An investigation into stress, inflammatory and apoptotic pathways in the placenta in labour, pre-eclampsia and fetal growth restriction

Abdulsid, Akrem (2018) An investigation into stress, inflammatory and apoptotic pathways in the placenta in labour, pre-eclampsia and fetal growth restriction. PhD thesis, University of Glasgow.

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Abstract

The mechanisms that are involved in maintaining a human pregnancy to term, and the switches that lead to a normal labour and pregnancy outcome or indeed an adverse outcome such as miscarriage, pre-eclampsia (PE), fetal growth restriction (FGR) or preterm labour, are complex but the role of the placenta is crucial to them all. Heat shock proteins (HSPs) play an important role in regulating signal transduction, inhibition of apoptosis and cellular proliferation and differentiation. Apoptosis has been implicated in both PE and labour. In the apoptotic pathway, HSPs act at several stages to prevent cell death initiated by stress-induced damage. Toll-like receptors (TLR) are the principal signalling molecules through which mammals sense infection. Since these pathways play a role in oxidative stress and inflammation, both features of labour and PE, the hypothesis was that placental stress pathways (HSP70, HSP27, HSF1-2, small HSPs family, DNAJAB, HSP60, GRP78, HSP90AA, HSP90B1 and HSP105), inflammatory mediates (TLRs, MyD88 and NF-κB) and apoptotic markers (caspases (CASP) 3, 7 and 9) pathways expression would alter during labour and pregnancies complications such as PE and FGR and this would vary in different placental zones perhaps due to the variation in blood supply to each zone or link to ischemic-reperfusion (IR) injury and are some zones of placenta more susceptible to IR injury. HSPs, TLRs and caspases were examined in placentas obtained from women who delivered by caesarean section (non-labour) and were not in labour and compared to the equivalent zone of placentas obtained from women who delivered vaginally (labour) following an uncomplicated labour. Samples were obtained from 12 sites within each placenta: 4 equally spaced apart pieces were sampled from the inner, middle and outer placental regions. The expression was investigated by Western blotting (HSP70, HSF1, HSP27 and P-HSP27) and real time PCR (HSP families, HSF1-2, TLRs, MyD88, NF-κB and caspases).
The first aim of this study was to examine the spatial expression of HSPs, caspases and TLR genes in placentas. Two HSF1 forms were observed in all samples which, according to the data sheet represent the phosphorylated (P-HSF1) and non-phosphorylated (NP-HSF1) forms. HSP70, HSF1 and HSP27 are expressed in a spatial manner in the placenta with the highest expression being in the middle zone in the labour group (HSF1, both forms) and in both the labour and the non-labour groups (HSP70 and HSP27). HSF1 mRNA expression was spatially distributed in the non-labour group with highest expression being in the outer zone. No spatial differences were noticed in both P-HSP27 protein expression and mRNA expression of other HSPs families, HSF2, TLRs, MyD88, NF-κB and caspases3, 7 and 9 genes.
Human parturition involves interaction of hormonal, neurological, mechanical stretch and inflammatory pathways and the placenta plays a crucial role. The second aim of this study was to determine these genes expression in the non-labour group compare to the labour group at the inner, middle and outer placental zones. When non-labour groups were compared with the labour group at the three zones the changes found can be summarized as follows:
HSP70 protein expression was increased in the labour group compared to the non-labour group and in contrast to that, HSP27 protein and mRNA expression were increased in the non-labour group both at the middle zone.
Phosphorylated-HSP27 (P-HSP27) expression was increased in the non-labour group compared to the labour group at the inner (all ser sites) and outer (ser 15 and 78) zones. No differences were found at the middle zone. HSF1 protein expressions (both forms) was increased in the non-labour, group compared to the non-labour group, paradoxically, HSF1 mRNA expression was increased in in the labour group at the inner zone.
HSPB6, HSP32 and DNAJB1 mRNA expression was increased in the non-labour group compared to the labour group at the middle zone. HSPB2, HSP90AA and HSP90B1 mRNA expression was increased in the labour group compared to the non-labour group at the outer zone. TLR5 and CASP7 mRNA expression was increased in labour group compared to the non-labour group at the middle zone. TLR9-10 mRNA expression was neglectable. No other changes were found for any other genes.
