- Title
- The role of the (pro)renin receptor pathway in placentation; implications for pregnancies complicated by preeclampsia
- Creator
- Morosin, Saije Kim
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2021
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- This thesis describes the role of the (pro)renin receptor (ATP6AP2/(P)RR) and soluble (pro)renin receptor (sATP6AP2/s(P)RR) in human placentation, specifically in trophoblast syncytialisation. Syncytialisation is a process of cell fusion where mononuclear cytotrophoblast cells fuse and form a multinucleated syncytiotrophoblast. The syncytiotrophoblast is responsible for regulating the exchange of nutrients, gases and wastes between the fetus and the mother, while also secreting hormones such as human chorionic gonadotropin (hCG). Dysregulation and/or damage of the syncytiotrophoblast has been associated with pregnancy complications including preeclampsia and intrauterine growth restriction (IUGR). Therefore, it is important to know how the syncytiotrophoblast develops in order to truly understand and identify the underlying placental pathophysiology of pregnancy complications such as preeclampsia and IUGR. The work in this thesis aimed firstly to determine if ATP6AP2 is involved in trophoblast syncytialisation. This is because ATP6AP2 can activate Wnt/beta-catenin and ERK/1/2 MAPK signalling, both of which are associated with syncytiotrophoblast formation. In spontaneously syncytialising primary human trophoblast cells, isolated directly from human placenta, I showed that ATP6AP2 does not play a role in placental syncytialisation. Alternatively, I have shown that syncytialisation increases expression of components of the renin-angiotensin system (RAS) including; renin (REN), angiotensinogen (AGT) and angiotensin II receptor type 1 (AGTR1). Thus, identifying a role for the tissue RAS, rather than ATP6AP2 in regulating trophoblast syncytialisation. Throughout this thesis I have also characterised the secretion of sATP6AP2 in syncytialising placental trophoblast cells. Soluble ATP6AP2 levels are elevated in the plasma of women with preeclampsia and IUGR, however the source of the increased maternal sATP6AP2 levels in pregnancy had not been elucidated. I have successfully confirmed, for the first time, that the placenta can secrete sATP6AP2, by showing that primary human trophoblast cells secrete sATP6AP2 and that its levels decrease as trophoblasts syncytialise. This is significant as it provides evidence that elevations in maternal sATP6AP2 levels that are seen in pregnancy complications, like preeclampsia and IUGR, could be coming from the placenta. Preeclampsia is associated with a dysfunctional syncytiotrophoblast that releases pro-inflammatory and anti-angiogenic factors into the maternal circulation that contribute its pathogenesis. This research has identified the possibility that in preeclampsia this damaged syncytiotrophoblast may also secrete greater amounts of sATP6AP2 into the maternal circulation, which could damage the maternal endothelium and contribute to the maternal endothelial dysfunction and renal damage, which is characteristic of preeclampsia. Since sATP6AP2 was secreted by placental trophoblasts it was important to also determine how it was cleaved. I have shown that neither Furin nor site 1 protease (MBTPS1), which have been identified as the proteases responsible for sATP6AP2 cleavage in other cell types, cleave sATP6AP2 in the placenta. However, I demonstrated that a protease that is also inhibited by the broad protease inhibitor, DEC-RVKR-CMK, is likely to be involved. In addressing the initial aims in this thesis, I employed another common model of syncytialisation; forskolin-induced syncytialisation of BeWo choriocarcinoma cells. However, I showed clear differences between BeWo cells and primary human trophoblasts in terms of their expression of the RAS and also in the role of ATP6AP2 in syncytialisation. I have shown that BeWo cells express little to no REN nor AGTR1, whereas primary trophoblasts not only express both but their expression is increased with syncytialisation. This indicates that BeWo cells have only a limited capacity to activate tissue RAS signalling via the Ang II/AGTR1 pathway. Therefore, signalling in BeWo cells is likely occurring via the pathways that ATP6AP2 can exert on its own, such as via the Wnt/beta-catenin signalling pathway, rather than the activation of the RAS. Furthermore, I have shown that while ATP6AP2 is not involved in syncytialisation of primary trophoblasts, ATP6AP2 is involved in BeWo cell syncytialisation. This supports the hypothesis that the BeWo cell RAS is mainly functional via ATP6AP2 signalling that is independent of RAS activation, especially because ATP6AP2 activates the Wnt/beta-catenin signalling pathway, which has also been previously shown to be important in BeWo cell syncytialisation. My research in this area highlights that using forskolin-induced syncytialisation of BeWo choriocarcinoma cells as a placental model may not be physiologically relevant, especially in terms of the role of the RAS. I have also provided a new perspective into the pathogenesis of choriocarcinoma involving the ATP6AP2 and sATP6AP2. I have shown that ATP6AP2 is involved in BeWo choriocarcinoma cell syncytialisation and in this way could be involved in changing the cellular profile from invasive cytotrophoblast cells to a non-invasive syncytiotrophoblast. I have also shown that BeWo cells secrete sATP6AP2 and that this reflects both cellular ATP6AP2 levels and syncytialisation. We therefore propose that sATP6AP2 could be used as a biomarker of the invasive potential of choriocarcinoma. Finally, we have shown that Furin, and not MBTPS1, is responsible for sATP6AP2 cleavage in choriocarcinoma but other proteases (inhibited by DEC-RVKR-CMK) may also be involved. Lastly, the work presented in this thesis challenges the current dogma surrounding Furin in placental syncytialisation. Previously, Furin had been shown to be important for placental syncytialisation in first trimester placental explants and BeWo choriocarcinoma cells. However, our study shows evidence to the contrary. Term primary trophoblast and BeWo choriocarcinoma cell experiments show that Furin is not involved in syncytialisation in either cell type. I have highlighted the need for careful reporting of methods by authors and careful assessment of manuscripts by the research community, before accepting findings. It also reveals the need for a universal benchmark for measuring syncytialisation. Overall, this thesis provides novel insights into the function of the placental RAS and Furin in syncytialisation. This thesis has also characterised the production and secretion of sATP6AP2 from the placenta and has provided new insights into a potential role for ATP6AP2 and sATP6AP2 in the pathogenesis of choriocarcinoma. The research in this thesis both bridges gaps in the literature and builds foundations for further research in this area.
- Subject
- placenta; (pro)renin receptor; soluble (pro)renin receptor; preeclampsia
- Identifier
- http://hdl.handle.net/1959.13/1425538
- Identifier
- uon:38266
- Rights
- Copyright 2021 Saije Kim Morosin
- Language
- eng
- Full Text
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