Helen Bischof

Dysregulated flow-mediated vasodilatation in the human placenta in fetal growth restriction

A correlation was found between in vivo umbilical artery Doppler velocimetry and resistance to fetal‐side flow in the human ex vivo dually perfused placenta, highlighting that the fetoplacental vascular bed is a key site of resistance to umbilico‐placental flow in pregnancy. We discovered high resistance and poor flow‐mediated vasodilatory responses in placentas from pregnancies associated with fetal growth restriction (FGR). Endothelial cells isolated from the FGR placentas and grown in static and flow culture showed a dysregulated phenotype, with biochemical signalling demonstrating a failed compensatory response to high blood‐flow resistance.

Cell free hemoglobin in the fetoplacental circulation: A novel cause of fetal growth restriction?

Cell free hemoglobin impairs vascular function and blood flow in adult cardiovascular disease. In this study, we investigated the hypothesis that free fetal hemoglobin (fHbF) compromises vascular integrity and function in the fetoplacental circulation, contributing to the increased vascular resistance associated with fetal growth restriction (FGR). Women with normal and FGR pregnancies were recruited and their placentas collected freshly postpartum. FGR fetal capillaries showed evidence of erythrocyte vascular pacκing and extravasation. Fetal cord blood fHbF levels were higher in FGR than in normal pregnancies (P < 0.05) and the elevation of fHbF in relation to heme oxygenase‐1 suggests a failure of expected catabolic compensation, which occurs in adults. During ex vivo placental perfusion, pathophysiological fHbF concentrations significantly increased fetal‐side microcirculatory resistance (P < 0.05). fHbF sequestered NO in acute and chronic exposure models (P <0.001), and fHbF‐primed placental endothelial cells developed a proinflammatory phenotype, demonstrated by activation of NF‐κB pathway, generation of IL‐1ä and TNF‐ä (both P < 0.05), uncontrolled angiogenesis, and disruption of endothelial cell flow alignment. Elevated fHbF contributes to increased fetoplacental vascular resistance and impaired endothelial protection. This unrecognized mechanism for fetal compromise offers a novel insight into FGR as well as a potential explanation for associated poor fetal outcomes such as fetal demise and stillbirth.—Brooκ, A., Hoaκsey, A., Gurung, R., Yoong, E. E. C., Sneyd, R., Baynes, G. C., Bischof, H., Jones, S., Higgins, L. E., Jones, C., Greenwood, S. L., Jones, R. L., Gram, M., Lang, I., Desoye, G., Myers, J., Schneider, H., Hansson, S. R., Crocκer, I. P., Brownbill, P. Cell free hemoglobin in the fetoplacental circulation: a novel cause of fetal growth restriction? FASEB J. 32, 5436–5446 (2018). www.fasebj.org

189 Altered platelet function and thrombosis in fetal growth restriction

Background Fetal growth restriction (FGR) affects 3%–8% of pregnancies and is associated with increased risk of stillbirth and perinatal morbidity, in addition to increased risk of cardiovascular disease later in life. In severe cases of FGR, vascular resistance in the placenta is increased and blood flow reduced, compromising oxygen and nutrient exchange between mother and fetus. Hypoxia and endothelial dysfunction, have both been demonstrated in FGR, and both have the ability to alter thrombosis and haemostasis. The aim of this study was to determine whether there is increased platelet activation and thrombosis in FGR which may contribute to its pathogenesis. Methods Dual ex-vivo perfusions were performed on placentas from healthy pregnancies (n=10) and those affected by FGR (n=11). Following perfusion (~5 hour), placental cross-sections were processed and stained with H and E and Martius Scarlet Blue for blind-randomised histological analysis or immunofluorescence to analyse levels of fibrin and tissue factor. Fetal blood samples were collected from the umbilical vein immediately following delivery (n=10 healthy; n=8 FGR) and platelet function analysed using the PFA200 Results Placental sections from pregnancies affected by FGR exhibited significantly more thrombi (p<0.01) within the fetal vasculature than healthy placental sections. Thrombi could be observed in both large conduit and small resistance vessels, with many small vessels completely occluded. In FGR, total fibrin deposition in placental tissue was significantly increased (p<0.001), as was fibrin expression specifically located within fetal vessels (p<0.05). In contrast, tissue factor levels remained unaltered. Blood samples from fetal growth restricted babies demonstrated increased closure time in platelet function assays (122±12.6 s) compared to healthy controls (90.10±5.46 s), and platelet counts were significantly reduced (p<0.05). The red blood cell count, white blood cell count and mean platelet volume were not significantly different. Conclusions These data indicate that in FGR there is increased platelet activation and thrombosis in fetoplacental vessels, which may contribute to increased vascular resistance through the occlusion of small resistance arteries. Anti-thrombotic therapies, which cross the placenta may therefore be beneficial in FGR to prevent placental thrombosis and improve pregnancy outcome.

