Endothelial Barrier Function Modulation by PKCDelta

描述（由申请人提供）：内皮单层通透性的增加是由肌动球蛋白丝的形成和收缩介导的，导致细胞-细胞连接的破坏和细胞间隙的形成。其他变化包括细胞-细胞和细胞-细胞外基质接触处的粘附力增加。虽然在理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知之甚少。我们已经证明PKC（过表达）通过增强局灶粘连增强微血管内皮基底屏障功能。此外，抑制剂研究支持PKC通过稳定局灶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。我们的数据表明，PKC通过RhoA途径调节应力纤维和局灶粘连形成，从而调节内皮屏障功能。此外，我们的数据显示，PKC抑制减少了细胞骨架和局灶黏附动力学，这种效应在暴露于凝血酶后被逆转。此外，凝血酶诱导的屏障功能障碍加重了rotlerin预处理。结果还表明PKC通过p190RhoGAP调控RhoA活性。最后，我们证明了PKC抑制可诱导体内肺水肿。因此，我们的工作证明了PKC（同种异构体）在体外和体内调节内皮屏障功能完整性方面的关键作用。本研究的总体目标是阐明PKC调节内皮细胞基底单层通透性的分子机制。强调细胞- ecm复合物和基础通透性维持机制的研究是提出的研究的独特和新颖方面。目的一：确定PKC在局点黏附组装/拆除内皮基础屏障功能和受体兴奋剂诱导的屏障功能障碍中的作用；目的二：确定PKC是否通过调节p190RhoGAP活性来维持基础水平的活性RhoA来维持内皮屏障的完整性；目的三：确定PKC在体内维持肺血管屏障功能中的作用。了解维持肺内皮屏障完整性的机制可能有助于开发急性肺损伤的治疗方法，以限制肺损伤的程度并加速肺功能的正常恢复。原理描述：受伤或外伤时，肺部血管渗漏，由血管输送的液体渗漏到肺部，造成呼吸困难。目前，没有有效的治疗方法来预防或解决这种情况。我们希望鉴定在维持非泄漏状态中起重要作用的蛋白质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.