Prolonged hypoxia upregulates vascular endothelial growth factor messenger RNA expression in ovine fetal membranes and placenta

OBJECTIVE: In ovine fetuses, 4 days of hypoxia resulted in a large increase in urine flow, without the development of polyhydramnios, which suggests that intramembranous absorption of the amniotic fluid was enhanced. Because vascular endothelial growth factor is speculated to be a regulator of intramembranous absorption through increases of membrane vascularity and fluid transport, we hypothesized that hypoxia upregulated vascular endothelial growth factor gene expression in the fetal membranes. STUDY DESIGN: Five near-term ovine fetuses that were subjected to 4 days of hypoxia and 5 age-matched time controls were studied. On day 4, the amnion, chorion, and placenta were collected for cellular localization and quantification of vascular endothelial growth factor messenger RNA and for the determination of vascular endothelial growth factor molecular forms that were expressed. The data were analyzed statistically with the use of t tests and 2-factor analyses of variance. RESULTS: Vascular endothelial growth factor messenger RNA was expressed in the fetal membranes localized to the amniotic epithelium and chorionic cytotrophoblast, and to the villous cytotrophoblast of the placenta. In hypoxic fetuses, vascular endothelial growth factor messenger RNA levels in these cell layers were significantly increased compared with the controls. Five vascular endothelial growth factor molecular forms were identified with vascular endothelial growth factor(164) being the most abundant form expressed. The pattern of expression of the forms was not altered by hypoxia. CONCLUSION: In the near-term ovine fetus, hypoxia induced vascular endothelial growth factor messenger RNA expression in the amnion, chorion, and placenta. This was associated with an increase in intramembranous absorption of amniotic fluid. We speculate that the increased intramembranous absorption was mediated by a vascular endothelial growth factor-induced increase in the transport of amniotic fluid into the fetal membranes.     

Hypoxia modulation of caveolin-1 and vascular endothelial growth factor in ovine fetal membranes

During normal pregnancy, amniotic fluid is absorbed from the amniotic compartment into fetal blood through the intramembranous blood vessels in the fetal membranes. It has been hypothesized that this transport process is mediated by transcytosis of caveolae-like vesicles. Because fetal hypoxia increases intramembranous absorption, the authors explore the effects of hypoxia on the gene expression of caveolin-1, a structural protein of caveolae, in ovine fetal membranes and cultured amnion cells. Near-term ovine fetuses were rendered hypoxic for 4 days. Caveolin-1 mRNA and protein levels were significantly reduced in the amnion and chorion but not in the placenta. In cultured ovine amnion cells incubated in 2% oxygen for 24 hours, hypoxia did not significantly alter caveolin-1 mRNA or protein expression. Vascular endothelial growth factor mRNA levels were increased in response to hypoxia in the fetal membranes as well as in cultured amnion cells. The results indicate that hypoxia does not augment but instead down-regulates or has no effect on caveolin-1 gene expression in the amnion and chorion, suggesting that caveolin-1 may play a role as a negative regulator of amnion transport function under hypoxic conditions.     

Developmental expression of vascular endothelial growth factor (VEGF) receptors and VEGF binding in ovine placenta and fetal membranes

The receptor tyrosine kinases, kinase-insert domain-containing receptor (KDR) and fms-like tyrosine kinase (Flt-1), and their ligand vascular endothelial growth factor (VEGF) are essential for the development and maintenance of placental vascular function during pregnancy. To further understand the role of VEGF in mediating angiogenesis and vascular permeability during development, the cellular localization of KDR and Flt-1 mRNA and protein, and the distribution of(125)I-VEGF binding sites in placenta, chorion and amnion of ovine fetuses were examined at three different gestational ages. In placentae at 62, 103 and 142 days, the predominant site of KDR mRNA and protein, and VEGF binding was the maternal vascular endothelium. In addition, a specific, although weak, signal for KDR mRNA was found in the maternal epithelium. At 103 and 142 days but not 62 days gestation, KDR mRNA and protein as well as VEGF binding sites were abundantly present in the endothelium of villous blood vessels. In the fetal membranes at 62, 103 and 142 days gestation, KDR mRNA and protein were expressed in the amniotic epithelium and intramembranous blood vessel endothelium, where binding of(125)I-VEGF was strong. There was no KDR mRNA or VEGF binding in the chorionic cytotrophoblast. Flt-1 expression was not detectable in placentae or fetal membranes at the three ages studied.In summary, the results demonstrated that VEGF receptors are present in the maternal and fetal vasculatures of the ovine placenta. This expression is consistent with a capillary growth-promoting function of KDR and its ligand VEGF. Further, the presence of KDR and VEGF binding sites in ovine fetal membranes suggests a role for VEGF in promoting intramembranous vascularity and permeability throughout gestation.     