167?TRPV4; a Potential New Target to Increase Placental Blood Flow in Fetal Growth Restricted Pregnancies?

Introduction Fetal growth restriction (FGR) is a pregnancy complication that predisposes to an increased risk of neonatal morbidity and mortality. A low birth weight is also associated with increased risk of cardiovascular disease and metabolic syndrome in adulthood. While the aetiology of FGR is unknown, reduced fetoplacental blood flow is thought to be a major contributing factor. Normal fetal growth requires a high-flow low-resistance fetoplacental circulation, maintained by vasodilatation. One of the most potent stimulators of vasodilatation is shear stress. Understanding the mechanisms that regulate shear stress-mediated vasodilatation in the placenta may reveal novel therapeutic targets in pregnancies complicated by increased vascular resistance such as FGR. The aim of this study was to investigate whether the potential shear stress sensor TRPV4 was present on the fetoplacental endothelium and involved in regulating vascular tone. Methods TRPV4 expression in placental vessels was determined using immunohistochemistry. To examine endothelial specific expression, Western blots were performed on lysates from human placental artery endothelial cells (HPAECs). TRPV4 mediated Ca2+responses were measured in FURA2 loaded HPAECs using a FlexStation3 and single cell Ca2+ imaging. Nitric oxide and PGI2 release from HPAECs was measured following TRPV4 stimulation and placental vascular reactivity measured using wire myography. Results Shear stress induced elevation of [Ca2+]i in HPAECs was largely dependent upon Ca2+ influx, indicating the involvement of a Ca2+ permeable cation channel. TRPV4 was expressed by placental vessels and more specifically was shown to be expressed by HPAECs. Stimulation of TRPV4 (GSK1016790A 0.1–100µM) resulted in a dose-dependent transient Ca2+ influx in HPAECS which was abolished by EGTA (4 mM). Incubation of HPAECS with GSK1016790A (0.5 µM) induced nitric oxide and PGI2 generation indicating that TRPV4 is capable of regulating vascular tone in the placenta. Furthermore, application of GSK1016790A (0.5 µM) to pre-constricted chorionic plate arteries stimulated a 51.9% relaxation of intact vessels and 28.2% relaxation of endothelial denuded vessels, demonstrating that TRPV4 mediates both endothelial dependent and independent vasodilatation of placental vessels. Conclusion TRPV4 stimulates vasodilatation of placental vessels through a variety of signalling pathways and may offer a novel therapeutic target in pregnancies complicated by increased vascular resistance.

PM.09 The Effect of Glucocorticoids on Angiogenesis in the Human Placenta

Background Fetal growth restriction (FGR) is associated with glucocorticoid (GC) excess in human pregnancies and animal models. In mice and rats, GC treatment reduces placental angiogenesis and dysregulates the expression of angiogenic factors. It is not known whether GCs reduce angiogenesis in the human placenta. Hypothesis Glucocorticoid excess in the human placenta inhibits angiogenesis by dysregulating angiogenic factors. Methods Human umbilical vein endothelial cells (HUVECs) and human placental artery endothelial cells (HPAECs) were treated with hydrocortisone (HC), prednisolone (PRED) and dexamethasone (DEX) for 24–48 hours. Tube-like structure (TLS) formation on matrigel, cell migration, proliferation and apoptosis were assessed. Chorionic plate arteries (CPAs) from normal placentas (n = 10) were cultured for 48 hours with HC or DEX. mRNA expression of six angiogenic factors were quantified using real-time Q-PCR with normalisation to TBP. Results Pilot studies in HUVECs (n = 3, p < 0.05) and subsequent experiments in HPAECs (n = 7, p < 0.05–0.01) treated with 10–1,000 nM HC, PRED and DEX showed reduced TLS formation and cell migration compared to vehicle control cells. GCs had no effect on cell proliferation, apoptosis or viability. HC and DEX treatment reduced the expression of fibroblast growth factor-2 (FGF-2) (p < 0.001), interleukin-8 (p < 0.001), VEGF-A (p < 0.01), VEGF-C (p < 0.01), matrix metalloproteinase-16 (p < 0.01), matrix metalloproteinase-1 (p < 0.05) and CCL-2 (p < 0.05). Discussion GCs reduced tube formation and cell migration, key facets of angiogenesis, in HUVEC and HPAEC models. These findings indicate that GCs inhibit human placental angiogenesis, which could contribute to the pathogenesis of FGR. The downregulation of specific angiogenic factors by GCs identifies putative mechanistic pathways involved.