Villota Villafuerte, Stephany D.
(2019)
The effect of gestational diabetes, and its treatment, on the expression of Occludin, VE-cadherin & Plakoglobin splice variants in the human placental endothelial barrier.
PhD thesis, University of Nottingham.
Abstract
The placenta is essential for the development, survival and growth of the embryo, as it forms the critical interface between the fetus and the mother. Gestational diabetes mellitus (GDM) is a type of diabetes typically detected during the second or third trimester of pregnancy. Reduced occupancy of tight and adherens junctional molecules (Occludin and VE-cadherin) and disrupted junctions are a feature of human placental vessels under a diabetic milieu. Differential expression of gene splice variants and resultant protein isoforms may be behind the changes in junctional molecules and was the central hypothesis of this PhD study. The role of angiogenic growth factors and epigenetic control, if any, were also investigated in order to elucidate mechanisms behind gene expression changes. Human placental tissue from normal pregnancies were compared with those complicated by GDM where glucose levels were treated by a change of diet or by medication (metformin).
Placental and umbilical cord tissue was collected from 40 term singleton pregnancies delivered by caesarean section. Gene expression was determined by qPCR; protein expression by immunoblotting, and junctional occupancy by the systematic sampling of immuno-positive vessels.
Diet-controlled GDM placental samples showed differential expression of Occludin gene and protein: Occludin variant 2 was reduced (1.5-fold), while Occludin variant 3 was increased (3.3-fold) compared to normal. Occludin isoform-A levels (fully functional isoform) were decreased (1.4-fold), and lower percentage of vessels showed junctional Occludin (12%) compared to normal. Additionally, VE-cadherin gene and protein expression remained unchanged between normal and GDM placental samples. Finally, Plakoglobin gene expression was higher in diet-controlled GDM samples (1.6-fold) compared to normal. In metformin-controlled GDM placental samples, Occludin, VE-cadherin and Plakoglobin gene and protein expression were similar to normal placental samples.
We explored if differential expression of angiogenic factors was behind the downregulation of Occludin found in GDM placentae. Assessment of the gene expression of angiogenic/permeability factors (VEGF-A, Ang-1, Ang-2 and MMP-9) with their receptors (VEGFR-2 and Tie-2) revealed similar expression of these factors between normal and GDM placental samples (p > 0.05). Total VEGF-A protein expression was downregulated (1.3-fold) in diet-controlled GDM placental samples compared to normal.
An ex vivo model (placental chorionic explants cultured under hyperglycaemic conditions (15 mM D-glucose) for 3-hours (n= 4), or 24-hours (n= 4)) showed that hyperglycaemia did not cause downregulation of Occludin gene expression or junctional localisation in the placental vasculature compared to control (p > 0.05).
To determine the mechanism behind the changes in Occludin expression, our study examined whether GDM affected the expression of epigenetic markers in placenta. Quantification of the DNA methylation levels of Occludin promoter showed no significant change between normal and GDM placental pregnancies. Additionally, miR181a-5p expression was higher (2.1-fold) in diet-controlled GDM placental samples compared to normal. To confirm the effects of miR-181a-5p in Occludin gene expression, miR-181a-5p was overexpressed in HUVEC. Results showed downregulation of Occludin variant 1 (3.2-fold) and variant 2 (1.8-fold) gene expression in the presence of miR181a-5p.
In conclusion, our study showed that the junctional molecules Occludin, VE-cadherin and Plakoglobin presented several gene splice variants and protein isoforms in human placental samples. Altered gene expression of Occludin and Plakoglobin were found in diet-controlled GDM placentae. Downregulation of Occludin was in part the result of overexpression of miR-181a-5p. The stable expression of the target genes in the metformin-controlled GDM group may not be a result of the corrective effects of metformin upon glucose. Our data suggests that, despite good glucose control, the adoption of lifestyle changes alone during a GDM pregnancy (diet-controlled) may not be enough to prevent an alteration in the expression of Occludin.
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