Control of endothelial cell function and arteriogenesis by MEG3:EZH2 epigenetic regulation of integrin expression.

Epigenetic processes involving long non-coding RNAs regulate endothelial gene expression. However, the underlying regulatory mechanisms causing endothelial dysfunction remain to be elucidated. Enhancer of zeste homolog 2 (EZH2) is an important rheostat of histone H3K27 trimethylation (H3K27me3) that represses endothelial targets, but EZH2 RNA binding capacity and EZH2:RNA functional interactions have not been explored in post-ischemic angiogenesis. We used formaldehyde/UV-assisted crosslinking ligation and sequencing of hybrids and identified a new role for maternally expressed gene 3 (MEG3). MEG3 formed the predominant RNA:RNA hybrid structures in endothelial cells. Moreover, MEG3:EZH2 assists recruitment onto chromatin. By EZH2-chromatin immunoprecipitation, following MEG3 depletion, we demonstrated that MEG3 controls recruitment of EZH2/H3K27me3 onto integrin subunit alpha4 (ITGA4) promoter. Both MEG3 knockdown or EZH2 inhibition (A-395) promoted ITGA4 expression and improved endothelial cell migration and adhesion to fibronectin in vitro. The A-395 inhibitor re-directed MEG3-assisted chromatin remodeling, offering a direct therapeutic benefit by increasing endothelial function and resilience. This approach subsequently increased the expression of ITGA4 in arterioles following ischemic injury in mice, thus promoting arteriogenesis. Our findings show a context-specific role for MEG3 in guiding EZH2 to repress ITGA4. Novel therapeutic strategies could antagonize MEG3:EZH2 interaction for pre-clinical studies.

Β-blockers activate autophagy on infantile hemangioma-derived endothelial cells in vitro.

The mechanisms underlying the success of propranolol in the treatment of infantile hemangioma (IH) remain elusive and do not fully explain the rapid regression of hemangiomatous lesions following drug administration. As autophagy is critically implicated in vascular homeostasis, we determined whether ß-blockers trigger the autophagic flux on infantile hemangioma-derived endothelial cells (Hem-ECs) in vitro. MATERIAL AND METHODS: Fresh tissue specimens, surgically removed for therapeutic purpose to seven children affected by proliferative IH, were subjected to enzymatic digestion. Cells were sorted with anti-human CD31 immunolabeled magnetic microbeads. Following phenotypic characterization, expanded Hem-ECs, at P2 to P6, were exposed to different concentrations (50 µM to 150 µM) of propranolol, atenolol or metoprolol alone and in combination with the autophagy inhibitor Bafilomycin A1. Rapamycin, a potent inducer of autophagy, was also used as control. Autophagy was assessed by Lysotracker Red staining, western blot analysis of LC3BII/LC3BI and p62, and morphologically by transmission electron microscopy. RESULTS: Hem-ECs treated with either propranolol, atenolol or metoprolol displayed positive LysoTracker Red staining. Increased LC3BII/LC3BI ratio, as well as p62 modulation, were documented in ß-blockers treated Hem-ECs. Abundant autophagic vacuoles and multilamellar bodies characterized the cytoplasmic ultrastructural features of autophagy in cultured Hem-ECs exposed in vitro to ß-blocking agents. Importantly, similar biochemical and morphologic evidence of autophagy were observed following rapamycin while Bafilomycin A1 significantly prevented the autophagic flux promoted by ß-blockers in Hem-ECs. CONCLUSION: Our data suggest that autophagy may be ascribed among the mechanisms of action of ß-blockers suggesting new mechanistic insights on the potential therapeutic application of this class of drugs in pathologic conditions involving uncontrolled angiogenesis.

Long Non-Coding RNA Regulation of Epigenetics in Vascular Cells.

The vascular endothelium comprises the interface between the circulation and the vessel wall and, as such, is under the dynamic regulation of vascular signalling, nutrients, and hypoxia. Understanding the molecular drivers behind endothelial cell (EC) and vascular smooth muscle cell (VSMC) function and dysfunction remains a pivotal task for further clinical progress in tackling vascular disease. A newly emerging era in vascular biology with landmark deep sequencing approaches has provided us with the means to profile diverse layers of transcriptional regulation at a single cell, chromatin, and epigenetic level. This review describes the roles of major vascular long non-coding RNA (lncRNAs) in the epigenetic regulation of EC and VSMC function and discusses the recent progress in their discovery, detection, and functional characterisation. We summarise new findings regarding lncRNA-mediated epigenetic mechanisms-often regulated by hypoxia-within the vascular endothelium and smooth muscle to control vascular homeostasis in health and disease. Furthermore, we outline novel molecular techniques being used in the field to delineate the lncRNA subcellular localisation and interaction with proteins to unravel their biological roles in the epigenetic regulation of vascular genes.