Dr Saije Endacott

Introduction: Fetal growth restriction (FGR) is a leading cause of infant morbidity and mortality. Approximately 60% of FGR cases result from placental dysfunction, often due to defective remodelling of the uterine vasculature and subsequent exposure to hypoxia/reoxygenation that induces oxidative stress. Angiotensin-converting enzyme 2 (ACE2) counteracts the ACE-driven axis of the renin-angiotensin system and is reduced in FGR placentae. We aimed to investigate the role of ACE2 in protecting against placental oxidative stress induced via a hypoxia/reoxygenation event. Methods: Term placental explants were exposed to normoxia (8% O2) for 6 hrs or were treated with media alone or recombinant human (rh)ACE2 and exposed to a hypoxia/reoxygenation insult (1 hr hypoxia (1% O2), 5 hrs normoxia). Oxidative stress markers, and ACE and ACE2 mRNA, protein, or activity were assessed. Results: ACE2 mRNA expression was increased with hypoxia/reoxygenation compared with normoxia (p=0.045). Hypoxia/reoxygenation significantly increased placental mRNA expression of the oxidative enzymes NOX4 and NOX5 compared with normoxia (p=0.021 and 0.023). NOX5 protein was not significantly different between normoxic controls and hypoxia/reoxygenation; however, rhACE2 significantly reduced NOX5 protein levels (p=0.015). Antioxidant activity of SOD decreased (p=0.028), while CAT increased with hypoxia/reoxygenation (p=0.010). Placental Nrf2 and NQO1 mRNA expression increased with rhACE2 compared with hypoxia/reoxygenation alone (p=0.038 and 0.013). Conclusion: We have characterised the redox-sensitive changes that occur in the placenta due to defective placentation and hypoxia/reoxygenation and have shown for the first time the role of placental ACE2 in mitigating oxidative insult associated with hypoxia/reoxygenation.

Preeclampsia is classified as new-onset hypertension coupled with gross endothelial dysfunction. Placental (pro)renin receptor ((P)RR) and plasma soluble (P)RR (s(P)RR) are elevated in patients with preeclampsia. Thus, we aimed to interrogate the role (P)RR may play in the pathogenesis of preeclampsia. Human uterine microvascular endothelial cells (HUtMECs, n = 4) were cultured with either; vehicle (PBS), 25¿100 nM recombinant s(P)RR, or 10 ng/ml TNF-a (positive control) for 24 h. Conditioned media and cells were assessed for endothelial dysfunction markers via qPCR, ELISA, and immunoblot. Angiogenic capacity was assessed through tube formation and adhesion assays. Additionally, pregnant rats were injected with an adenovirus overexpressing s(P)RR from mid-pregnancy (day 8.5), until term (n = 6¿7 dams/treatment). Maternal and fetal tissues were assessed. HUtMECs treated with recombinant s(P)RR displayed increased expression of endothelial dysfunction makers including vascular cell adhesion molecule-1, intracellular adhesion molecule-1, and endothelin-1 mRNA expression (P = 0.003, P = 0.001, P = 0.009, respectively), along with elevated endothelin-1 protein secretion (P < 0.001) compared with controls. Recombinant s(P)RR impaired angiogenic capacity decreasing the number of branches, total branch length, and mesh area (P < 0.001, P = 0.004, and P = 0.009, respectively), while also increasing vascular adhesion (P = 0.032). +ADV rats exhibited increased systolic (P = 0.001), diastolic (P = 0.010), and mean arterial pressures (P = 0.012), compared with -ADV pregnancies. Renal arteries from +ADV-treated rats had decreased sensitivity to acetylcholine-induced relaxation (P = 0.030), compared with -ADV pregnancies. Our data show that treatment with s(P)RR caused hypertension and growth restriction in vivo and caused marked endothelial dysfunction in vitro. These findings demonstrate the significant adverse actions of s(P)RR on vascular dysfunction that is characteristic of the preeclamptic phenotype. (Figure presented.)

