The Pathology Change Of Placenta Bed And The Effect Of MMP-2, TIMP-2, HIF-1α And VWF In The Pathogenesis Of Pre-eclampsia

Pre-eclampsia, a common type of hypertensive disordercomplicating pregnancy ,which is a kind of idiosyncratic disease inpregnancy, has been considered as one of the most significant reasons forthe death of pregnant women and perinatal babies. So far ischemicuterus-placenta has been recognized as an important cause ofpre-eclampsia, as well as the main pathologic changes. Effectiveestablishment of uterus-placenta circulation is thought of to be a factorthat trophoblast cells can normally invade placenta bed, yet completedand intuitionistic comparative researches have been in absence. At presentthere are many investigations of the relationship between the invadingability of trophoblast cells and MMP-2 together with its inhibitor TIMP-2,but a lot of them remain disputation. Some studys showed that whenhypoxia being exist,the hypoxia-inducible factor 1a(HIF-1a) is overexpression. HIF-1a can result in blood vessel endothelium damnificationby many kinds of ways. The Von Willbrand Factor(vWF) is a importantmarker that blood vessel endothelium cells were harmed. Whethertrophoblast cells invading placenta bed too superficially and ischemic uterus-placenta will induce the changes of the expression of HIF-1a andvWF in placenta bed or not? Whether the change of the concentration ofvWF in placenta bed and in mother's peripheral blood of pre-eclampsiaare correlated or not? The study about those questions are quite limitednow.As a result, we selected pre-eclampsia as our experiment objects.Started with placenta bed, firstly, probing the changes of placenta bed inhistology and morphology, and then, comparing the changes of theexpression of MMP-2 and its inhibitors TIMP-2,HIF-1αin trophoblastcells and placenta bed, as well as the changes of vWF concentration inplacenta bed and in mother's peripheral blood of pre-eclampsia.At last,toapproach their role in the pathogenesy of pre-eclampsia Chapterâ… Study of trophoblast cells invading placenta bed as well asalteration of spiral arteries in pre-eclampsiaObjective: To investigate the change of the invading degree of thetrophoblast in the placenta bed as well as the spiral arteries ofpre-eclampsia and normal pregnancy, then to provide the verification ofhistomorphology for further investigation of the pathogenesy ofpre-eclampsia.Methods: Choosing 40 cases pre-eclampsia as patient groups, 20 casesout of them were pre-eclampsia(mild) (group B) ,the remaining 20 caseswere pre-eclampsia(severe) (group C). The other 20 cases normalpregnancy were chosen as normal group(group A). The methods of HEstaining and immunohistochemistry staining (SP method) were used toobserve the depth and the density of the trophoblast invading placentabed as well as the spiral arteries lumen area, the thickness and themicrovessel density of spiral arteries in the decidual and the superficialmyometrial segments.Results:1. Comparison between the depth and density of trophoblast invadingplacenta bed:1.1. The depth of trophoblast invading the basal decidual segment was no significant difference among the three groups (p＞0.05). The significantdifference was mainly in the superficial myometrial segment of placentabed. From basal decidual-superficial myometrial segment to 3LPF, thedepth of the trophoblast invading the placenta bed in thepre-eclampsia(severe) were the lowest than that of the pre-eclampsia(mild)and the normal group (p＜0.05). Among them ,from the juncture of basaldecidual segment and superficial myometrial segment to 1LPF, there wasno significant difference between the pre-eclampsia(mild) and the normalgroup (p＞0.05). From 2LPF to 3LPF, there was significant differencebetween the pre-eclampsia(mild) and the normal group (p＜0.05).1.2. The density of trophoblast invading in≤1/2 basal decidual segmentof placenta bed did not show that it was significant difference among thethree groups (p＞0.05).In＞1/2 basal decidual segment, there wassignificant difference between the pre-eclampsia(severe) and thepre-eclampsia(mild), and there was significant difference between thepre-eclampsia(severe) and the normal group. But we can not see anysignificant difference between the pre-eclampsia(mild) and the normalgroup (p＞0.05). Comparison in the density of trophoblast invading inplacenta bed from the juncture of basal decidual segment and superficialmyometrial segment to 1LPF, 2LPF, 3LPF, shows us the lowest in thepre-eclampsia(severe) among the three groups, while thepre-eclampsia(mild) was the lower, the normal group was the highest. There