Mechanism Of RhoC Protein Regulating Human Trophoblast Migration And Clinical Data Analysis Of Patients With Preeclampsia

Background and Objective:Preeclampsia / eclampsia have been considered as severe obstetric diseases which seriously affect the maternal and perinatal health. The incidence and mortality of PE is high, but its pathogenesis is not clear. Therefore the main clinical treatment of PE is usually "symptomatic treatment" and "timely termination of pregnancy" with poor effects. According to the recently formed and widely accepted "two stages theory", the invasion barrier of placental trophoblast in early pregnancy is the initial factor of PE, while the endothelial dysfunctions and systemic immune-inflammations caused by placenta derived pathogenic factors which enter into the maternal blood in middle and late pregnancy are the central nodes of PE onset. At the same time, the bad placenta formation caused by the insufficiency of invasive migration of trophoblast can also lead to abortion and fetal intrauterine growth restriction. On the contrary, if the invasive migrations of trophoblast are excessive, it may cause hydatidiform mole, choriocarcinoma and placenta implantation. Therefore, the elucidation of the inherent mechanism of human trophoblast cell invasive migration and its regulating factors will become the key to solve these diseases.The invasive migration of trophoblast cell is a biological process with multi-steps, which involves "cell polarization, pseudopod formation at leading edges, cell adhesion with extracellular matrix in the target direction, cell tail departure, decomposition of adhesion structure and cell body contraction forward". This process is precisely regulated by a lot of internal and external factors, and the underlying molecular mechanism is also not clear. But the evidence has shown that in the dynamic process of cell migration, cytoskeletal reorganization and depolymerization of adhesion structure get throughout this process, and becomes the dynamical foundation and prerequisite conditions of cell invasive migration. It is also a key target to control and intervene in the migration of trophoblastic cells. Our colleagues have found in previous studies that, Rho A and Rho C, the members of Rho subfamily protein of small G protein, can play a molecular switch role in the intrinsic migration and induced migration of human trophoblast. Rho A realizes this regulation mainly through the interference of the orderly restructuring of microfilament skeleton. The regulation of RhoC on trophoblast migration depends on an unknown mechanism other than microfilament. Studies have shown that, RhoC are closely related to body cell migration, especially the distant metastasis of tumor cells. In addition, this molecule appears late than Rho A in molecular evolution, and there is almost no effect of RhoC on the basic life activities of the cells, such as apoptosis as well as substance and energy metabolism. Thus it has a good drug development and application prospects. However, the regulating mechanism of Rho C to human trophoblast migration has yet to be proved.Previous studies have shown that, Rho C plays a regulatory role mainly through its downstream Rho associated coiled coil forming protein kinase(ROCK), and activation of Rho C may change the microtubule cytoskeleton structure. The microtubule can also have cross-talk with the important adhesion structure of cell, focal adhesion, which affects cell adhesion and migration. But its acting model and signal pathway has not been reported. Combined with the regulating mechanisms of cell microtubule stability, we hypothesize that microtubule associated protein 4(MAP4) may be a key molecule through which ROCK affects the microtubule polymerization of trophoblast cell, but the underlying mechanism is not clear. Therefore, the experimental section of this study mainly focuses on the mechanism of the regulation of Rho C/ROCK to microtubule cytoskeleton and focal adhesion structure which relates to human trophoblast cell migration.However, the current clinical treatments for PE are very limited, and the research and development of new treatment that aimed to the initiation factor during early pregnancy will take a long time. The high incidence and adverse pregnancy outcomes of PE raise more urgent and realistic requirements for early prediction and prevention. According to the "two stages theory" of PE, a large number of adverse factors(including IL-2, TNF- alpha, sFlt-1 and other inflammatory factors as well as syncytiotrophoblast microvesicles etc.) were released into maternal circulation. So the patients are subjected to serious systemic inflammatory response, and their coagulation and fibrinolysis system was severely affected. The blood coagulation index and platelet parameters play important role in predicting PE. Many clinical studies in the past brought patients with comorbidities and complications of pregnancy into analysis, which often leads to uncertain disturbances, or even totally opposite conclusions. Therefore, in the section of clinical data analysis of this study, we set strict inclusion criteria for patients with PE, and the trends of coagulation indices and platelet parameters as well as their value for the prediction of PE were preliminarily studied.In addition, with regard to the "two stages theory", maternal multi organ damages in patients with PE are caused by the pathogenic factors such as the placenta-derived inflammatory factors and syncytiotrophoblast microvesicles that were released into maternal circulation in