Maternal and Fetal Plasma Protein Changes in Fetal Growth Restriction

Fetal Growth Restriction (FGR) is caused by impaired maternal-fetal exchange of oxygen and nutrients causing fetal hypoxia and starvation. A functional failure of the placenta is the underlying cause, however the pathophysiology remains unknown. The fetus adapts by limiting growth, reducing demand for metabolic substrates. Monitoring the fetal size is the primary clinical method of FGR detection, though it does not distinguish a constitutionally small fetus from a pathological. Proteomic profiling of fetal and maternal plasma was therefore undertaken for discovery of biomarkers and pathological mechanisms. As a model of hepatic secreted fetal plasma proteins, HepG2 cell secretion changes in hypoxia were also investigated. Profiling mother’s plasma revealed altered expression of vascular regulatory proteins VCAM-1 and haptoglobin. VCAM-1 positively correlated to placental size. Profiling of HepG2 secreted proteins in hypoxia revealed increased angiogenic protein PAI-1, and the growth inhibitor IGFBP-1. Fetal plasma PAI-1 levels were found to be oxygen dependent, and the levels determinant of plasma’s in vitro angiogenic potency. For IGFBP-1, increased phosphorylation was found at four discrete sites, leading to increased affinity for IGF-I, and mitigation of IGF-I stimulated cell proliferation in vitro. Increased VCAM-1 relative to placental size in FGR has potential as a marker of placental health. Fetal plasma PAI-1 levels mediating angiogenesis is a newly discovered mechanism in FGR. PAI-1’s hypoxia-dependent hepatic induction and consequent angiogenic effect may have significance to placental maldevelopment. Discovery of increased IGFBP-1 phosphorylation in hypoxia, and its inhibition of IGF-I mediated proliferation, may be an adaptive mechanism limiting fetal growth in FGR

Increased Umbilical Cord PAI-1 Levels in Placental Insufficiency Are Associated with Fetal Hypoxia and Angiogenesis

In intrauterine growth restriction (IUGR), a subset of pregnancies undergoes placental vascular dysregulation resulting in restricted blood flow and fetal hypoxemia. Altered transcription of hypoxic regulated plasminogen activator inhibitor 1 (PAI-1) has been associated with pregnancy complications and angiogenic regulation. Here we assessed circulating PAI-1 as an indicator of placental insufficiency. Venous umbilical PAI-1 of hypoxemic (VpO2 20 versus 35 mmHg, p<0.0001) placental insufficient pregnancies (resistance index 0.9 versus 0.63, p<0.05) (n=18) was compared to controls (n=12). PAI-1 was increased (~10-fold, p<0.001) and had a positive predictive ratio of 6.7. Further, PAI-1 levels correlated to blood oxygen (r=-0.68, p<0.0001). The plasma’s angiogenic potency measured in vitro was associated with umbilical cord blood PAI-1 levels (r=0.65, p<0.01). This association was attenuated by PAI-1 inhibiting antibody (p<0.001). The results demonstrate PAI-1 as a potential marker of placental insufficiency and identify its close association with pathological hypoxia and angiogenesis in a subset of growth restricted pregnancies

Experimental Zika Virus Infection in the Pregnant Common Marmoset Induces Spontaneous Fetal Loss and Neurodevelopmental Abnormalities.

During its most recent outbreak across the Americas, Zika virus (ZIKV) was surprisingly shown to cause fetal loss and congenital malformations in acutely and chronically infected pregnant women. However, understanding the underlying pathogenesis of ZIKV congenital disease has been hampered by a lack of relevant in vivo experimental models. Here we present a candidate New World monkey model of ZIKV infection in pregnant marmosets that faithfully recapitulates human disease. ZIKV inoculation at the human-equivalent of early gestation caused an asymptomatic seroconversion, induction of type I/II interferon-associated genes and proinflammatory cytokines, and persistent viremia and viruria. Spontaneous pregnancy loss was observed 16-18 days post-infection, with extensive active placental viral replication and fetal neurocellular disorganization similar to that seen in humans. These findings underscore the key role of the placenta as a conduit for fetal infection, and demonstrate the utility of marmosets as a highly relevant model for studying congenital ZIKV disease and pregnancy loss

Publisher Correction: Experimental Zika Virus Infection in the Pregnant Common Marmoset Induces Spontaneous Fetal Loss and Neurodevelopmental Abnormalities.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper

Increased Umbilical Cord PAI-1 Levels in Placental Insufficiency Are Associated with Fetal Hypoxia and Angiogenesis

