Natasha de Alwis

Preeclampsia is a serious pregnancy complication characterised by maternal vascular dysfunction, placental dysfunction, and organ injury, with no effective treatment currently available. Kampo, a system of Japanese traditional medicine comprising standardised herbal formulations, could target pathophysiological pathways driving preeclampsia. We evaluated the effects of select Kampo formulations on markers of preeclampsia using primary human trophoblasts, placental explants, human umbilical vein endothelial cells (HUVECs), and uterine microvascular endothelial cells (UtMVECs). Twelve formulations were initially screened in HUVECs, and six formulations advanced for further study. TNFα was used to induce endothelial dysfunction, and angiogenic, antioxidant, inflammatory, and vascular dysfunction markers were assessed. Overall, Kampo formulations had minimal effect on sFlt-1 expression and only modest effects on sFlt-1 secretion by primary human trophoblast. In contrast, several formulations consistently increased placental growth factor (PlGF) expression and secretion, upregulated HMOX1 in trophoblasts, and enhanced PlGF secretion from placental explants. In endothelial cells, Kampo treatment partially reversed TNFα-induced dysfunction, demonstrated by reduced VCAM1 expression, and additional endothelial cell type-dependent effects on ET-1 and inflammatory pathways. These findings indicate that selected Kampo formulations modulate key pathways involved in the pathophysiology underpinning preeclampsia and warrant further investigation as potential therapeutic candidates.

Preeclampsia is a life-threatening pregnancy complication, resulting in >70,000 maternal deaths and >500,000 fetal and neonatal deaths worldwide each year. Despite its prevalence and devastating consequences, there is still no medical therapy to halt disease progression and thus a vital need for novel treatments remains. The urgent demand for effective therapies for preeclampsia first necessitates a deeper understanding of the pathogenesis that underpins the condition. Understanding the coordinated interactions between the maternal vasculature and circulating pathogenic factors released from the placenta is a key focus in preeclampsia research. Extracellular vesicles are membrane-bound vesicles that act as essential mediators of cell–cell signalling. Extracellular vesicles are proposed to play a crucial role as regulators in placental–maternal vascular communication. This ­review explores contemporary literature investigating the role of placental-derived extracellular vesicles and their contribution to preeclampsia. Importantly, we provide a summary of key literature that should assist decision making for selection of models to study the role of extracellular vesicles in preeclampsia progression and their potential as therapeutic targets.

Background: Preeclampsia is a leading cause of maternal and fetal morbidity and mortality, with obesity recognised as a significant risk factor. However, the direct contribution of obesity to the pathophysiology underpinning preeclampsia remains unclear. Objectives: This study aimed to develop and characterise a diet-induced obese mouse model with superimposed preeclampsia to better understand the impact of obesity on disease pathogenesis. Methods: Female mice were fed either standard rodent chow or a high-fat diet from weaning. At 8 weeks of age, mice were mated. Pregnant mice were treated with L-N^G-Nitro arginine methyl ester (L-NAME; to block nitric oxide production) from gestational day (D)7.5 to D17.5 to induce a preeclampsia-like phenotype. Blood pressure was measured on D14.5 and D17.5, followed by the collection of maternal and fetal tissues for histological, biochemical, and molecular analyses. Results: Obese dams exhibited significantly increased body, fat pad, and liver weights compared to lean controls. While L-NAME induced hypertension in the control mice, contrary to expectations, the L-NAME-induced hypertension was partially attenuated in obese dams, with significantly lower systolic and diastolic blood pressures at D14.5 and reduced systolic pressure at D17.5. Fetal weights were comparable between groups, however, placentas were significantly heavier with obesity. Endothelial function, inflammatory markers, and renal gene expression patterns suggested distinct physiological adaptations in obese preeclamptic-like mice. Conclusions: These findings challenge the prevailing assumption that obesity drives hypertension, endothelial dysfunction, and inflammatory markers. The differential vascular and physiological responses observed in the obese dams highlight the complexity of obesity–preeclampsia interactions and underscore the need for refined preclinical models to disentangle mechanistic contributions. This work has implications for personalised management strategies and targeted therapeutic interventions in obese pregnancies at risk of preeclampsia.

Amniotic fluid (AF) is a valuable source of extracellular vesicles (EVs) derived from the fetoplacental unit. Preclinical and clinical studies have highlighted promising applications of AF-EVs and their role in cellular communication, yet our understanding of AF-EV physiology is limited. This study aimed to examine the physiological importance of AF-EVs in fetal development from the second trimester to term gestation. We obtained AF samples from routine second-trimester amniocentesis and prelabour Caesarean section at term. We isolated EVs using a combination of differential centrifugation, filtration, and ultracentrifugation and characterised them using nanoparticle tracking analysis, cryoelectron microscopy, and Western blotting. The differential EV proteome was analysed using label-free proteomics. We assessed the second trimester and term AF-EV properties through an enrichment analysis. The EV size and protein enrichment difference revealed a gestational-age-dependent variation in the predominant EV subtype. Second-trimester-derived EVs were enriched in ectosomes, while term EVs contained a significant proportion of exosomes. We identified several morphologies of AF-EVs, including unilamellar, multilamellar, multicompartmental and granular-centred EVs, across gestations. Proteomics analysis of AF-EVs identified 4137 proteins with high confidence, of which 1099 exhibited significant differential expression between the two groups. Second-trimester-enriched AF-EV proteins represented molecule assembly processes, metabolism and organogenesis. At term, AF-EV proteins corresponded to impending newborn functions such as immunity and digestion. In conclusion, we provide compelling evidence that EV biogenesis and secretion in the fetoplacental unit undergo significant alterations across gestation, revealing a complex and dynamic physiology and intercellular communication that adapts to the needs of the developing fetus. Abstract Figure: Graphical abstract This figure summarises the study’s main findings, including the shift in the predominant EV subtype, the different organs and biofluids represented by the enriched proteins at each gestation, and the main biological pathways.

