Double-Stranded RNA Attenuates the Barrier Function of Human Pulmonary Artery Endothelial Cells

Zoltán Bálint,Attila G Végh,Akos Heinemann,Viktoria Konya,Chandran Nagaraj,György Váró,Horst Olschewski,István A Krizbai,Andrea Olschewski,Diana Zabini,Imola Wilhelm,Csilla Fazakas,Rory Edward Morty

doi:10.1371/journal.pone.0063776

Abstract

Circulating RNA may result from excessive cell damage or acute viral infection and can interact with vascular endothelial cells. Despite the obvious clinical implications associated with the presence of circulating RNA, its pathological effects on endothelial cells and the governing molecular mechanisms are still not fully elucidated. We analyzed the effects of double stranded RNA on primary human pulmonary artery endothelial cells (hPAECs). The effect of natural and synthetic double-stranded RNA (dsRNA) on hPAECs was investigated using trans-endothelial electric resistance, molecule trafficking, calcium (Ca2+) homeostasis, gene expression and proliferation studies. Furthermore, the morphology and mechanical changes of the cells caused by synthetic dsRNA was followed by in-situ atomic force microscopy, by vascular-endothelial cadherin and F-actin staining. Our results indicated that exposure of hPAECs to synthetic dsRNA led to functional deficits. This was reflected by morphological and mechanical changes and an increase in the permeability of the endothelial monolayer. hPAECs treated with synthetic dsRNA accumulated in the G1 phase of the cell cycle. Additionally, the proliferation rate of the cells in the presence of synthetic dsRNA was significantly decreased. Furthermore, we found that natural and synthetic dsRNA modulated Ca2+ signaling in hPAECs by inhibiting the sarco-endoplasmic Ca2+-ATPase (SERCA) which is involved in the regulation of the intracellular Ca2+ homeostasis and thus cell growth. Even upon synthetic dsRNA stimulation silencing of SERCA3 preserved the endothelial monolayer integrity. Our data identify novel mechanisms by which dsRNA can disrupt endothelial barrier function and these may be relevant in inflammatory processes.

Highlights

Endothelial function is essential for vascular integrity
Primary human pulmonary artery endothelial cells (hPAECs) were incubated with double stranded RNA (dsRNA) or polycytidylic acid (Poly I):C up to 24 hours and the transendothelial electric resistance (TEER) was measured (Figure 1A)
Significant decrease in Transendothelial Electrical Resistance Measurement (TEER) was observed already after 18 hours of treatment suggesting that both, dsRNA and Poly I:C disrupted the integrity of the endothelial monolayer

Summary

Introduction

Endothelial function is essential for vascular integrity. The endothelium provides a barrier, regulates vascular tension, and is involved in angiogenesis and haemostasis. Endothelial cells express toll-like receptor 3 (TLR3) [3] which is activated by dsRNA [4,5]. The activation of TLR3 affects cell homeostasis [6,7] and causes changes at functional [8,9], as well as inflammatory gene expression level [10]. DsRNA induces a signaling cascade [11,12] leading to TLR3 receptor upregulation [4,13]. At organ level, repeated and long-term administration of synthetic dsRNA causes inflammation [14,15] and leads to impairment of lung function in mice [16,17,18]. The biological activity of circulating extracellular RNA is poorly understood

Methods

Results

Conclusion