The vascular endothelium controls the flux of macromolecules from the vascular space to the interstitium. Understanding the mechanisms that maintain and tighten the endothelial barrier would aid in the elucidation of therapeutic interventions to counter a loss of barrier activity and tissue edema. Both sphingosine 1-phosphate (S1P) and cAMP-enhancing agents increase barrier function. Proposed mechanisms for tightening the barrier include increases in cell-cell or cell-matrix adhesion, and/or a decrease in contractile activity. The increased barrier activity of S1P is independent of cAMP and protein kinases A and G and is not affected by inhibition of myosin light chain (MLC) kinase and extracellular signal-regulated kinase. Furthermore, S1P and cAMP have opposing effects on MLC phosphorylation, cellular tension, and actin stress fibers. We propose that the barrier-enhancing activity of S1P and cAMP is directed at the intercellular junctions. Alternatively, S1P and cAMP may function via different mechanisms. Preliminary data indicate that S1P increases the localization of vinculin, an alpha-catenin/actin linker protein, at cell-cell contacts and increases the association of alpha-catenin with actin. Preliminary data indicate that S1P decreases the peripheral localization of IQGAP1, which competes with alpha-catenin for binding to beta-catenin, increases the activity of Rac1, and increases the interaction of IQGAP1 with Rac1. These findings suggest that S1P increases barrier activity by enhancing adherens junction integrity. Preliminary data also indicate that S1P and cAMP reverse the barrier-decreasing effect of tumor necrosis factor (TNF). We propose that the adherens junction is a common target for S1P and cAMP. These mediators increase barrier function by strengthening the adherens junction via addition of linker proteins such as vinculin and via removal of IQGAP1 by activated Rac1. The three Specific Aims are: S1P and cAMP 1) increase barrier function by enhancing the attachment of alpha-catenin to actin via addition of linker proteins; 2) increase barrier function by removal of IQGAP1 from beta-catenin via activation of Rac1; and 3) rescue the barrier-disrupting effect of a phorbol ester, PMA, and a cytokine, TNF. Experimental approaches include the demonstration of protein interactions by immunofluorescence microscopy and immunoprecipitation and immunoblotting; functional analyses of adherens junctional integrity and endothelial barrier function; and infections with adenoviral recombinants to assess the importance of linker proteins (such as vinculin), alpha-catenin, IQGAP1, and Rac1. The biological significance is the identification of the cellular targets activated by S1P and cAMP that function to tighten the junctions between cells.
