Ethanol Induces Enhanced Vascularization Bioactivity of Endothelial Cell-Derived Extracellular Vesicles via Regulation of MicroRNAs and Long Non-Coding RNAs

Tek N Lamichhane,Lampouguin Yenkoidiok Douti,Steven M Jay,Christopher A Leung

doi:10.1038/s41598-017-14356-2

Abstract

Extracellular vesicles (EVs), such as exosomes, have been identified as regulators of vascular remodeling and have promise as therapeutics for vascularization applications. Towards development of EVs as therapeutics, it has been demonstrated that physiological stimuli of angiogenic phenotypes in EV-producing cells can enhance the potency of EVs for vascularization. The goal of this study was to assess whether ethanol, which induces angiogenic phenotypes in endothelial cells, could be employed to enhance endothelial-derived EV vascularization bioactivity. The results indicate that ethanol conditioning of endothelial cells increases the ability of endothelial EVs to induce a pro-vascularization response. This response is due in part to increased CD34 expression in recipient endothelial cells that may result from downregulation of microRNA-106b in EVs isolated from ethanol-conditioned producer endothelial cells. Further, ethanol-induced upregulation of long non-coding RNAs (lncRNAs) HOTAIR and MALAT1 in endothelial EVs was observed to play a significant role in mediating pro-angiogenic effects of these vesicles. Overall, these studies validate ethanol conditioning as a method to enhance the bioactivity of endothelial EVs via regulation of EV-associated microRNAs (miRNAs) and, especially, lncRNAs. Further, the results suggest that alcohol consumption may activate endothelial EVs towards a pro-vascularization phenotype, which could have implications for alcohol-induced tumor angiogenesis.

Highlights

Size distribution in the presence or absence of the indicated concentrations of EtOH in the medium was assessed via NanoTracking Analysis (NTA) using a NanoSight LM10 (n = 3)
Concentrations of ethanol beyond 100 mM were found to induce significant cell toxicity in human umbilical vein endothelial cells (HUVECs) (Fig. 1A), 100 mM was used as a maximum ethanol level in most experiments
The inclusion of ethanol in the culture medium did not appear to affect the structural integrity of produced Extracellular vesicles (EVs), as mean diameters (Fig. 1B) and protein expression levels (Fig. 1C,D) were found to be similar over the range between 0-200 mM ethanol for both HUVEC and human dermal microvascular endothelial cell (HDMEC) EVs

Summary

Introduction

We report that ethanol increases the vascularization bioactivity of endothelial cell EVs through at least two distinct mechanisms: downregulation of anti-angiogenic miRNA cargo (miR-106b) and upregulation of pro-angiogenic long non-coding RNA (lncRNA) cargo (MALAT1 and HOTAIR). These findings have implications for generation of EVs for therapeutic vascularization applications and may shed light on the role of EVs in alcohol-induced angiogenesis in cancer and other physiological settings

Objectives

Methods

Results

Conclusion