Ontogeny of aquaporins 1 and 3 in ovine placenta and fetal membranes

A sensitive and highly reproducible method has been used to show that Aquaporin 3 (AQP(3)) mRNA is present in the ovine placenta and chorion from at least 60 days of gestation (term=145-150d) with levels increasing substantially (>16 fold) at 100 days, and remaining constant thereafter. By immuno- and hybridization histochemistry, the epithelial cells expressing AQP(3)were found to be the trophoblast cells. Some AQP(3)was expressed in fibroblasts of the amnion and allantois but none was expressed in the epithelia of these membranes. AQP(1)was expressed in endothelial cells of fetal and maternal blood vessels but not in any epithelial cell of the ovine placenta and fetal membranes. The level of AQP(3)expression is consistent with known ovine placental permeabilities to water, glycerol and urea.     

Isolated placental vessel response to vascular endothelial growth factor and placenta growth factor in normal and growth-restricted pregnancy

Vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF) cause vasodilation. We examined the vasomotor response of isolated placental vessels to VEGF and PlGF in normal (group I) and intrauterine growth retardation (IUGR)-complicated pregnancy (group II). Rings of vessels were prepared in vitro and mounted on the vessel myograph plunged in tissue bath. The magnitude of dilation to increased doses of VEGF and PlGF has been studied. VEGF is a more potent vasodilator than PlGF. Both, VEGF- and PlGF-induced vasorelaxation was diminished in the IUGR (group II) nearly by half, compared to control (group I). Relative placental nitric oxide deficiency, or decreased sensitivity to VEGF and PlGF may contribute to the development of high impedance fetoplacental circulation.     

妊娠高血压综合征患者胎盘组织中血管内皮生长因子的表达

目的　探讨血管内皮生长因子（VEGF）在妊娠高血压综合征（妊高征）患者胎盘组织中的表达及对妊高征患者胎盘滋养细胞功能和绒毛毛细血管网形成的影响。方法　采用免疫组织化学SP法检测21例正常孕妇（对照组）,60例妊高征患者（妊高征组,其中轻、中、重度妊高征患者各20例）胎盘组织VEGF的表达强度。结果　VEGF主要表达于胎盘绒毛滋养细胞;中度及重度妊高征患者胎盘绒毛滋养细胞VEGF表达强度均明显低于对照组及轻度妊高征患者(P＜0.05),而轻度妊高征患者与对照组比较,差异无显著性(P＞0.05),各组间蜕膜组织VEGF表达强度差异无显著性(P＞0.05)。　结论　VEGF在胎盘中主要由绒毛滋养细胞分泌,妊高征患者VEGF分泌减少,可能是引起局部胎盘绒毛及血管病变的原因之一。     

缺氧诱导因子1α及其靶基因在子痫前期患者胎盘组织中的表达

目的探讨缺氧诱导因子1α(HIF-1α)及其靶基因血管内皮细胞生长因子(VEGF)、可溶性血管内皮细胞生长因子受体1(sFlt-1)基因在子痫前期患者胎盘组织中的表达。方法采用免疫组化链霉菌抗生物素蛋白-过氧化物酶连接(SP)法对20例重度子痫前期患者(子痫前期组)和15例正常妊娠妇女(对照组)的胎盘组织中的HIF-1α、VEGF、sFlt-1蛋白表达进行检测;同时应用RT- PCR技术对两组孕妇胎盘组织中HIF-1α、VEGF、sFlt-1 mRNA水平进行检测,并进行相关性分析。结果(1)子痫前期组HIF-1α蛋白表达(+++)者9例(45%,9/20),sFlt-1蛋白表达(+++)者11例(55%,11/20),对照组分别为2例(13%,2/15)和3例(20%,3/15),两组分别比较,差异均有统计学意义(P＜0．05);子痫前期组VEGF蛋白表达(+++)者3例(15%,3/20),明显低于对照组的7例(47%,7/15),两组比较,差异有统计学意义(P＜0．01)。(2)子痫前期组HIF-1αmRNA、sFlt-1 mRNA水平分别为0．604±0．013、0．898±0．041,对照组分别为0．208±0．007、0．559±0．244,两组分别比较,差异均有统计学意义(P＜0．05);子痫前期组VEGF mRNA表达水平虽高于对照组,但差异无统计学意义(P＞0．05)。子痫前期组VEGF mRNA/sFlt-1 mRNA比值(0．439±0．009)低于对照组(0．824±0．011),两组比较,差异有统计学意义(P＜0．05)。(3)子痫前期组HIF-1αmRNA的表达与sFlt-1 mRNA表达呈正相关(r=0．577,P＜0．05),与VEGF mRNA/sFlt-1 mRNA比值呈负相关(r= -0．376,P＜0．05)。结论HIF-1α在子痫前期患者胎盘组织中表达水平明显升高,主要是通过调节VEGF、sFlt-1基因的转录,而影响滋养细胞的浸润和胎盘血管重铸,参与子痫前期的发病。     

