Despite being an extremely thin single-cell layer, the endothelium performs exceedingly well in preventing blood fluids from leaking into the surrounding tissues. However, under specific pathological conditions this cell layer can be affected, compromising the barrier’s integrity. Vascular leakage is a hallmark of many cardiovascular diseases and albeit its medical importance no specialized therapies are available to prevent it or reduce it (1). Rho (Ras homology) GTPases are known as key regulators of different aspects of cell behavior, such as cell shape, migration, tension, division and contraction. The activity of Rho proteins is regulated by conformational changes induced by binding of guanine nucleotides, being GTP-bound Rho the active form and GDP-bound Rho the inactive (2). The regulators of this activation-inactivation cycle are: ca 70 GAPs (GTPase Activating Proteins) responsible for the inactivation process and approximately 80 GEFs (Guanine nucleotide Exchange Factors) controlling the activation. Interestingly, studies have shown that they can exert both positive and negative effects on the endothelial barrier’s integrity (3). Knowledge about both the precise mechanism of this regulation and the individual contribution of these specific regulatory proteins remains fragmentary. In order to identify suitable targets for intervention with the underlying RhoA-mediated signaling, we propose to study all known regulators of RhoGTPase activity. RNA interference screens will be carried out with small interfering RNA (siRNA) libraries targeting RhoGAPs, RhoGEFs and effectors, using human umbilical vein endothelial cells (HUVECs). With this approach we will be able to: test the effect of depletion on cellular morphology and endothelial barrier integrity using Electric Cell-Substrate Impedance Sensing (ECIS) technology and permeability assays; and also test the effect of depletion on RhoGTPase activity using an automated microscope (Cellomics Array Scan), as well as Rho G-LISAs. Positive candidates will be further studied using in-depth FRET-based biosensors in order to determine their role in regulation of RhoGTPase activity. The outcome of this project will provide detailed information on cellular RhoGTPase activity measured in space and time, as well as valuable insight regarding both the regulatory mechanism of these important proteins and the vulnerable loci in the endothelium. This mechanistic understanding will unveil unanticipated pathways suitable for therapeutic intervention, through which specific molecular targets for stabilization of the endothelial barrier will be identified, limiting episodes of vascular leakage.