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Modulation of Trophoblast Cell Death by Oxygen and EGF

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Abstract

Background

Preeclampsia, a maternal hypertensive disease, is characterized by shallow invasion of the maternal spiral arterioles resulting in hypoxia/reperfusion type insult; however, the molecular mechanism is unknown. The aim of this study was to determine the mechanism of altered oxygen tension or inhibition of phosphatidyl-inositol-3-kinase (PI3K) on trophoblast survival and to investigate the effect of epidermal growth factor (EGF) on maintaining cellular integrity.

Materials and Method

We have used flow cytometry, immunoblotting, and fluoroimmunocytochemistry to study apoptosis in a characterized, spontaneously transformed first trimester extravillous-like trophoblast cell line that exhibits many characteristics of in vivo trophoblast.

Results

Time-dependent exposure of first trimester extra-villous-like trophoblast to all oxygen tensions tested promoted dissipation of the mitochondrial membrane potential (ψm) and resulted in a significant increase in cell death by 48 hr as determined by dual staining flow cytometry. Western blot analysis revealed expression of cleaved caspase-3 and caspase-9 increased with time with hypoxia and hyperoxia promoting the greatest elevation indicating that longer duration of exposure to a change in oxygen tension causes increased apoptosis via a mitochondrial-mediated pathway. Disruption of the anti-apoptotic PI3K pathway by LY294002 (40 µM), its specific inhibitor, caused further significant dissipation of the ψm (p < 0.01) and cleavage of caspase-3. EGF was able to maintain the ψm and to prevent cleavage of caspase-3 even in the presence of LY294002, indicating that its survival effects were independent of the PI3K pathway.

Conclusions

These results suggest that inhibition of the PI3K/Akt pathway can sensitize first-trimester trophoblastlike cells into oxygen-induced cell death and that EGF exerts its anti-apoptotic effect independently of PI3K/Akt.

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Acknowledgments

We are grateful to Dr D.A. Kniss (Ohio State University, Columbus, Ohio, USA) for the generous gift of spontaneously transformed trophoblast cell lines. Thanks also go to Dr O. Genbacev (California, USA) for provision of the anti-HLA-G antibody for characterization studies. We greatly appreciate the assistance with immunofluorescence (Dr B. Bussolati, Turin, Italy) and flow cytometry (Dr M Rahman, Birmingham, UK, and Mr. R. Bird, IDRL Birmingham, UK).

This work was funded by the British Heart Foundation Grant Numbers FS/1998079 and RG/98/0003.

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Authors and Affiliations

  1. Department of Reproductive & Vascular Biology, The Medical School, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK

    Jonathan Perkins & Asif Ahmed

  2. Reproductive Biology & Genetics Group, The Medical School, University of Birmingham, Birmingham, UK

    Justin St. John

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Correspondence to Asif Ahmed.

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Perkins, J., St. John, J. & Ahmed, A. Modulation of Trophoblast Cell Death by Oxygen and EGF. Mol Med 8, 847–856 (2002). https://doi.org/10.1007/BF03402090

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