Novel roles for protein S-glutathionylation in angiogenesis and preeclampsia

Abstract

Background: Preeclampsia (PE) is a severe syndrome complicating 2-8% pregnancies, characterised by multi-system vascular defects and excess levels of the anti-angiogenic factor sFlt1 in the placenta and maternal circulation. Oxidative stress is a hallmark in PE, yet antioxidant supplementation failed to prevent PE and sometimes worsened pregnancy outcomes. Preeclamptic placenta are associated with a global reversal of S-glutathionylation, a common oxidative post-translational modification (oxPTM) reversed by Glutaredoxin (Glrx), and Glrx up-regulation promotes PE-like symptoms in a mice model of pregnancy. Although S-glutathionylation is emerging as an important redox-switch in angiogenesis by modulating various targets in the VEGF pathway, its role has not been investigated in the context of PE. We aimed to identify the molecular basis for how S-glutathionylation reversal may contribute to PE by altering placental angiogenic signals.

Methods: A combination of in vitro studies and exon-level microarray analysis from mice tissues was used to assess the effects of Glrx over-expression in placental angiogenic signals and identify putative redox targets involved in PE. Next, redox-resistant mutants of the splicing factor U2AF2 were constructed and expressed in primary endothelial cells (EC) to investigate the regulatory roles of U2AF2 oxPTM in angiogenesis. Finally, induced pluripotent stem cell (iPSC)-derived trophoblasts were implemented into a three-lane OrganoPlate® to create a 3D placenta-on-a-chip system replicating early placental events.

Results: Glrx up-regulation functionally disrupted EC 3D angiogenic sprouting, trophoblast migration and syncytialisation, and altered angiogenic balance in a cell type specific manner. Importantly, Glrx up-regulation caused an isoform-specific elevation of the PE marker sFlt1 e15a in EC and trophoblasts. In parallel, a genome-wide exon-level profiling of Glrx-overexpressing mice placenta revealed widespread changes in alternative splicing events. The splicing factor U2AF2 was identified as a putative S-glutathionylation target directly relevant to splicing and PE, and in vitro studies confirmed that preventing U2AF2 oxPTM inhibited EC migration and modulated Flt1 splicing to favour sFlt1-e15a expression. Genome-wide transcriptome profiling identified multiple angiogeniesis-related splicing events under U2AF2 oxPTM regulation, and the regulation of cell migratory functions appeared as a pathway of interest in PE. Trophoblasts were successfully generated from iPSC and replicated angiogenic responses to hypoxia as seen in primary trophoblasts. When differentiated into a 3D OrganoPlate® platform, iPSC-derived trophoblasts spontaneously formed a leak-tight physical barrier showing fusogenic and endocrine activities, closely resembling the placental barrier.

Conclusion: A novel molecular mechanism was identified by which S-glutathionylation reversal leads to sFlt1-e15a elevation and endothelial dysfunction through the modulation of U2AF2 splicing activity. These findings highlighted the pivotal role of oxPTM in regulating angiogenic signals under oxidative stress conditions, and provided a new potential route causing placental anti-angiogenic response in PE. The novel iPSC-derived 3D placenta-on-a-chip platform developed appears as a promising system to further explore functional roles of redox signalling in early placenta and boost therapeutic development.

Date of Award	2022
Original language	English
Sponsors	European Union
Supervisor	Colin Murdoch (Supervisor) & Michael Ashford (Supervisor)