Role of Protein Kinase D1 in the regulation of endothelial permeability after blunt chest trauma.

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The isolated chest trauma is one of the most critical injuries associated with a 10-20% mortality rate. During chest trauma, pulmonary contusions occur in 75% of cases. Acute lung injury (ALI) and its more severe form, the acute respiratory distress syndrome (ARDS) are characterized by acute respiratory failure with widespread damage to cells and structures of the alveolar and endothelial capillary membranes. Our preliminary work has suggested a novel role for Protein Kinase D1 (PKD1) in the regulation of endothelial barrier function. We show that PKD is potently activated by Interleukin-8 (lL-8) and thrombin, barrier destabilizing agents released during trauma or trauma associated coagulopathy. Knockdown of PKD1 or inhibition of PKD impaired leakage of albumin through HUVEC monolayers and in chorioallantois membrane (CAM) assays. PKD1 depletion also impaired transendothelial passage of human neutrophils. We also show that PKD1 depletion enhanced stability of VE-Cadherin adhesion complexes and their linkage to F-actin, via the PKD-substrate Cortactin. In addition, we have identified p120-Catenin as a novel PKD substrate implicated in endothelial permeability. Our proposal therefore aims to investigate the role of PKD1 in the control of endothelial barrier stability after blunt chest trauma. The following three aims will be investigated: Aim1: Role of PKD1 in the modulation of endothelial barrier dysfunction after trauma. We will subject mice to a blunt chest trauma and pre-treat animals with PKD-inhibitor to study if vascular leakage, edema formation, lung tissue damage and neutrophil infiltration can be ameliorated. We will further investigate vascular permeability in response to cytokines and anaphylatoxins released during trauma using the CAM model and we will generate a conditional PKD1 knockout in the endothelium of mice to analyze the endothelial barrier after blunt chest trauma. Aim2: Molecular regulation of endothelial integrity by PKD1 in the context of trauma. We will investigate PKD activation by thrombin transduced by PAR-1 receptor activation. We will determine specific involvement of G-protein subunits, Rho-GTPases, RhoGEF-proteins and PKC isoforms. We will also investigate activation pathways of PKD1 by other stimuli controlling endothelial barrier function, such as IL-8. Aim3: Role of Cortactin and the novel PKD substrate p120-Catenin in the modulation of endothelial barrier stability and leukocyte transmigration. We will investigate the molecular composition of VE-cadherin adhesion complexes following inhibition or depletion of PKD1 and the contribution of the PKD-substrates Cortactin as well as p120-Catenin to the regulation of endothelial barrier stability and neutrophil transmigration. With the herein proposed aims we will explore if inhibition of PKD activity will help to ameliorate adverse effects after blunt chest trauma by stabilizing the endothelial barrier and how effects are controlled on a molecular level.