Co-localization of vascular endothelial growth factor and its two receptors flt-1 and kdr in the mink placenta

Placental angiogenesis plays an important role in placental development and morphogenesis. Vascular endothelial growth factor (VEGF) is a well-known angiogenic growth factor, which has previously been localized in different epitheliochorial and haemochorial placenta types. In the present study VEGF and its Flt-1(VEGFR-1) and KDR (VEGFR-2) receptors were immunolocalized in the endotheliochorial mink placenta throughout gestation. VEGF, Flt-1 and KDR co-localized to fetal and maternal microvascular endothelial cells, but with a temporal difference, displaying KDR in endothelial cells throughout gestation, whereas the VEGF and Flt-1 maternal endothelial cell staining was most intense during late gestation. Additionally, KDR was found in vascular related mesenchymal cells. The VEGF-receptors were also localized in non-endothelial cells, e.g. the uterine luminal and glandular epithelium as well as the trophoblast. Our results are in agreement with former studies, showing the different effects of the Flt-1-and KDR receptors in respect of angiogenesis. More importantly, the present study of the endotheliochorial placenta localizes the VEGF-ligand-receptor system in non-endothelial cells, and thereby strengthen the hypothesis that VEGF, apart from its well-established angiogenic properties, must also have additional functional roles in the establishment and development of the placenta.     

Vascular endothelial growth factor, placenta growth factor and their receptors in isolated human trophoblast

The expression of the angiogenic growth factors, vascular endothelial cell growth factor (VEGF) and placenta growth factor (PIGF) was demonstrated in isolated human term cytotrophoblast and in vitro differentiated syncytiotrophoblast. RNase protection assays demonstrated VEGF expression in both cytotrophoblast and syncytiotrophoblast while prominent PIGF expression was detected in both types of trophoblast by Northern blot analyses. VEGF expression increased approximately eightfold in trophoblast cultured under hypoxic conditions (1 per cent O₂) yet PIGF expression decreased 73 ± 5.5 per cent in the same trophoblast. These results suggest distinct regulatory mechanisms govern expression of VEGF and PIGF in trophoblast. Characterization of the VEGF/PIGF receptors, KDR and flt-1, revealed the presence of flt-1 mRNA in isolated cytotrophoblast and in vitro differentiated syncytiotrophoblast. KDR was not detected in the isolated trophoblast. Exogenous rhVEGF induced c-Jun N-terminal kinase (JNK) activity in the normal trophoblast indicating that the flt-1 receptors on trophoblast are functional. Trophoblast-derived VEGF/PIGF could act in a paracrine fashion to promote uterine angiogenesis and vascular permeability within the placental bed. In addition, presence of functional flt-1 on normal trophoblast suggests that VEGF/P1GF functions in an autocrine manner to perform an as yet undefined role in trophoblast invasion, differentiation, and/or metabolic activity during placentation.     

Reductions of vascular endothelial growth factor and placental growth factor concentrations in severe preeclampsia

OBJECTIVE: The aim of this study was to determine whether plasma concentrations of vascular endothelial growth factor and placental growth factor are altered in women with severe preeclampsia. STUDY DESIGN: We performed a case-control study to compare plasma concentrations of vascular endothelial growth factor and placental growth factor between women with severe preeclampsia and normotensive women admitted for delivery. Twenty-one women with severe preeclampsia were matched for gestational age and ethnicity with 21 normotensive women. Vascular endothelial growth factor and placental growth factor concentrations were measured with a specific antigen-capture enzyme-linked immunosorbent assay. RESULTS: Women with severe preeclampsia demonstrated significantly lower plasma concentrations of both vascular endothelial growth factor (6.36 +/- 3.96 pg/mL vs 18.65 +/- 5.98 pg/mL; P <.0001) and placental growth factor (138 +/- 119 pg/mL vs 531 +/- 340 pg/mL; P <.0001) than did women with normotensive pregnancy. Logistic regression analysis showed an independent association between plasma vascular endothelial growth factor concentration and plasma placental growth factor concentration and preeclampsia. CONCLUSION: Patients with severe preeclampsia had decreased maternal serum concentrations of both vascular endothelial growth factor and placental growth factor.     