was significant difference among the three groups (p＜0.001).1.3. It's negative correlation between the depth and the density oftrophoblast invaded placenta bed and the pathogenetic condition degreeof pre-eclampsia(p＜0.05).2. Comparison in the change of the spiral arteries of placenta bed:2.1. The average spiral arteries lumen area in decidual segment and thesuperficial myometrial segment of placenta bed was the smallest in thepre-eclampsia(severe) among the three groups, while the thepre-eclampsia(mild) was the smaller, the normal group was the largest.The significant difference among the three groups was abserved(p＜0.01) .There was significant difference among group comparison(p＜0.001) .The average thickness of the spiral arteries was thickest inthe pre-eclampsia(severe) among the three groups, while thepre-eclampsia(mild) was the thicker, the normal group was the thinnest.The significant difference among the three groups was abserved (p＜0.05).There was significant difference among group comparison (p＜0.01)2.2. The incidence rate of the physiological and pathological change ofthe spiral arteries in the basal decidual segment was no significantdifference among the three groups (p＞0.05). But the incidence rate ofthe spiral arteries without physiological chages was higher in thepre-eclampsia(severe) (39.67%) than that of the normal group(p＜0.05).The incidence rate of the physiological changes in the superficial myometrial segment of placenta bed was the lowest in thepre-eclampsia(severe) among the three groups, while the pre-eclampsia(mild) was the higher, the normal group was the highest. We cannot seeany significant difference among the three groups (P＜0.001). Theincidence rate of the pathological changes was the highest in thepre-eclampsia(severe) among the three groups, while thepre-eclampsia(mild) was the higher, the normal group was the highest.There was significant difference among the three groups (P＜0.001).2.3. It's positive correlation between the physiological change of thespiral arteries, the depth and density of trophoblast invading placenta bed(p＜0.05), and it was negative correlation between the pathologicalchange of the spiral arteries,the depth and density of trophoblast invadingplacenta bed (p＜0.05). It was negative correlation between thephysiological change and the pathogenetic condition degree ofpre-eclampsia (p＜0.05) ,and it was positive correlation between thepathological change and the pathogenetic condition degree ofpre-eclampsia (p＜0.05). Furthermore, trophoblast invasion in myometrialspiral arteries was noted in 62.50% of the normal group and 27.50% ofpreeclampsia (severe) (P＜0.001).3. Comparison in the microvessel density of placenta bed:The microvessel density in the basal decidual segment and thesuperficial myometrial segment of the pre-eclampsia(severe) was the lowest among the three groups, while the pre-eclampsia(mild) was thehigher, the normal group was the highest. It showed significant differenceamong the three groups (P＜0.001).Conclusion:1. The depth of the trophoblast invading placenta bed of pre-eclampsiawere superficialer than normal pregnancy.2. The changes of depth and the density of the trophoblast invadingplacenta bed existed in superficial myometrial segment mainly. It wasclosely related to pathogenetic condition degree of pre-eclampsia.3. The pathological changes of the spiral arteries was mainly insuperficial myometrial segment and was closely related to the depth andthe density of trophoblast invading placenta bed.4. The microvessel density of placenta bed decreased beginning frombasal decidual segment in pre-eclampsia. It's probably that themicrovessels developmen of placenta bed to be impaired inpre-eclampsia. Chapterâ…¡Changes and effects of MMP-2, TIMP-2 in trophoblast cellsand placenta bed of pre-eclampsiaObjective: To investigate the expression of MMP-2 and TIMP-2 introphoblast and placenta bed from pre-eclampsia and normal pregnancy,then to analyze their effect on the pathogenesis of pre-eclampsia.Methods: The patient groups and normal group were split as the same asPart 1.To compare the level of MMP-2 and TIMP-2 in trophoblast andplacenta bed among the three groups. Trypsin digestion and percolldiscontinuous density gradient were applied to purify trophoblast fromplacenta bed. It is identified and assayed by immunohistochemistrystaining for human cytokeratin 7. The radioimmunoassay was used todetermine progesterone level in the supernatant liquid of the culturedtrophoblast. The immunohistochemistry staining method was applied todetect the expression of MMP-2 protein and TIMP-2 protein in