In intrauterine growth restriction (IUGR), a subset of pregnancies undergoes placental vascular dysregulation resulting in restricted blood flow and fetal hypoxemia. Altered transcription of hypoxic regulated plasminogen activator inhibitor 1 (PAI-1) has been associated with pregnancy complications and angiogenic regulation. Here we assessed circulating PAI-1 as an indicator of placental insufficiency. Venous umbilical PAI-1 of hypoxemic (VpO2 20 versus 35 mmHg, p<0.0001) placental insufficient pregnancies (resistance index 0.9 versus 0.63, p<0.05) (n=18) was compared to controls (n=12). PAI-1 was increased (~10-fold, p<0.001) and had a positive predictive ratio of 6.7. Further, PAI-1 levels correlated to blood oxygen (r=-0.68, p<0.0001). The plasma’s angiogenic potency measured in vitro was associated with umbilical cord blood PAI-1 levels (r=0.65, p<0.01). This association was attenuated by PAI-1 inhibiting antibody (p<0.001). The results demonstrate PAI-1 as a potential marker of placental insufficiency and identify its close association with pathological hypoxia and angiogenesis in a subset of growth restricted pregnancies

Quantitative 2-D gel electrophoresis-based expression proteomics of albumin and IgG immunodepleted plasma

Proteomic analysis of plasma is challenging because of its large dynamic range, which prevents the detection of low abundance proteins. Immunodepletion of high abundance proteins, such as albumin and IgG, has emerged as a favored technology to overcome this problem; however its suitability in quantitative expression proteomics has not yet been adequately addressed. In this study, albumin and IgG immunodepletion was evaluated by ELISAs and the reproducibility of depletion was tested with 2-DGE. Depletion of plasma resulted in removal of 62 \ub1 1.2% of the total protein, 93 \ub1 1.4% of the albumin (0.43 \u3bcg/\u3bcL, residual), and 94 \ub1 1.5% of the IgG (0.21 \u3bcg/\u3bcL, residual). These results were confirmed by immunoblotting. Computerized image analysis of 2-D gels using Progenesis SameSpots software revealed an enhancement in the number of visible spots (675\u20131325), with 10 \ub1 6% inter-gel variability in spot density. LC\u2013ESI-MS/MS identification of newly resolved protein spots further validated the procedure. An innovative application of the software employed led to identification of 11 proteins lost non-specifically during depletion. This study demonstrates the effectiveness of immunodepletion of albumin and IgG in quantitative 2-DGE-based differential analysis of plasma proteins.Peer reviewed: YesNRC publication: Ye

Hypoxia and Leucine Deprivation Induce Human Insulin-Like Growth Factor Binding Protein-1 Hyperphosphorylation and Increase Its Biological Activity

Fetal growth restriction is often caused by uteroplacental insufficiency that leads to fetal hypoxia and nutrient deprivation. Elevated IGF binding protein (IGFBP)-1 expression associated with fetal growth restriction has been documented. In this study we tested the hypothesis that hypoxia and nutrient deprivation induce IGFBP-1 phosphorylation and increase its biological potency in inhibiting IGF actions. HepG2 cells were subjected to hypoxia and leucine deprivation to mimic the deprivation of metabolic substrates. The total IGFBP-1 levels measured by ELISA were approximately 2- to 2.5-fold higher in hypoxia and leucine deprivation-treated cells compared with the controls. Two-dimensional immunoblotting showed that whereas the nonphosphorylated isoform is the predominant IGFBP-1 in the controls, the highly phosphorylated isoforms were dominant in hypoxia and leucine deprivation-treated cells. Liquid chromatography-tandem mass spectrometry analysis revealed four serine phosphorylation sites: three known sites (pSer 101, pSer 119, and pSer 169); and a novel site (pSer 98). Liquid chromatography-mass spectrometry was used to estimate the changes of phosphorylation upon treatment. Biacore analysis indicated that the highly phosphorylated IGFBP-1 isoforms found in hypoxia and leucine deprivation-treated cells had greater affinity for IGF-I [dissociation constant 5.83E (times 10 to the power)--0 m and 6.40E-09 m] relative to the IGFBP-1 from the controls (dissociation constant approximately 1.54E-07 m). Furthermore, the highly phosphorylated IGFBP-1 had a stronger effect in inhibiting IGF-I-stimulated cell proliferation. These findings suggest that IGFBP-1 phosphorylation may be a novel mechanism of fetal adaptive response to hypoxia and nutrient restriction