Investigating metabolomic biomarkers of hypoxic ischaemic encephalopathy

Background An early objective biomarker to predict the severity of hypoxic-ischaemic encephalopathy (HIE) and identify infants suitable for intervention remains elusive. This thesis aims to progress metabolomic markers of HIE through a pipeline of biomarker discovery and validation by employing a novel untargeted mass spectrometry metabolomic method. Methodology Term infants with perinatal asphyxia were recruited, all having umbilical cord blood (UCB) drawn and biobanked within three hours of birth. HIE was defined by Sarnat score at 24hours and continuous multichannel-EEG. Infant neurodevelopment was assessed at 36-42 months using the Bayley Scales of Infant and Toddler Development Ed. III (BSID-III). Untargeted metabolomic analysis of UCB was performed using direct injection FT-ICR mass spectrometry (DI FT-ICR MS). Putative metabolite annotations and lipid classes were assigned and pathway analysis was performed. Results Untargeted metabolomic analysis: Thirty enrolled infants were diagnosed with HIE, including 17 mild, 8 moderate, and 5 severe cases. Pathway analysis revealed that ΔHIE was associated with a 50% and 75% perturbation of tryptophan and pyrimidine metabolism respectively, alongside alterations in amino acid pathways. Significant metabolite alterations were detected from six putatively identified lipid classes including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids and prenol lipids. Outcome prediction: Metabolite model scores significantly correlated with outcome R=0.429 (model A) and R=0.549 (model B) respectively. Model B demonstrates the potential to predict both severe outcome (AUROC of 0.915) and intact survival (AUROC of 0.800). The effect of haemolysis: On average 5% of polar and 1.5% of non-polar features were altered between paired haemolysed and clean samples. However unsupervised multivariate analysis concluded that the preanalytical variability introduced by haemolysis was negligible compared with the inherent biological inter-individual variability. Conclusion This research has employed untargeted metabolomics to identify potential early cord blood biomarkers of HIE and has performed the technical validation of previously proposed markers.

MicroRNA expression and function in neonatal hypoxic ischaemic encephalopathy

Hypoxic ischaemic encephalopathy (HIE) is a devastating neonatal condition which affects 2-3 per 1000 infants annually. The current gold standard of treatment - induced hypothermia, has the ability to reduce neonatal mortality and improve neonatal morbidity. However, to be effective it needs to be initiated within the therapeutic window which exists following initial insult until approximately 6 hours after birth. Current methods of assessment which are relied upon to identify infants with HIE are subjective and unreliable. To overcome this issue, an early and reliable biomarker of HIE severity must be identified. MicroRNA (miRNA) are a class of small non-coding RNA molecules which have potential as biomarkers of disease state and potential therapeutic targets. These tiny molecules can modulate gene expression by inhibiting translation of messenger RNA (mRNA) and as a result, can regulate protein synthesis. These miRNA are understood to be released into the circulation during cellular stress, where they are highly stable and relatively easy to quantify. Therefore, these miRNAs may be ideal candidates for biomarkers of HIE severity and may aid in directing the clinical management of these infants. By using both transcriptomic and proteomic approaches to analyse the expression of miRNAs and their potential targets in the umbilical cord blood, I have confirmed that infants with perinatal asphyxia and HIE have a significantly different UCB miRNA signature compared to UCB samples from healthy controls. Finally, I have identified and investigated 2 individual miRNAs; both of which show some potential as classifiers of HIE severity and predictors of long term outcome, particularly when coupled with their downstream targets. While this work will need to be validated and expanded in a new and larger cohort of infants, it suggests the potential of miRNA as biomarkers of neonatal pathological conditions such as HIE.

Therapeutically targeting mitochondrial redox signalling alleviates endothelial dysfunction in preeclampsia

Aberrant placentation generating placental oxidative stress is proposed to play a critical role in the pathophysiology of preeclampsia. Unfortunately, therapeutic trials of antioxidants have been uniformly disappointing. There is provisional evidence implicating mitochondrial dysfunction as a source of oxidative stress in preeclampsia. Here we provide evidence that mitochondrial reactive oxygen species mediates endothelial dysfunction and establish that directly targeting mitochondrial scavenging may provide a protective role. Human umbilical vein endothelial cells exposed to 3% plasma from women with pregnancies complicated by preeclampsia resulted in a significant decrease in mitochondrial function with a subsequent significant increase in mitochondrial superoxide generation compared to cells exposed to plasma from women with uncomplicated pregnancies. Real-time PCR analysis showed increased expression of inflammatory markers TNF-α, TLR-9 and ICAM-1 respectively in endothelial cells treated with preeclampsia plasma. MitoTempo is a mitochondrial-targeted antioxidant, pre-treatment of cells with MitoTempo protected against hydrogen peroxide-induced cell death. Furthermore MitoTempo significantly reduced mitochondrial superoxide production in cells exposed to preeclampsia plasma by normalising mitochondrial metabolism. MitoTempo significantly altered the inflammatory profile of plasma treated cells. These novel data support a functional role for mitochondrial redox signaling in modulating the pathogenesis of preeclampsia and identifies mitochondrial-targeted antioxidants as potential therapeutic candidates.