Abstract 20376: Inhibition of Farnesylation of Ras-GTPases Ameliorates Inflammation-Induced Loss of Endothelial Barrier Integrity and Leukocyte Transmigration

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Abstract

Background and Aims: The family of small GTPases (Rho- and Ras-GTPases) is known to regulate endothelial cytoskeleton dynamics and hence barrier integrity. The role of Rho GTPases (RhoA and Rac1) in endothelial barrier integrity is well characterized; however, little is known about the role of Ras-GTPases. The activity of small GTPases is regulated by several mechanisms, including post-translational prenylation (farnesylation or geranylgeranylation). The Ras-GTPases are preferentially post-translationally farnesylated by farnesyl transferases (FTs). The aim of the present study was to investigate whether inhibition of farnesylation of Ras-GTPases protects loss of endothelial barrier integrity induced by inflammatory mediators.

Methods and Results: Treatment of human primary endothelial cells (ECs; HUVEC and lung microvascular ECs) with pharmacological inhibitor of farnesyl transferases (FTIs), LB42708 (LB) and tipifarnib, stabilized basal endothelial barrier function (albumin-flux) and abrogated thrombin-induced hyperpermeability in a concentration- and time-dependent manner reaching maximum at a concentration of 2 μmol/L after 12 h of incubation (n=5; p<0.05 for all experiments). These concentrations of the FTIs reduced farnesylation of all the Ras isoforms but not the geranylgeranylation of Rho GTPases. Likewise, LB abrogated the TNFα-induced leucocyte transendothelial migration (Trans-well assay). Interestingly, FTIs treatment caused an enhanced translocation of VE-cadherin to cell-cell junctions (confocal microscopy) without exerting any effect on the actin cytoskeleton or endothelial contractile machinery; mainly regulated by Rho GTPases. The Ca2+-switch experiments demonstrated an inhibition of VE-cadherin internalization. These data were reproduced by lentiviral-mediated stable over-expression of the prenylation-dead mutants of Ras isoforms but not RhoA or Rac1.

Conclusion: The novel data of the present study demonstrate that FTIs protect endothelial barrier function against inflammatory mediator-induced endothelial hyperpermeability. We propose that FTIs are novel drug candidates for the treatment of edematous conditions (such as lung edema) where endothelial barrier integrity is compromised.

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