The Study On The Effect Of Pigment Epithelium-derived Factor (PEDF) On The Pathogenesis Of Preeclampsia

Research BackgroundHypertensive disorder complicating pregnancy is the systemic disease which is idiopathetic in the gestational period, the morbidity in China is9.4%, while it is7%～12%abroad. Up to now, the disease is still the main reason for the high incidence and mortality rate in the pregnant women and fetus. Preeclampsia is the most frequent type of the disease. It is shown by the epidemiologic survey, that morbidity rate of preeclampsia is5-10%. The etiological factors and process is not yet full clear to date, at present the main reasons are considered as the immuno-unbalance, superficial embedded placenta, the lesions of vascular endothelial cell, the poorly developed vascular net of placenta and decidua and so on. The research has shown that there is the defective vascular remoldeling of uterine decidua in preclampsia, the decreased amount of chorionic villus, angiodysplasia in chorionic villus, and interstitial fibrosis, which induce the hypoxia of placenta and wide lesions of vascular endothelial cell. It lead the functional lesion of multiorgan and multisystem finally.Normal placental vascular development depends upon the complex interactions between angiogenic inducers and inhibitors within the placenta.Pigment epithelium-derived factor (PEDF) is one of the most potent angiogenic inhibitors identified to date., which is a multifunctional50kDa secreted glycoprotein. Not only pigment epithelium-derived factor (PEDF) could inhibit the formation of new vessels, but also it can reverse the vessels which have been formative. And it is selective to inhibiting the formation of vessels, it inhibits the production of pathologic new vessels, while not affect the formation of physiologic vessels. The findings that pigment epithelium-derived factor(PEDF) is multifunctional protein have been identified to date, it has the function to give the nutrition to nerve, to resist the tumor, to reject the new vessels, to resist the vasopermeability, and it also has the personality of antioxygen and adjusting the cytopoiesis of adipose cells. In the year of2008, Beth and so on,who are American scholars, demonstrated that pigment epitheliumderived factor (PEDF), a potent inhibitor of angiogenesis, is expressed in both the vasculature and trophoblasts of placentas obtained from women with normal pregnancies. Pigment epitheliumderived factor (PEDF) is the factor responsible for inducing vascular quiescence and it is essential to maintain both vascular quiescence and to ensure normal vascular integrity.A lot of previous study has indicated that, there are vascular endothelial growth factor(VEGF) and its receptor(VEGF-R) expressed in the placentas and maternal serum in the whole pregnanct periods. VEGF is an effective inducer of the production of vessels. After the combination of VEGF and its receptor, it could regulate the differentiation of vascular endothelial cells by the paracrine secretion, and promote the hyperplasy、migration and invasion of endothelial cells. Meanwhile VEGF could increase the permeability of vessels, which induced a lot of plasma protein extravasation, to afford the temporary base which is necessary to promote the information of vessels. VEGF induces the expression of anti-apoptosis material named Bcl-2on the endothelial cells, and inhibite the apoptosis of endothelial cells.So it is evident that VEGF is essential to promote the information of vessels and matain the function of vascular endothelial cells. And VEGF also plays an important role in the implantation of embryo and the developtment of placental vessels. In the early trimester of pregnancy, the level of VEGF in placentas and serum occupies the relative elevated condition. One reason is that there is relative hypoxia stasis in the early trimester of pregnancy, which induces the up-regulation of the level of VEGF by a series of signal transduction pathways. The other reason is to adapt the physiologyical changes of pregnancy, because it needs adequate sufficient blood and oxygen supply in the growth and development of fetus and placentas.The study has showed that there is intimate correlation between the desecended level of VEGF in the earlier pregnant period and the pathogenesis of preeclampsia.The obvious low level of VEGF in the placenta tissues and maternal peripheral blood-serum, which maybe one of the etiological factors for preeclampsia disease. PEDF could induce the apoptosis of vascular endothelial cells and inhibit the formation of new vessels by the signal transduction pathway, which is mediated by Fas/FasL. Furthermore, PEDF could inhibit the up-regulation of VEGF by the angiotonin Ⅱ. So PEDF is the natural negative regulator to VEGF. There is no report about whether PEDF, similarity like VEGF. involves in the pathogenesis of preeclampsia.The implantation of blastocyst to the endometrium and myometrium is completed by the well differentiated trophoblast cells in normal pregnancy. It is essential important for the regulation to the trophoblast cells during the formation of normal placenta, the dysfunction of trophoblast cell is main cause to a series of pathogenic pregnancy, such as intrauterine growth retardation (IUGR) and pre-eclampsia. Human first trimester trophoblast cell is the cell,which has the invasive and destructive characteristics, similarity to tumor cell, but not tumorigenicity and metastasis. It has been shown in research,that the decreased ability of migration