Objective: To determine the levels of soluble (pro)renin receptor (s(P)RR) in women carrying Aboriginal and/or Torres Strait Islander (First Nations) babies and investigate whether s(P)RR levels change in women who have complicated pregnancies. Study Design: Cross-sectional analysis of data (2010¿2018). Data/samples were from the Gomeroi Gaaynggal Study, a longitudinal cohort study based on Gomeroi/Kamilaroi lands (Tamworth), NSW, Australia. Third trimester samples (blood/urine) were collected from pregnant women carrying a First Nations baby (N = 188). Methods/Main outcome measures: Plasma s(P)RR and markers of kidney function (plasma: creatinine, urea and cystatin C; urinary: creatinine, protein, albumin, angiotensinogen, nephrin and Na/K) were measured by enzyme-linked immunosorbent assay or standardised pathology procedures as needed. Results: Soluble (P)RR was detected in plasma of women in the cohort (median: 19.86 ng/mL; IQR: 12.52¿26.8). Soluble (P)RR levels correlated positively with maternal plasma creatinine (P = 0.0001) and gestational age in the third trimester (P = 0.002). Levels of s(P)RR tended to positively correlate with urinary protein/creatinine (P = 0.04) and nephrin/creatinine (P = 0.03). Soluble (P)RR levels tended to be higher in women who birthed prematurely (P = 0.06). Soluble (P)RR levels did not change with other pregnancy complications or outcomes (preeclampsia, GDM or small or large for gestational age birth). Conclusions: Soluble (P)RR is present in the plasma of pregnant women carrying First Nations babies and is correlated with known urinary biomarkers of renal function. Increased maternal s(P)RR levels may be associated with increased risk of preterm birth.

The (pro)renin receptor ((P)RR) is a multi-functional protein that can be proteolytically cleaved and released in a soluble form (s(P)RR). Recently, the (P)RR and s(P)RR have become of interest in pregnancy and its associated pathologies. This is because the (P)RR not only activates tissue renin angiotensin systems, but it is also an integral component of vacuolar-ATPase, activates the wingless/integrated (Wnt)/ß-catenin and extracellular signal regulated kinases 1 and 2/mitogen-activated protein kinase signalling pathways, and stabilises the ß subunit of pyruvate dehydrogenase. Additionally, s(P)RR is detected in plasma and urine, and maternal plasma levels are elevated in pregnancy complications including fetal growth restriction, preeclampsia and gestational diabetes mellitus. Therefore, s(P)RR has potential as a biomarker for these pregnancy pathologies. Preliminary functional findings suggest that s(P)RR may be important for regulating fluid balance, inflammation and blood pressure, all of which contribute to a successful pregnancy. The (P)RR and s(P)RR regulate pathways that are known to be important in maintaining pregnancy, however their role in the physiological context of pregnancy is poorly characterised. This review summarises the known and potential functions of the (P)RR and s(P)RR in pregnancy, and how their dysregulation may contribute to pregnancy complications. It also highlights the need for further research into the source and function of s(P)RR in pregnancy. Soluble (P)RR levels could be indicative of placental, kidney or liver dysfunction and therefore be a novel clinical biomarker, or therapeutic target, to improve the detection and treatment of pregnancy pathologies.

Angiotensin-converting enzyme 2 (ACE2) is a membrane-bound protein containing 805 amino acids. ACE2 shows approximately 42% sequence similarity to somatic ACE but has different biochemical activities. The key role of ACE2 is to catalyze the vasoconstrictor peptide angiotensin (ANG) II to Ang-(1-7), thus regulating the two major counterbalancing pathways of the renin-angiotensin system (RAS). In this way, ACE2 plays a protective role in end-organ damage by protecting tissues from the proinflammatory actions of ANG II. The circulating RAS is activated in normal pregnancy and is essential for maintaining fluid and electrolyte homeostasis and blood pressure. Renin-angiotensin systems are also found in the conceptus. In this review, we summarize the current knowledge on the regulation and function of circulating and uteroplacental ACE2 in uncomplicated and complicated pregnancies, including those affected by preeclampsia and fetal growth restriction. Since ACE2 is the receptor for SARS-CoV-2, and COVID-19 in pregnancy is associated with more severe disease and increased risk of abnormal pregnancy outcomes, we also discuss the role of ACE2 in mediating some of these adverse consequences. We propose that dysregulation of ACE2 plays a critical role in the development of preeclampsia, fetal growth restriction, and COVID-19-associated pregnancy pathologies and suggest that human recombinant soluble ACE2 could be a novel therapeutic to treat and/or prevent these pregnancy complications.

