Abstract 5527: Double-Stranded RNA Causes Leakage in Human Pulmonary Artery Endothelial Cell Monolayer

Abstract

Proper function of endothelial cells is necessary to maintain vessel integrity and to regulate the uptake of circulating substances. Increased levels of circulating RNA may result from excessive cell damage or cancer. We investigated the effect of double-stranded RNA (dsRNA) on monolayer permeability, mechanical properties and intercellular junctions of primary human pulmonary artery endothelial cells (hPAECs). Primary hPAECs were used to investigate shape and mechanical changes caused by Poly I:C (synthetic dsRNA) and dsRNA incubation by means of in situ atomic force microscopy in time. To assess junctional changes, vascular endothelial cadherin staining was performed on control and 24h Poly I:C or dsRNA treated hPAECs. For visualizing cytoskeletal rearrangements, the F-actin network was stained with phalloidin. The cellular barrier properties of hPAECs, as a read-out for cell function, were assessed by measuring changes in the trans-endothelial electric resistance. The in situ atomic force microscopy topographies show a remarkable cell shape change after 2 hours of incubation with Poly I:C. The cell height is increased and they become more elongated. Furthermore, after 6 hours the cell-cell contacts are disrupted and the surface of the culturing dish appeared on the topographies. The local force measurements showed a time dependent stiffening of hPAECs under Poly I:C incubation. Parallel to this effect a 2-fold increase in the Young moduli of the cells was observable, which was further raised to 5-fold increase compared to the initial state, at 24 hours of incubation. The 24h Poly I:C incubated cells presented a decrease in VE-cadherin staining, pointing to loosened cell-cell contacts. The phalloidin staining showed an increased signal at the cell boundaries, confirming the cytoskeletal rearrangement. The electrical resistance of the monolayer was decreased by 50% after 24h Poly I:C or dsRNA treatment, whereas the first, significant drop in the resistance was already observed after 2 hours. In conclusion, our data suggest that double-stranded RNA causes endothelial leakage in human pulmonary endothelial cell monolayer and contributes to barrier disruption as well as endothelial dysfunction.