BACKGROUND: Preeclampsia is a serious condition of pregnancy, complicated by aberrant maternal vascular dysfunction. CNP (C-type natriuretic peptide) contributes to vascular homeostasis, acting through NPR-B (natriuretic peptide receptor-B) and NPR-C (natriuretic peptide receptor-C). CNP mitigates vascular dysfunction of arteries in nonpregnant cohorts; this study investigates whether CNP can dilate maternal arteries in ex vivo preeclampsia models. METHODS: Human omental arteries were dissected from fat biopsies collected during cesarean section. CNP, NPR-B, and NPR-C mRNA expression was assessed in arteries collected from pregnancies complicated by preeclampsia (n=6) and normotensive controls (n=11). Using wire myography, we investigated the effects of CNP on dilation of arteries from normotensive pregnancies. Arteries were preconstricted with either serum from patients with preeclampsia (n=6) or recombinant ET-1 (endothelin-1; vasoconstrictor elevated in preeclampsia; n=6) to model vasoconstriction associated with preeclampsia. Preconstricted arteries were treated with recombinant CNP (0.001–100 µmol/L) or vehicle and vascular relaxation assessed. In further studies, arteries were preincubated with NPR-B (5 µmol/L) and NPR-C (10 µmol/L) antagonists before serum-induced constriction (n=4–5) to explore mechanistic signaling. RESULTS: CNP, NPR-B, and NPR-C mRNAs were not differentially expressed in omental arteries from preeclamptic pregnancies. CNP potently stimulated maternal artery vasorelaxation in our model of preeclampsia (using preeclamptic serum). Its vasodilatory actions were driven through the activation of NPR-B predominantly; antagonism of this receptor alone dampened CNP vasorelaxation. Interestingly, CNP did not reduce ET-1–driven omental artery constriction. CONCLUSIONS: Collectively, these data suggest that enhancing CNP signaling through NPR-B offers a potential therapeutic strategy to reduce systemic vascular constriction in preeclampsia.

Objective: Low molecular weight heparins, including enoxaparin, have been associated with increased non-invasive prenatal testing (NIPT) failures due to low fetal fraction of cell-free (cf) DNA. This study investigated the effect of enoxaparin and antiplatelet therapies on placental tissue in vitro, aiming to determine effects on cfDNA release. Method: First- and third-trimester human placental explants were treated with graded concentrations of enoxaparin, aspirin, clopidogrel, prasugrel and ticagrelor. Concentrations of cfDNA in the culture media were quantified and relative differences between treated and control samples were assessed using the Student’s T-test. Expression of apoptosis genes BAX and BCL2 was assessed in placental explants by RT-qPCR. Results: Enoxaparin treatment significantly increased cfDNA release by placental explants (p<0.01). This effect was more pronounced in the first trimester samples. Increase in cfDNA with enoxaparin was not associated with altered expression of apoptosis genes. There was no significant difference in cfDNA release in placental explants cultured with aspirin or newer anti-platelet agents. Conclusions: Our in vitro results suggest that NIPT failures associated with enoxaparin therapy are unlikely to be mediated by reduced placental cfDNA release. Antiplatelet agents do not have a significant impact on cfDNA release or apoptosis.

: In brief: Preterm birth is the leading cause of perinatal morbidity and mortality, and new therapies that delay preterm birth and improve neonatal outcomes are urgently needed. This study investigates whether ticagrelor inhibits uterine contractility and inflammation in preclinical in vitro, ex vivo (human) and in vivo (mouse) studies, to explore the potential of repurposing ticagrelor for the prevention of preterm birth. Preterm birth remains a significant global health challenge, affecting approximately 10% of pregnancies and resulting in one million deaths globally every year. Tocolytic agents, used to manage preterm labour, have considerable limitations including lack of efficacy, and adverse side effects, emphasising the urgent need for innovative solutions. Here, we explore repurposing an antiplatelet cardioprotective drug, ticagrelor, as a potential treatment to prevent preterm birth. Ticagrelor has demonstrated pleiotropic actions beyond platelet inhibition, including relaxant effects on smooth muscle cells and anti-inflammatory effects in models of diabetes and sepsis. As preterm birth is underscored by inflammatory processes triggering uterine contractions, these actions position ticagrelor as an attractive candidate for prevention or delay of preterm birth. Utilising primary human myometrial tissue, human myometrial cells, and a mouse model of preterm birth, we investigated ticagrelor’s potential as a safe and effective therapy for preterm birth. We showed that ticagrelor did not reduce the frequency or strength of spontaneous muscle contractions of ex vivo myometrial tissue nor did it reduce in vitro inflammation-induced contractility in myometrial cells. Additionally, ticagrelor did not exhibit the anticipated anti-inflammatory effects in myometrial cell culture experiments. In our mouse model of preterm birth, ticagrelor neither improved the preterm birth rate or fetal survival outcomes. Gene expression of pro-inflammatory cytokines and contraction-associated proteins in postpartum mouse uteri were unaltered by ticagrelor. In conclusion, ticagrelor is not a strong candidate to continue investigations in clinical trial for the treatment of preterm labour and prevention of preterm birth.

