Endothelial Barrier Function Modulation by PKCdelta

PKCdelta 调节内皮屏障功能

• 批准号: 6359745
• 负责人: Elizabeth O Harrington
• 金额: $ 25.2万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6359745
• 关键词: actins; calcium flux; cell adhesion; cell cell interaction; enzyme activity; focal adhesion kinase; guanosinetriphosphatases; immunofluorescence technique; immunoprecipitation; intercellular connection; microfilaments; mitogen activated protein kinase; myosins; phosphorylation; protein kinase C; protein protein interaction; thrombin; tissue /cell culture; vascular endothelium; vascular endothelium permeability

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.

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）和多系统器官衰竭导致肺水肿。 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。 鉴定调节血管通透性变化的细胞内机制可能会导致治疗策略，以控制败血症或其他急性肺损伤原因引起的组织损伤。 我们的初步结果表明，蛋白激酶CDELTA（PKCDELTA）的过表达可预防内皮单层渗透性的凝血酶诱导的增加。该提议的假设是，PKCDELTA通过调节细胞骨架连接络合物的组织来通过调节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期间，内皮单层渗透率随着EC收缩和内皮细胞 - 细胞相互作用的破坏而随着间皮细胞间隙的形成而增加。 EC收缩是通过细胞内钙动员，Rho GTPase激活，肌球蛋白轻链磷酸化，肌动蛋白微丝重组和局灶性粘附复合物（FA）形成而发生的。 内皮细胞 - 细胞连接的破坏与粘附间间隙形成点处的粘附连接（AJ）蛋白复合物的重新分布同时发生。 尚不完全了解杂志剂促进EC收缩和FA形成以及AJ络合物拆卸的细胞内机制。许多炎症和血栓形成剂激活PKC，增加单层内皮渗透性。 该提案的总体目的是确定PKCDELTA调节内皮屏障功能的机制。 使用稳定过表达PKCDELTA和矢量控制的微血管EC，我们将确定：i）内皮屏障功能降低是否是由于AJ或FA与PKCDELTA的稳定和缔合； ii）delta pkcdelta是否通过调节肌动蛋白应力纤维形成和/或功能的水平来调节EC缩回来调节内皮屏障功能； iii）PKCDELTA是否通过改变MAPK SignalTransDuction途径来降低内皮屏障功能。 PKCDELTA降低内皮单层通透性的分子机制的阐明可能导致治疗剂的发展，这些治疗剂可防止内皮屏障功能障碍。