INTEGRIN SIGNALLING IN VASCULAR ENDOTHELIUM

血管基底膜的细胞外基质蛋白组分 深刻影响内皮细胞的形态和生长行为。我们的 中心假说是FAK(ppl25FAK，粘着斑激酶)是 对细胞骨架的组织和促进 血管内皮细胞的迁移。拟议中的研究将检验 FAK信号转导在血管形成和重塑中的作用 灶性粘连。第一个具体目标(A)将侧重于 改变FAK表达对内皮细胞黏附和迁移的影响。 FAK与潜在底物相互作用的调制将是 通过微量注射FAK片段或抗FAK抗体， 通过基因转染法或反义寡核苷酸法。FAK交互 与其他焦点黏附蛋白的结合，以及激活状态和 将在这些细胞中研究FAK的磷酸酪氨酸含量。第二 具体目标(B)是确定FAK与启动的关系 整合素介导的细胞内游离钙振荡 内皮细胞黏附。数字化视频显微镜，荧光钙 指示剂和细胞内钙通量的笼式介体将被使用 研究FAK表达正常和改变的内皮细胞。这个 第三个特定目标(C)是确定纤维连接蛋白所需的结构域 在局部黏附过程中通过FAK的内皮细胞信号转导 集合。这将通过研究FAK的激活和酪氨酸来实现 磷酸化与局灶性黏附形态平行。内皮细胞 在与各种纤维连接蛋白黏附的过程中，细胞将被成像 具有免疫荧光、干涉反射和激光的结构域 扫描共聚焦显微镜。 血管内皮细胞功能障碍是VITAL发病机制的核心 因创伤、炎症和败血症导致的器官衰竭。了解 血管内皮细胞支持微血管完整性的方式 可能有助于危重儿童的治疗。这些研究可能 深入了解内皮过程中发生的信号机制 血管生长发育过程中细胞与细胞外基质的黏附 以及对创伤的反应。改变这些的治疗策略 机制可能对各种疾病具有深远的影响，如 先天性心脏病，败血症时循环衰竭，肺 血管疾病在急性呼吸衰竭和脑缺血后 精神创伤。长期目标包括开发治疗药物 针对黏附蛋白表达和信号的干预 转导通路。

Extracellular matrix protein components of vascular basement membranes profoundly affect endothelial cell morphology and growth behavior. Our central hypothesis is that FAK (ppl25FAK, focal adhesion kinase) is essential to the organization of the cytoskeleton and the facilitation of migration in vascular endothelial cells. The proposed studies will test the role of FAK signal transduction during the formation and remodeling of focal adhesions. The first specific aim (A) will focus on the effects of altered FAK expression on endothelial cell adhesion and migration. Modulation of FAK interactions with potential substrates will be accomplished by microinjection of FAK fragments or anti-FAK antibodies, and by cDNA transfection or antisense oligonucleotides. FAK interactions with other focal adhesion proteins, and the activation state and phosphotyrosine content of FAK will be studied in these cells. The second specific aim (B) is to identify the relationship of FAK to the initiation of cytosolic free calcium oscillations during integrin-mediated endothelial cell adhesion. Digitized video microscopy, fluorescent calcium indicators, and caged mediators of intracellular calcium flux will be used to study endothelial cells with normal and altered FAK expression. The third specific aim (C) is to identify the domains of fibronectin required for endothelial cell signalling through FAK during focal adhesion assembly. This will be done by studying FAK activation and tyrosine phosphorylation in parallel with focal adhesion morphology. Endothelial cells will be imaged during the process of adhesion to various fibronectin domains with immunofluorescence, interference reflection, and laser scanning confocal microscopy. Vascular endothelial dysfunction is central to the pathogenesis of vital organ failure due to trauma, inflammation, and sepsis. Understanding the ways in which the vascular endothelium supports microvascular integrity may facilitate the treatment of critically ill children. These studies may provide insights into signalling mechanisms that occur during endothelial cell adhesion to extracellular matrix during vascular growth, development, and responses to trauma. Therapeutic strategies that modify these mechanisms may have far reaching implications for disorders as diverse as congenital heart disease, circulatory failure during septicemia, pulmonary vascular disease in acute respiratory failure, and cerebral ischemia after trauma. Long range goals include the development of therapeutic interventions targeting adhesion protein expression and signal transduction pathways.