Young Investigator Abstract Session - Vascular

Background: Endothelial cell (EC) maladaptation to disturbed blood flow at predilection sites of atherosclerosis is characterized by inflammation and defective regeneration. The RNase Dicer is essential for the production of microRNAs, which play a crucial role in EC maladaptation. EC-derived Dicer promotes atherosclerosis and suppresses KLF4, Notch1, and Wnt signaling. While Dicer increases EC inflammation by miR-103-mediated suppression of KLF4, the role of Dicer-regulated Wnt and Notch1 signaling in EC regeneration is unclear. Purpose: We test the hypothesis that Dicer impairs EC regeneration through miRNA-mediated suppression of long non-coding RNAs (lncRNAs) that promote Wnt and Notch1 signaling. Methods: Microarrays, rapid amplification of cDNA ends and K4-K36 domain analysis of lncRNAs was performed. MiRNA binding sites were predicted by RNAHybrid. Endothelial GW182 was immunoprecipitated to study miRNA-mediated lncRNA targeting. Antisense oligonucleotides to inhibit miRNAs and to block the interaction between a specific miRNA and one of its targets (target site blocker, TSB) were used in vitro. Flow cytometry was performed to study apoptosis, quantitative PCR to analyze gene expression, and immunofluorescence staining to assess Ki67, Notch1 and b-catenin expression in vitro and in Apoe-/mice with a conditional knockout of Dicer in ECs (EC-Dicer-/mice). Results: In EC-Dicer-/mice, endothelial Notch1 and b-catenin activation and EC proliferation were increased in atherosclerotic arteries. The novel lncWDR59 was the most significantly upregulated lncRNA in EC-Dicer-/mice and its sequence contained a putative miR-103 binding site. Inhibition of miR-103 increased the expression of lncWDR59 in ECs and overexpressing miR-103 increased the enrichment of lncWDR59 in the RNA-induced silencing complex. Blocking the interaction between miR-103 and lncWDR59 by TSBs promoted EC proliferation, reduced apoptosis, upregulated the expression of the arterial marker SOX17, and increased Notch1 and b-catenin activity. Inhibiting Notch1 and b-catenin activity decreased EC proliferation and increased apoptosis. Blocking Notch1 but not silencing b-catenin abolished the TSB-mediated increase of EC proliferation and SOX17 expression. Conclusions: MiR-103 targets the novel lncRNA lncWDR59 in ECs and, thereby, impairs EC proliferation by inhibiting Notch1 activity. This mechanism may contribute to the pro-atherogenic effects of Dicer and blocking the interaction between miR-103 and lncWDR59 might be a promising therapeutic strategy against atherosclerosis.