Endothelial Barrier Function Modulation by PKCDelta

说明(申请人提供)：肌动球蛋白细丝的形成和收缩导致细胞-细胞连接中断和细胞间隙的形成，导致内皮单层通透性增加。其他变化包括细胞-细胞和细胞-细胞外基质接触处粘附力的增加。虽然在了解调节屏障功能障碍的分子机制方面取得了很大进展，但对维持内皮屏障完整性所需的细胞内信号转导知之甚少。我们已经证明，PKC(过度表达)通过增加局部粘连增强微血管内皮细胞的基础屏障功能。此外，抑制剂研究支持PKC(通过稳定焦点粘连和肌动球蛋白细丝来增强未受刺激的内皮单层的屏障功能)的作用。我们的数据表明，PKC通过RhoA途径调节应力纤维和焦点黏附形成，从而调节内皮屏障功能。此外，我们的数据显示，PKC(抑制)减弱了细胞骨架和局部黏附动力学；这一效应在暴露于凝血酶时被逆转。此外，rotlerin预处理可加重凝血酶诱导的屏障功能障碍。结果还表明，PKC通过p190RhoGAP调节RhoA活性。最后，我们证明了抑制PKC(。因此，我们的工作已经证明，在体外和体内，PKC(异构体)在调节内皮屏障功能完整性方面发挥着至关重要的作用。这项建议的总体目标是阐明PKC(调节内皮细胞基础单层通透性)的分子机制。对细胞-ECM复合体的重视和对维持基础渗透性的机制的研究是拟议研究的独特和新颖的方面。目的I：确定PKC(在局部黏附组装/分解中的作用)在内皮细胞基础屏障功能和激动剂诱导的屏障功能障碍中的作用；目的II：确定PKC(通过调节p190RhoGAP活性来维持基础水平的活性RhoA是否维持内皮屏障的完整性)；目的III：确定PKC(在体内维持肺血管屏障功能中的作用)。了解维持肺内皮细胞屏障完整性的机制可能有助于开发治疗急性肺损伤的方法，以限制肺损伤的程度，并加速恢复正常的肺功能。描述：肺内血管在受伤或创伤时发生渗漏，导致血管输送的液体渗漏到肺部，导致呼吸困难。目前，还没有有效的治疗方法来预防或解决这种情况。我们希望，对维持非渗漏状态重要的蛋白质的识别将有助于发现治疗肺水肿患者的方法。

DESCRIPTION (provided by applicant): Increased endothelial monolayer permeability is mediated by actomyosin filament formation and contraction resulting in disruption of cell-cell junctions and formation of intercellular gaps. Additional changes include increases in adhesive forces at cell-cell and cell-extracellular matrix contacts. While much progress has been made in understanding molecular mechanisms regulating barrier dysfunction, little is known about the intracellular signaling necessary for maintenance of endothelial barrier integrity. We have shown that PKC( overexpression enhanced microvascular endothelial basal barrier function by augmenting focal adhesions. In addition, inhibitor studies support a role for PKC( in enhancing barrier function in unstimulated endothelial monolayers by stabilizing focal adhesions and actomyosin filaments. Our data suggests that PKC( modulates endothelial barrier function through modulation of stress fiber and focal adhesion formation through a RhoA pathway. Also, our data shows that PKC( inhibition diminishes cytoskeletal and focal adhesion dynamics; effects which were reversed upon exposure to thrombin. Additionally, thrombin-induced barrier dysfunction was exacerbated by pretreatment with rottlerin. Results also suggest that PKC( regulates RhoA activity through p190RhoGAP. Finally, we demonstrate an induction of lung edema in vivo upon inhibition of PKC(. Thus, our work has demonstrated a crucial role for the PKC( isoform in regulating endothelial barrier function integrity in in vitro and in vivo. The overall goal of this proposal is to elucidate the molecular mechanisms by which PKC( regulates endothelial basal monolayer permeability. The emphasis on cell-ECM complexes and the investigation of mechanisms of maintenance of basal permeability are unique and novel aspects of the proposed research. Aim I: To determine the role of PKC( in focal adhesion assembly/disassembly in endothelial basal barrier function and agonist-induced barrier dysfunction; Aim II: To determine if PKC( maintains endothelial barrier integrity by preserving a basal level of active RhoA through modulation of p190RhoGAP activity; and Aim III: To determine the role of PKC( in maintaining lung vascular barrier function in vivo. Understanding mechanisms of maintenance of pulmonary endothelial barrier integrity may be useful in developing therapies for acute lung injury to limit the extent of lung injury and speed recovery to normal lung function. Lay Description: Blood vessels in the lung become leaky upon injury or trauma, resulting in fluid transported by the blood vessels to leak into the lung causing difficulty breathing. Currently, no effective treatments for preventing or resolving this condition are available. We hope that identification of proteins important in maintaining a non-leaky state will assist in discovering a treatment for patients suffering from lung edema.