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Furthering the understanding of the redox control of soluble Epoxide Hydrolase and Protein Kinase G in the cardiovascular system

Student thesis: Doctoral ThesisDoctor of Philosophy

Redox regulation of proteins represents an important control mechanism that can finely tune cell homeostasis or responses to stress. Two proteins regulated in this way include soluble epoxide hydrolase (sEH) and protein kinase G (PKG). sEH hydrolyses epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs), which are less potent in terms of their ability to dilate blood vessels and lower blood pressure (BP) or induce angiogenesis. Thus inhibitors of sEH, which include lipid electrophiles, that adduct to C521, increase EET levels. As sepsis is a time of oxidative stress when lipid electrophiles can be generated, the hypothesis that vasodilation and hypotension at this time is mediated by C521-dependent inhibition of sEH was explored. Wild-type (WT) or ‘redox-dead’ C521S sEH knock-in (KI) mice, that are resistant to lipid electrophile-induced inhibition, were subjected to sepsis. There was no difference in BP, heart rate (HR) or activity. Analysis of isolated mesenteries or aortae by myography following sepsis did not identify any differences between genotypes. Similarly, there were no differences in plasma markers of organ damage or metabolic acidosis. Comprehensive comparison of the plasma inflammatory responses of each genotype identified a significant increase solely in granulocyte-colony stimulating factor (G-CSF), together with a similar trend in IL-17A in WT compared to KI. Overall it was concluded that inhibition of sEH by electrophilic lipids is unlikely to mediate blood pressure lowering during sepsis.
The attenuated increase in G-CSF and IL-17A in the C521S sEH KI was notable as it is important in vasculogenesis and angiogenesis, in which increased EETs have been implicated. Postischaemic revascularisation in the model of hindlimb ischaemia failed to identify differences between genotypes. Furthermore, the capillary/myofibre ratio in the gastrocnemius muscle, as well as G-CSF levels therein, were also similar between genotypes.
Recombinant sEH was also studied with a library of nitro-alkene fatty acids with the position of the double bond or electron-withdrawing nitro moiety altered, potentially allowing for more potent inhibitors of the hydrolase to be identified than previous studies with 10-nitro-oleic acid. Notably it was determined that low concentration of many of the electrophiles actually stimulated sEH, before inhibiting at higher concentrations.
Accumulating evidence suggests that C117 and C195 in PKG can be oxidised to from an intraprotein disulfide bond within the high affinity cGMP binding pocket, and this directly mediates oxidant-induced activation. Moreover, recent study has shown that nitroxyl (HNO) can induce intradisulfide-mediated PKG activation. To determine the potential physiological impact of this modification, C195S PKG KI mice were generated and characterised for their haemodynamic function. Basal BP, HR and cardiac function were not different between each group. When each genotype was exposed to a sepsis protocol, no differences in indices of well-being or haemodynamic parameters were observed. Effect of HNO donors, NCA and CXL-1020, on BP and HR was investigated in vivo. While NCA administration resulted in BP lowering in both genotypes to the similar extent, CXL-1020 resulted in attenuated BP only in WT mice and not KI animals, although the differences were not statistically significant and more experiments are required. Although cGMP-dependent vasorelaxation is intact in KI mice, the impact of the C195S mutation on cGMP binding requires further examination. Notably, KI mice basally have enlarged caecum and increased whole gut transit time, which was also reported in mice deficient in the nitric oxide-cGMP pathway.
Original languageEnglish
Awarding Institution
Award date2018


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