and invasion for the trophoblast cell plays an important role in preeclampsia. It was reported by literacy that PEDF could down-regulate the level of expression of matrix metalloproteinase (MMP-9),and then decrease the invasion ability of tumor cells, so it has the antineoplastic function.At present,the study has confirmed that the decreased expression level of matrix metalloproteinase induced the invasion ability of trophoblast cells, so to involve in the pathogenesis of preeclampsia. But it is still need to be verificated whether PEDF could also depressed the invasion ability of trophoblast cells and involve in the pathogenesis of preeclampsia by the similar mechanism,Therefore, we infer that pigment epithelium-derived factor(PEDF) may play an important role in the onset of preeclampsia disease, while it has not yet been reported all over the world. So our research is to begin with the factors which affect the the generations of the placental vascular beds. It will investigate the expression of pigment epithelium-derived factor (PEDF) in the placentas of preeclampsia women, and furthermore, it will study the effect of pigment epithelium-derived factor(PEDF) to the preeclampsia disease on the deck of vascular endothelial cells and trophoblast cells. It will provide the base of searching for the reason and mechanism for the pathogenesis of preeclampsia disease. [Objective] To investigate the expression of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor(VEGF) in the placentas of preeclampsia patients, and analyze the relationship between the vasculopathy in placentas with PEDF and VEGF.[Method] A study was performed in60cases of pregnant women with preeclampsia in the obstetrical department of Nanfang Hospital from Octorber2011to January2013, in which30cases were mild preeclampsia(mPE) and30cases were severe preeclampsia(sPE).40normal pregnant women who also be hospitalized and delivered contemporitely were selected as control group. The expression of PEDF, VEGF and MVD in placentas of the norma pregnant group were assayed by using western blot and immunofluorescence double labeling method, mild preeclampsia group and sever preeclampsia group, the relationship between PEDF,VEGF and MVD was analyzed. One-Way ANOVA is adopted to analyze the datas in the multiple groups, LSD test is applied to analyzed the comparison between two groups, the pearson coefficient is used in the correlation between two variables[Results](1) PEDF and VEGF are both expressed on the trophoblast cells and vascular endothelial cells in the placentas, mainly expressed in the cytoplasm and cell membrane, and the two factors almost were expressed in the same positon.(2) The expression of PEDF in preeclampsia group obviously increased and was significantly higher than that in normal group (P<0.05), which in severe preeclampsia group was significantly higher than that in mild preeclampsia group (P <0.05).(3) compared to normal group, the expression of VEGF in preeclampsia group was significantly lower (P<0.05),but there was no difference between mild preeclampsia group and severe preeclampsia group (P>0.05).(4) The microvessel density (MVD) reduced in preeclampsia group,which was significantly lower than that in normal group(P<0.05), and in severe preeclampsia group it was lower than that in mild preeclampsia group (P<0.05).(5) The expression of PEDF was negatively correlated with the expression of VEGF and MVD (r=-0.365, P<0.05; r=-0.655, P<0.05)in preeclampsia group, and the expression of VEGF was positively correlated with the expression of MVD(r=0.448,P<0.05).[Conclusion] PEDF and VEGF were together expressed in the trophocyte and vascular endothelial cell of the placenta. The expression of PEDF in preeclampsia placenta was increased, while VEGF and MVD expression was reduced, both of which were negatively correlated with PEDF. It indicates that PEDF may be involved in pathogenesis of preeclampsia, which possibly through affecting the placental vascular reconstruction.[Objective] To detect the expression of pigment epithelium-derived factor (PEDF) and its mRNA in the first trimester human trophoblast cells(HTR-8/SVneo) in the deficiency oxygen surroundings. then investigate the effect of hypoxia on the first trimester human trophoblast cells, therefore study the effect of PEDF on the preeclampsia disease.[Method] the first trimester human trophoblast cells (HTR-8/SVneo) were cultured in the deficiency environment, observe the morphologicalchanges of trophoblast cells by inverted microscope. Then detect the expression of pigment epithelium-derived factor (PEDF) and its mRNA in the first trimester human trophoblast cells by using the technology of cell immunohistochemistry and real-time fluorescent quantitative PCR.To detect the apoptosis and the proliferation activity of all the trophoblast cells by the technology of TUNNEL and CKK-8, respectively. And compare the invasion ability of all the trophoblast cells using transwell plate. One-Way ANOVA for Factorial design datas is adopted to analyze the datas in the multiple groups, Independent-Samples T Test is applied in the two groups comparison, the spearman coefficient is used in the correlation between two variables.[Result](1)the growth status of HTR8/SVneo cell under the1%oxygen concentration:HTR-8/SVneo cells were very sensitive to hypoxia, the shapes of cells changed from polygon or Fusiform shape to round shape, the prominence shrinked,and some of them disappeared, some cells assembled and fused together.