Pre-clinical characterisation of mitochondrial antioxidants as novel therapeutics for pre-eclampsia

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Williamson, Rachel
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University College Cork
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Pre-eclampsia, a pregnancy specific condition associated with significant morbidity and mortality, is characterised by high blood pressure accompanied by and/or proteinuria, acute kidney injury (AKI), liver dysfunction fetal growth restriction (FGR) after 20 weeks’ gestation in the second half of pregnancy. It affects 2-5% of pregnancies worldwide. There is overwhelming evidence showing oxidative stress plays a significant role in the aetiology of this disorder. The mitochondria are known to be a dominant cellular producer of reactive oxygen species. Mitochondrial dysfunction acts as a pathogenic mediator of oxidative stress in pre-eclampsia and modulates the clinical characteristics of this syndrome. There is evidence that mitochondrial dysfunction and the innate immune system both play roles in the pathophysiology of this disease. Mitochondrial damage-associated molecular patterns (DAMPs) such as mtDNA bind specific pattern recognition receptors such as Toll-like receptor 9 (TLR9) on the surface of immune cells, particularly neutrophils, subsequently activating them and triggering an innate immune response. Currently there is no treatment for pre-eclampsia except delivery of the placenta and the baby, with the resultant risk of iatrogenic prematurity and significant neonatal morbidity and mortality. As a result, intensive research endeavours have focused on defining the molecular mechanisms of pre-eclampsia and thereby expediting identification of new therapeutic targets for this condition. In pregnancies affected by pre-eclampsia there is an exaggerated oxidative stress phenotype with a correspondent deficient antioxidant response. The therapeutic potential of vitamin antioxidants in the prevention of pre-eclampsia was supported by extensive research, however, ultimately, they were unsuccessful in clinical trials. The ongoing hypothesis is that these vitamin antioxidants were sequestered in the cytosol and didn’t reach the mitochondria to alleviate the excess oxidative stress. Therefore, there is a need for the development of a specific mitochondrial-targeted antioxidant as a potential therapy for pre-eclampsia. This body of research provided evidence of mitochondrial dysfunction and a deficient antioxidant response in women who developed pre-eclampsia as early as 15 weeks’ gestation. However, there was no association found between lifestyle factors on mitochondrial dysfunction in the study cohort. Furthermore, there was increased activation of TLR9, a mediator of mtDNA induced innate inflammation in the same cohort, albeit at a later point in gestation. Additionally, there was a significant increase in the production of a number of neutrophil activation markers; calprotectin, myeloperoxidase and Interleukin-8 in women who developed pre-eclampsia. In the next study, I examined if L-ergothioneine, a water-soluble amino acid, and potential mitochondrial targeted antioxidant could mediate oxidative stress in an in vitro Jeg-3 placental cell line. In this novel study, L-ergothioneine exhibited cytoprotective effects against H2O2-induce cell death and significantly reduced exaggerated mitochondrial-specific ROS production in part by altering mild uncoupling. The protective effects of pre-treatment with L-ergothioneine were more pronounced when compared with another mitochondrial targeted antioxidant, MitoTempo and the non-mitochondrial targeted antioxidant N-acetylcysteine. The final study examined the potential therapeutic effects of L-ergothioneine in vivo using the reduced uterine perfusion pressure (RUPP) rat model of pre-eclampsia. In this study, treatment with L-ergothioneine significantly reduced blood pressure and rescued fetal growth restriction in RUPP rats. Furthermore, sFlt-1, an anti-angiogenic factor evident in preeclampsia, was significantly decreased in rats treated with L-ergothioneine. Additionally, using a novel ratiometric mass spectrometry probe, I showed that mitochondrial H2O2 production in vivo was significantly increased in the kidney from the RUPP rats and that pre-treatment with L-ergothioneine significantly reduced mitochondrial H2O2 production in vivo. In summary, this thesis has provided evidence of mitochondrial dysfunction in women who develop pre-eclampsia. Additionally, this work has shown that mitochondrial dysfunction activates a neutrophil-driven innate immune response in women with pre-eclampsia which is mediated in part by activation of the TLR9 receptor. Furthermore, this novel work established that L-ergothioneine, a unique antioxidant directly targets the mitochondria and reduces exaggerated mitochondrial specific ROS production in both in vitro and in vivo models of pre-eclampsia. Additionally, in the in vivo model of pre-eclampsia, treatment with Lergothioneine positively modulates some of the detrimental clinical characteristics evident in women with pre-eclampsia, highlighting the therapeutic potential of this mitochondrialtargeted antioxidant in the treatment of pre-eclampsia.
Pre-eclampsia , Mitochondrial dysfunction , Mitochondrial antioxidant , Therapeutic treatment
Williamson, R. 2019. Pre-clinical characterisation of mitochondrial antioxidants as novel therapeutics for pre-eclampsia. PhD Thesis, University College Cork.
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