late pregnancy. Various clinical evidences have proved that kidney is usually damaged earlier and more heavily among the multi organs. Therefore, in the section of clinical data analysis of this study, we also retrospected the pathology and the conditions of maternal and fetal of patients with PE / eclampsia, who take renal biopsy during prenatal period or 1 week after delivery.Materials and Methods:In the section of cytology experiments of this study, we took human trophoblast cell line JEG-3 as the main research platforms. The lentiviral vectors of over- and low-expressing Rho C protein were constructed, respectively. The corresponding lentiviral packages were used to infecting JEG-3 cells in order to realize the regulation of RhoC gene of human trophoblast in vitro. Then the Transwell chamber migration assay and wound healing assay were used to detect the ability variation of natural migration and horizontal migration in trophoblast with different expression level of RhoC, respectively. The invasion assay of Transwell chamber combined matrigel was used to detect the impact of different levels of RhoC protein on cell invasive function. The CCK-8 cell proliferation assay was used to detect the impact of different levels of Rho C protein on cell proliferation function of cells cultured under normal conditions and low serum condition. Then we used Western blot assay to detected the expression of ROCK I and ROCK II proteins, which are downstream target molecules of Rho C, in both Rho C high- and low-expressing cells, and the expression differences of MAP4 protein which can regulate microtubule polymerization. In order to analyze the polymerization and depolymerization level of microtubule in trophoblast with different levels of RhoC expression, the tubulin proteins of polymeric state and free state were firstly separated, and then they were detected by Western blot assay, meanwhile the changes of microtubule structure were also observed by confocal laser scanning microscope. Finally, the expression of vinculin protein in JEG-3 cells with different levels of RhoC was detected also by Western blot assay, and the arrangement characteristics of adhesion plaque structures were observed by confocal laser scanning microscope.In the first section of the analysis of patients with PE, 173 pregnant women were enrolled: 53 women who were diagnosed with mild preeclampsia(mPE), 41 with severe preeclampsia(sPE) and 79 normal pregnancies. All of these pregnant women were nulliparous, and at last they were delivered between January 2007 and December 2013, in the Department of Obstetrics and Gynecology, Daping Hospital, Third Military Medical University. However, women with pre-existing renal disease, insulin-dependent diabetes, asthma requiring steroidal treatment, chronic hepatitis(with or without hepatic dysfunction), severe trauma history, anticoagulant drug-use history, oral contraceptive-use history, smoking history, idiopathic thrombocytopenic purpura(ITP), HELLP syndrome, gestational thrombocytopenia(GT) or any hematological diseases were all excluded. Then the coagulation function and platelet parameters of each pregnant woman were collected. The results were taken into comparative and statistics analysis. The statistical significance of parametric variables among the different groups was performed using multiple comparisons performed by one-way analysis of variance(ANOVA) and non-parametric variables by the Kruskal-Wallis test with post-hoc analysis. Pairwise comparisons were applied to compare the same index of one subject at early and late gestational stages, either by Fisher’s LSD for parametric variables or by the Wilcoxon test for non-parametric variables. The receiver-operating characteristic(ROC) curves were generated to estimate the utility of each parameter as a tool for predicting the severity of PE. The predictive value of the indicators was examined through the binary regression analysis at last.In the second section of the analysis of patients with PE, we retrospected eight renal biopsies which were performed on patients because of the progress of disease or the essentiation of diagnosis and treatment, during pregnancy or immediately after delivery, between Jan. 2007 and Jan. 2012, in the Department of Nephrology, Daping Hospital, Third Military Medical University. None of the patients had a history of diabetes mellitus, systemic lupus erythematosus, chronic hypertension, nephropathy, or recent urinary tract infection. The clinical findings of patients, major serological indices, maternal and neonatal outcomes, and renal histopathologic and immunofluorescent characteristics were reviewed for each case.Results:1. Section of cytology experiments(1) In this study, bioinformatics technology, RNA interference technology combined with genetic engineering technique were all used to construct the lentivirus vector of Rho C high-expressed and Rho C interference successfully. These two lentiviral packages can successfully and efficiently infect 293 T cell model and JEG-3 trophoblast cells. JEG-3 trophoblast with continuously and steadily high- and low- expressing Rho C were identified and selected out. Their RhoC protein expression levels were significantly higher or lower than the empty vector virus infected trophoblast and normal control group trophoblast, respectively.