Endothelial Barrier Function Modulation by PKCdelta

微血管通透性增加导致急性肺损伤肺水肿、成人呼吸窘迫综合征（ARDS）和多系统器官衰竭。内皮细胞-细胞接触的破坏是微血管通透性增加的可能原因。确定调节血管通透性变化的细胞内机制可能会导致控制脓毒症或其他原因引起的急性肺损伤的组织损伤的治疗策略。我们的初步结果表明，蛋白激酶Cdelta （PKCDELTA）的过度表达可以防止凝血酶诱导的内皮单层通透性增加。该提议的假设是PKCDelta通过调节细胞骨架连接复合物的组织来减弱内皮单层通透性的变化。内皮细胞（EC）通常形成紧密粘附的单层。在炎症或血栓形成反应中，由于EC收缩和内皮细胞间相互作用的破坏，内皮单层通透性随着内皮细胞间隙的形成而增加。EC收缩发生在细胞内钙动员、Rho GTPase激活、肌球蛋白轻链磷酸化、肌动蛋白微丝重组和局灶粘连复合体（FA）形成。内皮细胞-细胞连接的破坏伴随着粘附连接（AJ）蛋白复合物在细胞间隙形成点的重新分布而发生。致肿剂促进EC收缩和FA形成以及AJ复合物分解的细胞内机制尚不完全清楚。许多炎症和血栓形成因子激活PKC，增加单层内皮通透性。该建议的总体目标是确定PKCDELTA调节内皮屏障功能的机制。使用稳定过表达PKCDELTA和载体控制的微血管EC，我们将确定：I)内皮屏障功能的降低是否由于AJ或FA与PKCDELTA的稳定和关联；II) PKCDELTA是否通过在肌动蛋白应激纤维形成和/或功能水平上调节EC收缩来减弱内皮屏障功能；III) PKCDELTA是否通过改变MAPK信号转导途径降低内皮屏障功能。阐明PKCDELTA降低内皮单层通透性的分子机制可能有助于开发抗内皮屏障功能障碍的治疗药物。

Increased microvascular permeability causes pulmonary edema in acute lung injury, adult respiratory distress syndrome (ARDS), and multisystem organ failure. Disruption of endothelial cell-cell contacts is a possible cause of increased microvascular permeability. Identification of intracellular mechanisms which regulate changes in vascular permeability may lead to therapeutic strategies for controlling tissue damage due to sepsis or other causes of acute lung injury. Our preliminary results indicate that overexpression of protein kinase Cdelta (PKCDELTA) protects against thrombin-induced increases in endothelial monolayer permeability. The hypothesis of this proposal is that PKCDelta blunts changes in endothelial monolayer permeability by modulating the organization of cytoskeleton junctional complexes. Endothelial cells (EC) normally form a tightly adherent monolayer. During an inflammatory or thrombogenic response endothelial monolayer permeability increases with the formation of interendothelial cell gaps as a result of both EC contraction and disruption of endothelial cell-cell interactions. EC contraction occurs by intracellular calcium mobilization, Rho GTPase activation, myosin light chain phosphorylation, actin microfilament reorganization, and focal adhesion complex (FA) formation. Disruption of endothelial cell-cell junctions occurs concomitantly with the redistribution of the adherens junction (AJ) protein complexes at the points of intercellular gap formation. The intracellular mechanisms by which edemagenic agents promote EC contraction and FA formation and the disassembly of the AJ complexes are not completely understood. A number of inflammatory and thrombogenic agents activate PKC, increasing monolayer endothelial permeability. The overall objective of this proposal is to determine the mechanism by which PKCDELTA modulates endothelial barrier function. Using microvascular EC which stably overexpress PKCDELTA and vector control, we will determine: I) whether diminished endothelial barrier function is due to the stabilization and association of AJ or FA with PKCDELTA; II) whether delta PKCDELTA blunts endothelial barrier function by modulating EC retraction at the level of actin stress fiber formation and/or function; and III) whether PKCDELTA reduces endothelial barrier function by altering MAPK signaltransduction pathway. The elucidation of the molecular mechanisms by which PKCDELTA decreases endothelial monolayer permeability may lead to the development of therapeutic agents which are protective against endothelial barrier dysfunction.