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

蛋白激酶 D1 在钝性胸部创伤后调节内皮通透性中的作用。

基本信息

批准号：
376202546
负责人：
Dr. Tim Eiseler
金额：
--
依托单位：
Klinik für Innere Medizin I
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2017
资助国家：
德国
起止时间：
2016-12-31 至 2020-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/376202546?language=en
关键词：
Role Protein Kinase D1 regulation

项目摘要

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.

孤立性胸部创伤是最严重的伤害之一，死亡率为10-20%。在胸部创伤中，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）的特征在于急性呼吸衰竭，肺泡和内皮毛细血管膜的细胞和结构受到广泛损伤。我们的初步工作表明蛋白激酶D1（PKD 1）在调节内皮屏障功能中的新作用。我们表明PKD被白细胞介素-8（IL-8）和凝血酶（在创伤或创伤相关凝血病期间释放的屏障去稳定剂）有效激活。PKD 1的敲低或PKD的抑制损害了白蛋白通过HUVEC单层和在绒毛尿囊膜（CAM）测定中的渗漏。PKD 1耗竭也损害了人中性粒细胞的跨内皮通道。我们还表明，PKD 1耗竭增强VE-钙粘蛋白粘附复合物的稳定性，并通过PKD-底物Cordyn连接到F-肌动蛋白。此外，我们已经确定p120-连环蛋白作为一种新的PKD底物牵连内皮通透性。因此，我们的建议旨在研究PKD 1在钝性胸部创伤后控制内皮屏障稳定性中的作用。以下三个目标将被调查：目的1：PKD 1在创伤后内皮屏障功能障碍的调制中的作用。我们将使小鼠遭受钝性胸部创伤并用PKD抑制剂预处理动物以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模型进一步研究血管通透性对创伤期间释放的细胞因子和过敏毒素的反应，我们将在小鼠内皮中产生条件性PKD 1敲除，以分析钝性胸部创伤后的内皮屏障。目的2：创伤背景下PKD 1对内皮完整性的分子调控。我们将研究凝血酶通过PAR-1受体活化转导的PKD活化。我们将确定G蛋白亚基，Rho-GTP酶，RhoGEF蛋白和PKC亚型的具体参与。我们还将研究PKD 1的激活途径，通过其他刺激控制内皮屏障功能，如IL-8。目的3：Cordyn和新型PKD底物p120-Catenin在调节内皮屏障稳定性和白细胞迁移中的作用。我们将研究PKD 1抑制或耗竭后VE-钙粘蛋白粘附复合物的分子组成，以及PKD底物Cordyn和p120-Catenin对内皮屏障稳定性和中性粒细胞迁移的调节的贡献。根据本文提出的目标，我们将探索抑制PKD活性是否有助于通过稳定内皮屏障来改善钝性胸部创伤后的不良反应，以及如何在分子水平上控制这些影响。