Epigenetic mechanisms implicated in the programming of endothelial dysfunctión in foetuses with intrauterine growth restrictión

Abstract

The association between reduced foetal growth and adult health has risen as a feasible explanation for non-Mendelian diseases, such as hypertension and type-2 diabetes. Epigenetic mechanisms (i.e. DNA methylation, histone post-translational modifications) have been broadly suggested to be involved in this process, 'recording' normal and abnormal perinatalstimuli and shaping long term gene expression in the cardiovascular system. In this context, cultured human umbilical endothelial cells (HUVEC) derived from pregnancies with intrauterine growth restriction (IUGR) present an altered expression of proteins related withthe L-arginine/nitric oxide metabolism (i.e. eNOS and arginase-2), suggesting an early onset of endothelial dysfunction. This project studied whether arginase-2 participates in the IUGR placental vascular dysfunction, counteracting with eNOS activity in umbilical arteries andveins, as well as chorionic arteries. Additionally, the expression of both proteins in cultured endothelial cells from chorionic (PLAEC), umbilical arteries (HUAEC), and HUVEC wasanalysed. In order to determine the participation of epigenetic mechanisms, the effect of DNA methylation and histone acetylation activities on basal eNOS and arginase-2 expression was studied, as well as the presence of altered methylation status in the NOS3 (eN OS) and ARG2 (arginase-2) promoters. In IUGR vessels eNOS-dependent relaxation was reduced, being improved by arginase-inhibition. Arginase-2 protein expression was increased IUGR HUVEC, without changes in HUAEC and PLAEC. Additionally, eNOS expression was reduced in HUVEC and augmented in HUAEC and PLAEC; however there was a generalized reductionin the in vitro eNOS activation (Ser1177-P-eNOS). Inhibition of heritable DNA methylation restored to normal values the eNOS expression in IUGR EC; however it further increased the arginase-2 expression in IUGR HUVEC, without effects on HUAEC. The DNA methylation pattem in NOS3 promoter showed a limited number of CpGs differentially methylated innormal HUAEC, PLAEC and HUVEC, with differences between arteries and vein endothelial cells. Noteworthy, the changes in NOS3 promoter DNA methylation in IUGR endothelial cells occurred in the same CpGs that were differentially methylated in normal arteries (HUAEC andPLAEC) and HUVEC. In contrast, DNA methylation pattem in ARG2 promoter wascomparable in normal endothelial cells, but changes in DNA methylation in IUGR cells were similar only between HUAEC and PLAEC. Inhibition of histone deacetylation reduced eN OS mRNA and protein levels, and increased arginase-2 expression in IUGR HUAEC. Altogetherthese data showed that arginase-2 and eNOS expression are differentially regulated in IUGR endothelial cells, being their expression basally controlled by epigenetic mechanisms thatoperate in a cell- and gene- specific manner.