Disulfiram inhibits placental soluble FMS-like tyrosine kinase-1 and soluble endoglin secretion independent of the proteasome

Highlights

• Disulfiram, an alcoholism treatment, has been reported as a proteasome inhibitor.

• Disulfiram significantly reduced cytotrophoblast and HUVEC sFLT-1.

• Disulfiram quenched cytotrophoblast soluble endoglin secretion.

• Disulfiram did not affect the proteasome of primary cytotrophoblasts and HUVECs.

Abstract

Preeclampsia is associated with intermittent placental hypoxia, inflammation and the release of antiangiogenic factors, namely sFLT-1 and sEng. These factors cause maternal endothelial dysfunction and the manifestation of clinical disease. Disulfiram is a dehydrogenase inhibitor used to treat alcoholism and has been suggested as a proteasome inhibitor. Inhibiting the proteasome has been previously shown to reduce FLT-1 gene expression. Thus, we aim to investigate whether disulfiram alters the secretion of sFLT-1 and sEng and reduces endothelial dysfunction.

Methods and results

We assessed the effects of disulfiram on primary cytotrophoblast and human umbilical vein endothelial cells (HUVECs). Disulfiram significantly reduced mRNA expression of membrane bound FLT-1 and sFLT-1 variants in primary cytotrophoblasts, which translated into a significant reduction in the protein secretion of sFLT-1. Additionally, sFLT-1 was reduced in primary HUVECs treated with disulfiram, whilst sEng was only reduced in primary cytotrophoblasts. Next, we investigated the effect of disulfiram on endothelial dysfunction using primary HUVECs treated with 5% preeclamptic serum ± disulfiram. Serum from preeclamptic women induced endothelial dysfunction evidenced by increased mRNA expression of vascular cell adhesion molecule-1 (VCAM-1) and adhesion of peripheral blood mononuclear cells (PBMCs) to HUVECs. The addition of disulfiram reduced VCAM-1 mRNA expression, however did not affect the adhesion of PBMCs to endothelial cells. Lastly, we assessed the effects of disulfiram on the 20S subunit of the proteasome and found disulfiram did not inhibit this subunit in either primary cytotrophoblast or HUVECs.

Conclusions

Disulfiram quenches sFLT-1 and sEng via mechanisms independent of the 20S subunit of the proteasome. Understanding of the mechanisms by which disulfiram inhibits antiangiogenic secretion may reveal insights into the pathogenesis and potential therapeutic targets for preeclampsia.

Introduction

Preeclampsia is a hypertensive disorder of pregnancy characterized by maternal endothelial dysfunction and end organ damage. Today, it remains a serious pregnancy complication, claiming the lives of 60,000 mothers annually with far greater rates of neonatal mortality [1]. Although avidly investigated, no medical therapeutics are available, and delivery of the placenta remains the only means of halting disease progression. It is widely accepted that inadequate placental invasion and implantation results in hypoperfusion, reduced oxygen availability and a state of placenta hypoxia during preeclampsia [2]. This is believed to result in excessive and pathological release of anti-angiogenic factors, soluble FMS-like tyrosine kinase-1 (sFLT-1) [3] and soluble endoglin (sEng) [4] from the preeclamptic placenta. These placental factors are thought to inflict the wide spread maternal endothelial dysfunction, which is central to disease pathogenesis. Whilst it is known that sFLT-1 arises from the alternative splicing of FLT-1 and its expression is positively regulated by hypoxia [3], the actual pathways regulating placental production remain poorly understood. Thus, identifying mechanisms involved in the regulation of sFLT-1, sEng and subsequent endothelial dysfunction may uncover therapeutic leads.

Disulfiram, has been used in the treatment of alcoholism for over 60 years and has recently gained research focus as a potential proteasome inhibitor and cancer treatment [5], [6]. The proteasome comprises a large ATP dependant multi-subunit protease, which is involved in the degradation of proteins that regulate important cell functions including cell cycle, transcription, apoptosis, and angiogenesis. Importantly, Mezquita et al., reported inhibiting the proteasome significantly decreased FLT-1 gene expression in macrophages and endothelial cells [7]. Given proteasome inhibition reduces total FLT-1 gene expression and sFLT-1 is central to the pathogenesis of preeclampsia, we hypothesised that disulfiram, which is thought to potently inhibit the proteasome, may inhibit placental sFLT-1 expression. We therefore undertook functional studies in primary human tissues to examine the role of disulfiram in the regulation of the anti-angiogenic molecules central to preeclampsia.