Proteasomal activity in placentas from women with preeclampsia and intrauterine growth restriction: implications for expression of HIF-alpha proteins.

Hypoxia-inducible transcription factor-1alpha and -2alpha (HIF-alpha) proteins and regulated genes are increased in preeclamptic (PE) placentas. Although placental hypoxia likely stabilizes HIF-alpha proteins, we previously reported that there is also a defect in oxygen-dependent reduction of HIF-alpha proteins in PE relative to normal pregnant (NP) placentas that could contribute to their over-expression. After a 4-h exposure to 2% oxygen, placental villous explants were exposed to 21% oxygen over 90 min. As assessed by Western analysis, the defective oxygen-dependent reduction of HIF-1alpha protein in villous explants from PE placenta was unaffected by the protein synthesis inhibitor, cycloheximide. However, after incubation with the proteasomal inhibitor, clasto-lactacystin, oxygen-dependent reduction of HIF-1alpha protein was markedly and similarly impaired in the villous explants from both normal and PE placentas. Thus, impairment of protein degradation rather than increased synthesis causes inadequate oxygen-dependent reduction of HIF-1alpha protein in PE placentas. Immunoprecipitation studies revealed comparable association of HIF-1alpha with von Hippel Lindau (VHL) protein in placentas from NP and PE women. Furthermore, prolyl hydroxylase-3 protein was appropriately upregulated in the PE placentas as determined by Western analysis paralleling the increases of HIF-alpha proteins. These results suggest that molecular events leading to the formation of the HIF-1alpha:VHL:ubiquitin ligase complex are most likely not impaired in PE placentas. Finally, proteasomal trypsin, chymotrypsin, and peptidyl glutamyl-like activities were significantly reduced by approximately 1/3 in PE placentas by using specific peptide substrates coupled to a fluorescent tag. Unexpectedly, however, they were even further decreased in placentas from normotensive women delivering growth restricted babies >37 weeks gestation-placentas which do not have elevated HIF-alpha proteins. In conclusion, accumulation of HIF-alpha proteins in PE placentas may occur as a consequence of both increased formation secondary to relative ischemia/hypoxia and reduced degradation after reperfusion/oxygenation consequent to proteasomal dysfunction. In contrast, in placentas from normotensive women delivering growth restricted babies >37 weeks gestation, proteasomal activity, albeit markedly reduced, is adequate to cope with degradation of HIF-alpha proteins, which have not been increased by an hypoxic environment.

Rajakumar A ，Michael HM ，Daftary A ，Jeyabalan A ，Gilmour C ，Conrad KP ... -  《PLACENTA》

Evidence for the functional activity of hypoxia-inducible transcription factors overexpressed in preeclamptic placentae.

Placentas from women with preeclampsia overexpress the hypoxia-inducible transcription factor proteins, HIF-1alpha and -2alpha (Rajakumar 2001, Biol Reprod 64; p499-506 and p1019-1020). As a first step in evaluating whether HIF-alpha overexpressed in preeclamptic placentae is capable of transactivation, we tested its ability to bind to the DNA hypoxia response element (HRE). Six pairs of normal and preeclamptic placentae obtained by cesarean section were investigated. Three biopsy sites per placenta were analyzed. We first confirmed HIF-1alpha protein overexpression in the preeclamptic placentae using Western analysis. The ratios of the arbitrary densitometry units for HIF-1alpha protein from the preeclamptic and normal placentae (PE/NP) in the three biopsy sites were: 1.9 +/- 0.3, 1.7 +/- 0.2 and 1.8 +/- 0.2, each p < 0.05 vs 1.0. (A ratio of >1.0 indicates that HIF-1alpha protein expression in placentas of women with PE exceeds that in placentas of NP women.) Conventional methods for extracting nuclear proteins and subsequent analysis by electrophoretic mobility shift assay were not suited for the frozen, archived samples (data not shown). Therefore, we employed DNA affinity chromatography using a biotinylated oligonucleotide representing the HRE of the erythropoietin gene coupled to streptavidin-coated Dynabeads. The HRE-bound proteins were then characterized by Western blot analysis. The PE/NP ratios of HRE-bound HIF-1alpha in the three biopsy sites from the six pairs of normal and preeclamptic placentae were 1.7 +/- 0.2, 2.1 +/- 0.4 and 2.4 +/- 0.5, each p < 0.05 vs 1.0. Having established DNA-binding potential at least in vitro, we subsequently analyzed three proteins that have been shown to be regulated by HIF-alpha as downstream, molecular markers of HIF-1alpha activity in vivo. VEGF receptor Flt-1 and Flk-1 play key roles in angiogenesis. Tyrosine hydroxylase is the rate-limiting enzyme in catecholamine synthesis. All three genes contain functional HRE in their promoter sequences. Total proteins were extracted from the same biopsy samples that were used for total and HRE-bound HIF-1alpha. Using specific antibodies we performed Western analysis and the levels of these three proteins were quantitated. The Flt-1 and tyrosine hydroxylase proteins were significantly higher, and Flk-1 significantly lower in placentae from preeclamptic compared to normal pregnancies. In summary, HIF-1alpha protein overexpressed in preeclamptic placentae is capable of binding to its DNA recognition sequence in vitro, and modulates gene expression in vivo.