PE is associated with maternal and placental oxidative stress. The third aim was to determine the expression of genes of the same pathways described above in the placenta in pregnancies complicated with PE and FGR compare to the normal pregnancies. HSPs, TLRs and caspases were examined in placentas obtained from non-labouring women who delivered by caesarean section (normal pregnancy and PE) and compared to women who delivered vaginally (normal pregnancy and PE). Samples were obtained from 12 sites within each placenta: Four equally spaced apart pieces were sampled from the inner, middle and outer, however, due to time constraints only the inner and middle zones were investigated in PE group (except HSP27 and HSP70). Samples from FGR placentas were obtained from 8 sites within each placenta represent inner and outer zones. Western blotting and qRT-PCR was used for the analysis as required.
When PE (non-labour and labour) and FGR (labour only) groups were compared with the controls (non-labour and labour) at the two zones, the changes found can be summarized as follows: HSP70 was increased in the non-labour-PE group compared to the non-labour control group at the inner zone. No other differences were found. Non- Phosphorylated-HSF1 (NP-HSF1) protein expression and HSF1 mRNA expression were increased in the PE group (non-labour and labour) compared to the control group (non-labour and labour) at the inner zone. HSP27 and P-HSP27 protein expression was increased in the PE (labour) compared to the control (labour) at the middle zone. No changes were found in the mRNA expression of HSP27. There was a significant increase in P-HSP27 protein expression in labour PE compared to the labour control at the inner (ser 78 and 82) and middle (ser 15) zones.
CASP7 and CASP9 mRNA expression was increased in the non-labour control compared to the non-labour PE at the inner (CASP7) and middle (CASP9) zones. CASP3 mRNA expression was increased in the labour PE compared to labour control at the middle zone.
There was decreased in mRNA expression of HSPB6, TLR5 and TLR1 in the PE group non-labour compared to the controlnon-labour at the inner (HSPB6) and middle (TLR1 and TLR5) zones.
Towards the end of the PhD time preliminary experiments were performed to determine if different zones of the placenta were more susceptible to heat shock and IR injury in vitro. The markers mentioned above were used and pieces of placenta from each zone were exposed to 2% oxygen then 8% oxygen for different time periods to create an IR model. The number of experiments were not sufficient to make definitive conclusions but the pilot data suggest this should be explored more in future studies include more placenta tissue exposed to IR injury and heat shock to understand how placental stress might affect placental function.
Obesity is characterized by a chronic, low-grade inflammation and an impaired intracellular stress defence system. Obesity increases the risk for PE. The last aim was to screen for different HSPs and CASPs families in placentas obtained from 4 BMI groups (non-labour). In general, HSPs expression was reduced whereas caspases expression was increased in the morbid obesity group (BMI>40) compared to the normal BMI group. This indicates reduced cellular protection and increased cellular damage respectively in placentas from obese women.
In summary this thesis has reiterated the importance of controlled sampling of the placenta for studying pregnancy disorders. The results show that spatial changes in inflammatory and stress markers occur in both labour and PE. Trying to make sense of the data is very difficult however the preliminary data from the in vitro studies suggests that further studies exposing different zones of the placenta to stress may help to understand the process that take place during stress. Clearly the bigger aim would be, one day, to try and decide which of the pathways might be targeted for therapy for pre-term labour and PE.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Supported by funding from the Libyan Embassy.
Keywords: HSP, TLR, CASPs, PE, FGR, labour, obesity and oxidative stress.
Subjects: R Medicine > RZ Other systems of medicine
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Medicine, Dentistry & Nursing
Funder's Name: The Royal Society (ROYSOC)
Supervisor's Name: Lyall, Professor Fiona
Date of Award: 2018
Depositing User: Dr Akrem Abdulsid
Unique ID: glathesis:2018-9138
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 29 May 2018 09:26
Last Modified: 21 Jun 2018 16:01
URI: http://theses.gla.ac.uk/id/eprint/9138
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