This study aimed to determine if the (pro)renin receptor (ATP6AP2) changes the cellular profile of choriocarcinomas from cytotrophoblast cells to terminally syncytialised cells and ascertain whether this impacts the invasive potential of choriocarcinoma cells. Additionally, we aimed to confirm that FURIN and/or site 1 protease (MBTPS1) cleave soluble ATP6AP2 (sATP6AP2) in BeWo choriocarcinoma cells and determine whether sATP6AP2 levels reflect the cellular profile of choriocarcinomas. BeWo choriocarcinoma cells were treated with ATP6AP2 siRNA, FURIN siRNA, DEC-RVKR-CMK (to inhibit FURIN activity), or PF 429242 (to inhibit MBTPS1 activity). Cells were also treated with forskolin, to induce syncytialisation, or vehicle and incubated for 48 h before collection of cells and supernatants. Syncytialisation was assessed by measuring hCG secretion (by ELISA) and E-cadherin protein levels (by immunoblot and immunocytochemistry). Cellular invasion was measured using the xCELLigence real-time cell analysis system and secreted sATP6AP2 levels measured by ELISA. Forskolin successfully induced syncytialisation and significantly increased both BeWo choriocarcinoma cell invasion (P < 0.0001) and sATP6AP2 levels (P= 0.02). Treatment with ATP6AP2 siRNA significantly inhibited syncytialisation (decreased hCG secretion (P= 0.005), the percent of nuclei in syncytia (P= 0.05)), forskolin-induced invasion (P= 0.046), and sATP6AP2 levels (P < 0.0001). FURIN siRNA and DEC-RVKR-CMK significantly decreased sATP6AP2 levels (both P < 0.0001). In conclusion, ATP6AP2 is important for syncytialisation of choriocarcinoma cells and thereby limits choriocarcinoma cell invasion. We postulate that sATP6AP2 could be used as a biomarker measuring the invasive potential of choriocarcinomas. Additionally, we confirmed that FURIN, not MBTPS1, cleaves sATP6AP2 in BeWo cells, but other proteases (inhibited by DEC-RVKR-CMK) may also be involved.

Introduction: In the placenta, the (pro)renin receptor (ATP6AP2) is localised to the syncytiotrophoblast. ATP6AP2 can activate the placental renin-angiotensin system (RAS), producing Angiotensin II (Ang II) which, acting via the angiotensin II type 1 receptor (AGTR1), is important for placental development and function. ATP6AP2 can also independently stimulate intracellular signalling pathways known to regulate trophoblast syncytialisation. We proposed that ATP6AP2 plays a role in trophoblast syncytialisation. Methods: Primary trophoblast cells were isolated from human placentae and transfected with an ATP6AP2 siRNA, a negative control siRNA or vehicle and allowed to spontaneously syncytialise. Syncytialisation was determined by secretion of human chorionic gonadotrophin (hCG) and by decreased CDH1 (E-cadherin) levels. Expression of RAS mRNAs and proteins were measured by qPCR and immunoblotting, respectively. Results: Primary trophoblast cells spontaneously syncytialised in culture. Syncytialisation did not affect ATP6AP2 mRNA or protein levels. However, the expression of REN, AGT and AGTR1 mRNAs were increased (P = 0.02, P = 0.01 and P = 0.03, respectively). ATP6AP2 siRNA had no effect on syncytialisation. Discussion: In primary trophoblasts, syncytialisation was associated with increased expression of the RAS. hCG was increased during syncytialisation and is known to stimulate REN and possibly AGT, however further experiments are needed to confirm that this was the mechanism via which the RAS was activated. Therefore, syncytialisation of primary trophoblasts may involve hCG-induced RAS activation and downstream activation of signalling pathways and growth factors, which can be stimulated via the interaction of Ang II with AGTR1. Nevertheless, it appears that the (pro)renin receptor is not involved.

Introduction: Placental development occurs in a low oxygen environment, which stimulates angiogenesis by upregulating vascular endothelial growth factor A (VEGFA), plasminogen activator inhibitor-1 (SERPINE1) and the angiopoietin-2/-1 ratio (ANGPT2/1). At this time, Angiotensin II type 1 receptor (AT1R) is highly expressed. We postulated that the early gestation placental oxygen milieu, by stimulating the angiotensin (Ang) II/AT1R pathway, increases expression of proliferative/angiogenic factors. Methods: HTR-8/SVneo cells were cultured in 1%, 5% or 20% O2 with the AT1R antagonist (losartan) for 48 h. mRNA and protein levels of angiogenic factors were determined by qPCR and ELISA. Angiogenesis and cell viability were assessed by HUVEC tube formation and resazurin assay. Results: Culture in low oxygen (1%) increased angiogenic VEGFA, SERPINE1 and placental growth factor (PGF) mRNA and VEGFA and SERPINE1 protein levels, and reduced anti-angiogenic ANGPT1, endoglin (ENG) and soluble fms-like tyrosine kinase-e15a (sFlt-e15a) mRNA (all P = 0.0001). At 1% oxygen, losartan significantly reduced intracellular VEGFA and SERPINE1 levels and secreted VEGF levels (P = 0.008, 0.0001 and 0.0001). HUVEC tube formation was increased in cells grown in HTR-8/SVneo conditioned medium from 1 to 5% cultures (all P = 0.0001). HUVECs cultured in medium from losartan treated HTR-8/SVneo cells had a reduced number of meshes, branching points and total branching length (P = 0.004, 0.003 and 0.0002). At 1% oxygen, losartan partially inhibited the oxygen-induced increase in cell viability (P = 0.0001). Discussion: Thus, AT1R blockade antagonised the low oxygen induced increase in pro-angiogenic factor expression and cell viability. Our findings highlight a role for an oxygen-sensitive Ang II/AT1R pathway during placentation.