Tiam1 and Rac1 are required for platelet-activating factor-induced endothelial junctional disassembly and increase in vascular permeability.

It is known that platelet-activating factor (PAF) induces severe endothelial barrier leakiness, but the signaling mechanisms remain unclear. Here, using a wide range of biochemical and morphological approaches applied in both mouse models and cultured endothelial cells, we addressed the mechanisms of PAF-induced disruption of interendothelial junctions (IEJs) and of increased endothelial permeability. The formation of interendothelial gaps filled with filopodia and lamellipodia is the cellular event responsible for the disruption of endothelial barrier. We observed that PAF ligation of its receptor induced the activation of the Rho GTPase Rac1. Following PAF exposure, both Rac1 and its guanine nucleotide exchange factor Tiam1 were found associated with a membrane fraction from which they co-immunoprecipitated with PAF receptor. In the same time frame with Tiam1-Rac1 translocation, the junctional proteins ZO-1 and VE-cadherin were relocated from the IEJs, and formation of numerous interendothelial gaps was recorded. Notably, the response was independent of myosin light chain phosphorylation and thus distinct from other mediators, such as histamine and thrombin. The changes in actin status are driven by the PAF-induced localized actin polymerization as a consequence of Rac1 translocation and activation. Tiam1 was required for the activation of Rac1, actin polymerization, relocation of junctional associated proteins, and disruption of IEJs. Thus, PAF-induced IEJ disruption and increased endothelial permeability requires the activation of a Tiam1-Rac1 signaling module, suggesting a novel therapeutic target against increased vascular permeability associated with inflammatory diseases.

Adenosine deaminase inhibition attenuates microvascular dysfunction and improves survival in sepsis.

The ability of increased endogenous adenosine to mitigate microvascular derangements in sepsis was studied. Pentostatin (2'-deoxycoformycin), an inhibitor of adenosine deaminase, was administered to mice immediately after induction of sepsis by cecal ligation and puncture. Intravital video microscopy of cremasteric postcapillary venules was performed. Leukocyte rolling and adhesion were significantly increased in septic mice compared with control mice. Treatment of septic mice with pentostatin significantly decreased leukocyte rolling and adhesion (6.02 +/- 0.09 versus 1.72 +/- 0.12 rolling cells/min, 2.07 +/- 0.04 versus 0.62 +/- 0.05 adherent cells/100 microm per minute; p < 0.001). Albumin leakage (ratio) was significantly attenuated in septic animals treated with pentostatin (0.42 +/- 0.05 versus 0.21 +/- 0.04; p < 0.01). Circulating levels of interleukin-6, tumor necrosis factor-alpha, and soluble tumor necrosis factor type II receptor were decreased in septic mice treated with pentostatin. Survival was significantly improved at 48 hours in mice treated with pentostatin. These results suggest an important role for adenosine in modulating both leukocyte-dependent and -independent mechanisms of endothelial injury in sepsis. Exploiting the advantageous action of endogenous adenosine represents a potentially useful and novel therapeutic approach for the treatment of sepsis.