Endothelial Barrier Function Modulation by PKCDelta

描述（由申请方提供）：内皮单层通透性增加由肌动球蛋白丝形成和收缩介导，导致细胞-细胞连接破坏和细胞间间隙形成。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传导知之甚少。我们已经发现PKC过表达通过增加局灶性粘连增强微血管内皮基础屏障功能。此外，抑制剂研究支持PKC的作用（通过稳定局灶性粘连和肌动球蛋白丝来增强未刺激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粘着斑形成来调节内皮屏障功能。此外，我们的数据表明，PKC抑制减少细胞骨架和局灶性粘附动力学;这种作用在暴露于凝血酶时被逆转。此外，凝血酶诱导的屏障功能障碍加剧了预处理与罗特勒。结果还表明，PKC β通过p190 RhoGAP调节RhoA活性。最后，我们证明了在体内抑制PKC后诱导肺水肿（.因此，我们的工作已经证明了在体外和体内PKC（亚型）在调节内皮屏障功能完整性中的关键作用。这一建议的总体目标是阐明PKC（调节内皮细胞基底单层通透性的分子机制。强调细胞-ECM复合物和基础渗透性维持机制的研究是所提出的研究的独特和新颖的方面。目标一：确定PKC在内皮基础屏障功能和激动剂诱导的屏障功能障碍中的粘着斑组装/分解中的作用;目的II：确定PKC是否通过调节p190 RhoGAP活性保持活性RhoA的基础水平来维持内皮屏障完整性;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恢复正常肺功能。铺设说明：肺中的血管在受伤或创伤时变得渗漏，导致由血管输送的流体渗漏到肺中，引起呼吸困难。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，对维持非渗漏状态重要的蛋白质的鉴定将有助于发现肺水肿患者的治疗方法。

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.