Abstract 240: RhoGTPases and RNAi: A Large-scale Screen Using Ecis® to Measure Endothelial Integrity in Real-time

Abstract

Introduction: Vascular leakage is a hallmark of many cardiovascular diseases and albeit its importance no specialized therapies are available to prevent or reduce it. RhoGTPases, their on/off switchers GEFs and GAPs and downstream effectors are known regulators of different cellular processes and studies have shown their positive and negative effects on the integrity of the endothelial barrier. Still, with data available on only a few family members, precise knowledge about this mechanism remains fragmentary. We hypothesize that there is a tight spatiotemporal regulation of a yet unknown combination of these regulatory proteins and that a systematic study of all members will provide more information on this mechanism. Using state-of-art RNAi and Electric Cell-substrate Impedance Sensing we measured the silencing effect of given genes on the endothelial barrier. Methods: A selective RNAi library of 270 siRNAs targeting RhoGTPases, GAPs, GEFs and Effectors was ordered. HUVECs of 12 donors were isolated, pooled and plated on 96 wells ECIS arrays. Before reaching confluence, cells were transfected and 72h later stimulated with 1U/ml of thrombin. Positive and negative controls were carefully chosen and three independent screens were carried out on different pools of cells. Trans endothelial electrical resistance was measured in real-time and recorded throughout the whole procedure. Results: Analysis of 3 critical time-points (72h post-transfection, direct thrombin response and recovery phase) confirmed already known key players such as Gef-H1, Larg or RhoA, which supports the reliability of this technique and its accuracy. However, it also revealed more than 10 new candidates involved in the regulation of the endothelial barrier. A few promoted a tighter endothelial barrier, some showed a protective effect upon thrombin challenge and others an intense disruptive response to the stimulus. Further characterization studies will be carried out and validation is ongoing. Conclusions: Using a novel screening approach we identified previously unknown candidate regulators involved with endothelial barrier formation, dysruption and restoration. These new molecular targets provide opportunities for future pharmacological intervention.