Gestational diabetes mellitus (GDM) is associated with hyperglycaemia, endothelial inflammation, altered DNA methylation and mitochondrial (mt) gene expression in the fetoplacental vasculature. Adenosine regulates inflammation and DNA methylation, in which the DNA methyltransferase 1 (DNMT1) and S-adenosylhomocysteine hydrolase (SAHH) are involved [1]. Adenosine is regulated by adenosine kinase (AK) and human equilibrative nucleoside transporter 1 (hENT1), which are likely to be altered in the fetoplacental vasculature from GDM [2]. A cytoplasmic AK (c-AK) and nuclear AK (n-AK) isoforms of AK and at least three groups (D, C, and B) of hENT1 transcriptional variants, i.e., hENT1TV-D, hENT1TV-C, or hENT1TV-B, have been described [3]. AK inhibition leads to a decrease in TNFα-induced endothelial inflammation [4]. Whether this phenomenon is present in fetoplacental vasculature in GDM is unknown. This study aims to determine the expression of AK isoforms and hENT1 TVs and the role of AK in TNFα-induced endothelial inflammation, mt gene expression and DNA methylation in human umbilical vein endothelial cells (HUVECs) exposed to a high extracellular D-glucose. The investigation conforms to the principles outlined in the Declaration of Helsinki. HUVECs from normal pregnancies were provided by the Endothelial Cell Facility at UMCG. HUVECs were isolated (collagenase digestion) and cultured in RPMI medium (20% FCS, 5.5 mmol/L D-glucose (basal glucose, BG)). At ~70% confluency, cells were exposed to 25 mmol/L D-glucose (high glucose, HG) and incubated (24 h) with TNFα (2 ng/mL), ABT-702 (2 µmol/L, AK inhibitor) or both. The mRNA expression was evaluated by RTq-PCR. GAPDH and β-actin were used as housekeeping. Values are mean ± S.D., compared by non-parametric t-test or ANOVA, P<0.05. hENT1TV-D (relative expression (RE) = 0.012 ± 0.0037), hENT1TV-C (RE = 0.0038 ± 0.0013) and hENT1TV-B (RE = 0.00011 ± 0.00004) mRNA were differentially expressed. The RE of n-AK was higher than c-AK (RE = 0.00026 ± 0.0003 and 0.000007 ± 0.000005, respectively). HG increased the expression total hENT1 and hENT1TV-C, DNMT1 and SAHH expression (1.1 ± 0.1, 1.2 ± 0.03, 1.1 ± 0.1 and 1.14 ± 0.08 fold, respectively), tends to increase mRNA expression of ICAM-1, IL-8 and E-selectin and tends to reduce cytochrome c oxidase I (COX1), with no changes in total AK, n-AK, and mt-ribosomal 12S and 16S. TNFα associated with increased n-AK and total AK mRNA expression and perhaps hENT1TV-D and with decreased expression of total hENT1, COX1, 12S, and 16S in BG and HG HUVECs. ABT-702 prevented (~50%) TNFα-increased ICAM-1, IL-8, and E-selectin and increased total AK, n-AK, total hENT1, DNMT1, and SAHH mRNA expression in BG and HG, and hENT1TV-D in HG treated cells. In conclusion, AK and differential expression of hENT1 transcriptional variants could be a new target to prevent GDM-associated pathophysiological and epigenetic alterations in the fetoplacental vasculature.