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

孤立性胸部创伤是与10-20%死亡率相关的最严重伤害之一。在胸部外伤中，75%的病例发生肺挫伤。急性肺损伤（ALI）及其更严重的形式，急性呼吸窘迫综合征（ARDS）的特点是急性呼吸衰竭，肺泡和内皮毛细血管膜的细胞和结构广泛受损。我们的初步工作提示了蛋白激酶D1 （PKD1）在调节内皮屏障功能中的新作用。我们发现，创伤或创伤相关凝血病期间释放的屏障不稳定剂白介素-8 （il -8）和凝血酶能有效激活PKD。在绒毛尿囊膜（CAM）试验中，敲低PKD1或抑制PKD可损害白蛋白通过HUVEC单层渗漏。PKD1缺失也会损害人中性粒细胞的跨内皮传递。我们还发现，通过pkd底物接触，PKD1缺失增强了VE-Cadherin粘附复合物及其与F-actin连接的稳定性。此外，我们已经确定p120-Catenin是一种新的PKD底物，与内皮通透性有关。因此，我们的建议旨在研究PKD1在钝性胸外伤后内皮屏障稳定性控制中的作用。以下三个目标将被研究：目的1:PKD1在创伤后内皮屏障功能障碍调节中的作用。我们将对小鼠进行钝性胸部创伤，并用pkd抑制剂对动物进行预处理，研究是否可以改善血管渗漏、水肿形成、肺组织损伤和中性粒细胞浸润。我们将使用CAM模型进一步研究创伤过程中释放的细胞因子和过敏毒素对血管通透性的影响，我们将在小鼠内皮中产生条件敲除PKD1，以分析钝性胸部创伤后的内皮屏障。目的2：创伤背景下PKD1对内皮完整性的分子调控。我们将研究凝血酶通过PAR-1受体激活介导的PKD激活。我们将确定g蛋白亚基、Rho-GTPases、rhogef蛋白和PKC亚型的特异性参与。我们还将研究通过其他刺激控制内皮屏障功能（如IL-8）激活PKD1的途径。目的3：皮质蛋白和新的PKD底物p120-Catenin在内皮屏障稳定性和白细胞转运调节中的作用。我们将研究抑制或耗尽PKD1后VE-cadherin粘附复合物的分子组成，以及pkd底物皮质蛋白和p120-Catenin对内皮屏障稳定性和中性粒细胞迁移的调节作用。在本文提出的目的下，我们将探讨抑制PKD活性是否有助于通过稳定内皮屏障来改善钝性胸外伤后的不良反应，以及如何在分子水平上控制这种作用。

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.