Abstract 17419: Differential Roles of Rap1 Isoforms in Regulation of Vascular Permeability and Endothelial Cell Barrier
Dynamic regulation of vascular permeability is critical for normal vessel function as evidenced in pathological conditions such as edema or inflammation; yet underlying mechanisms are not fully understood. Small G protein Rap1 has been implicated in promoting endothelial cell-cell (EC) junction formation upon elevation of cellular cAMP but also in promoting angiogenic responses to VEGF. However, what role Rap1 plays in regulation of vascular permeability in vivo is not known. The project's objective was to investigate the function of two Rap1 isoforms, Rap1A and 1B, in the formation and maintenance of adherens junctions (AJ) in ECs and in regulation of vessel permeability in vivo. To accomplish this, we used Cre-lox P method and Tie2 promoter to generate endothelial-specific knockout mice deficient in Rap1A (EC-Rap1A KO), Rap1B (EC-Rap1B KO) or partially depleted of both isoforms (EC-Rap1A KO; Rap1B+/-). We found that vessel leakage of Evans blue dye was significantly increased only in lungs from EC-Rap1A KO; Rap1B+/- mice but not from EC-Rap1B KO mice suggesting that Rap1A is a main isoform promoting vessel barrier. Using bone marrow chimeras we showed that vessel barrier protection by Rap1 is EC-autonomous. In vitro, confocal microscopy revealed that Rap1A-deficiency in ECs led to altered organization of AJs with dispersed β-catenin distribution across AJs in contrast to narrow distribution in control or Rap1B KO ECs, suggesting that Rap1A may regulate endothelial barrier properties by spatially organizing AJ proteins. However, the kinetics of barrier reformation was not altered in Rap1A-deficient ECs, but was slower in Rap1B-deficient ECs, as evidenced by diminished cell monolayer electrical resistance measured by electric cell-substrate impedance sensing (ECIS) and slower VE-cadherin fluorescence recovery at AJs after junction disruption with EGTA. Recovery of β-catenin at cell-cell contacts was slower in Rap1B-depleted ECs also after AJs disruption using VEGF, suggesting that Rap1B may play a role in regulating AJs formation kinetics. Overall, these results suggest that two Rap1 isoforms may play differential roles in ECs: Rap1A is required for vascular barrier maintenance, while Rap1B is involved in dynamic regulation of AJs formation.
- © 2011 by American Heart Association, Inc.