Rajakumar A ，Brandon HM ，Daftary A ，Ness R ，Conrad KP ... -  《PLACENTA》

Extra-placental expression of vascular endothelial growth factor receptor-1, (Flt-1) and soluble Flt-1 (sFlt-1), by peripheral blood mononuclear cells (PBMCs) in normotensive and preeclamptic pregnant women.

The soluble VEGF receptor, sFlt-1 (otherwise referred to as sVEGFR-1), has been implicated in the pathogenesis of preeclampsia. The preeclamptic placenta has been previously demonstrated to produce high levels of the soluble VEGF receptor. Here we tested the hypothesis that peripheral blood mononuclear cells (PBMCs) may also represent an additional source for circulating sFlt-1 during normal and preeclamptic pregnancies. We first demonstrate that preeclamptic placentae show five-fold increased Flt-1 and sFlt-1 mRNA levels. We also show that the Flt-1 and sFlt-1 levels are eight-fold higher in preeclamptic placentae if we collect biopsies without rinsing them in saline to remove excess blood. Cultured villous explants from women with preeclampsia failed to show the increased amount of Flt-1 and sFlt-1 mRNA that was observed in the placental biopsies of normal pregnancy and preeclampsia. Under normoxic conditions the Flt-1 and sFlt-1 mRNA levels in the explants were 3.11+/-0.6 fold in normal pregnancy and 3.6+/-0.4 fold in women with preeclampsia (p = NS by ANOVA). However, the same villous explants showed hypoxic induction of Flt-1 mRNA (NP 3.96+/-0.4 fold, p = NS and PE 5.24+/-0.6 fold, p < 0.05 by ANOVA). We analyzed Flt-1 and sFlt-1 protein levels in the peripheral blood mononuclear cells (PBMCs) to analyze the possibility of an extra-placental sFlt-1 source. Our results indicate that PBMCs of pregnant women are capable of expressing variable amounts of Flt-1 proteins. PBMCs from pregnant women exposed to hypoxia show up-regulation of HIF-1alpha and Flt-1 proteins. PBMCs obtained from women with preeclampsia (n = 9) produced significantly higher amounts of sFlt-1 under normal tissue culture conditions (104.6+/-14.3 pg/ml vs. 46.23+/-5.03 pg/ml, p < 0.05 by ANOVA) and much higher concentrations under hypoxia (196.74+/-26.3pg/ml vs. 83.3+/-13.6pg/ml, p < 0.05 by ANOVA) than PBMCs from normal pregnant women (n = 11). Moreover, analysis of PBMCs from a different group of women with a history of preeclampsia showed persistent abnormality of Flt-1 women one year post-partum. The present study indicates that Flt-1 dysregulation in PBMCs of pregnant women resulting in over-expression of sFlt-1 could be an additional (extra-placental) source of sFlt-1 that contributes to the pathogenesis of preeclampsia.

Rajakumar A ，Michael HM ，Rajakumar PA ，Shibata E ，Hubel CA ，Karumanchi SA ，Thadhani R ，Wolf M ，Harger G ，Markovic N ... -  《PLACENTA》

Expression of von Hippel Lindau (pVHL) protein in placentae from normal pregnant women and women with preeclampsia.

