Cellular localization of vascular endothelial growth factor in ovine placenta and fetal membranes

To further understand the role of vascular endothelial growth factor (VEGF) in mediating angiogenesis and vascular permeability during development in the sheep placenta and fetal membranes, we examined the localization of VEGF mRNA and protein in placental, chorionic and amniotic tissues by in situ hybridization and immunohistochemistry in ovine fetuses at 62, 102 and 141 days gestation (term=150 days). In the placenta, VEGF mRNA expression and VEGF protein immunostaining were strong in cytotrophoblasts surrounding the villi. In addition, VEGF protein was localized in smooth muscle cells around fetal and maternal blood vessels and in the maternal epithelium. There was no apparent difference in placental VEGF mRNA or protein levels associated with advancing gestation. In the fetal membranes, VEGF mRNA was detected in the amniotic epithelium and the chorionic cytotrophoblastic cell layer. The intensity of the hybridization signals in both amnion and chorion appeared low at 62 days, moderate at 102 days and high at 141 days gestation. VEGF protein was detected in amniotic epithelium and chorionic cytotrophoblasts at all gestational ages studied. The increase in VEGF gene expression in fetal membranes as term approaches suggests that during fetal development VEGF may promote the vascularity and permeability of the microvessels which perfuse the fetal membranes, as well as permeability of the amniotic membrane itself. Thus VEGF may participate in the regulation of amniotic fluid volume.     

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.     

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.     

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.     

Ki-67 and vascular endothelial growth factor expression in uterine leiomyosarcoma

OBJECTIVE: Uterine leiomyosarcomas (LMS) are rare female neoplasms of high malignant potential. The importance of different prognostic parameters in these malignancies remains controversial. The aim of the present study was to evaluate the prognostic value of Ki-67 and vascular endothelial growth factor (VEGF) in uterine LMS. METHODS: Ki-67 and VEGF expression was assessed using immunohistochemistry from paraffin-embedded tissue in 20 patients with uterine LMS. RESULTS: Ki-67 was expressed in 10 of 20 cases (50%) of uterine LMS. A statistically significant positive correlation was observed between vascular space involvement and Ki-67 expression (P = 0.05). There was no statistically significant relationship between Ki-67 expression and clinical stage (P = 0.38), recurrent disease (P = 0.86), and age (P = 1.0). An expression of VEGF could be observed in 1 of 20 (5%) cases. The median disease-free survival was 13 months (range: 3-23 months). Eleven (55%) patients died of the disease with a median overall survival of 47.4 months (range: 1-227 months), resulting in a 5-year-overall survival rate of 45%. In a univariate analysis a statistically significant shortened disease-free survival in patients with Ki-67 positive tumors could be observed (P = 0.01). CONCLUSIONS: Our results suggest that the significant expression of Ki-67 in uterine LMS, correlating with vascular space involvement and being associated with a shortened disease-free survival, may be an indicator of the biological aggressiveness of these rare tumors.     

Lysyl oxidases: expression in the fetal membranes and placenta

The cross-linking of the connective tissues in the fetal membranes and placenta is important for their tensile strength and elasticity. We have studied the expression of lysyl oxidase (LOX) because it is the classical enzyme responsible for the cross-linking of collagen and elastin. We have also studied the two recently described, genetically distinct lysyl oxidase-like genes and proteins, lysyl oxidase-like (LOXL) and lysyl oxidase-like 2 (LOXL2), of unknown functions.Specific antisera have been used for immunolocalization in fetal membranes and placentae from early pregnancy terminations and after caesarean section at both preterm and term, prior to labour. In addition, the steady state mRNA levels of the three genes has been quantitated in separated amnion, chorion, decidua and placentae collected at term before labour. The immunocytochemistry shows that the spatial expression of the three lysyl oxidases is similar in early pregnancy in both the fetal membranes and placentae. However, by preterm this pattern had diverged and becomes greatest at term. The expression of the genes found at term was similar to the results of protein expression obtained by immunocytochemistry, with the exception of LOXL which had high placental gene expression, but low levels of immunolocalized protein. Thus by term, LOX was expressed predominantly in the amniotic epithelium, with little expression in the placenta, while LOXL showed highest gene expression in the placenta and lowest expression in the amnion. LOXL2 expression was again different and was expressed predominantly in the chorionic cytotrophoblast of the membranes with low expression in both the amnion and placentae. These results suggest that these three members of the lysyl oxidase family may have similar roles in early pregnancy during the development of the placenta and fetal membranes, but their divergence as pregnancy advances to term, may reflect changes in substrate specificity and connective tissue composition.     

