S-glutathionylation, a redox-switch involved in placental angiogenic balance and preeclampsia

Objectives: Preeclampsia (PE) is a severe pregnancy complication associated with increased oxidative stress and elevation of the anti-angiogenic factor sFlt-1 in the maternal circulation. Yet antioxidant therapy has failed, in some cases worsening pregnancy outcomes. S-glutathionylation is a common oxidative post-translational modification (oxPTM) modulating protein activity and enzymatically reversed by glutaredoxin (Grx). Although the importance of S-glutathionylation as a “redox-switch” is emerging in cardiovascular diseases, its role has not been investigated in the context of PE. We aimed to investigate the molecular basis for how thiol modification removal may contribute to pregnancy-induced vascular complications by altering angiogenic signalling at the maternofoetal interface.

Methods: We combined physiological in vivo assessment with bioinformatics proteomic analysis and Affymetrix exon-level microarray analyses to investigate the role of protein oxPTM in the development of PE phenotype. In vitro studies using primary endothelial cells and iPSC-derived trophoblasts investigated the role of oxPTM in angiogenic signalling and early-pregnancy events in 3D models replicating key placental functions.

Results: Overexpressing Grx transgenic mice (TG) and littermate controls (WT) underwent timed pregnancy. TG mice developed gestational hypertension, kidney dysfunction and elevated plasma sFlt-1 compared to WT. Adenoviral Grx overexpression disrupted endothelial cell sprouting in a 3D organoplate®, inhibited trophoblast migration and syncytialisation. Grx altered angiogenic balance in a cell-type specific manner by increasing sFlt1:PlGF ratio in endothelial cells and syncytiotrophoblasts while decreasing it in extra-villous trophoblasts. A genome-wide exon-level profiling of TG vs WT mice placenta revealed a global alteration of alternative splicing events. Bioinformatic analysis identified redox-sensitive targets directly relevant to splicing and PE. Of significance, S-glutathionylation removal was found to disrupt the spliceosome machinery consequently affecting Flt-1 splicing.

Conclusion: Grx-dependent removal of ox-PTMs regulates angiogenic pathways in the placenta via the modulation of redox-sensitive targets in the spliceosome machinery, which may promote the vascular defects associated with PE phenotype