Gestational diabetes mellitus (GDM) is a condition of pregnancy defined by new-onset hyperglycemia. GDM is associated with impaired maternal endothelial and vascular reactivity. Endothelin-1 (ET-1) is a potent vasoconstrictor that contributes to endothelial dysfunction, however, its abundance and actions in GDM are unclear. Maternal plasma was obtained from pregnancies complicated by GDM (n = 24) and gestation-matched controls (n = 42); circulating ET-1 levels were assessed by ELISA. Human omental arteries from healthy pregnancies and those complicated by GDM were dissected from omental fat biopsies and collected at cesarean section. mRNA expression of ET-1 and its receptors, ET_A and ET_B, in addition to vascular cell adhesion molecule-1 (VCAM1) and intercellular adhesion molecule-1 (ICAM1) were assessed by qPCR (n = 28). Using wire myography, we investigated vascular constriction to ET-1 (10⁻¹¹–10⁻⁴ M) in omental arteries from pregnancies complicated by GDM, compared to gestation-matched controls (n = 7). GDM cases were stratified by clinical management, diet intervention (n = 5), or insulin treatment (n = 6). Additionally, arteries from healthy pregnancies were treated with insulin (1 mU/mL (n = 7) and 10 mU/mL (n = 5)) or vehicle control. Vasoactive response to ET-1 was measured via wire myography. Circulating ET-1 levels and mRNA expression of the ET-1 system in omental arteries were not found to be significantly different between pregnancies complicated by GDM compared to healthy controls. However, we found insulin treatment during pregnancy and in ex vivo models reduced ET-1 vasoconstriction of maternal vasculature in GDM. These data suggest insulin may improve vascular function in GDM, however, further investigation is needed to define the role of ET-1 in pregnancy.

Preeclampsia is associated with an increased lifelong risk of cardiovascular disease (CVD). It is not clear whether this is induced by persistent systemic organ and vascular damage following preeclampsia or due to a predisposition to both conditions that share cardiovascular pathophysiology. Common to both CVD and preeclampsia is the dysregulation of corin and its proteolytic product, atrial natriuretic peptide (ANP). ANP, a hypotensive hormone converted from pro-ANP by corin, is involved in blood pressure homeostasis. While corin is predominantly a cardiac enzyme, both corin and pro-ANP are significantly upregulated in the gravid uterus and dysregulated in preeclampsia. Relatively little is known about ANP function in the endothelium during a pregnancy complicated by preeclampsia. Here, we investigated the effect of ANP on endothelial cell proliferation and migration, markers of endothelial dysfunction, and receptor expression in omental arteries exposed to circulating preeclamptic toxins. ANP receptor expression is significantly upregulated in preeclamptic vasculature but not because of exposure to preeclampsia toxins tumour necrosis factor α or soluble fms-like tyrosine kinase-1. The supplementation of endothelial cells with ANP did not promote proliferation or migration, nor did ANP improve markers of endothelial dysfunction. The role of ANP in preeclampsia is unlikely to be via endothelial pathways.

Intrauterine growth restriction (IUGR), predominantly caused by placental insufficiency, affects partitioning of nutrients to the fetus. The system A sodium-coupled transporters (SNAT or SLC38), of types A1, A2, and A4, control non-essential amino acid uptake and supply. Here, we aimed to investigate the expression of these transporters across different placental disease cohorts and cells. To determine disease impact, transporter expressions at the gene (qPCR) and protein (western blots) level were assessed in gestationally matched placental tissues. Early (<34 weeks), and late (34–36 weeks) onset IUGR cases with/out preeclampsia were compared to preterm controls. We also investigated level of transporter expression in primary trophoblasts under glucose deprivation (n = 6) and hypoxia conditions (n = 7). SLC38A4 protein was significantly downregulated in early preterm pregnancies complicated with IUGR with/out preeclampsia. There were no differences in late preterm IUGR cohorts. Furthermore, we demonstrate for the first time in primary trophoblast cells, that gene expression of the transporters was sensitive to and induced by glucose starvation. SLC38A4 mRNA expression was also significantly upregulated in response to hypoxia. Thus, SLC38A4 expression was persistently low in early preterm IUGR pregnancies, regardless of disease aetiology. This suggests that gestational age at delivery, and consequently IUGR severity, may influence loss of its expression.