LUNG VASCULAR BARRIER PRESERVATION BY PLATELET PRODUCTS

血管通透性的改变是多种过程的定义特征，包括动脉粥样硬化、炎症、缺血/再灌注损伤和血管生成。管理的机制，增加血管通透性正在紧张的调查。 然而，很少有人知道的过程中，决定屏障保护或恢复。血小板和血小板衍生产物对于维持内皮细胞屏障的完整性是必不可少的，并且与血管稳态和病理生物学密切相关。 我们已经表明，鞘氨醇1-磷酸（Sph 1-P）是一个磷酸化的脂质血管生成因子释放激活血小板连接特异性内皮分化基因（Edg）受体，刺激内皮细胞趋化性和血管生成反应。 Sph 1-P占血清中强内皮细胞趋化活性的大部分，并且在增强成纤维细胞生长因子诱导无血管小鼠角膜中血管生成的能力方面是显著有效的。 重要的是，Sph 1-P产生快速，持续，剂量依赖性增加的人肺动脉和肺微血管内皮细胞的屏障完整性。此外，Sph 1-P有效地逆转由水肿剂凝血酶引起的屏障功能障碍。 Sph 1-P增强屏障功能的确切机制尚不清楚，然而，我们的数据强烈暗示了内皮细胞细胞骨架动力学在这种反应中的重要作用。 Sph 1-P介导的屏障增强依赖于肌动蛋白丝重排和Rac GTP酶依赖性的已知细胞骨架调节蛋白如corneum、p21相关激酶（PAK）、LIM激酶和cofilin向皮质肌动蛋白细胞骨架的募集。在这个建议中，我们将研究Sph 1-P诱导的屏障增强的分子基础，并有针对性的皮质细胞骨架与细胞粘附蛋白，促进血管完整性的相互作用。 SA编号1将表征剪切应力后Sph 1-P介导的皮质细胞骨架重排，并评估corneum、PAK和cofilin参与。 SA 2号将研究Sph 1-P诱导的粘附分子（β/γ连环蛋白复合物）通过血小板内皮细胞粘附分子（PECAM）和酪氨酸磷酸化与肌动蛋白细胞骨架相互作用的改变。 SA编号3将定义活化的p125粘着斑激酶（FAK）和Rho/Rac GTP酶在Sph 1-P介导的粘着斑结构/功能调节中的作用。 最后，SA编号4将使用反义策略、PKC过表达构建体、药理学和豆蔻酰化肽抑制剂以及永生化稳定PKC过表达细胞系来确定屏障保护性PKC δ同种型在Sph 1-P介导的细胞骨架重排中的作用。 考虑到在多种血管病理学中观察到的伴随血管渗漏的严重生理紊乱，Sph 1-P输注可能为这些破坏性疾病提供一种新的治疗干预。

Alterations in vascular permeability are a defining feature of diverse processes including atherosclerosis, inflammation, ischemia/reperfusion injury and angiogenesis. Mechanisms which govern increases in vascular permeability are under intense investigation. However, little is known about processes which determine barrier protection or restoration. Platelets and platelet-derived products are essential to maintaining the integrity of the endothelial cell barrier and are intimately involved in vascular homeostasis and pathobiology. We have shown that sphingosine 1-phosphate (Sph 1-P) is a phosphorylated lipid angiogenic factor released from activated platelets which ligates specific endothelial differentiation gene (Edg) receptors to stimulate endothelial cell chemotaxis and angiogenic responses. Sph 1-P accounts for the majority of the strong endothelial cell chemotactic activity of blood serum, and was strikingly effective in enhancing the ability of fibroblast growth factor to induce angiogenesis in the avascular mouse cornea. Importantly, Sph 1-P produced rapid, sustained, and dose-dependent increases in the barrier integrity of human pulmonary artery and lung microvascular endothelial cells. Furthermore, Sph 1-P potently reversed barrier dysfunction elicited by the edemagenic agent, thrombin. The exact mechanisms by which Sph 1-P enhances barrier function are unknown, however, our data strongly implicate an essential role of endothelial cell cytoskeletal dynamics in this response. Sph 1-P-mediated barrier enhancement was dependent upon actin filament rearrangement and Rac GTPase-dependent recruitment to the cortical actin cytoskeleton of known cytoskeletal regulatory proteins such as cortactin, p21-associated kinase (PAK), LIM kinase and cofilin. In this proposal we will examine the molecular basis of Sph 1-P- induced barrier enhancement and have targeted cortical cytoskeletal interactions with cellular adhesive proteins which promote vascular integrity. SA number 1 will characterize the Sph 1-P-mediated rearrangement of the cortical cytoskeleton following shear stress and assess cortactin, PAK, and cofilin involvement. SA number 2 will investigate Sph 1-P-induced alterations in zona adherens (beta/gamma catenin complex) interaction with the actin cytoskeleton via platelet-endothelial cell adhesion molecule (PECAM) and tyrosine phosphorylation. SA number 3 will define the role of activated p125 focal adhesion kinase (FAK) and Rho/Rac GTPases in Sph 1-P-mediated regulation of focal adhesion structure/function. Finally, SA number 4 will define the role of the barrier-protective PKC delta isotype in Sph 1-P-mediated cytoskeletal rearrangement using antisense strategies, PKC over-expression constructs, pharmacologic and myristoylated peptide inhibitors, and immortalized stable PKC over-expressing cell lines. Given the profound physiologic derangements which accompany the vascular leak seen in multiple vascular pathobiologies, Sph 1-P infusion may provide a novel therapeutic intervention for consideration in these devastating disorders.