Investigating the role of protein S-glutathionylation in sFlt-1 regulation and preeclampsia phenotype

Background: Elevation of the anti-angiogenic factor soluble Flt-1 (sFlt-1) is pivotal in the development of the preeclampsia phenotype of hypertension and kidney dysfunction during pregnancy. Likewise, oxidative stress is explicitly linked to preeclampsia with high levels measurable both in the placenta and maternal circulation. Yet antioxidant therapy has failed, in some cases worsening pregnancy outcomes. The underlying molecular mechanisms affected by redox signalling in preeclampsia remain unclear. Reactive oxygen/nitrogen species (RONS) form diverse oxidative post-translational modifications (ox-PTMs) on redox-sensitive cysteines thiols. Ox-PTMs can regulate intracellular signalling by either potentiating or inhibiting protein activity. One of these, S-glutathionylation is a common thiol modification enzymatically reversed by glutaredoxin (Glrx) and is important in transducing redox signalling in cardiovascular disease. Glrx expression is increased in preeclamptic patients. We aimed to investigate the role of Glrx in preeclampsia, combining physiological in vivo assessment with redox proteomic analysis and in vitro studies to identify the molecular basis for how thiol modification removal may contribute to pregnancy-induced vascular complications.

Results: Overexpressing Glrx transgenic mice (TG) and littermate controls (WT) underwent timed pregnancy. During pregnancy, TG mice developed hypertension, kidney dysfunction and elevated plasma sFlt-1 compared to WT. Bioinformatics analysis identified several redox-sensitive targets directly relevant to preeclampsia and sFlt-1 regulation. In vitro studies investigated the effects of S-glutathionylation on protein activity in those targets, and consequences on sFlt-1 release. Of significance, S-glutathionylation removal was found to disrupt the spliceosome machinery consequently affecting Flt-1 splicing. Co-immunoprecipitation assays revealed that S-glutathionylation removal disrupted binding between spliceosome partners, promoting sFlt-1 release.

Conclusion: These data support the proposed hypothesis that Glrx’s removal of oxPTMs regulates sFlt-1 expression which may promote the ‘preeclampsia phenotype’ of hypertension and kidney dysfunction. Further studies will need to assess impact of imbalance in redox homeostasis during pregnancy on longterm endothelial and cardiac function. This study highlights how iodo-tandem mass tag proteomics can be used to profile redox sensitive pathways and determine how oxPTMs regulate specific proteins to control angiogenic pathways.