SFlt-1and Hypoxia:Important Linkers Between Endothelial Dysfunction And Oxidative Stress In Preeclampsia Placenta

Preeclampsia(PE) is an unique multi-organs affected disease of pregnancy, the mainclinical manifestations included high blood pressure (≥140/90mmHg) andproteinuria(≥0.3g/24h) after20weeks of gestation. We can only take somesymptomatic treatment for this disease, such as spasmolysis, depressurization andtermination of pregnancy, however, the efficacy and prognosis is not satisfied. So PEis still one of the leading causes of death of maternal and perinatal infants.So far, thepathogenesis of preeclampsia is not yet completely understood, one of the mostwidely recognized and accepted mechanisms is abnormal trophoblast cells invadinginto the spiral artery of uterus. This poor rebuilding of vessels can cause endothelialdysfunctionand oxidative stress damage in placenta, which results in the excessiveapoptosis reaction in trophoblast cells of placenta. This study suggests that there is aclose connection between the endothelial dysfunction and oxidative stress damage,and the linkers between are hypoxia and sFlt-1, forming a vicious circle and finallycausing preeclampsia. Therefore, in this study we verified this vicious circle inpreeclampsia from three aspects:1.To validate that sFlt-1is closely related withpreeclampsia from the angle of clinical serum samples and mice.2. To verify thevicious cycle hypothesis by a series experiments, including: HUVEC treated bysFlt-1, HTR-8/SVneo hypoxia treated, and co-culture of both the two cells.3. Tostudy the effect of the vicious circle to the apoptosis reaction of trophoblast cells. Objective:1.To validate that sFlt-1is closely related with preeclampsia from theangle of clinical serum samples and mice.Methods:1.The patients were chosed form the peoples’ hospital of Jiangsu provincewith preeclampsia (n=32) and normal pregnant women (n=35). The PE group wasdivided into preterm preeclampsia (PP,<34weeks, the average gestational age33.2±0.4weeks, n=14) and term preeclampsia (TP,≥34weeks, the average gestationalage36.2±0.2weeks, n=18). For gestational age matched, the normal group wasalso divided into preterm normal pregnancy (PNP,<34weeks,33.7±0.9weeks, n=17) and term normal pregnancy (TNP,≥34weeks,37.8±0.3weeks, n=18). Thecomparison was divided into two groups (PP vs. PNP; TP vs. TNP). The levels ofsFlt-1were tested in the serum of patients by ELISA.2.The ICR mice weresuccessively intraperitoneal injected with sFlt-1or saline from the8thday ofpregnancy for8days. The blood pressure and urine protein were tested. The plasmasFlt-1level was detected by ELISA. The sFlt-1mRNA were analyzed by RT-PCR.The morphology of the placenta, uterus and kidney were observed by HE staining.And the ultrastructure of placenta and kidney were further observed under electronmicroscope.Results:1.There were two groups of comparisons (PP vs. PNP) and (TP vs. TNP).There were no significant differences in age, BMI, gestational age, smoking in boththe two groups, and there were significant differences in systolic blood pressure,diastolic blood pressure, proteinuria and birth weight in the two groups.2.PlasmasFlt-1level (PP vs. PNP,45.07±22.32ng/mL vs.14.59±2.64ng/mL, p <0.01),(TP vs. TNP,26.23±10.75ng/mL vs.12.95±7.44ng/Ll, p <0.01). The plasmasFlt-1levels were significantly higher in preeclampsia pregnant women than thenormal control groups.3.The sFlt-1mRNA level of sFlt-1-treated mice was significantly higher than the saline group in placenta, so was the plasma sFlt-1level(2.5±0.13ng/mL vs.0.9±0.03ng/mL, p <0.05).4. The systolic blood pressure,mean arterial pressure, urine protein of sFlt-1treated pregnant mice weresignificantly increased than the saline-treated mice (p <0.05). There were nodifferences in the number and weight of fetus between the two groups (p>0.05).5.There were some changes in morphology of placenta, uterus and kidney of the sFlt-1treated pregnant mice: endothelial injury in the vessels of placenta and oxidativestress in the trophoblast cells, thinner endometrium in uterus, vascular fracture inglomerulus, local thickened basement membrane of the glomerular endothelial cellsand fusion of foot processes.Conclusion: sFlt-1is closely related with preeclampsia from the angle of clinicalserum samples and mice.Part2: sFlt-1and hypoxia are important linkers of the viciouscircle between endothelial dysfunction and oxidative stressObjective: To find whether sFlt-1and hypoxia are important linkers of the viciouscircle between endothelial dysfunction and oxidative stress.Methods:1.After umbilical vein endothelial cells (HUVEC) treated by sFlt-1for48h, we tested the ability of formation of blood vessels to observe the role of sFlt-1in angiogenesis and the expression of eNOS by Western blot.2.After trophoblastcells HTR-8/SVneo treated under hypoxia for48h, the sFlt-1secretion and mediumoxidatie stress index malondialdehyde (MDA), superoxide dismutase (SOD) weredetected.3.The sFlt-1over-expressed HTR-8/SVneo cells were co-cultured withHUVEC cells for48h, and the ability of formation of blood vessels and the expression of eNOS were tested to observe the role of trophoblast derived sFlt-1inangiogenesis.Results:1.The tube-like structures were damaged and the expression of eNOS wassignificantly decreased by sFlt-1.2.After been hypoxic-treated for48h, the sFlt-1mRNA and protein were significantly elevated. Meanwhile, the oxidant MDA wasfound to be increased and antioxidant SOD was decreased.3.The sFlt-1secreted byHTR-8/SVneo cells significantly impaired the structure of the tubes formed byHUVEC cells and the expression of eNOS was greatly reduced by the HTR-8/SVneosecreted sFlt-1.Conclusion: sFlt-1and hypoxia are important linkers of the vicious circle betweenendothelial dysfunction and oxidative stress.Part3: sFlt-1and hypoxia promote apoptosis reaction introphoblast cells in preeclampsiaObjective: To verify whether sFlt-1and hypoxia promote apoptosis reaction introphoblast cells in preeclampsia. Methods:1. HTR-8/SVneo cells were treatedwith sFlt-1and/or hypoxia. The rate of apoptosis was detected by flow cytometrytechnology and the apoptosis related proteins, such as Bax, Bcl-2and Caspase-3were detected by Western blot.2. Normal placenta villus tissues were cultured andtreated by sFlt-1and/or hypoxia for48h, and the apoptosis related proteins, such asBax, Bcl-2and Caspase-3were also detected by Western blot.3.The expression ofapoptosis related proteins Bax, Bcl-2and Caspase-3were studied in normal andpreeclampsia placenta. Results:1. The apoptotic HTR-8/SVneo cells were significant increased by sFlt-1or hypoxia. And a more significant increased apoptosis ratio by13.1%±1.4%wasobserved under both sFlt-1and hypoxia conditions. Apoptic protein Bax andCaspase-3were upregulated by sFlt-1or hypoxia, especially under sFlt-1andhypoxia conditions. On the contrary, the anti-apoptotic protein Bcl-2was downregulated.2.Apoptic protein Bax and Caspase-3were upregulated by sFlt-1orhypoxia in placenta villus tissues, especially under sFlt-1and hypoxia conditions.On the contrary, the anti-apoptotic protein Bcl-2was also down regulated in placentavillus tissues.3. An imbalance of apoptotic and anti-apoptotic protein was alsopresented, with Bax and Caspase-3up-regulated and Bcl-2down-regulated in earlyand late onset preeclampsia placenta.Conclusion: sFlt-1and hypoxia promote apoptosis reaction in trophoblast cells inpreeclampsia.