placentabed. We adopt reverse transcription-polymerase chain reaction to detectthe quantity of MMP-2mRNA and TIMP-2mRNA secreted by thecultured trophoblast. Zymography about gelatinase was used to detect theactivity of MMP-2 in the cultured trophoblast and the placenta bed.Results:1. Compared the progesterone level in supernatant liquid of the cultured trophoblast show that it was the lowest in the pre-eclampsia (severe)among the three groups, the pre-eclampsia (mild) in the middle level andthe normal group was the highest. There was significant differenceamong the three groups (p＜0.01).2.Compared the levels of MMP-2, TIMP-2 in the cultured trophoblast:2.1 The gray scale of MMP-2 protein in the cultured trophoblast showedthat it was the highest in the pre-eclampsia (severe) among the threegroups followed by the pre-eclampsia (mild) and the normal group wasthe lowest. There was significant difference among the three groups (p＜0.001). The comparison in the level of TIMP-2 protein in the culturedtrophoblast showed that it was no significant difference among the threegroups (P＞0.05).2.2 The expression of MMP-2mRNA in the cultured trophoblast showedthat it was the highest in the pre-eclampsia (severe) among the threegroups followed by the pre-eclampsia (mild) and the normal group wasthe lowest. There was significant difference among the three groups (p＜0.001). The comparison in the level of TIMP-2mRNA in the culturedtrophoblast showed that it was no significant difference among the threegroups (P＞0.05).3. Compared the levels of MMP-2, TIMP-2 in the placenta bed:3.1 The immunohistochemistry integration of MMP-2 protein in the basaldecidual segment of placenta bed showed that it was no significant difference among the three groups(p＞0.05). But the comparison in theimmunohistochemistry integration of TIMP-2 protein in the basaldecidual segment of placenta bed told us that it was the highest in thepre-eclampsia (severe) among the three groups,followed by thepre-eclampsia (mild) and the normal group was the lowest. We foundsignificant difference among the three groups (p＜0.001). The comparisonin the immunohistochemistry integration of MMP-2 protein in thesuperficial myometrial segment of placenta bed showed that it was thelowest in the pre-eclampsia (severe) among the three groups, then thepre-eclampsia (mild) listed in the middle and the normal group was thehighest. There was significant difference among the three groups (p＜0.001). By comparison in the immunohistochemistry integration ofTIMP-2 protein in the superficial myometrial segment of placenta bed,we found that it was the highest in the pre-eclampsia (severe) among thethree groups followed by the pre-eclampsia (mild), and the normal groupwas the lowest. There was significant difference among the three groups(p＜0.001).3.2 The level of MMP-2mRNA in the placenta bed and the culturedtrophoblast showed that it was the lowest in the pre-eclampsia (severe)among the three groups, the pre-eclampsia (mild) was in the middle leveland the normal group was the highest. Significant difference was foundamong the three groups (p＜0.001). Compared the level of TIMP-2mRNA in the cultured trophoblast showed that it was no significant differenceamong the three groups (P＞0.05).While comparison in the level ofTIMP-2mRNA in the placenta bed told us it was the highest in thepre-eclampsia (severe) among the three groups followed by thepre-eclampsia (mild), and the normal group was the lowest. There wassignificant difference among the three groups (p＜0.001).4. Compared the correlation of the expression of MMP-2 between thecultured trophoblast and the placenta bed, then the expression of TIMP-2between the two too:4.1 It's positive correlation between the expression of MMP-2 protein inthe cultured trophoblast and the placenta bed,the correlation coefficientwas r₁=0.79, (p＜0.001). It's positive correlation between the expressionof MMP-2mRNA in the cultured trophoblast and the placenta bed too,thecorrelation coefficient was r₂=0.73, (p＜0.001).4.2 It's positive correlation between the expression of TIMP-2 protein inthe cultured trophoblast and the placenta bed,the correlation coefficientwas r₃=0.23, (p＜0.05). It's positive correlation between the expression ofTIMP-2mRNA in the cultured trophoblast and the placenta bed too,thecorrelation coefficient was r₄=0.21, (p＜0.05). The correlative degree ofTIMP-2 expressing between the cultured trophoblast and the placenta bedwas lower than that of MMP-2.5. Compared the activity of MMP-2 in the cultured trophoblast and the placenta bed: the activity of MMP-2 in the placenta bed and the