Purinergic signalling involvement in the regulation of the human placental vasculature

Fodor, P. (2022) Purinergic signalling involvement in the regulation of the human placental vasculature. PhD thesis, University of Nottingham.

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Abstract

Pregnancy involves a physiological series of actions which necessitate a coordinated transformation involving numerous organ systems. The human placenta supports and protects the growing fetus by facilitating an adequate oxygen and selective nutrient transfer to the fetus whilst functioning as a selective barrier. This notably vascularised fetal organ lacks neuronal innervation, therefore the regulation of the blood flow is adjusted via released vasoactive mediators within the local microcirculation. The role of adenosine 5’-triphosphate (ATP) and associated nucleotides in the control of systemic vascular tone has attracted much attention over the past few decades. However, few studies have assessed the role of purines at the level of the fetoplacental circulation. Here, extracellular nucleotides and their corresponding receptors may act as paracrine and autocrine signals in regulating the microvascular contractility of the low resistance placental blood vessels, potentially involving the P2X7-NLRP3-IL-1β pathway. The hypothesis of this investigation was that purinergic receptor agonists as well as danger associated molecular patterns (DAMPs) such as D-glucose and pathogen associated molecular patterns (PAMPs) for instance lipopolysaccharide (LPS) regulate the blood flow at the level of the chorionic plate and stem villous vasculature of the human placenta via their interaction with the endothelial and smooth muscle cells. This process is mainly achieved by involving the P2X7 receptor in the autoregulation of the blood flow and vessel tone which is altered by DAMPs, PAMPs and inflammation.

Human placental collected after written informed consent, from healthy pregnancies (N) and those complicated by preeclampsia (PE) and gestational diabetes mellitus (GDM) were used. All healthy and GDM were from elective Caesarean section at term (≥37 weeks). PE placenta samples fell within 33 and 39 weeks of gestation. The P2X7 receptor, Nod-like receptor protein-3 (NLRP3) inflammasome and procaspase-1 protein expression levels in stem villous artery lysates (N=8, PE=8 patients) were assessed using western blotting. No statistically significant differences in the relative P2X7 receptor levels were found between the two study groups using a Mann Whitney test (P=0.3823). The relative NLRP3 inflammasome levels did not significantly differ between the two study groups in the case of both 74kDa isoform (unpaired T-test) and 92kDa isoform (Mann Whitney test). The relative procaspase-1 levels between healthy and PE groups did not significantly differ (P=0.8785); Mann Whitney test. Chorionic plate artery (CPA), stem villous artery (SVA) and trophoblast tissue originating from N=10 healthy patients were incubated with RPMI media (control) with added antibiotics (4h), 10mM ATP (4h), 10μg/mL LPS (3.5h) + 10mM ATP (0.5h) and 10μg/mL LPS (3.5h) + 300μM 3’-O-(4-Benzoyl)benzoyl ATP (BzATP) (0.5h) and the released levels of IL-1β, IL-6 and IL-18 were measured using enzyme-linked immunosorbent assay. LPS+ATP condition induced the highest secretion of IL-1β in all tissue types studied. There were no statistically significant differences in IL-18 levels between control vs. ATP, control vs LPS+ATP and control vs LPS+BzATP in CPA, SVA and trophoblast tissue. The P2X7 receptor protein expression (N=3) in trophoblast tissue lysates incubated with 0, 5.5, 10, 20 and 30mM D-glucose for 4 and 48 hours was also assessed using western blotting. No statistically significant differences in P2X7 receptor levels were found between the 0h control and the D-glucose different concentrations at both timepoints using Dunnett’s T3 multiple comparison tests. IL-1β release also showed no statistically significant differences

between the D-glucose groups (Dunn’s multiple comparison tests). However, the measured LDH levels in the same media were notably decreased in 20mM and 30mM D-glucose subgroups at 48 hours and showed multiple statistically significant differences between 5.5mM vs. 20mM (*P=0.0101), 5.5mM vs. 30mM (*P=0.0254) and between 0mM vs. 20mM (*P=0.0395) D-glucose treated trophoblast tissue using the same statistical test.

The functional responses of resistance sized CPA’s (≤520μm) were investigated using wire myography. The vasoactive effects of ATP (10-5M to 10-2M) in the presence and absence of the P2X7 receptor antagonist A740003 (10-6M) were investigated using resistance chorionic plate arteries (n=5) originating from N=5 placental samples. ATP gave rise to spontaneous concentration-dependent vasoconstriction responses in the absence of A740003. Addition of A740003 with subsequent ATP stimulation elicited a notably downward shifted concentration response curve implying a marked loss of ATP efficacy. The exogenous application of BzATP on isolated CPA’s originating from healthy patients elicited a weak vasoconstriction response characterised by a particularly low efficacy of less than 25% of the U46619 standard. The absence of robust and reproducible responses to BzATP of isolated human placental chorionic plate arteries are due to the lack of involvement of the P2X7 receptor in the contractility of these blood vessels.

This study also provides evidence of a completely established in vitro model of a human chorionic vessel via the formation of a human umbilical vein endothelial cells (HUVEC) monolayer on transwell membrane inserts for the duration of 48 hours post- seeding followed by the coculture of HUVEC with chorionic plate artery smooth muscle cells (CPA-SMC) for a further 26 hours prior to the start of the permeability assay. The permeability data highlighted that CPA-SMC’s do not significantly alter the tightness of the bilayer compared to monoculture of HUVEC cells during all timepoints of the tracer leakage assay.

The P2X7 receptor and CD31 immunolocalization in placental villous tissue originating from N=12 healthy and N=6 diet managed GDM patients was investigated using immunohistochemistry. There was a significant downregulation of the P2X7 receptor surface expression in GDM vascular profiles (double labelled with CD31) compared to normal (**P=0.0063); Mann-Whitney test. The correlation between P2X7 downregulation and the endothelial monolayer permeability remains to be elucidated.

In conclusion, these studies suggest that extracellular ATP may act on various P2X receptors giving rise to a local constriction of superficial placental blood vessels denoting that circulating nucleotides are particularly involved in the regulation of the feto-placental blood flow. These results suggest that the CPA-SMC’s were unable to induce a notable alteration in the vascular permeability levels of established HUVEC monolayers which may denote that during the coculture of both primary cell types, a limited EC-SMC cross talk regarding vascular permeability took place. Further in vitro studies are necessary to fully understand the complicated interplay between isolated primary cells in co-culture conditions and to elucidate the potential involvement of multiple purinergic signalling pathways in the regulation of the human placental barrier function including the role of the P2X7 receptor in controlling the vascular permeability at the level of the chorionic plate arteries.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Leach, L.
Alexander, S.
Keywords: Human, placenta, vasculature, purinergic signalling, inflammation, pharmacology
Subjects: W Medicine and related subjects (NLM Classification) > WQ Obstetrics
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Graduate Entry Medicine and Health
UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 67143
Depositing User: Fodor, Paul
Date Deposited: 31 Jul 2022 04:40
Last Modified: 31 Jul 2022 04:40
URI: http://eprints.nottingham.ac.uk/id/eprint/67143

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