Endothelial Barrier Function Modulation by PKCdelta

微血管通透性增加会导致急性肺损伤、成人呼吸窘迫综合征(ARDS)和多系统器官衰竭时的肺水肿。内皮细胞-细胞接触的中断可能是微血管通透性增加的一个原因。识别调节血管通透性变化的细胞内机制可能导致控制因脓毒症或其他原因引起的急性肺损伤而造成的组织损伤的治疗策略。我们的初步结果表明，蛋白激酶C增量(PKCDELTA)的过度表达可以对抗凝血酶诱导的内皮单层通透性的增加。这一建议的假设是，PKCDelta通过调节细胞骨架连接复合体的组织来钝化内皮细胞单层通透性的变化。内皮细胞(EC)通常形成一个紧密贴壁的单层。在炎症或血栓形成反应中，由于内皮细胞收缩和内皮细胞-细胞相互作用的中断，内皮细胞单层通透性增加，形成内皮细胞间隙。EC收缩通过细胞内钙动员、Rho GTP酶激活、肌球蛋白轻链磷酸化、肌动蛋白微丝重组和局部黏附复合体(FA)的形成而发生。内皮细胞-细胞连接的破坏伴随着黏附连接(AJ)蛋白复合体在细胞间缝隙形成点的重新分布。水肿剂促进EC收缩和FA形成以及AJ复合体的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。这项建议的总体目标是确定PKCDELTA调节内皮屏障功能的机制。利用稳定高表达PKCDELTA的微血管内皮细胞和载体对照，我们将确定：1)内皮屏障功能减弱是否是由于AJ或FA与PKCDELTA的稳定和结合；2)βPKCDELTA是否通过在肌动蛋白应激纤维形成和/或功能水平调节EC收缩而钝化内皮屏障功能；3)PKCDELTA是否通过改变MAPK信号转导途径而降低内皮屏障功能。阐明PKCDELTA降低血管内皮细胞单层通透性的分子机制可能有助于开发抗内皮细胞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.