culturedtrophoblast showed that it was the lowest in the pre-eclampsia (severe)among the three groups, the pre-eclampsia (mild) was in the middle level,and the normal group was the highest. There was significant differenceamong the three groups (p＜0.001).Conclusion:1. The low expression of MMP-2mRNA and MMP-2 protein, the lowactivity of MMP-2, the high level of the expression of TIMP-2mRNA andTIMP-2 protein in the placenta bed and the cultured trophoblast frompre-eclampsia that it's probably the important pathogenetic reason for thepre-eclampsia.2. MMP-2 is probably secreted by trophoblast,but TIMP-2 is probablysecreted by cells from placenta bed cells such as decidual cells. Chapterâ…¢The expression of HIF-1αprotein in trophoblast andplacenta bed and the concentration change of vWF inplacenta bed and mother's peripheral blood ofpre-eclampsiaObjective: To investigate the expression of HIF-1αprotein introphoblast and placenta bed, the concentration of vWF in placenta bedand mother's peripheral blood from pre-eclampsia and normal pregnancy,then to investigate together with the effect of HIF-1αprotein and vWF inthe pathogenesis of pre-eclampsia.Methods: The patient groups and normal group were split as the same asPart 1. Immunohistochemistry staining (SP method) method and westernblotting method were used to detect the expression of HIF-1αprotein inthe cultured trophoblast and placenta bed. ELISA method was adopted todetect the concentration of vWF in homogenate of placenta bed andmother's peripheral blood.Results:1. Compared the level of HIF-1αprotein in the cultured trophoblast andthe placenta bed:1.1 The gray scale that the HIF-1αprotein were detected in the culturedtrophoblast by immunohistochemistry staining method showed that the expressed result of HIF-1αprotein was the lowest in the pre-eclampsia(severe) among the three groups, the pre-eclampsia (mild) listed in themiddle and the normal group was the highest, there was significantdifference among the three groups (p＜0.001).1.2 The result detected by western blot assay showed that the expressionof the HIF-1αprotein was the highest in the pre-eclampsia (severe)among the three groups, followed by the pre-eclampsia (mild), the normalgroup was the lowest, there was significant difference among the threegroups (p＜0.001).1.3 The expression of the HIF-1αprotein in placenta bed detected bywestern blot assay showed that it was the highest in the pre-eclampsia(severe) among the three groups,the pre-eclampsia (mild) was listed inthe middle and the normal group was the lowest,there was significantdifference among the three groups (p＜0.001).1.4 It's positive correlation between the expression of HIF-1αprotein introphoblast and placenta bed of pre-eclampsia (r₁=0.62,p＜0.001).2. Compared the concentration of vWF in homogenate of placenta bed and inmother's peripheral blood the placenta bed:2.1.The concentration of vWF in homogenate of placenta bed showed thehighest was in the pre-eclampsia (severe), followed by the pre-eclampsia(mild), and the normal group was the lowest, there was significantdifference among the three groups (p＜0.001). 2.2. The concentration of vWF in mother's peripheral blood told us that itwas the highest in the pre-eclampsia (severe), then the pre-eclampsia(mild) in the middle and the normal group was the lowest, there wassignificant difference among the three groups (p＜0.001).2.3 It's positive correlation between the concentration of vWF inmother's peripheral blood and homogenate of placenta bed frompre-eclampsia (r₂=0.71,p＜0.001).2.4 It's positive correlation between the concentration of vWF inmother's peripheral blood and the pathogenetic condition degree ofpre-eclampsia (r₃=0.69,p＜0.001).3. Compared the correlation of HIF-1αprotein in placenta bed and theconcentration of vWF in homogenate of placenta bed from pre-eclampsia:It's positive correlation between the two (r₄=0.73,p＜0.001)。Conclusion:1. It was higher expression of HIF-1αprotein in trophoblast and placentabed from pre-eclampsia. This indicates that it is the importantpathogenetic reason for pre-eclampsia.2. The concentration of vWF was higher in homogenate of placenta bedand mother's peripheral blood from pre-eclampsia. It's positivecorrelation between the concentration of vWF in mother's peripheralblood and homogenate of placenta bed. It's positive correlation betweenthe concentration of vWF in mother's peripheral blood and pathogenetic condition degree of pre-eclampsia.3. It's positive correlation between the expression of HIF-1αprotein inplacenta bed and the concentration of vWF in homogenate of placentabed from pre-eclampsia.