(2) cell immunohistochemistry results showed that, PEDF were expressed in each group, the immunological reaction products were expressed mainly in kytoplasm and cell membrane; compared to the normal oxygen concentration, there was no difference on the expression of PEDF in cytotrophoblast cell within hypoxia24hours (P>0.05), but the expression of PEDF increased after under hypoxia48hours, where there is statistical significance (P<0.05), and with the prolonged hypoxia, the expression of PEDF increased more obviously (P<0.05).(3) the real time fluorescent quantitation PCR results showed that, compared to the normal oxygen concentration, there was no difference on the expression of PEDF mRNA in cytotrophoblast cell within hypoxia24hours (P>0.05); but the expression of PEDF mRNA increased after under hypoxia48hours, where there is statistical significance (P<0.05), and with the prolonged hypoxia, the expression of PEDF mRNA increased more obviously (P<0.05).The above results showed that expression of PEDF protein and mRNA reacted to hypoxia as time dependent.(4) to detect apoptosis by the TUNNEL method:compared to the normal oxygen concentration, there was no difference on the amount of apoptosis in cytotrophoblast cell within hypoxia48hours (P>0.05); but the apoptosis cells increased after under hypoxia72hours, where there is statistical significance (P<0.05)(5) to detect the proliferation activity of trophoblast cells by the technology of TUNNEL:there was no difference on the proliferation activity each group (P>0.05).(6) to detect the invasion ability by Transwell:there were disparate trophoblast cells,which infiltrated to the membrane below chamber. compared to the normal oxygen concentration, the infiltrated cells obviously decreased under hypoxia48hours and72hours, where there is statistical significance (P<0.05).(7) The expression of PEDF protein and PEDF mRNA in HTR-8/Svneo trophoblast cells of different groups were postively correlated with the number of apoptosis cells respectively (r=0.772, P<0.05; r=0.839, P<0.05), while the expression of PEDF protein was positively correlated with the expression PEDF mRNA((r=0.876,P<0.05).(8) The expression of PEDF protein in HTR-8/Svneo trophoblast cells of different groups were negatively correlated with the invasion ability of cells respectively(r=-0.615, P<0.05).[Conclusions] Our results infer that hypoxia can induce the obvious increased expression of pigment epithelium-derived factor (PEDF) protein, as well as its mRNA. And with the prolonged hypoxia time, the tendency of the expression level also increase significantly. Hypoxia could induce the apoptosis of trophoblast cell, and inhibit the invasion activity obviously on the trophoblast cell. The prolonged hypoxia time is followed by the decreased invasion activity.But PEDF do not affect the proliferation of trophoblast cells. [Objective] To construct the plasmid with PEDF gene (pcDNA-PEDF), then transfect the plasmid to the the first trimester human trophoblast cells, detect the apoptosis of trophoblast cells, then study the effect of the proliferation and invasion activity on the trophoblast cells by PEDF.[Methods] The first trimester human trophoblast cells were transfected by the the plasmid with PEDF gene, detect the expression of PEDF protein and its mRAN in the trophoblast cells after transfected24hours, through the the technology of cell-mediated immunochemistry and real time fluorescent quantitative PCR. To detect the apoptosis and the proliferation activity of all the trophoblast cells by the technology of TUNNEL and CKK-8, respectively. And compare the invasion ability of all the trophoblast cells using transwell plate. One-Way ANOVA is adopted to analyze the datas in the multiple groups, LSD test is applied to analyzed the comparison between two groups, the spearman coefficient is used in the correlation between two variables[Result]](1)Compared to the normal control group, after transfected24hours by the PEDF plasmid, the expression of PEDF protein and its mRAN in the HTR8/SVneo cytotrophoblast were increased obviously, where there is statistic significance (P<0.05),while there was no difference between the level in negative control group and normal control group(P>0.05).(2)After the transfection24hours, the apoptosis of trophoblast cell increased, while there was no difference on the proliferation activity in each group(P>0.05).(3) Compared to the normal control group, the trophoblast cells reduced which filtrated to the below chamber of transwell membrane and were transfected by the plasmid with PEDF gene (P<0.05), while there was no difference on the proliferation activity in each group (P>0.05).The result showed the cell invasion activity reduced obviously.(4) The expression of PEDF protein and PEDF mRNA in HTR-8/Svneo trophoblast cells of different groups were postively correlated with the number of apoptosis cells respectively (r=0.970, P<0.05; r=0.745, P<0.05), while the expression of PEDF protein was positively correlated with the expression PEDF mRNA(r=0.672,P<0.05).(5)The expression of PEDF protein was negatively correlated with the invasion ability of the cytoblast(r=-0.882, P<0.05).[Conclusions] Pigment epithelium-derived factor (PEDF) is very essential to maintain the normal function of the first trimester human trophoblast cells. The increase of PEDF expression could increase the apoptosis of trophoblast cells and reduce their invasion ability. The expression of PEDF protein is negatively correlated with the invasion ability of the trophoblast cells.