disulfiram significantly reduced mRNA expression of the sFLT-1 variants, sFLT-1-i13 and sFLT-e15a. This translated into a significant and dose dependent reduction in the protein secretion of sFLT-1. In primary trophoblast disulfiram did not inhibit the 20S subunit of the proteasome, evidenced by no change in cellular fluorescence following addition of Suc-LLVY-AMC. Treating primary HUVECs with serum from preeclamptic women induced endothelial dysfunction with significantly increased mRNA expression of VCAM-1 as well as the adhesion of PBMCs to HUVECs. Disulfiram reduced VCAM-1, with expression reduced below control levels, however, did not alter the adhesion of PBMCs to primary HUVECs. Conclusions We have identified disulfiram to quench placental sFLT-1 secretion via a mechanism independent of inhibiting the 20S subunit of the proteasome. Understanding of the mechanisms by which disulfiram inhibits sFLT-1 secretion may assist in identifying therapeutic targets. Interestingly, disulfiram also reduced markers of endothelial dysfunction.