Research On Rho/ROCK Molecules Involving In Syncytiotrophoblast Microvillous Membranes Shedding And Its Relationship With Severe Preeclampsia

Background and objective：Severe preeclampsia （sPE） is one of the major obstetric complications, which severelythreatens maternal and fetal health. The current researches have not yet accurately predictedthe incidence of sPE. In addition, there is no substantial prospect based on anatomicalpathology of precisely regulating trophoblast invasion to sPE. Thus, intervening andblocking the pathophysiological process of sPE is more significant in clinical treatment.The serum or plasma of sPE patients can interfere with the function of vascular endothelialcells, suggesting that there is an important factor which leads to clinical symptoms in sPEpatients blood. Clinical symptoms quickly disappear after the expulsion of the placenta,indicating that the adverse factors of sPE in peripheral blood are mainly from the placenta.Syncytiotrophoblast microvillous membrane（STBM） is one of placenta derived adversefactors, which is membrane vesicle-like structures produced by the syncytiotrophoblastapoptosis or activation, and currently is considered as an important factor causing sPE.STBM shedding off to the maternal blood circulation can act on the peripheral vascular,leading to malfunctions of vascular endothelial cells, while affecting the maternal immunesystem by regulating the body’s inflammatory cytokine release and cellular immunity.Studies have shown that the concentration of STBM significantly increases in peripheralblood of sPE patients, and is even more obvious in early-onset sPE. However, themechanism of STBM shedding is unclear. It is noteworthy that, STBM can be measured inperipheral blood of normal pregnant women, but can not be measured in peripheral blood ofthe non-pregnant women. It suggests that the release of STBM is a normal physiologicalphenomenon in the process of normal pregnancy. The raise of STBM shedding whichmediated by certain fators leads to sPE. Domestic and international researches suggest thatthe main stimulus of STBM shedding is hypoxia. Rho/ROCK signal pathway includes Ras homologue protein family and Rho-associated protein kinase, which extensively involve in cellular signal transduction andparticipates in cellular proliferation, apoptosis and migration by activating downstreammolecules. A small GTPase, including RhoA, RhoB, RhoC, have thought to be themolecular switch to mediate signals, which effect the downstream target molecules toregulate actin cytoskeleton reconstruction, and participate in cell ahesion, cell polarity andcell migration. It has been confirmed that the Rho/ROCK signal pathway is involved inthe process of the incidence of sPE, and furthermore, RhoA and ROCKII proteins werehighly expressed in placenta of sPE patients. Previous studies have demonstrated thathypoxia can activate the Rho/ROCK signal pathway, in particular, increasing theexpression of RhoB and ROCKI. In view of the important pathological environment whichthe placenta is low perfusion in sPE, we speculate that Rho/ROCK is likely involved in theraise of the STBM shedding which mediated by hypoxia in sPE, and the two isoforms ofROCK possibly play different roles. However, so far, the subtype expression of Rho andROCK in placental tissue of sPE are still unclear, nor to be understood their relationshipwith STBM shedding and their possible mechanism.Our research was carried out based on the experimental platform of hypoxic culturedplacental villi in vitro, using experimental techniques of electron microscopy, immuno-histochemistry, Western blot, and ELISA to explore the mechanism of hypoxia-mediatedSTBM shedding which involved in Rho/ROCK protein.Materials and methods：1. Subjects and groups40puerperae, whose cesarean deliveries were carried out （from January,2009toSeptember,2010） in Daping Hospital and Xinqiao Hospital of The Third Military MedicalUniversity, were selected for our research all informed consents have been completed.40puerperae were averagely divided into two groups which were severe preeclampsiagroup（sPE） and normal pregnancy group（NP）. The maternal age and gestational age of twogroups had no statistically significant difference （P>0.05）. All patients had no otherpregnancy complications.2. Specimen collection and pretreatmentUnder aseptic condition, we scissored about1.0cm³non-calcified part in the center of placentas maternal surface, of which placental deciduas were abandoned. The tissues wererinsed with PBS, and then were dried by placing on sterile gauzes. Some specimens werefrozen in liquid nitrogen canister, and others were fixed with2.5%glutaraldehyde or4%paraformaldehyde.3. Hypoxic cultured placental villi in vitro1.0cm³non-calcified part of placentas, of which deciduas and vessels were abandoned,and rinsed with sterile saline, cut into0.1cm³each piece, and then weighted. Each hole isabout60mg. All pieces were inoculated into24-well cell culture plates, in which adding5mL medium （DMEM/F12+10%FBS+anti）, and were respectively cultured at37℃,5%O₂（volume fraction）+95%N2mixture hypoxic boxes and37℃,5%CO₂and95%constanttemperature incubator. Organizations and supernatant were respectively collected after0.5h,1h,2h,3h,6h,12h. The experimental group was divided into normoxic （Normoxia） andhypoxia group （Hypoxia）.4. Observed samples of TEMPlacental tissue samples were fixed with2.5%glutaraldehyde after2h, and then werefiexed with2%osmium tetroxide. Fisrtly, samples were embedded by epoxyresin and cutinto ultrathin sections with uranyl acetate and citrate lead double staining. Each slice withacceleration voltage80KV, the magnification was10000fold. Structure changes of place-ntal syncytiotrophoblast microvilli were observed.5. Calculating method of stereological parametersNetwork test system, designed by Image Pro was used to do analysis. The intervalpoint of the side nets was20pixels. Before the test, we used electron microscope photoscale to calibrate the grid, and took syncytiotrophoblast as a reference, using the abovementioned test system to test points hitting the microvilli and inclusive space, counting theintersection points of the test line and the microvilli, and respectively testing the numberdensity of syncytiotrophoblast microvilli （NV, n/μm³ï¼‰, surface density （Sv, μm2/μm³ï¼‰ andvolume density （Vv, μm³/μm³ï¼‰, calculating the stereological parameters which were tested.6. Detecting the location of RhoA, B, C and ROCKI, II by immunohistochemistryPlacental tissue fixed with4%formaldehyde for24h, were embedded in paraffin andthen sliced. Expressions of RhoA, RhoB, RhoC, ROCKI and ROCKII proteins of placentaltissue were tested by immunohistochemical ABC method. All procedures were strictly completed in accordance with immunohistochemical reagent instructions. Primary antibody（Rabbit polyclonal to RhoA, RhoB, RhoC, ROCKII,1:1500; Rabbit polyclonal to ROCKI,1:500） was kept in4℃overnight, and we replaced primary antibody with PBS as negativecontrol. Samples were stained by DAB staining and hematoxylin staining, and then routinedehydrated, transparentized, dried. Brown particles, observed by light microscope, wereconsidered as positive reaction.7. Detecting the expression of RhoA, B, C and ROCKI, II by Western-blotWe extracted protein from the placenta, and operated by SDS-PAGE, to do electrophor-esis （100mV,1.5h） and closure （5%skim milk for1h）. Proteins were incubated withmonoclonal antibody （Rabbit polyclonal to RhoA, RhoB, RhoC and1:2000; the Rabbitpolyclonal to ROCKI, ROCKII,1:300） and peroxidase-labeled secondary antibody,displaying bands by applying enhanced chemiluminescence. We used Quantity one4.62software to analyze gray values, and took the ratio of the targeted bands and the internalreference bands as the relative content of the targeted protein.8. Detecting the level of STBM shedding by ELISAThe level of STBM shedding was tested by application of the STBM surface mar-ker-the human placental alkaline phosphatase （PLAP） ELISA kit.Results:1. The stereological parameters of placental syncytiotrophoblast microvilliThe number density, surface density and volume density of trophoblastic microvilli inthe NP were higher than sPE group; the number of syncytiotrophoblast microvill weresignificantly less than the NP group （P <0.05）, as well as Hypoxia group when comparedwith matched Normoxia group （P <0.05）.2. Detecting the location of RhoA, B, C and ROCKI, II by immnohistochemistryRhoA, RhoB, RhoC, ROCKI and ROCKII proteins were expressed in syncytiotro-phoblast cells, cytotrophoblast, endothelial cells and stromal cells in the cytoplasm in thefour groups （brown particles as a positive expression）, but mainly in the syncytitrophoblastcytoplasm.3. Detecting the expression of RhoA, B, C and ROCKI, II by Western-blotRhoA, RhoB, ROCKI, ROCKII proteins appeared to have much higher expression insPE group compared with the NP group （P <005）;0.5h RhoA, RhoB, ROCKI, ROCKII protein expression began to increase,3h increased significantly, reached a peak at6h,followed by a small decline at12h, and RhoA, RhoB, ROCKI, ROCKII proteins was morehighly expressed in Hypoxia group than the Normoxia group （P <0.05）; whereas the levelof RhoC was unaffected （P>0.05）.4. Detecting the level of STBM sheding by ELISAThe levels of0.5h STBM sheding began to increase, reached a peak at6h, followed bya small decline at12h, and the levels of STBM in the Hypoxia group were increased whencompared with matched Normoxia group （P <0.05）.Conclusions:1. By TEM and Image Pro analysis, the stereological parameters of syncytiotrophob-last microvilli in placenta of sPE were significantly lower, when compared with normalpregnancy, while application of low oxygen conditions analoged the micro-environment ofsPE, in order to verifying the scientific hypothesis that hypoxia can cause the raise ofSTBM shedding form syncytiotrophoblast surface and “proving experimental basis for theSTBM of peripheral circulation to forecaste the incidence and prognosis of sPE”. Inaddition, it is important significance to the prevention of sPE.2. By Western-blot and immnohistochemistry analysis，RhoA, RhoB, ROCKI, ROCKII proteins appeared to have much higher expression in sPE and Hypoxia groups,whereasRhoC was unaffected. It suggested that RhoA, RhoB, ROCKI, ROCKII involved in thechanges of the pathological process and Biological functions of the syncytiotrophoblast insPE.3. By the Spearman correlation analysis, the levels of RhoB, ROCKI, and ROCKIIproteins expressing and the levels of STBM shedding were positively correlated in sPE andHypoxia group, thus, RhoB/ROCK signaling molecules may play an important role insPE.