Study On Placental Growth Factor In The Pathogenesis Of Hypertensive Disorder Complicating Pregnancy And Its Relationship With Nitric Oxide

Placenta growth factor (PLGF) is a new member of the cysteine-knot family of growth factors and has significant amino acid homology with vascular endothelial growth factor (VEGF). The regulatory function of PLGF for trophoblast cell and endothelial cell by autocrine and paracrine patterns plays an important role in the development of placentas. So research on PLGF in normal placentas is a focus.Hypertensive disorder complicating pregnancy (HDCP) is a common disease during pregnancy, and its certain etiological factors are unclear. At present, it has been widespreadly accepted for the theory of placenta hypoxia and ischemia. PLGF is highly expressed in placenta, with the primary site of synthesis within the placenta being trophoblast. Its special receptor is a fms-like tyrosion kinase (Flt-1), which locates in the vascular endothelial cells and trophoblasts. The presence of this receptor in trophoblasts and vascular endothelial cells coupled with the high-level expression of PLGF by trophoblasts suggests a key autocrine and paracrine function for this growth factor. The abnormal expression of PLGF and Flt-1 probably plays an important role in the development of HDCP. However, there have few documents on PLGF in HDCP placentas.Nitric oxide (NO) is an endothelium derived relaxing factor. NO plays an essensial role in modulating placental blood circulation. It has been confirmed that low level of NO has a close relationship with HDCP. VEGF binding to Flt-1 induces NO releasing. However, whether PLGF binding to Flt-1 mediates NO production is unclear.Based on the above backgrounds, we investigated the roles of PLGF in normal and HDCP placentas tissue, as well as in human umbilical vein endothelial cells (HUVEC). First, we detected the expressions of PLGF, Flt-1 and NO in placentas, and analysised the relationship between PLGF and NO. Then, we investigated the role of PLGF-1 in inducing angiogenesis and NO production in HUVEC through primary culture. HUVEC were stimulated by PLGF-1, and then the proliferation of HUVEC and NO releasing were studied, as well as the molecul mechanisms of the proliferation of HUVEC and NO releasing mediated by PLGF-1.The study will contribute to the step research on the pathogenesis of HDCP and provide the theory instruction for controlling the disease.Study on PLGF in the genesis of HDCP and its relationship with NO46 women with HDCP and 20 normal pregnant women were studied. Among 46 women, there were 23 severe preeclampsia patients, 1 eclampsia woman and 1 preeclampsia superimposed upon chronic hypertension. The protein levels of PLGF and Flt-1 were determined by immunohistochemistry and Western blot method. The expressions of PLGF mRNA and Flt-1 mRNA in placentas were assessed by the reverse transcriptional-polymerase chain reaction (RT-PCR). The concentrations of NO in placental tissues were assayed by nitrite reductase. Our studies showed that: (1) The distributions of PLGF and Flt-1 in placentas of HDCP group were similar to normal one, which were in the cytoplasm of villous syncytiotrophoblast, villous blood vessels and villous stroma, and in some cases, Flt-1 was expressed in vascular smooth muscle cell. (2) The expressions of PLGF and Flt-1 were significantly decreased in mild and severe preeclampsia placentas compared to the normal ones (p<0.05) . There were no differences between the gestational hypertension placentas and normal one. (3) The transcription levels of the PLGF mRNA in mild and severe preeclampsia placentas were significantly lower than that of normal groups (3.33±0.39 vs 4.87±0.60; 1.97±0.29 vs 4.87±0.60, p<0.01) . However, the expression levels of Flt-1 mRNA were higher in preeclampsia placentas compared to control as assessed by RT-PCR (2.14±0.42 vs 1.55±0.49; 2.81±0.39 vs 1.55±0.49,p<0.05) . (4) The concentrations of NO in placental tissues in preeclampsia groups were significantly lower than those in normal one{ (8.20±5.56) μ mol/g vs (18.10±7.12) μ mol/g; ( 6.46±2.25) μ mol/g vs (18.10±7.12) μ mol/g,p<0.05}. (5) The expressions of PLGF in HDCP placentas were correlated to the concentrations of NO. (r=0.54,p<0.05).The analysis of placental growth factor-1 in mediating HUVEC proliferation and NO productionHUVEC were collected by trypsin digestion for primary culture. The protein levels of Flt-1 were determined by Western blot. The expressions of Flt-1 mRNA in HUVEC were assessed by RT-PCR. We examined the proliferation of HUVEC with or without PLGF-1 regulation by MTT method. The cells were treated respectively by PLGF-1 or PLGF-1 plus PD98059, inhibitor of mitogen-activated protein kinase (MAPK/MEK). The cells or culture media were collected in the different time for ERK protein analysis or NO detection. The protein levels of ERK were determined by Western blot. The concentration assession of NO was done by nitrate reductase. Our studies showed that: (1) Flt-1 mRNA and protein were expressed in HUVEC. (2) PLGF-1 stimulated HUVEC proliferation and induced NO releasing. (3) PLGF caused a time-dependent transient increase in phosphorylation of extracellular signal-regulated kinase-1/2(ERK-1/2) (4) PLGF-mediated NO releasing was inhibited by PD98059. In conclusion, the study got the following results1.We found that the expressions of PLGF were significantly decreased in mild and severe preeclampsia placentas compared to the normal ones(p<0.05) .It is suspected that the secretory restriction of PLGF is due to the placental bed hypoxia or ischemia. In turn, the decreased PLGF levels in HDCP placentas affects trophoblast invasion and angiogenesis, following that placenta hypoxia or ischemia goes a step further.2.Our findings also showed that the expressions of Flt-1 mRNA were higher in placentas with preeclampsia compared to controls as assessed by RT-PCR. Howerer, the expressions of Flt-1 protein were significantly decreased in HDCP placentas compared to the normal controls. We suspected that it may be due to the abnormal translation and expression of Flt-1 protein, although the transcription of Flt-1 mRNA in placentas with preeclampsia was increasing.3.In our study, we found that NO concentration in HDCP placenta was significantly lower than that of the normal controls. The low level of NO in placentas may increase fetal-placenta cycle resistance, thus result in the decreasing of placental blood flow and placenta hypoxia, and induce HDCP.4.We found PLGF level was correlated with NO concentration in HDCP placentas. It can be thought that the decreasing of PLGF results in HDCP not only by weaking trophoblast cells proliferation and invasion but also by affecting NO contents. It needs further study to be validated.5.We found that the Flt-1 protein and mRNA were expressed in HUVEC; PLGF-1 binding to Flt-1 stimulated HUVEC proliferation and induced NO releasing; PLGF caused a time-dependent transient increase in phosphorylation of extracellular signal-regulated kinase-1/2(ERK-1/2); PLGF-mediated NO releasing was inhibited by PD98059. Taken together, these data showed that the proliferation of HUVEC and NO releasing by PLGF occurs via activation of Flt-1, which results in activation of ERK-1/2 pathways.This study investigates the role of PLGF in normal pregnancy and HDCP placentas and provides data for clarifying the pathology of HDCP.