(2) Under normal cell culture conditions, the increased Rho C protein expression in trophoblast could significantly improve the ability of natural migration, horizontal migration, invasion and proliferation. And the decreased RhoC protein expression could significantly impress the ability of natural migration, horizontal migration, invasion and proliferation. But in low serum culture conditions, the expression levels of RhoC in trophoblast would not have a significant impact on cell proliferation.(3) The expression levels of RhoC protein in trophoblast were highly consistent with that of ROCK I and ROCK II, which are downstream target molecules of Rho C. It suggested that the biological effects in cells of RhoC might exert through ROCK I and ROCK II molecules. And in trophoblast, the expression of MAP4 protein was highly consistent with the changes of the expression level of ROCK I and ROCK II, which suggested that Rho C might achieve the regulation of cell migration through phosphorylation effects of ROCK I and ROCK II on MAP4.(4) In trophoblast, Rho C protein could regulate the polymeric and free state of microtubules. After the expression of Rho C was increased, the polymeric tubulin was significantly promoted with a significant reduction of the free tubulin, and the polymerization of microtubule structure was enhanced in morphology, as well as the microtubules arranged in order. When the expression of RhoC was decreased, the polymeric tubulin was significantly impressed with a significant up-regulation of the free tubulin, and the microtubule structure tended to depolymerize in morphology, as well as the microtubules arranged disorderly.(5) In trophoblast, changes in the expression levels of Rho C protein and vinculin were opposite. Increased Rho C expression leaded to the decrease of vinculin and enhanced dissociation of focal adhesion in morphology. While decreased Rho C expression resulted to the increase of vinculin and enhanced polymerization of focal adhesion in morphology.2. Section 1 of clinical data analysis of patients with PE(1) Normal late pregnancy shows a physiological hypercoagulable state with decreased levels of activated partial thromboplastin time(APTT), prothrombin time(PT), and thrombin time(TT) and platelet count(Pt Count), as well as an increased level of FIB and mean platelet volume(MPV) compared to early pregnancy. This result may be caused by platelet consumption and aggregation followed by a secondary regeneration.(2) In the third trimester, PE patients present with a super-hypercoagulable state. The activation of thrombopoiesis and fibrin production results in the consumption of platelets and FIB. The onset of PE, in particular sPE, mainly results in the prolongation of APTT and TT, the augmentation of D-dimer(DD) and the enlargement of MPV, which indicated complex disorders in both endogenous and exogenous coagulative pathway.(3) Among the blood coagulation parameters and platelet indices, MPV is considered to be not only an ideal early monitoring index for the onset of PE(with optimal cut-off level of >8.95 fL), but also a potential marker for predicting the severity of PE in early pregnancy(with optimal cut-off level of >9.95 f L).3. Section 2 of clinical data analysis of patients with PE(1) Endotheliosis, vacuolation of podocytes, proliferation of mesangial cells, and protein casts in the renal tubule lumens were the most common pathologic characteristics of renal biopsies from pregnant women with PE/eclampsia.(2) The deposition of complement components(C4) and immunoglobulin(Ig M) may contribute to serious renal lesions.(3) The BUN/SCr ratio may be a new marker for severe PE.(4) Eclampsia can occur in patients with a blood pressure of 135/93 mm Hg, which is only slightly elevated. This should be paid more attention in clinical work.(5) To minimize complications, renal biopsy in pregnancy should only be recommended for definitive diagnosis and improvement of therapeutic strategy, and should be performed based on the general conditions of the patient, including blood pressure and coagulation function.Conclusions:(1) RhoC plays regulating role in natural migration, horizontal migration and invasion of trophoblast. Its underlying mechanism may be regulating the expression of MAP4 molecules through its downstream target proteins ROCK I and ROCK II, and then affecting microtubule polymerization and reconstruction of focal adhesion structures of cell. These results laid certain foundation for the further clarifying the related cytoskeleton mechanisms of Rho subfamily proteins regulating trophoblast migration, and provided experimental basis for developing new regulating target of trophoblast migration to intervene about the occurrence of PE.(2) Normal pregnancy causes a maternal physiological hypercoagulable state in late pregnancy. It reflects both consumption and activating production of platelets. PE may trigger complex disorders in both endogenous and exogenous coagulative pathway and shows a super-hypercoagulable state. At the same time, the platelets and FIB are consumed, which casuses multi-organ diseases and bleeding tendency. And then it leads to the pathogenesis and further aggravation of PE. MPV may serve as early predicting markers for the onset and severity of PE. It provides primary basis for the researches on the characteristics of coagulation and early prediction.(3) Endotheliosis, vacuolation of podocytes, proliferation of mesangial cells, and protein casts in the renal tubule lumens were the most common pathologic characteristics of renal biopsies from pregnant women with PE/eclampsia. Immune depositions of C4 and IgM are major contributors to renal lesions and proteinuria in preeclamptic patients. These results provide some clinical data for further study on the organic injury and its mechanism.