Impact of the diabetic environment on the fetal vasculature: role of microRNA-101 and Enhancer of Zeste Homologue 2

Gestational diabetes mellitus (GDM) is a specific form of diabetes affecting about 7% of all pregnancies. GDM usually reverses after delivery, but altered glucose homeostasis harms the mother. Moreover, since it translates into fetal hyperglycemia, GDM can affect the healthy development of the fetus, with consequences that can be experiencedin uteroand/or at different stages of post-natal life. In particular, the child from a GDM pregnancy has higher chance of developing type 2 diabetes (T2D) and cardiovascular diseases (CVD). Several clinical trials and epidemiological studies in patients with either type 1 or 2 diabetes show that exposure to prolonged hyperglycemia leaves a long-lasting impression on vascular cells leading to the development of vascular complications which persist after glycemic control is achieved. The same studies also suggested that a good glycemic control can delay the onset of vascular complications in diabetic subjects.The term “epigenetic” refers to heritable changes in the cellular phenotype that do not involve changes in the genotype. There are different kind of epigenetic modifications including methylation and acetylation of histones, and DNA methylation, and these modifications influence gene expression. Although the precise mechanistic insight is still unclear, epigenetic regulation might be involved in the long-lasting effect of diabetes on the vascular system.In this study, we focused our attention on the methyltransferase Enhancer of Zeste Homologue 2 (EZH2), that is part of the Polycomb Repressor Complex 2 (PRC2) and plays an essential role in the epigenetic maintenance of the trimethylation of lysine 27 on histone3 (H3K27me3). It has been recently shown that alternative splicing of the EZH2 locus yields two major transcriptional variants: EZH2α and EZH2β.The two isoforms can each not only regulate distinct targets, but also share gene expression networks, making the regulation of transcription based on the deposition of the H3K27 histone mark more complex than previously anticipated.MicroRNAs (miRNAs) are single-stranded, non-coding RNAs of 19-25 nucleotides that normally bind the 3’-untranslated region of target mRNAs and contribute to the post-transcriptional regulation of gene expression. A link has been shown between EZH2 and some miRNAs. In particular, miR-101 reportedly affects endothelial function and angiogenesis by targeting and inhibiting EZH2 under normoglycemic conditions.The aim of our study was to investigate the impact of GDM on fetal endothelial biology. To this end, we isolated human umbilical vein endothelial cells (HUVECs) from the umbilical cords of GDM and control healthy pregnancies. These cells represent a good cell model allowing for non-invasive analyses of the effect of the uterine environment on the fetal endothelium. The first objective was to investigate the impact of GDM on the functional capacity of HUVECs, with particular focus on apoptosis, migration, proliferation and tube formation. Next, we evaluated the expression of EZH2 (both α and β isoforms) and miR-101 to see whether they affect endothelial function in diabetic conditions. We first found that cultured GDM-HUVECs demonstrated decreased survival and functional capabilities in comparison with HUVECs obtained from healthy non-diabetic mothers. Additionally, we have identified a novel underlying mechanism involving miR-101 and EZH2 in GDM-elicited endothelial dysfunction. In particular, we have found that in HUVECs, EZH2 is both a target of, and a transcriptional repressor for, miR-101. Notably we found that among the two recently identified EZH2 isoforms, EZH2β is the only one which is both a target of GDM and a mediator of some of the GDM-related damaging effects on HUVECs.