Abstract 455: Increased Endothelial Permeability in RhoGDI-1 Null Mice Due to the Activation of RhoA Signaling Cascade

Introduction Increased vascular permeability is a hallmark of inflammation that occurs in acute and chronic diseases such as atherosclerosis, acute respiratory distress syndrome, and diabetes. Rho family of small GTPases have a major role in the regulation of endothelial permeability via their action on cytoskeletal organization and integrity of interendothelial junctions. Guanine nucleotide dissociation inhibitors (GDIs) are a class of regulators known to form a complex with the GDP-bound form of the small RhoGTPases and thus inhibiting their activation. Hypothesis We hypothesized that removal of RhoGDI-1 disrupts the equilibrium between the small RhoGTPases with profound effects on the endothelial barrier function. Methods We used in-vivo lung perfusion studies, small GTPases pull-down assay, western blotting, electron and confocal microscopy to evaluate the role of RhoGDI-1 in endothelial barrier function. Results We studied the lung microcirculation to address the in-vivo role of RhoGDI-1 signaling in endothelial barrier regulation. We found that endothelial permeability in the lungs of RhoGDI-1 null mice was two-fold higher than that in the lungs of wild type mice. Electron microscopy showed that interendothelial junctions in lung capillaries are open in RhoGDI-1 −/ − mice. The opening of the interendothelial junctions was also seen after down-regulation of endogenous RhoGDI-1 protein with siRNA in HUVECs and staining for ZO-1 and VE-cadherin proteins. Although previous in vitro studies showed that the affinities of RhoGDI-1 for all three major small RhoGTPases (RhoA, Rac1 and Cdc42) are equal, we determined that the amount of activated form of RhoA, but not Rac1 or Cdc42 was significantly increased in RhoGDI-1 −/ − mice. As a consequence myosin light chain-2 is hyperphopsphorylated in the lungs of these mice and pretreatment with a Rho-kinase inhibitor restored the lung endothelial permeability to almost normal levels in the transgenic mice. Conclusion RhoGDI-1 is essential for maintaining normal endothelial barrier function in vivo through the regulation of the amount of activated RhoA, suggesting that therapies which target endothelial RhoGDI-1 or downstream effectors of RhoA may be beneficial in re-establishing endothelial barrier.