Therapeutic Interventions to Target Fetoplacental Vascular Dysregulation in Fetal Growth Restriction

Abstract

Fetal 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.