Loss of lung vascular endothelial barrier integrity leads to increased vascular permeability and alveolar flooding, and contributes to the morbidity and mortality associated with ARDS. Thrombin activates G protein-coupled receptor PAR-1 in endothelial cells and induces actin stress fiber formation and the resultant increase in transendothelial permeability. The activation of the alpha subunit of the heterotrimeric G13 protein by thrombin in endothelial cells is critical for the activation of Rho proteins, and the subsequent increased endothelial permeability response. We have shown that Galpha-13 protein interacts with the actin-binding protein, radixin, which is critical for the assembly of focal adhesions and actin filaments. Thus, radixin may be an essential effector in signaling the Galpha-13-induced Rho activation via the Rho guanine nucleotide dissociation inhibitor, RhoGDI. In Project 3, we will address a new and potentially important signaling pathway by which Galpha-13-dependent Rho activation results in increased lung endothelial permeabihty. We will define the upstream regulation of Rho involving radixin and the signaling pathways mediating the increase in endothelial permeability. In response to inflammatory mediators such thrombin, the actin cytoskeleton alterations and the increased endothelial permeability are typically reversible within hours. The reversibility ot the response restores endothelial barrier integrity; however, its molecular and cellular bases are poorly understood. Protein kinase A, PKA, a kinase linked to enhancing endothelial barrier function is usually activated by cyclic AMP. We have discovered that both thrombin and Galpha-13 can stimulate PKA via two novel mechanisms that do not require cAMP. One mechanism is dependent on the interaction of Galpha-13 with radixin, which activates the reversal of the permeability response. Another mechanism is dependent on the stimulation of the NF-kappaB signaling pathway via mitogen-activated protein kinases. The end result is that PKA may phosphorylate Galpha-13, and thereby inhibit Rho activation. We will address the concept that this is a key mechanism for the down-regulation of Galpha-13 activity and the reversal of the permeability response. Another possibility to be addressed is that Galpha-13 induces the phosphorylation of vasodilator-stimulated phosphoprotein, VASP, which can prevent actin polymerization and thus re-establish the endothelial barrier. Project 3 will test the general hypothesis that signaling complexes formed by PAR-1 activation of Galpha-13 signal the loss of endothelial barrier function and sequentially activate signals that lead to endothelial barrier recovery, and the reversal of the permeability-increase. We believe that the proposed studies will generate novel information elucidating the regulation of lung vascular permeability. Moreover, with the unraveling of the signals regulating the reversal of the increase in lung vascular permeability, it will be possible to identify novel targets tor therapies whereby the inappropriate increase in endothelial permeability can be controlled reducing the vascular """"""""leak"""""""" associated with ARDS.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
2P01HL060678-06
Application #
6967981
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
2005-03-01
Project End
2010-02-28
Budget Start
2005-03-01
Budget End
2006-02-28
Support Year
6
Fiscal Year
2005
Total Cost
$370,204
Indirect Cost
Name
University of Illinois at Chicago
Department
Type
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
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