The hypoxia inducible transcription factors, HIF-1alpha and -2alpha proteins, are overexpressed in placentae from women with preeclampsia (Biol Reprod 2001;64:499-506; Biol Reprod 2001;64:1019-1020). Normally, these proteins are regulated in an oxygen-dependent manner being rapidly degraded by the ubiquitin-mediated proteasomal pathway. Recent studies have shown that the tumor suppressor protein, von Hippel Lindau (VHL), targets HIF for ubiquitinylation under nonhypoxic conditions. The objectives of the present work were: (1) to investigate VHL protein expression in normal pregnant (NP), preeclamptic (PE), and preterm (without PE) placentae, (2) to test whether VHL protein is hypoxia inducible in term and first trimester placental villous explants, and (3) to analyze the ontogeny of VHL protein expression in the human placenta. To begin evaluating the potential contribution of VHL to HIF overexpression in preeclamptic placentae, we analyzed the levels of the VHL protein in both normal and preeclamptic placentae (n=7 each). We hypothesized a deficiency of VHL protein in preeclamptic placentae. Eight biopsy sites were tested in each placenta and protein extracts were made. Western analysis was performed using VHL specific antibodies. Human renal adenocarcinoma (ACHN) cell extracts and extracts from COS-7 cells transfected with a VHL expression vector were used as positive controls. In a total of 112 biopsy sites that were analyzed (56 each for normal and preeclamptic placentae), the composite densitometry ratios (PE/NP) for the long (28 kDa) and short (19 kDa) forms of VHL were 1.09+/-0.2 and 1.16+/-0.11, respectively (both p=NS vs 1.0). A ratio of 1.0 indicates equal expression by preeclamptic and normal placentae. The same placentae exhibited composite densitometry (PE/NP) ratios of 1.97+/-0.23 and 1.68+/-0.20 for HIF-1alpha and -2alpha proteins, respectively (both p<0.05 vs 1.0). In a separate analysis, the protein expression of the short form of VHL was also comparable among NP, PE and preterm (n=6) placentae. VHL immunoreactivity was localized to cells within the basal plate and the syncytiotrophoblast. Despite induction of HIF proteins by hypoxia in first and term villous explants, there was no significant upregulation of VHL proteins. Finally, the expression of both the short and long forms of VHL protein decreased with gestational age (both p<0.05 by ANOVA), and in villous tissue from first trimester placentae VHL immunoreactivity was predominantly localized to the cytotrophoblast. These results suggest that (1) deficiency of VHL protein does not account for HIF-alpha overexpression in preeclamptic placentae, (2) VHL protein is not regulated by hypoxia in either first trimester or term placental villous explants, and (3) VHL protein expression in the placenta decreases as a function of gestational age.

Rajakumar A ，Doty K ，Daftary A ，Markovic N ，Conrad KP ... -  《PLACENTA》

Adriana and Luisa Castellucci Award lecture 2001. Hypoxia inducible factor-1: oxygen regulation of trophoblast differentiation in normal and pre-eclamptic pregnancies--a review.

During early pregnancy, trophoblast differentiation occurs in an environment of relative low oxygen tension which is essential for normal embryonic and placental development. At around 10-12 weeks' gestation, when the intervillous space opens to maternal blood, there is an increase in Po(2). This increase correlates with the time of maximal trophoblast invasion into the maternal decidua, which allows extravillous trophoblast cells to access and remodel the maternal spiral arteries. Hypoxia Inducible Factor 1(HIF-1) is a transcription factor which activates gene transcription in response to varying oxygen concentration of cells. HIF-1 is a heterodimer composed of the inducible HIF-1alpha and the constitutively expressed HIF-1beta/ARNT subunits. Using villous explants, we have demonstrated that the oxygen-regulated events of early trophoblast differentiation are in part mediated by TGFbeta(3), an inhibitor of trophoblast differentiation, via HIF-1alpha. Pre-eclampsia is a disease of pregnancy that is characterized by shallow trophoblast invasion. Recently, we have reported that TGFbeta(3) is over-expressed in pre-eclamptic pregnancy and that its down-regulation restores invasive capability to trophoblast cells. Because TGFbeta(3) is downstream of HIF-1alpha, in the present study we investigated the expression of HIF-1alpha in pre-eclamptic placentae and age-matched controls using in situ hybridization and histochemical analyses. We found that HIF-1alpha mRNA and protein expression are abnormally elevated in pre-eclamptic placental tissue when compared to normal placental tissue. We conclude that pre-eclampsia may result from a developmental failure of oxygen to increase or of trophoblast cells to respond and/or sense an increase in oxygen. This will prevent the normal TGFbeta3 down-regulation and will lead to poor trophoblast invasion predisposing the pregnancy to pre-eclampsia.

Caniggia I ，Winter JL 《PLACENTA》