Amniotic levels of nitric oxide and vascular endothelial growth factor in pregnancy with subsequent intrauterine fetal death

OBJECTIVE: Nitric oxide (NO) and vascular endothelial growth factor (VEGF) regulate angiogenesis and seem involved in the early stages of placentation. If angiogenesis is reduced, this may lead to poor placentation and fetal death. This study was aimed to determine whether VEGF and NO are associated to subsequent fetal death. STUDY DESIGN: We retrospectively assessed NO and VEGF on midtrimetster amniotic fluid from seven women who had subsequently had intrauterine fetal death before 20 weeks, and compared the results with those of 14 controls matched for age and gestation. All women had undergone amniocentesis for maternal age. All were at 16 weeks of gestation. None had shown chromosomal abnormalities. Results (mean+/-S.D.) were tested for statistics with Student's t-test with significance at P<0.05. RESULTS: Women with subsequent fetal death had both amniotic NO and VEGF lower than women with normal pregnancy (NO 3.28+/-1.20 microg/mg creatinine versus 6.02+/-1.57 microg/mg creatinine, P<0.05; VEGF 210.10+/-69.55 pg/ml versus 255.05+/-88.66 pg/ml). CONCLUSIONS: An early reduction of both NO and VEGF may be responsible of an impaired placental vascular development and endothelial regulation that may lead to fetal death.     

Preterm premature rupture of membranes: vascular endothelial growth factor and its association with histologic chorioamnionitis

OBJECTIVE: We hypothesize that vascular endothelial growth factor, a known angiogenic and permeability factor that is locally expressed in fetal membranes and decidua, may be the primary regulator in the pathway that eventually leads to preterm premature rupture of membranes. Our objective was to test the hypothesis that, both in the presence and in the absence of histologic chorioamnionitis, there is an increased expression of the vascular endothelial growth factor gene and its receptor Flt-1 in the human fetal membranes. STUDY DESIGN: Membranes were sampled from a region that was distinct as the rupture site from three groups of patients with preterm premature rupture of membranes. Groups 1 and 2 differed only in the length of the latency period from rupture of the membranes to delivery. Group 3 included preterm patients with intact membranes, who acted as control subjects. All patients who were selected for the study lacked clinical signs of chorioamnionitis and were delivered by cesarean delivery. Tissue samples were analyzed for interleukin-6 gene expression by Northern blot analysis and for the presence of interleukin-6 protein by immunocytochemistry. The expression of vascular endothelial growth factor and Flt-1 genes was analyzed by in situ hybridization. RESULTS: All tissue samples from group 1 and five tissue samples from group 2 (designated as group 2A) showed expression of the interleukin-6 gene and the presence of interleukin-6 protein in the fetal membranes (P <.001) and were therefore identified as inflamed. Five tissue samples from the patients in group 2 (designated as group 2B) and all control tissue samples showed neither evidence of interleukin-6 gene expression nor the presence of its protein and therefore were identified as not inflamed. Vascular endothelial growth factor and Flt-1 gene expression were increased significantly in the fetal membrane and decidua samples that were obtained from the noninflamed tissues from group 2B (P <.005) yet showed further enhancement in expression in the inflamed tissues. CONCLUSION: The expression patterns of vascular endothelial growth factor and Flt-1 genes are indicative of a molecular pathologic condition of fetal membranes, regardless of their inflammatory status, which suggests their role as a primary regulator of preterm premature rupture of membranes.     

HIF-1α、VEGF及MVD在子痫前期不同发病类型胎盘的表达

目的:探讨缺氧诱导因子1α(HIF-1α)、靶基因血管内皮生长因子(VEGF)及微血管密度(MVD)在子痫前期患者胎盘组织的表达及与胎盘血管病变的关系。方法:用链酶菌抗生物素蛋白-过氧化物酶连接(SP)法检测早发型,晚发型重度子痫前期患者和正常妊娠妇女(对照组)胎盘组织中HIF-1α、VEGF的表达,并计数胎盘微血管密度(MVD)值,并进行相关性分析。结果:(1)子痫前期患者胎盘组织中HIF-1α阳性表达率明显高于正常对照组,差异有统计学意义(P<0.05),早发型、晚发型子痫前期组HIF-1α与正常对照组的差异有统计学意义(P<0.05),早发型组HIF-1α阳性表达率高于晚发型组,两者差异有统计学意义(P<0.05);(2)子痫前期患者胎盘组织中VEGF阳性表达率低于正常对照组,两者差异有统计学意义(P<0.05),而早发型组VEGF阳性表达率低于晚发型组,差异亦有统计学意义(P<0.05);(3)正常对照组,晚发型子痫前期组,早发型子痫前期组MVD计数依次递减,子痫前期组与正常对照组比较有统计学差异(P<0.05),早发型组与晚发型组比较有统计学差异(P<0.05);(4)子痫前期组和正常对照组胎盘中HIF-1α表达与VEGF及MVD值的变化呈显著负相关(r=-0.562,P<0.05;r=-0.622,P<0.05),VEGF及MVD值的变化则呈显著正相关(r=0.718,P<0.05)。结论:HIF-1α在子痫前期患者胎盘组织中表达升高,通过下调靶基因VEGF,引起VEGF降低,影响胎盘血管重铸,参与子痫前期的发生和发展。