Saudi Cultural Missions Theses & Dissertations
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Item Restricted Therapeutic Interventions to Target Fetoplacental Vascular Dysregulation in Fetal Growth Restriction(The University of Manchester, 2024-06-21) Almohammadi, Lujain; Brownbill, PaulFetal growth restriction (FGR) affects 3-7% of pregnancies and is a condition where the fetus fails to reach its genetic growth potential, often due to placental dysfunction. This can lead to stillbirth and in survivors increased risk of morbidity and ill health in later life. FGR is associated with reduced fetoplacental blood flow and elevated placental oxidative stress. Nitric oxide (NO) plays a crucial role in increasing blood flow, but in FGR, NO bioavailability and endothelial nitric oxide synthase (eNOS)-dependent vasodilation are impaired. Shear stress, which is the frictional force between the flowing blood and the endothelium, is a powerful stimulator of NO synthesis and vasodilation in several vascular beds. Flow mediated vasodilation (FMVD) is dysregulated in FGR. NOs is mainly produced by eNOS, using L-arginine as a substrate and various cofactors including tetrahydrobiopterin (BH4). Arginase-2 (ARG-2) competes with eNOS for L-arginine, thereby inhibiting NOS-dependent relaxation. BH4 is an essential cofactor for eNOS enzyme activity; without BH4, eNOS creates superoxide instead of NO. In this thesis, the focus was on investigating the effectiveness of (S-(2-boronoethyl)-L-cysteine) (BEC) as a potent and specific arginase inhibitor, along with BH4 both alone and in combination. Here two hypotheses were tested (a) Arginase inhibition by BEC, alone and with BH4, would augment NO production by the fetoplacental endothelium. (b) Arginase inhibition by BEC will reduce vascular tone and improve FMVD in normal pregnancy (NP) and FGR. The aim was to identify potential therapeutic strategies that could increase NO bioavailability, lower vascular resistance and improve FMVD in FGR. Two placental preparations were used to test the hypotheses (a) primary cultures of human placental arterial endothelial cells (HPAECs) (b) dual perfusion of the human placental cotyledon ex vivo. Experiments were performed on placentas from normal pregnancy (defined as the delivery of a singleton infant at term with an individualised birthweight ratio (IBR) between the 20th and 80th centiles) and FGR (defined as an infant with an IBR below the 3rd centile). Using HPAECs maintained in static culture conditions, the effects of BEC, BH4 and BEC+BH4 were investigated on the expression of cell stress related proteins (Proteome Profiler Human Cell Stress Array), the phosphorylation of protein kinases in the eNOS pathway (Human Phosphokinase Array) and on arginase activity (MAK384, Sigma-Aldrich kit). The effects of BEC and BH4 on the ratio of phospho-eNOS (S1177) to total eNOS, and on NO production (nitrite measured in culture medium after 48hr treatment using the Greiss reaction) were assessed in static conditions and in response to shear stress of 20dyn.cm2 (cells maintained under flow: Ibidi fluidic units). The effect of BEC on the fetoplacental vasculature of FGR was studied using the dual placental perfusion preparation. Baseline fetal inflow hydrostatic pressure (FIHP; a measure of fetoplacental vascular resistance), and the reduction in FIHP in response to incremental increases in flow rate (FMVD), were measured in NP and FGR. Initial studies to assess potential concentration-dependent effects of BEC and BH4 on HPAEC viability, showed that metabolism (MTT assay) was unaffected by BEC (0.125 µM -1250µM) but reduced by BH4 (at 0.2µM -20µM) (n=6 placentas: NP). BEC (12.5µM) reduced the expression of 15 pro-survival proteins, necessary for the regulation of cellular oxidative stress (expression <40% of control; pooled cell lysate from 3 placentas; NP); this effect was not reversed by BH4 (20 µM). BH4 alone increased the expression of proteins involved in cellular growth, angiogenesis, proliferation, activation of eNOS, regulation of inflammatory responses, and protection from reactive oxygen species (ROS). In general, neither BEC nor BH4 increased the expression of phosphorylated proteins involved in eNOS signalling. The ratio of active eNOS to total eNOS was also unaffected by BEC and BH4 in static culture conditions or under flow in NP (n=3-6) or FGR (n=2). Neither BEC nor BH4 increased NO production by HPAECs of NP (n=3-6) or FGR (n=2). However, BEC and BH4 failed to inhibit arginase enzyme activity in NP (n=6) or FGR (n=2). There was no evidence that BEC could lower fetoplacental vascular resistance or increase FMVD upon ex vivo placental perfusion in normal pregnancy (n=5). In the current study, reduced FMVD was not evident in FGR and BEC did not affect FIHP or FMVD (n=3) The limited number of samples used in this study precludes statistical analyses of some of the data and definitive conclusions cannot yet be made. The indication from the preliminary findings, that BEC failed to activate eNOS or stimulate NO production by HPAECs, could be related to its failure to inhibit arginase activity and/or its effect to lower the expression of anti-oxidant proteins. BH4 reduced cell metabolism but in general, increased the expression of proteins that have a cell protective effect. BH4 but did not elevate NO production alone or in concert with BEC. There was no evidence of the ability of the fetoplacental circulation to elicit a vasodilatory response upon perfusion with BEC