The effects of pravastatin on the normal human placenta: Lessons from <i>ex-vivo</i> models

<div><p>Introduction</p><p>Research in animal models and preliminary clinical studies in humans support the use of pravastatin for the prevention of preeclampsia. However, its use during pregnancy is still controversial due to limited data about its effect on the human placenta and fetus.</p><p>Methods</p><p>In the present study, human placental cotyledons were perfused in the absence or presence of pravastatin in the maternal reservoir (PraM). In addition, placental explants were treated with pravastatin for 5, 24 and 72 h under normoxia and hypoxia. We monitored the secretion of placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), soluble endoglin (sEng), endothelial nitric oxide synthase (eNOS) expression and activation and the fetal vasoconstriction response to angiotensin-II.</p><p>Results</p><p>The concentrations of PlGF, sFlt-1 and sEng were not significantly altered by pravastatin in PraM cotyledons and in placental explants compared to control. Under hypoxic conditions, pravastatin decreased sFlt-1 concentrations. eNOS expression was significantly increased in PraM cotyledons but not in pravastatin-treated placental explants cultured under normoxia or hypoxia. eNOS phosphorylation was not significantly affected by pravastatin. The feto-placental vascular tone and the fetal vasoconstriction response to angiotensin-II, did not change following exposure of the maternal circulation to pravastatin.</p><p>Conclusion</p><p>We found that pravastatin does not alter the essential physiological functions of the placenta investigated in the study. The relevance of the study lays in the fact that it expands the current knowledge obtained thus far regarding the effect of the drug on the normal human placenta. This data is reassuring and important for clinicians that consider the treatment of high-risk patients with pravastatin, a treatment that exposes some normal pregnancies to the drug.</p></div

Ferroptosis induces membrane blebbing in placental trophoblasts

Ferroptosis is a regulated, non-apoptotic form of cell death, characterized by hydroxy-peroxidation of discrete phospholipid hydroperoxides, particularly hydroperoxyl (Hp) forms of arachidonoyl- and adrenoyl-phosphatidylethanolamine, with a downstream cascade of oxidative damage to membrane lipids, proteins and DNA, culminating in cell death. We recently showed that human trophoblasts are particularly sensitive to ferroptosis caused by depletion or inhibition of glutathione peroxidase 4 (GPX4) or the lipase PLA2G6. Here, we show that trophoblastic ferroptosis is accompanied by a dramatic change in the trophoblast plasma membrane, with macro-blebbing and vesiculation. Immunofluorescence revealed that ferroptotic cell-derived blebs stained positive for F-actin, but negative for cytoplasmic organelle markers. Transfer of conditioned medium that contained detached macrovesicles or co-culture of wild-type target cells with blebbing cells did not stimulate ferroptosis in target cells. Molecular modeling showed that the presence of Hp-phosphatidylethanolamine in the cell membrane promoted its cell ability to be stretched. Together, our data establish that membrane macro-blebbing is characteristic of trophoblast ferroptosis and can serve as a useful marker of this process. Whether or not these blebs are physiologically functional remains to be established.Ministry of Education (MOE)Nanyang Technological UniversityPublished versionThe project was supported by the following National Institutes of Health (NIH) grants: P01 HD069316 (to Y.S.), R01 HD086325 (to Y.S., K.J.H., C.H.), U01 AI156924, R01 AI145406, P01 HL114453, R01 CA165065 (to V.E.K.), the 25 Club of MageeWomens Hospital (to Y.S.), a Magee-Womens Research Institute Postdoctoral Fellowship (to O.B.), the Jikei University School of Medicine Department of Obstetrics and Gynecology (to K.K.), Nanyang Technological University start-up grants M4082428.050 (to K.J.H.) and M4082352.050 (to C.H.), and Ministry of Education - Singapore, Academic Research Fund Tier 1 Award M4012229.050 (to C.H.). Deposited in PMC for release after 12 months

The effect of pravastatin on placental eNOS expression and activation in the human perfused cotyledon.

<p>Isolated cotyledons were perfused for 5 h in the absence (Cntl) or presence of 0.2 micromol/L pravastatin in the maternal (PraM) circulation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172174#pone.0172174.s002" target="_blank">S1 Fig</a>). A. a representative immunoblot demonstrating eNOS expression and activation, as detected by phosphorylation on serine 1177. B-C. The relative fold of eNOS and p-eNOS^Ser1177 expression is shown by mean ± SEM. D-E. The concentration of NO in collected samples were determined by measurements of nitrite and normalized to gram of perfused cotyledon. Cntl: n = 6; PraM: n = 11.</p

eNOS expression and activation in cultured placental explants exposed to various concentrations of pravastatin.

<p>Placental explants were incubated in the absence or presence of pravastatin (Pra; 1, 10, 200 or 2000 micromol/L) for 24 and 72 h (n = 5) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172174#pone.0172174.s003" target="_blank">S2 Fig</a>). A. a representative immunoblot demonstrating eNOS expression and activation, as detected by phosphorylation on serine 1177. B-C. The relative fold of eNOS and p-eNOS^Ser1177 expression is shown by mean ± SEM. D. The concentration of NO in collected samples were determined by measurements of nitrite and expressed relative to Cntl (untreated cultures).</p