Differential Proteomic Analysis Of Syncytiotrophoblast Extracellular Vesicles From Early-Onset Severe Pre-Eclampsia

Background and Objective:Hypertensive disorders complicating preganancy(HDCP) is a group of idiopathic diseases during pregnancy. The average morbidity of HDCP is 5 to 8 percent in China, and 10 to 16 percent of maternal deaths were reported to be due to HDCP. Pre-eclampsia(PE) / eclampsia is the most dangerous type of HDCP, which is a pregnancy-specific disease characterized by new-onset hypertension and proteinuria after 20 weeks gestation, even secondary maternal multiple organ dysfunction and fetal complications. PE is divided into mild and severe type according to the severity of the disease, and also divided into early-onset(﹤34 weeks’ gestation) and late-onset(≥34 weeks’ gestation) subtypes. The early-onset sPE is particularly harmful to maternal and perinatal health, both in short- and long term adverse effect. However, as the pathogenesis of PE remains not well illuminated and lacked the simple, effective, reliable and economical prediction methods, symptomatic treatment combined with termination of pregnancy is the only definitive treatment, and the outcome is not optimistic.New treatment strategies depend on the better understanding of etiology and pathogenesis. It is generally accepted ‘two-stage’ theory as well as the recently formed ‘three-stage’ etiology theory. Placenta derived pathogenic factors release is the key pathophysiological process of PE onset, and endothelial injury is the terminal pathway and the center part of the disease. Syncytiotrophoblast extracellular vesicles(STB-EVs) are membrane vesicle-like structures, which continuously shed and secreted from the placental syncytium into the maternal circulation. STB-EVs comprised syncytiotrophoblast microvesicles(STBM) and syncytiotrophoblast exosomes(STBE). STB-EVs ranged from 1.5 percent to 3 percent of the total microparticles in plasma. As a placenta derived factor, STB-EVs has been extensively studied more than twenty years. The release behavior of STB-EVs is a physiological phenomenon in normal pregnancy. STB-EVs can lead to maternal moderate inflammatory reaction and physiological hypercoagulable state, which is benefit for normal pregnancy. However, when the quantity and quality of STB-EVs released from the placenta were different from normal pregnancy, STB-EVs may have effects on vascular endothelial injury, proinflammatory, procoagulant and imbalance of immune tolerance. These may explain the principal pathophysiological processes and clinical features of PE. The differences of STB-EVs between PE and normal pregancy may be the important reason for the disease. It is generally believed that the number of STB-EVs from PE was significantly increased. At present most of the in vivo or in vitro / ex vivo experiments on STB-EVs were about phenomena, very few studies have been done to explore the specific mechanisms, this may due to the composition of STB-EVs was unclear and the essential factors about PE were unknown. Early-onset PE is generally considered as a primarily placental disease, and the etiology and pathogenesis is different with late-onset type. Very few studies have been performed to compare the comositon of STB-EVs between early-onset sPE and normal pregnancy, which may limit exploring the pathogenic mechanism of STB-EVs in PE.As proteins are the molecules that execute a vast array of functions, there may be multiple different proteins carried by STB-EVs between early-onset sPE and normal pregancy. Analysis of the different composition of STB-EVs may provide important guarantee for the further study of its specific mechanisms, early diagnosis and the evaluation of therapeutic effect. Furthermore, the essential proteins may result in targets for intervention and blockade that providing new therapeutic strategies.Differential proteomics focuses on the screening and identification of different proteins between various species and various states, which may have bright application in the early diagnosis, monitor and treatment of diseases. Isobaric tags for relative and absolute quantitation(i TRAQ) is a new methodology and widely applied in quantitative proteomics, which is characterized by high throughput, high sensitivity, low detection limit, powerful separation ability, wide analysis range, etc. We aim to establish a comparative proteome profile of the STB-EVs from early-onset sPE and normal pregnancies using 8-Plex i TRAQ quantitative proteomics, and further to study the role of sialic acid-binding Ig-like lectin 6(Siglec-6) in vascular endothelial injury and its related mechanism.Materials and Methods:1. Placentae were from early-onset s PE and normal pregnant women. STB-EVs were prepared from placentae by the in vitro explant culture combined with a four-step centrifugation/ultracentrifugation method. Then scanning electron microscopy, immunoelectron microscopy and western blotting technologies were applied to verify the morphology and immunity of STB-EVs.2. We used the i TRAQ quantitative proteomics technology combined with bioinformatics analysis to study the differential expression proteins of STB-EVs between early-onset s PE and normal pregnant women. Some differential expression proteins were verified by western blotting.3. We acquired plasma from non-pregnancy, early-onset sPE and normal pregnant women, before and after delivery. Firs of all, we applied western blotting to analysis the expression of Siglec-6 in the plasma between early-onset s PE and normal pregnant women before delivery. Then, we used ELISA to assay the