Regulatory mechanisms of endothelial barrier function: dissecting the critical role of Rho GTPases, ubiquitination and degradation

The most inner layer of blood vessels is formed by the endothelium, which consists of a monolayer of tightly connected endothelial cells and acts as a dynamic barrier by controlling passage of leukocytes, plasma and macromolecules from the circulation to the interstitial space. Dysfunction of the endothelial barrier is a hallmark of chronic inflammatory diseases and is characterized by vascular leakage, which leads to edema and compromises organ function. Tight cell-cell adhesion is mainly mediated by homotypic interactions of the transmembrane protein vascular endothelial (VE)-cadherin, which is intracellularly linked to the F-actin cytoskeleton via catenin proteins. The forces generated by the dynamics of the F-actin cytoskeleton regulate the strength of adhesion of the adherens junctions and thus, directly determine permeability across the endothelium. Due to their major role in controlling actin cytoskeleton dynamics, Rho GTPases are critical regulators of endothelial barrier function. Next to nucleotide binding and hydrolysis, post-translational modifications such as ubiquitination have been shown to regulate Rho GTPase signaling. With this thesis we improved our understanding of the regulation of Rho GTPases and other proteins, specifically by ubiquitination, in the context of endothelial integrity. In chapter 2, we reviewed the current knowledge on a subgroup of E3 ligases, which are based on Cullin3 complexes, controlling Rho GTPase function. Notably, Cullin3-based E3 ligases regulate not only Rho GTPases but also their regulators, in various cellular settings. Interestingly, ubiquitination by Cullin3 is involved in various vascular functions such as endothelial barrier function, angiogenesis and in regulation of blood pressure, both indicative of a diverse role of Cullin3-based E3 ligases contributing to fine-tuning Rho GTPases signaling and crucial for distinct physiological processes in the vessel wall. In chapter 3, we take this to a next level by describing the importance of ubiquitination of specifically RhoB, but not other Rho GTPases such as RhoA, to preserve the integrity of both quiescent and activated endothelium. In a subsequent proteomics screen, we identified two proteins, AAMP and MTSS1, as novel regulators of endothelial barrier function, which are, similar to RhoB, controlled by a short half-life, which we describe in chapter 4. In chapter 5 and 6 we identified novel aspects of Rac1 regulation by ubiquitination, RhoGDI binding and the RhoGAP HMHA1. In summary, this thesis gives insights into the mechanisms of ubiquitination and degradation of Rho GTPases, with special emphasis on their importance for endothelial barrier function. We further establish that ubiquitination of other proteins, besides Rho GTPases, is crucial to maintain the endothelial barrier. In addition, we report on novel aspects on regulation of Rac1 activity and ubiquitination. This thesis improves our understanding of the complex events controlling endothelial barrier function and may contribute to the development of strategies fighting pathological vascular leakage.