Expression of vascular endothelial growth factor messenger ribonucleic acid and protein in human preimplantation embryos

In contrast to a previous report by Krussel et al., with the inclusion of larger numbers of unfertilized oocytes and normal embryos and more sensitive immunofluorescence, this study shows that expression of vascular endothelial growth factor messenger ribonucleic acid can be detected from the oocyte to the blastocyst stage and that protein can be detected from the 3-cell stage to the blastocyst stage in human preimplantation embryos.     

Angiogenic growth factor expression in placenta

New blood vessel growth is generally a rare event in the healthy adult. However, a notable exception to this is the female reproductive tract where cyclic angiogenesis occurs. Striking new vessel growth and remodeling also occurs during placentation; thus angiogenesis is essential for reproductive success. Vascular endothelial growth factor is a potent stimulator of this process and its production and action is tightly regulated. Indeed the placenta is a rich source of a soluble variant of the flt-1 receptor which seems to protect the placenta from the effects of excess vascular endothelial growth factor. The balance between new vessel growth (in the placental villi for example) and endothelial cell loss in the spiral arteries within the decidua is a delicate one. This is influenced by the local production of promotors and inhibitors of endothelial cell activation. Perturbation of this may lead to maternal pathology during pregnancy.     

缺氧对滋养细胞VEGF分泌与基因表达调控作用的研究

目的:研究原代培养人早孕绒毛滋养细胞VEGF分泌、基因表达的特点,以及缺氧的调控作用。方法:原代培养早孕绒毛滋养细胞,研究正常和缺氧(氯化钴化学诱导)条件下细胞培养上清液中VEGF含量(ELISA法)以及滋养细胞VEGF mRNA表达(RT-PCR方法)特点。结果:(1)免疫组化技术证实培养细胞角蛋白阳性,波形蛋白阴性,纯度达95%以上;(2)正常培养的滋养细胞在培养12h后分泌产生的VEGF明显增加,至培养结束时的72h达到最高点;缺氧诱导滋养细胞大量分泌产生VEGF的时间提前至缺氧培养后6h,持续至72h无下降,VEGF水平明显高于正常,最高达到2200ng/ml,较正常上升47%;(3)滋养细胞VEGF mRNA表达于培养12h时开始明显上升,至48h时达峰值;缺氧则使VEGF mRNA表达显著上升的时间提前至6h,约12h达峰值。结论:早孕绒毛滋养细胞中存在VEGF基因转录并分泌至细胞外;缺氧诱导VEGF转录增强、分泌增加。     

The effects of hypoxia on growth cones in the ovine fetal brain

Objective: This study was designed to investigate the effects of hypoxia on neural process proliferation by studying its effects on growth cone tubulin and insulin-like growth factor (IGF)-I receptor content.

Methods: Six fetal lambs were catheterized in the brachial artery and vein. Maternal oxygenation was reduced in steps from a fractional inspired oxygen concentration (FiO₂) of 20% to 6% by addition of nitrogen to the inhaled gas mixture for a period of 4?h of reduced oxygen intake. Fetal arterial blood was sampled after the maternal FiO₂ and oxygen were stable for >?5?min at maternal FiO₂ of 20% to 6%. Controls were obtained from normoxic fetuses whose ewes had similar surgery and were kept at an FiO₂ of 20% throughout the experiment. Growth cones were isolated from the fetal cerebrum and cerebellum. α-Tubulin and IGF-I receptors were quantified by immunoblotting. Tubulin and IGF-I receptor mRNA expressions were quantified by real-time polymerase chain reaction.

Results: Maternal nitrogen breathing reduced fetal arterial pH from 7.32?±?0.06 to 6.99?±?0.02 (p?<?0.001). Hypoxia increased IGF-I receptors from 143?±?10 to 327?±?14 (p?<?0.001) and from 272?±?26 to 396?±?34 (p?<?0.001) fluorescence units/μg protein in the cerebrum and cerebellum, respectively. It also increased α-tubulin from 713?±?30 to 1873?±?126 (p?<?0.001) and from 780?±?34 to 2362?±?79 (p?<?0.001) fluorescence units/μg protein in the cerebrum and cerebellum, respectively. Expression of IGF-I receptor mRNA increased significantly in the hypoxic animals both in the cerebrum and the cerebellum, but there was no change in expression of α-tubulin mRNA.

Conclusions: This increase in IGF-I receptor expression and growth cone content may be an adaptive response to hypoxia to maintain neurite growth by facilitating binding of IGF-I. Hypoxia also increased the growth cone level of α-tubulin but did not increase its mRNA expression, which may indicate an inability to polymerize tubulin and build microtubules.