Page 229 - Haematologica - Vol. 105 n. 6 - June 2020

Placental extracellular vesicles induce preeclampsia
Significantly elevated plasma levels of pcEV have been consistently associated with the development and severity of preeclampsia.10,11
The pcEV found in patients with preeclampsia are het- erogeneous in their cells of origin, size, and cargo con- tents, and thus possess diverse activities, some of which may not be apparent or detectable in their parental cells. Despite this heterogeneity, extracellular vesicles (EV) from syncytiotrophoblasts are widely used as the surro- gate marker for detecting pcEV in the maternal circula- tion,12,13 even though there is no evidence in the literature that these pcEV directly cause preeclampsia. Placental cells vesiculate when the placenta is subjected to ischemic and hypoxic injuries that result in cell apoptosis and tissue necrosis.14-17 These injuries can also occur when trophoblasts are unable to infiltrate the wall of the placenta’s spiral artery to gradually replace the endothe- lium in a process called “blood vessel recasting”.14-17
Once released into the circulation, pcEV can cause endothelial injury,18-20 systemic inflammation,21,22 and coagulation dysfunction,23 all of which are hallmark events of preeclampsia. Despite extensive studies on the causes and mechanisms of placental injuries, key ques- tions remain as to whether pcEV released from injured placenta directly induce the hypertension and protein- uria that define preeclampsia or are merely the products of preeclampsia. If the former holds true, could accelerat- ing or enhancing the clearance of pcEV prevent preeclampsia or reduce its severity? Here we report the results of a study designed to answer these questions by analyzing blood samples from preeclamptic patients, studying new mouse models of preeclampsia, and con- ducting in vitro experiments.
Methods
Study of patients
Pregnant women were recruited from Tianjin Medical University General Hospital after they had given informed con- sent to participate in the study. Blood samples were collected from patients with preeclampsia at the diagnosis of their condition, before the administration of magnesium sulfate or anti-hyperten- sive medications, and analyzed for plasma levels of pcEV (Online Supplementary Methods). This study was approved by the Ethics Committee of Tianjin Medical University General Hospital.
Mouse models
We used three complementary models to study the role of pcEV in the development of preeclampsia (Online Supplementary Methods). The first model was designed to study whether increas- ing circulating pcEV in pregnant mice induced preeclampsia. For this study, we defined a mouse preeclampsia phenotype by hyper- tension, proteinuria, and kidney injury.1 Blood pressure (BP) was measured at baseline, 17-18 days post-coitum (dpc), and 7-10 days postpartum using a noninvasive mouse tail-cuff BP analyzer (CODA; Kent Scientific Co., Torrington, CT, USA).24 Urinary albu- min and creatinine concentrations in a pooled urine sample col- lected over a 24-h period were measured and their ratio was cal- culated to define proteinuria (Online Supplementary Methods). The second model was used to study whether pcEV induced hyperten- sion and proteinuria in non-pregnant C57BL/6J female mice. BP was measured 30 min after the pcEV infusion and 24 h urine sam- ples were analyzed for proteinuria. The third model was used to specifically investigate the role of EV clearance.
Flow cytometry
Levels of pcEV in plasma samples from women with a normal pregnancy or preeclampsia were measured using a fluorescein isothiocyanate-conjugated antibody against placental alkaline phosphatase (PLAP; LifeSpan Biosciences, Inc., Seattle, WA, USA).11,14,20,25 For the mouse study, we used syncytin as the marker for pcEV because PLAP is not expressed in mouse placenta (Online Supplementary Methods).26
Hematologic measurements
We used three tests to measure the procoagulant activity of pcEV: (i) a phosphatidylserine (PS)-dependent plasma-clotting assay that specifically measured microvesicle-mediated coagula- tion (Online Supplementary Methods);27 (ii) platelet activation; and (iii) detection of plasma levels of the anionic phospholipid PS- expressing EV. Platelet activation and PS+ EV were detected by, respectively, a phycoerythrin-CD62p antibody (eBiosciences) and allophycocyanin-annexin V (eBiosciences) using flow cytome- try.27,28
Vascular leakage
We used an Evans blue extravasation test to measure pcEV- induced vascular leakage in vivo (Online Supplementary Methods).28 We also measured the ability of pcEV to disrupt the integrity of cultured cells from the mouse endothelial line bEnd.3 (ATCC, Manassas, VA, USA) (Online Supplementary Methods).27,29
Microvesicle-induced vasoconstriction and changes in cerebral blood flow
We measured vascular wall tension using a modified protocol (Online Supplementary Methods).30 We also used non-invasive laser speckle contrast analysis (LASCA) technology to measure the impact of pcEV on cerebral blood flow. Cerebral blood flow was chosen because LASCA cannot accurately measure the blood flow of internal organs such as the kidney without surgery, which would have been a confounding injury that would have been dif- ficult to stratify.
Statisticalanalysis
Quantitative data are expressed as percentages for categorical (frequency) variables or as the mean ± standard error of the mean for continuous variables. For parametric data, a Shapiro-Wilk test was performed to test the distribution of the data. Data were ana- lyzed using a paired t-test or one-way or repeated-measures analy- sis of variance, as specified for each dataset. A P value of less than 0.05 was considered to be statistically significant. For multiple comparisons, the Holm-Sidak method was used to control for family-wise error rate (Sigma plot V. 11.2).
Results
Placenta-derived extracellular vesicles were detected in preeclamptic women and pregnant mice
Among the 17 pregnant women (all first pregnancies) recruited into the study, ten were diagnosed as having preeclampsia between 28 and 38 weeks of gestation. Their information is listed in Table 1. Patients were excluded from the study if they had baseline hypertension and dia- betes, developed pregnancy-associated diabetes, or were diagnosed as having eclampsia or HELLP syndrome. The women with preeclampsia developed hypertension and proteinuria and had earlier deliveries as compared to the women with normal pregnancies. Using PLAP as the surro- gate marker, pcEV were detected in peripheral blood sam-
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