Transforming growth factor (TGF)-beta is involved in endothelial cell dysfunction. However, its effect on microRNAs and downstream targets in this context is not known. Therefore, our objective was the identification, modulation and prevention of TGF-beta associated miRNA-deregulation with its linked changes in endothelial cell function, contributing to heart failure.

For target identification, arrays for miRNA-profiling and mRNA-profiling were performed. In-vitro endothelial cell function was quantified with scratch migration, tube formation and boyden chamber migration. Genomic changes were analysed with RT-PCR and Western-Blot. Target validation was performed with luciferase-assay. Target-modulation was performed with precursor or inhibitory miRNA-molecules, siRNA or lentiviral induced over-expression. Methylation status was measured via bisulfite conversion, whereas methylation inhibition was performed with 5’-Aza-2’-deoxycytidine.

miRNA profiling during TGF-beta-mediated endothelial cell dysfunction identified miR-30a-3p and its family to be strongly silenced. Supplementation of miR-30a-3p restored in-vitro endothelial cell function in TGF-beta treated cells. We identified and validated the epigenetic factor methyl CpG binding protein 2 (MeCP2) to be a direct and functional target of miR-30a-3p. Viral over-expression of MeCP2 yielded similar results on endothelial cell function as TGF-beta, suggesting that de-repression of MeCP2 after TGF-beta treatment is responsible for impaired endothelial cell function. Besides, siRNA-mediated silencing of MeCP2 revealed protective effects on endothelial cell function. Microarray transcriptome analysis of MeCP2 over-expressing endothelial cells identified several deregulated genes important for endothelial cell function. Further experiments identified that MeCP2 inhibits Sirt1, a crucial protein in endothelial cell function, by promoter hypermethylation. Methylation inhibition resulted in a prevention of hypermethylation-associated silencing.

In conclusion, TGF-beta impairs endothelial cell function by down-regulating miR-30a-3p and subsequent de-repression of MeCP2 mediated epigenetic silencing of Sirt1.