Placental Exosomes From Severe Preeclampsia Patients Disregulating Trophoblasts And Endothelial Cells Function

[Objective]Pre-eclampsia (PE) is a pregnancy-specific hypertensive syndrome that causes substantial maternal and fetal morbidity and mortality. The incomplete of placenta uterine spiral artery remodeling which is involved in trophoblasts is thought to be important to the pathogenesis in PE. High blood pressure caused by the systemic vascular spasm is associated with endothelial injury and dysfunction. Recently, Redman put forward a two-stage theory of the pathogenesis in PE In the first clinically silent stage, placental hypoperfusion develops because of insufficient trophoblast invasion into spiral arteries of the uterus early in pregnancy, which results in abnormal placentation. In the second stage, placental factors are released increasingly into the maternal circulation, leading to a systemic inflammatory response and endothelial dysfunction and clinical manifest disease. Accumulating evidences suggest that placenta-derived exosomes carry proteins and RNA molecules that in a redundant way influence pregnancy process. In this study, we tested the hypothesis that exosomes secreted from abnormal placenta may disregulate trophoblasts and endothelial cells function which in turn contribute to the aggravation of preeclampsia.[Methods]Serum samples were collected during the third trimester from normal pregnant women (n=15) and patients with severe PE (n=15). After sucrose density gradient centrifugation and two precipitation with PEG6000, fractions containing different densities were sequentially extracted from the top of the sample tube and analyzed by SDS-PAGE with exosomal marker proteins—anti-CD63, anti-CD81 and placenta-specific marker—anti-PLAP. Purified exosomes were labeled with PKH67 green fluorescent labeling kit and co-incubated with HTR-8/SVneo cells and HUVEC cells for 6h, and then observed by fluorescent microscopy. CCK-8 reagent was used to detect the influence of placental exosomes on the proliferation of HTR-8/SVneo cells. Western blot and qPCR analyzed the effects of placenta related exosome derived from PE serum on the expression of proliferation-associated molecule P-P65, MyD88, IL-6. The apoptosis of HUVEC cells were detected by-Cell Death Detection ELISA kit. Western blot analyzed the effects of placenta related exosomes derived from PE serum on endothelial cell nitric oxide — eNOS expression in HUVEC cells.[Results]As Western blot showed, after sucrose density gradient centrifugation of exosomes from the serum of the pregnant women, CD63, CD81, and PLAP were found mainly in 21%-34% density layer, demonstrating that serum placental related exosomes were mainly in 1.08-1.11g/mL. CCK-8 was used to detect the proliferation of HTR-8/SVneo cells, and the results showed that, compared with the normal control group, placental exosomes derived from serum of severe PE patients can significantly inhibit the proliferation of HTR-8/SVneo cells (OD450nm:1.09±0.21 Vs 0.86±0.21, P<0.05). Western blot showed exosomes from severe PE patients inhibited the expression of CCND1, activated NF-κB p65 inflammation signal pathway, and increased IL-6 mRNA levels. Cell Death Detection ELISA assay showed that placental exosomes derived from serum of severe PE patients promoted the apoptosis of HUVEC cells (relative change fold to normal groups:1.00±0.04 Vs 1.15±0.13, P=0.026); Western blot showed exosomes derived from serum of severe PE patients significantly inhibited the expression of eNOS in HUVEC cells.[Conclusions]Pregnancy-related exosomes secreted from placenta exist in maternal serum, while placental exosomes derived from serum of severe PE patients significantly inhibited the proliferation of HTR-8/SVneo cells, and activated NF-κB p65 inflammation signal pathway, resulting in sustained activation of inflammatory pathway in PE, causing the aggravation of PE. And also placental exosomes derived from serum of severe PE patients promoted apoptosis of HUVEC cells, inhibited eNOS expression, which may increased endothelial permeability, limited diastolic function, resulting in vascular endothelial dysfunction, and caused systemic symptoms of PE.