| Objective: To detect the expression of Krüppel-like factor8(KLF8)and matrix metalloproteinase-9(MMP-9) in human villous tissues duringnormal early pregnancy, placentas from patients with preeclampsia, and thehuman first trimester extravillous trophoblast cell line HTR8/SVneo cellsexposed to hypoxia-reoxygenation (H/R), then to explore the associationbetween KLF8and trophoblast invasion, oxidative stress and furtherinvestigate the roles of KLF8in the pathology of preeclampsia.Methods: A total of22early pregnancy human villous tissues of6-11weeks of gestation (Wks) were obtained from elective terminations ofapparently normal pregnancies from March2012to June2012in the FirstAffiliated Hospital of Chongqing Medical University; Twenty-two womenwith PE who delivered from September2011to March2012in the FirstAffiliated Hospital of Chongqing Medical University were chosen as PEgroup (n=22), and elective term cesarean sections group without previousonset of labor and perinatal complications as control group (n=20).Placentas were collected after the women delivered both from PE and control groups. And then immunohistochemical SP method were employedto detect the localization of KLF8protein. KLF8mRNA level wasdetermined by quantitative real-time PCR technique and western blotanalysis was used to quantify KLF8and matrix metalloproteinase-9(MMP-9) protein expression levels. HTR8/SVneo cells were exposed toH/R (8h at2%oxygen, followed by16h under standard culture conditionsfor two cycles) and standard culture conditions. Immunofluorescencestaining was employed to detect the subcellular localization of KLF8inHTR8/SVneo cells. Western blot analysis was used to quantify KLF8andmatrix metalloproteinase-9(MMP-9) protein expression levels andinvasion assay was performed to examine the invasive ability.Results:(1) There were KLF8immunoreactivity expressed in villoustissues from6-11Wks. It was primarily located in cyto-andsyncytio-trophoblast in the trophoblast layer of the chorionic villi. TheKLF8mRNA levels were gradually increased with gestational ages, peak at8-10gestational ages (highest at8Wks) and then decreased sharply. KLF8and MMP-9protein levels also shared the same pattern of expression inhuman villous tissues from early pregnancy: gradually increased from6Wks, peaked at8-10Wks (highest at8Wks) and then slightly decreased atthe end of the first trimester (11Wks). There was a positive correlationbetween the expression of KLF8and MMP-9protein in human villoustissues from early pregnancy (r=0.66, P<0.001).(2)There was no difference of KLF8protein distribution between placentas of the PE andcontrol group. It was mostly located in trophoblast, vascular endothelialcells and a few stromal cells. The KLF8mRNA levels were significantlydecreased in placentas of the PE groups (0.69±0.08) compared with that inthe control group (1.14±0.09, P<0.01). PE placentas also showed asignificant decrease in both KLF8(0.68±0.05) and MMP-9(0.21±0.03)protein levels relative to the normal control (0.94±0.06,0.34±0.03,P<0.01). There was a positive correlation between the expression of KLF8and MMP-9protein in human placentas (r=0.64, P<0.0001).(3) KLF8wasprimarilay located in nuclei of HTR8/SVneo cells exposed to standardculture conditions, however the levels of KLF8were increased in thecytoplasm in HTR-8/SVneo cells exposed to H/R. The in vitro Matrigelinvasion assay was shown that the percentage of invading cells treated withH/R was significantly inhibited compared with the untreated cells (P<0.01).HTR-8/SVneo cells exposed to H/R showed a significant decrease in bothKLF8(0.42±0.02) and MMP-9(0.13±0.01) protein levels relative to theuntreated control cells (0.75±0.02,0.19±0.01, P<0.01).Conclusion: The reduction of KLF8in the reoxygenation of hypoxictissues in both placentas from normal early pregnancies and latepregnancies complicated by PE and HTR-8/SVneo cells exposed by H/Rsuggests KLF8may mediate H/R-induced suppression of trophoblastinvasion as an oxygen tension sensor to participate in the pathologic process of preeclampsia. |
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