Mechanosensory and Sphingolipid Mediators of Inflammatory Signaling in Placental Dysfunction
Creators: Fakhr, Yuliya
Publisher: name: University of Alberta Library
Year: 2022

<p>Preeclampsia (PE) is a leading cause of maternal and fetal morbidity. Placental dysfunction plays a key role in PE and includes decreased syncytialization and increased inflammation and cell death. Tumor Necrosis Factor alpha (TNF-α) is a key factor in inducing this dysfunction. The controversy in using TNF-α inhibitors during pregnancy highlights the importance of identifying downstream mediators of its signaling. I investigated sphingolipids and mechanosensory mediators as potential targets. In other tissues, TNF-α signals via sphingosine kinase 1 (SphK1), a sphingosine 1-phosphate (S1P) synthesizing enzyme. TNF-α also intersects with S1P signaling by upregulating S1PR2 in other tissues. SphK1 hinders ST formation, and S1PR2 increases cell death and placental inflammation. Whether this occurs downstream of TNF-α signaling is unclear. Piezo1, a mechanosensory channel, regulates epithelial cell function via S1P. Other mechanosensory channels play a role in increasing both syncytialization and cell death. The role of Piezo1 in the placenta and the syncytium and its relationships to TNF-α and S1P remain unknown. I hypothesized that TNF-α will induce placental dysfunction by altering SphK1, S1PR2, and Piezo1 levels and activity. <br>
The first study investigated levels of SphK1 in placental biopsies from normal pregnancies and PE and the S1P regulatory enzyme levels in response to TNF-α in cultured human villous explants and primary human trophoblasts. Placental SphK1 was increased in PE. Only S1P phosphatase 1 increased at the end of the syncytial regeneration in explants, regardless of TNF-α treatment, suggesting a role for S1P degradation at the end of syncytialization. The expression of SphK1 in trophoblasts was increased in response to TNF-α, suggesting that elevated TNF-α in PE can upregulate SphK1 in trophoblasts but other factors are involved in SphK1 upregulation at the placental level. <br>
Next, I examined the syncytialization, cell death and syncytial shedding, and the release of inflammatory cytokines and growth factors from villous explants that were treated with TNF-α and/or the SphK1 inhibitor, PF-543. Increased cell death, shedding, interferon-α2, IFN-gamma-induced protein 10, fibroblast growth factor-2, and platelet-derived growth factor-AA release induced by TNF-α were reversed upon SphK1 inhibition. TNF-α also decreased IL-10 release, and inhibiting SphK1 reversed this effect. Inhibiting SphK1 alone decreased TNF-α release. Hence, inhibiting SphK1 can partially reverse the TNF-α-induced PE phenotype of the placenta.<br>
The third study investigated placental levels of S1PRs in placental biopsies from women with PE and in response to TNF-α in villous explants, in primary human trophoblasts, and in a choriocarcinoma cell line, BeWo. Placental S1PR1 remained unchanged in PE and in primary trophoblasts and BeWo cells in response to TNF-α. However, TNF-α muted the surge of S1PR1 at the end of the re-syncytialization phase in explants. Since S1PR1 signaling is generally protective, this suggests that TNF-α could induce its disruptive effect by decreasing placental S1PR1. Placental S1PR2 was higher PE, and S1PR2 was increased in primary trophoblasts treated with TNF-α but was decreased in treated BeWo cells. S1PR2 and S1PR3 increased in response to TNF-α at the end of explant re-syncytialization. Hence, BeWo cells are not an approriate model to study S1PRs. Since S1PR2 activity also decreased cell death and human placental lactogen, a marker for syncytial mass, high placental expression of S1PR2 in PE may be induced by TNF-α, and this increase in S1PR2 could be protective against TNF-α-induced placental damage.<br>
Finally, I examined the role of Piezo1 on syncytialization, cell death, and the release of inflammatory cytokines and growth factors from cultured villous explants using Yoda1, a Piezo1 pharmacological agonist. Piezo1 interaction with TNF-α and SphK1 was also examined. Piezo1 activation increased syncytial endocrine function but at higher concentrations also increased cell damage, inflammatory cytokine, and growth factor release. Piezo1 increased TNF-α and TNF-α increased Piezo1, but co-treating with Yoda1 and TNF-α decreased syncytialization. Piezo1 increased syncytial placental alkaline phosphatase release via SphK1. Placental Piezo1 expression was higher in PE. This showed that low Piezo1 expression and activity induced syncytialization via SphK1, whereas, high levels of expression and activity, found in PE, induced placental damage via TNF-α.<br>
This work shows the association between TNF-α, SphK1, S1PR2, and Piezo1 signaling and expression patterns in physiological placental functions and dysfunction. This provides the groundwork for sphingolipid and mechanosensory pharmaceutical targets to be investigated in lieu of TNF-α inhibitors.</p>