INTEGRIN SIGNALLING IN VASCULAR ENDOTHELIUM

血管基底膜的细胞外基质蛋白成分 深刻影响内皮细胞形态和生长行为。我们 中心假设是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。拟议的研究将测试 FAK信号转导在血管内皮细胞形成和重塑中的作用 局灶性粘连第一个具体目标（A）将侧重于以下方面的影响： 改变FAK表达对内皮细胞粘附和迁移的影响。 调节FAK与潜在底物的相互作用将是 通过显微注射FAK片段或抗FAK抗体完成， 以及通过cDNA转染或反义寡核苷酸。FAK相互作用 与其他粘着斑蛋白，和激活状态， 将在这些细胞中研究FAK的磷酸酪氨酸含量。第二 具体目的（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 内皮细胞粘附数字化视频显微镜，荧光钙 将使用细胞内钙流的指示剂和笼状介质 研究FAK表达正常和改变的内皮细胞。的 第三个具体目标（C）是确定所需的纤连蛋白结构域 在粘着斑过程中通过FAK的内皮细胞信号传导 组装件.这将通过研究FAK激活和酪氨酸 与粘着斑形态平行的磷酸化。内皮 细胞在与各种纤维连接蛋白粘附的过程中将被成像 免疫荧光、干涉反射和激光 扫描共聚焦显微镜 血管内皮功能障碍是生命体征发病机制的核心。 创伤炎症和败血症导致的器官衰竭了解 血管内皮支持微血管完整性的方式 可以帮助危重患儿的治疗。这些研究可能 提供深入了解发生在内皮细胞 在血管生长，发育， 和对创伤的反应治疗策略，修改这些 机制可能对多种疾病产生深远的影响， 先天性心脏病，败血症期间循环衰竭，肺 急性呼吸衰竭和脑缺血后的血管疾病 外伤长期目标包括开发治疗药物 针对粘附蛋白表达和信号传导的干预 转导途径。

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.