Markers and mechanisms of pre-eclampsia in pregnancies complicated by diabetes

Abstract

Pre-eclampsia complicates 2-8% of pregnancies worldwide. Women with diabetes have a 4-fold increased risk of pre-eclampsia compared to the general population. Mechanisms for pre-eclampsia development are not well understood; however, evidence indicates that an increased release of two anti-angiogenic factors, soluble fms-like tyrosine kinase (sFlt-1) and soluble endoglin (sEng) from placental trophoblasts into the maternal circulation promotes endothelial dysfunction associated with pre-eclampsia. The aim of this thesis was to investigate whether modified lipoproteins and/or elevated glucose have a role in pre-eclampsia in pregnancies complicated by diabetes. Trophoblast cell lines HTR8/SVneo and JAR were exposed to native LDL (N-LDL) vs. ‘highly-oxidized glycated’ LDL (HOG-LDL) (10-200 µg protein/ml) for 24 hours, with and without pre-treatment with 30 mM glucose for 72 hours. The mRNA expressions of sFlt-1 isoforms (sFlt-1-i13, sFlt-1-e15a), endoglin and MMP-14 were measured by RT-PCR, and levels of sFlt-1 and sEng in cell supernatants were determined by ELISA. JAR and BeWo cells were cultured on semi-permeable membrane inserts in Transwell plates for up to 16 days, then exposed to N-LDL vs. HOG-LDL. Transepithelial electrical resistance (TEER) was measured after 6, 12 and 24 hours of treatment and fluorescein-isothiocyanate (FITC)-dextran leakage was measured after 24 hours of treatment. At termination of experiment, tight junction (zonula occludens-1, ZO-1) immunostaining and TUNEL staining were performed. To determine the effects of dysglycaemia and pre-eclampsia on placental pathology, term placentae from two study cohorts were utilized. Pathology in placental sections was examined by performing haematoxylin and eosin (H&E) staining. Immunostaining of Apolipoprotein B100 (ApoB, a marker of LDL), oxidized LDL (Ox-LDL) and 4-hydroxynonenal (4-HNE) was performed. In our in-vitro trophoblast cell model, modified lipoproteins decreased trophoblast cell viability in a concentration dependent manner and increased release of two anti-angiogenic factors, sFlt-1 and sEng into cell supernatant. High glucose alone had no effect on trophoblast cell viability and anti-angiogenic factor release, however, in combination with modified LDL, high glucose amplified the effects on sFlt-1 and sEng, suggesting that hyperglycaemia in diabetes may not be sufficient to cause pre-eclampsia, and oxidative stress and modified lipoproteins may play a crucial role. Exposure of trophoblast cell monolayers to modified LDL caused diminished barrier integrity providing evidence for a role of modified lipoproteins in placental barrier dysfunction. Modified lipoproteins were present in all human term placentae. It is possible that oxidative stress is increased earlier in gestation in women with diabetes who develop pre-eclampsia compared to those who do not. We speculate that the placenta at term is an aged tissue in all pregnancies, regardless of glycaemia or pre-eclampsia. In conclusion, exposure of trophoblasts to modified lipoproteins may enhance release of anti-angiogenic factors and compromise the materno-foetal barrier, contributing to pre-eclampsia; these effects may be enhanced by elevated glucose. These findings may explain, in part, the high risk for pre-eclampsia in women with diabetes. The results of this thesis will, it is hoped, contribute to the development of a better understanding of the mechanisms underlying pre-eclampsia, particularly in women with diabetes.