Siglec-6 in the plasma from non-pregnancy, early-onset s PE and normal pregnant women, before and after delivery.4. We chose human umbilical vein endothelial cell line(HUVEC) for the main research platforms. The CCK-8 cell proliferation assay was used to detect the impact of different levels and different incubation times of Siglec-6 protein on HUVEC proliferation function. The wound healing assay and transwell chamber migration assay were used to detect the ability variation of horizontal migration and natural migration in HUVEC with different level of Siglec-6, respectively. The TUNEL assay was performed to detect the impact of different levels of Siglec-6 protein on cell apoptosis. ELISA analysises were used to dectect the secretion of IL-6, TNF-α and VEGF in HUVEC with different level of Siglec-6 protein. To explore the probalble mechanism of Siglec-6 in endothelial dysfunction, we used western blotting to analysis the expression of PI3 K and phosphorylation of AKT in HUVEC with Siglec-6.Results:1. The scanning electron microscopy and immunoelectron microscopy showed that purified STB-EVs were round, spherical, narrow, cup even irregular structures and heterogeneous in size, ranging from approximately 30 to 1700 nm, and they tend to clump together. The morphology and size distribution were consistent with previously described STB-EVs. Immunoelectron microscopy also demonstrated the trophoblast source of STB-EVs. Western blotting showed the presence of heat shock protein 70(Hsp70), tumor susceptibility gene 101(Tsg101) and Flotillin-1 proteins, which are commonly used as markers for EVs.2. We performed a differential proteomic analysis on STB-EVs derived from early-onset s PE and normal pregnant women, using i TRAQ quantitative proteomics technology. We identified 194 differentially expressed proteins in STB-EVs derived from early-onset sPE patients, 122 of which were up-regulated and 72 of which were down-regulated. Further bioinformatics analysis revealed that mitochondrion, transmembrane transport and transmembrane transporter activity were the most abundant categories in CC, BP and MF, respectively. Glycolysis/gluconeogenesis, citrate cycle, fatty acid elongation, steroid hormone biosynthesis and oxidative phosphorylation were the five significantly represented pathways. Up-regulated differentially expressed proteins correlated with inflammatory, coagulation, angiogenesis and immunoregulation may participate in the main pathophysiological processes of PE, and the candidate proteins are: S100-A8, C4b-B, CD63, ATP synthase subunit beta, c DNA FLJ14908 fis, endoglin,interleukin-27 subunit beta, F11 receptor, isoform CRA_a, dynamin-2, protein SEC13 homolog, calnexin, serpin B9, stomatin-like protein 2, phosphoinositide 3-kinase adapter protein 1 and dolichyl-diphosphooligosaccharide-protein glycosyltransferase 48 kDa subunit. Siglec-6 was the most up-regulated protein in the STB-EVs of early-onset s PE. Western blotting verified that four differentially expressed proteins(Siglec-6, calnexin, CD63 and S100-A8) expressed high levels in the early-onset s PE group and showed the same trends as the i TRAQ results.3. Western blotting demonstrated that Siglec-6 expressed high level in the plasma of early-onset s PE group. ELISA detected Siglec-6 in the plasma of non pregnancy was very low(0.08±0.0007 ng/ml). For early-onset sPE and normal pregnant women before delivery, the levels were 0.80±0.23 ng/ml and 0.21±0.04 ng/ml, respectively. After delivery, the expression of Siglec-6 decreased rapidly, the levels were 0.09±0.007 ng/ml and 0.09±0.01 ng/ml, which were close to the level of non pregnancy.4. The CCK-8 cell proliferation assay demonstrated that Siglec-6 promotes HUVEC proliferation, and the promotion effect peaked with Siglec-6 concentration of 2.0 ng/ml and stimulation time of 48h(P<0.05). The migration assay showed that Siglec-6(2.0 ng/ml) significantly reduced both the horizontal migration and the natural migration of HUVEC(P<0.05), and the wound healing assay also showed that Siglec-6 concentration of 5.0 ng/ml significantly reduced the natural migration more than the concentration of 2.0 ng/ml(P<0.05). Siglec-6(2.0 ng/ml, 48h) promoted the apoptosis of HUVEC. Siglec-6 promoted HUVEC to secrete IL-6 and TNF-α in a dose-dependent and time-dependent manner(P<0.05). Siglec-6 suppressed HUVEC to secrete VEGF, and the suppression was not related to the dose and stimulation time of Siglec-6. Western blotting showed that Siglec-6(2.0 ng/ml, 48h) significantly reduced the expression of PI3K(P<0.05), and also significantly reduced the phosphorylation of AKT.Conclusions:1. Proteins of STB-EVs derived from early-onset s PE were different from normal pregnant women. Siglec-6 was the most up-regulated differential expression protein in the STB-EVs of early-onset s PE.2. The level of Siglec-6 in the plasma of early-onset sPE was significantly increased, and it was about four times of normal pregnant women. After delivery, the expression of Siglec-6 decreased rapidly, which was close to the level of non pregnancy. Termination of pregnancy is the only definitive treatment for early-onset sPE, and the change of Siglec-6 in plasma provided an experimental evidence for it.3. Siglec-6 may injury vascular endothelium by inhibiting the proliferation and migration of endothelial cells, promoting endothelial cells apoptosis as well as secretion of IL-6 and TNF-α, and inhibiting the secretion of VEGF. These effects may be achieved by inhibiting the PI3K-AKT signaling pathway. Siglec-6 may be one of the key proteins in the pathogenesis of early onset s PE.