Phenotypic characterisation and molecular changes induced by gestational diabetes mellitus on human umbilical endothelial cells: focus on the KDM2B/miR-101/EZH2 pathway.

Background and aims: Gestational diabetes mellitus (GDM) is characterised by maternal hyperglycaemia that is first recognised during pregnancy. GDM results in chronic foetal hyperglycaemia and hyperinsulinaemia, with consequent changes in foetal endothelial gene expression and endothelial cell (EC) function. We aim to determine whether exposure to a diabetic intrauterine environment alters human umbilical vein EC (HUVEC) function, with further investigation into microRNA (miR) expression and epigenetic pathways. Methods: HUVECs were extracted from expecting mothers with and without GDM. We performed functional assays and focused miR and target gene analyses on extracted cell line-ages. Finally, we also studied 'healthy' HUVECs that were grown in high glucose conditions. Results: We observed a reduced capacity of closure on scratch assay, diminished capillary-like tube formation on Matrigel and decreased proliferation in response to foetal bovine serum (FBS), after a period of FBS-starvation, in the GDM-extracted lineages compared with the controls. On microRNA screening we found a tendency towards increased levels of miRs belonging to the miR-15 family. Specifically, miR-101 was significantly upregulated in the GDM-extracted lineages; hence we focused on this miR's molecular mechanisms in our target cell lineages. There was a strong positive correlation between miR-101 levels and apoptosis (Pearson r = 0.9, p value < 0.001). There was a decreased expression of Enhancer of Zester Homolog 2 (EZH2) in the GDM-extracted lineages. Confirming previous reports which have validated EZH2 as a target-gene of miR-101 in HUVECs, we found their expression levels were negatively correlated. EZH2 mRNA levels correlated positively with KDM2B and VEGF-A; conversely miR-101 levels correlated negatively with KDM2B. These data are in line with the described KDM2B/EZH2/miR-101/EZH2 axis. Anti-miR-101 treatment in GDM cells resulted in upregulation of EZH2, decreased apoptosis and an improved angiogenic phenotype, restoring the endothelial tube formation to the level seen in control lineages. As expected, in vitro exposure of "healthy" HUVECs to high glucose significantly impaired cellular function. Moreover, we found a concomitant upregulation of miR-101, which is in agreement with high miR-101 levels in GDM HUVECs.. Conclusion: These data identify a signalling pathway that links hyperglycaemia to miR-101 and its target gene EZH2, suggesting new targets in the fields of gestational diabetes and EC dysfunction induced by hyperglycaemia in utero. We characterise a novel mechanism regulating hyperglycaemia-impaired angiogenesis, with the possibility of restoring the angiogenic capacity of GDM cells in vitro by blocking miR-101 and subsequently upregulating its target EZH2. [ABSTRACT FROM AUTHOR]