Endothelial cells (EC) form a semi-permeable barrier between the interior space of blood vessels and the underlying tissues. In acute lung injury the EC barrier is weakened leading to increased permeability. Among the mechanisms regulating EC barrier function, Rho/VRad balance appears to be of major importance with RhoA activation being barrier-destructive and Rad activation is barrier-protective. This Project will elucidate the mechanisms underlying the barrier protective effects of Rad. We show that adenosine induces rapid increases in Rad activation and this correlates with a significant attenuation of LPS-induced EC permeability in vitro and in vivo. The ability of adenosine to enhance barrier function appears to be dependent on MLCP and correlates with increased levels of cAMP. However, the mechanistic links coupling increases in cAMP with the activation of Rad, MLCP stimulation and barrier protection are unknown. Thus, in this project we propose to investigate a novel mechanism of adenosine-induced EC barrier enhancement/protection via the coordinated activation, and interaction, of Rad and MLCP that is mediated by Epac1-Rap1- and protein kinase A (PKA)-signaling. We will also determine if the interactions between Rad and MYPT1 are involved in regulating MLCP activation and if the role of Epac1-Rap1 and PKA in this process. Finally, we will test the hypothesis that MLCP activation leads to EC barrier enhancement/protection via the dephosphorylation of the novel cytoskeletal proteins: the ERM proteins (ezrin, radixin, and moesin), and caldesmon. Thus, the overall objective of this project is to elucidate the molecular mechanisms through which Rad is involved in EC barrier enhancement/protection in response to adenosine. The detailed specific aims are: SA #1. To define the links between adenosine-induced Rad and MLCP activation and determine their role in EC barrier protection in vitro and in vivo SA #2. To define the involvement of MLCP cytoskeletal targets in Rad-induced EC barrier enhancement/protection in vitro and in vivo.
The overall goal of this Project is to develop a better understanding of the mechanisms by which Rad activity is stimulated by purinergic receptor agonists. Our overarching premise is that maintaining Rad signaling could alleviate the endothelial barrier disruption associated with acute lung injury (ALI). Emphasis is placed on understanding both novel mechanisms and on developing novel reagents to maintain and/or restore EC barrier function during ALI.
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