arginase inhibitor in NP or FGR, arginase activity was not assessed in this preparation. Based on the preliminary findings, it seems that BEC might not be an effective therapeutic intervention for improving fetoplacental blood flow in cases of FGR. It is possible that BEC could increase oxidative stress through off-target actions, which could explain its lack of effect. Further studies are needed to determine whether BH4 could be a viable strategy for enhancing the ability of placental endothelial cells (ECs) to generate NO in FGR and reduce vascular resistance to improve blood flow.19 0Item Restricted Causes and Consequences of Reduced Placental Glutamine and Glutamate Uptake into The Human Placenta in Fetal Growth Restriction(Saudi Digital Library, 2023-09-19) Alharthi, Turki; Desforges, M; Dilworth, M.R; Greenwood, S.LFetal growth restriction (FGR) describes a fetus that fails to achieve its genetic growth potential and is a major risk factor for stillbirth. Placental dysfunction is the underlying cause of the majority of cases of FGR that cannot be explained by fetal congenital and genetic abnormalities. In FGR, placental uptake of amino acid has been found to be reduced in both humans and animal models. Glutamine is a vital amino acid for fetal growth and is important for the citric acid cycle, ammonia metabolism and the biosynthesis of purines and pyrimidines. Glutamate is a precursor of neurotransmitters and glutathione and regulates intracellular pH levels. Placental uptake of glutamine and glutamate at initial rate (proxy of transporter activity) is reduced in FGR. This is associated with an increased expression of key proteins that transport glutamine and glutamate into the placenta. Thus, the mechanisms underpinning reduced glutamine and glutamate uptake in FGR remain unknown. Additionally, it is assumed that this reduced activity of glutamine and glutamate transporters will result in a reduction in intracellular levels of glutamine and glutamate within the placenta but this assumption remains unproven. FGR is associated with elevated placental oxidative stress (OS) and an agent that induces OS, tert-Butyl hydroperoxide (tBOOH), inhibits amino acid (alanine and methionine) uptake into BeWo cells (human placental cell line that originates from a choriocarcinoma). However, the possibility that OS underlies the reduced placental uptake of glutamine and glutamate in FGR has not been investigated. The key research questions of this thesis are: (i) does oxidative stress reduce the uptake of glutamine and glutamate into placental villous tissue? (ii) does a reduction in placental uptake of glutamine and glutamate at initial rate in FGR translate to reduced intracellular levels in the placenta? Oxidative damage in placental villous explants from normal pregnancy was induced by hydrogen peroxide (H2O2) and tBOOH (1 mM for a 48 hr period), confirmed by increased lipid peroxidation. tBOOH markedly reduced the transporter mediated 24 hr uptake of 14C glutamine and 14C glutamate into villous explants (<10% of control). tBOOH also significantly reduced explant human chorionic gonadotropin (hCG) secretion, increased interleukin 6 (IL-6) secretion and increased apoptosis, suggestive of cell stress, but did not affect lactate dehydrogenase (LDH) release or syncytiotrophoblast regeneration. Despite elevating OS, H2O2 did not affect glutamine and glutamate uptake or any of the other variables measured. Given the suggestion that 1 mM tBOOH increased cell stress, a follow up study was conducted to assess the effect of tBOOH at varying concentrations (100 μM, 500 μM and 1 mM) on glutamine and glutamate uptake, hCG and IL-6 secretion, LDH release and the expression of intracellular cell stress-related proteins in placental explants. Glutamate uptake was reduced to 79% of control by 100 μM tBOOH and 57 % by 500 μM. Glutamine uptake was unaffected by 100 μM and inhibited to 73 % of control by 500μM tBOOH. The inhibitory effect of tBOOH at 500 μM was not associated with a significant change in IL-6 secretion or LDH release. The expression of intracellular cell stress-related proteins was altered by 100 μM, 500 μM and 1 mM tBOOH to varying degrees. To assess whether the previously reported reduction in glutamine and glutamate transporter activity in FGR resulted in lower intracellular levels at steady state, uptake of 14C glutamine and 14C glutamate into placental villous tissue was assessed at 24 hr in FGR versus normal pregnancies. There was no significant difference in glutamine or glutamate uptake at 24 hr. In summary, tBOOH induced oxidative damage and reduced glutamine and glutamate 24 hr uptake (proxy measure of intracellular levels) into villous explants. However, causal links between elevated oxidative stress and decreased amino acid uptake remain to be proven, especially given the increased cell stress observed following 1 mM tBOOH treatment and the lack of effect with H2O2. In addition, the intracellular levels of glutamine and glutamate in the placenta, measured over 24 hr, was not lower in FGR compared to normal pregnancy. It is unclear whether this lack of reduction is due to methodological differences between the previous study demonstrating a reduced glutamine and glutamate transporter activity in FGR, or whether this challenges the assumption that reduced activity leads to reduced intracellular levels of amino acid over time. This knowledge is key given the importance of amino acid for both placental metabolism and fetal growth.12 0