Our major theme is that the layered structure of the endothelial barrier requires continuous activation of signaling pathways regulated by S1P and intracellular cAMP. when S1P or intracellular cAMP are elevated at the time of inflammatory stimulus they can buffer changes induced by inflammatory agents and maintain NG25 normal barrier stability. When endothelium is exposed to inflammatory conditions and subsequently exposed to elevated S1P or intracellular cAMP the same processes restore the functional barrier by first reestablishing the adherens junction then modulating tight junctions and glycocalyx. In more extreme inflammatory conditions loss of the inhibitory actions of Rac1 dependent mechanisms may promote expression of more inflammatory endothelial phenotypes by NG25 contributing to the up-regulation of RhoA dependent contractile mechanisms and the sustained loss of surface glycocalyx allowing access of inflammatory cells to the endothelium. vascular barriers including those of skeletal muscle skin lung and mesentery (Michel and Curry 1999 It follows that the initial state of the endothelial barrier must be taken into account when evaluating mechanisms that regulate permeability. We have previously stressed this idea based on the observation that RhoA dependent contractile mechanisms contribute to increased permeability in intact microvessels (single perfused microvessels and a mouse skin wound model) only after the vessels are subjected to injury (skin wound) or inflammatory conditions (Curry and Adamson 2010 Waschke has noted that activation of RhoA dependent mechanisms occur in endothelial cells that undergo changes in cell shape and orientation after exposure to inflammatory conditions (Spindler et al. 2010 These observations led to the suggestion that endothelial cells in culture that also have up-regulated contractile responses are better models of endothelial barriers exposed to chronic inflammatory conditions than normal vessels that have not been exposed to such disturbances. We extend this idea in this review focusing on the regulation of adherens junctions tight junctions and the glycocalyx. 3.2 Overview of signaling NG25 pathways In this section we briefly review the mechanisms regulating the stability of the barrier as deduced mainly from investigations of endothelial cells in culture and describe some Rabbit Polyclonal to FRS2. of the limitations of this current understanding when applied to intact microvessels. The two parts of Figure 2 summarize key signaling pathways activated by S1P (Fig 2A) and cAMP (Fig 2B) known to contribute to the regulation of the permeability barrier (Spindler et al. 2010 Wang and Dudek 2009 The action of the small GTPases Rac1 and Rap1 are understood to increase adhesion between adjacent endothelial cells and to NG25 stabilize the peripheral actin band. When the development of tension contributes to gap formation the small GTPase Rho A regulates actin-myosin force generation and this is modulated by the PKA dependent action of cAMP to attenuate MLCK. A full description is beyond the scope this review but some of the most important changes include the following. Activation of Rac1 or Rap1 dependent pathways are associated with reduced stress fibers and increased peripheral band actin as well as the peripheral localization of the actin binding protein cortactin and non-muscle myosin light chain kinase (Dudek et al. 2004 Garcia et al. 2001 NG25 Schlegel et al. 2008 The prevalence of cell-cell and cell-substrate junctions is also rapidly increased. Proteins of the adherens complex including VE-cadherin α- and β-catenin are enhanced at the cell periphery following activation (Lee et al. 2006 Mehta et al. 2005 Also components of tight junctions including ZO-1 occludin and claudin-5 become more localized and the number of tight junction “kissing points” has been shown to increase (Baumer et al. 2008 Lee et al. 2006 Localization of focal adhesion associated proteins such as paxillin and focal adhesion kinase (FAK) following S1P (Shikata et al. 2003 or specific activation of the epac/Rap1 pathway (Lorenowicz NG25 et al. 2008 has also been associated with improved endothelial barrier function. These changes to the cytoskeleton and adhesion complexes are nearly universally associated with improved barrier function. Understanding these pathways is an area of active research. In the following we focus on those aspects of the Rac1 and Rap1 dependent pathways that are.