SummaryThe endothelial barrier of the vasculature is of utmost importance for separating the blood stream from underlying tissues. This barrier is formed by tight and adherens junctions (TJ and AJ) that form intercellular endothelial contacts. TJ and AJ are integral membrane structures that are connected to the actin cytoskeleton via various adaptor molecules. Consequently, the actin cytoskeleton plays a crucial role in regulating the stability of endothelial cell contacts and vascular permeability. While a circumferential cortical actin ring stabilises junctions, the formation of contractile stress fibres, e. g. under inflammatory conditions, can contribute to junction destabilisation. However, the role of actin-binding proteins (ABP) in the control of vascular permeability has long been underestimated. Naturally, ABP regulate permeability via regulation of actin remodelling but some actin-binding molecules can also act independently of actin and control vascular permeability via various signalling mechanisms such as activation of small GTPases. Several studies have recently been published highlighting the importance of actin-binding molecules such as cortactin, ezrin/ radixin/moesin, Arp2/3, VASP or WASP for the control of vascular permeability by various mechanisms. These proteins have been described to regulate vascular permeability under various pathophysiological conditions and are thus of clinical relevance as targets for the development of treatment strategies for disorders that are characterised by vascular hyperpermeability such as sepsis. This review highlights recent advances in determining the role of ABP in the control of endothelial cell contacts and vascular permeability.

The intestinal epithelium forms a stable barrier protecting underlying tissues from pathogens in the gut lumen. This is achieved by specialized integral membrane structures such as tight and adherens junctions that connect neighboring cells and provide stabilizing links to the cytoskeleton. Junctions are constantly remodeled to respond to extracellular stimuli. Assembly and disassembly of junctions is regulated by interplay of actin remodeling, endocytotic recycling of junctional proteins, and various signaling pathways. Accumulating evidence implicate small G proteins of the Ras superfamily as important signaling molecules for the regulation of epithelial junctions. They function as molecular switches circling between an inactive GDP-bound and an active GTP-bound state. Once activated, they bind different effector molecules to control cellular processes required for correct junction assembly, maintenance and remodelling. Here, we review recent advances in understanding how GTPases of the Rho, Ras, Rab and Arf families contribute to intestinal epithelial homeostasis.

The intestinal epithelium constitutes a first line of defense of the innate immune system. Epithelial dysfunction is a hallmark of intestinal disorders such as inflammatory bowel diseases (IBDs). The actin cytoskeleton controls epithelial barrier integrity but the function of actin regulators such as cortactin is poorly understood. Given that cortactin controls endothelial permeability, we hypothesized that cortactin is also important for epithelial barrier regulation. We found increased permeability in the colon of cortactin-KO mice that was accompanied by reduced levels of ZO-1, claudin-1, and E-cadherin. By contrast, claudin-2 was upregulated. Cortactin deficiency increased RhoA/ROCK1-dependent actomyosin contractility, and inhibition of ROCK1 rescued the barrier defect. Interestingly, cortactin deficiency caused increased epithelial proliferation without affecting apoptosis. KO mice did not develop spontaneous colitis, but were more susceptible to dextran sulfate sodium colitis and showed severe colon tissue damage and edema formation. KO mice with colitis displayed strong mucus deposition and goblet cell depletion. In healthy human colon tissues, cortactin co-localized with ZO-1 at epithelial cell contacts. In IBDs patients, we observed decreased cortactin levels and loss of co-localization with ZO-1. Thus, cortactin is a master regulator of intestinal epithelial barrier integrity in vivo and could serve as a suitable target for pharmacological intervention in IBDs.

During inflammation, the disruption of the endothelial barrier leads to increased microvascular permeability. Whether tension along cell junctions contributes to histamine-induced endothelial barrier disruption remains unknown. Rapid Ca2+ influx induced by both histamine and thrombin was accompanied by endothelial barrier breakdown revealed as drop of transendothelial electric resistance in primary human microvascular endothelial cells. Interestingly, GLISA measurements revealed activation of RhoA but not inactivation of Rac1 at the time-point of barrier breakdown. FRET measurements showed activation of RhoA at intercellular junctions after both thrombin and histamine exposure. Breakdown coincided with increased stress fiber formation but not with translocation of vinculin, which was located along junctions in the resting state similar to postcapillary venules ex vivo. Moreover, increased tension at AJs was indicated by immunostaining with a conformation-sensitive antibody targeting the α18-subunit of α-catenin. Ca2+ chelation by BAPTA-AM and ROCK1 inhibition by Y27632 abolished both increase of tension along AJs as well as barrier dysfunction. Moreover, BAPTA-AM decreased RhoA activation following histamine stimulation, indicating a key role of Ca2+ signaling in barrier breakdown. Taken together, in response to histamine, Ca2+ via RhoA/ROCK activation along endothelial adherens junctions (AJs) appears to be critical for barrier disruption and presumably correlated with enhanced tension. However, vinculin appears not to be critical in this process.

The correct regulation of tissue barriers is of utmost importance for health. Barrier dysfunction accompanies inflammatory disorders and, if not controlled properly, can contribute to the development of chronic diseases. Tissue barriers are formed by monolayers of epithelial cells that separate organs from their environment, and endothelial cells that cover the vasculature, thus separating the blood stream from underlying tissues. Cells within the monolayers are connected by intercellular junctions that are linked by adaptor molecules to the cytoskeleton, and the regulation of these interactions is critical for the maintenance of tissue barriers. Many endogenous and exogenous molecules are known to regulate barrier functions in both ways. Proinflammatory cytokines weaken the barrier, whereas anti-inflammatory mediators stabilize barriers. Adrenomedullin (ADM) and intermedin (IMD) are endogenous peptide hormones of the same family that are produced and secreted by many cell types during physiologic and pathologic conditions. They activate certain G-protein-coupled receptor complexes to regulate many cellular processes such as cytokine production, actin dynamics and junction stability. In this review, we summarize current knowledge about the barrier-stabilizing effects of ADM and IMD in health and disease.