Endothelial Responses to Leukocyte Engagement

白细胞穿过内皮的迁移是炎症中的关键事件。该项目的目标是 了解白细胞粘附于内皮细胞所引发的信号， 白细胞穿过内皮。我们将重点关注这一过程的两个方面： 白细胞诱导的杯状结构，形成于内皮细胞和白细胞表面 通过内皮细胞-细胞连接。在第一个目标中，我们将检验白细胞 粘附到内皮细胞激活特异性Rho GTPases，这些都有助于形成 杯和内皮细胞-细胞连接的解体。我们将探索 白细胞粘附调节这些GTP酶，使用技术鉴定相关的鸟嘌呤核苷酸 交换因子（GEF）和GTP酶激活蛋白（GAP）。将特别关注SGEF， 其与ICAM-1在杯中共定位。SGEF激活RhoG，一种Rho蛋白， 膜皱褶我们还将研究RhoA和Rac 1下游的途径， 连接拆卸在第二个目标中，假设内皮连接受Rap 1调节， 将检测响应白细胞粘附的活性。我们将使用小鼠炎症模型， 研究Rap同种型在无Rapla或Raplb的小鼠内皮细胞中的作用。在 初步工作，我们已经表明，白细胞粘附刺激酪氨酸磷酸化， 内皮连接成分。在第三个目标中，我们将研究这种情况发生的途径， 是否响应Rac 1的活化和活性氧物质的产生。我们将 酪氨酸激酶和磷酸酶参与，并确定是否酪氨酸磷酸化的 VE-钙粘蛋白通过内吞作用导致其从连接处去除。几种受体酪氨酸磷酸酶 存在于内皮连接处。我们将测试这一假设，即这些可能相互作用，并受到抑制， 渗出白细胞，从而提高连接处的磷酸酪氨酸水平。因为白细胞 通过血管内皮屏障的迁移是炎症的关键步骤， 调节这一过程的信号通路可能揭示新的治疗目标， 控制炎症和炎症性疾病。

Leukocyte migration across the endothelium is a critical event in inflammation. The goals of this project are to understand the signals initiated by leukocyte adhesion to endothelial cells that promote passage of leukocytes across the endothelium. We will focus on two aspects of this process: the generation of leukocyte-induced cup-like structures that form on the surfaces of endothelial cells, and on leukocyte passage through endothelial cell-cell junctions. In the first aim, we will test the hypothesis that leukocyte adhesion to endothelial cells activates specific Rho GTPases and that these contribute both to formation of cups and to the disassembly of endothelial cell-cell junctions. We will explore the pathways by which leukocyte adhesion regulates these GTPases, using techniques to identify relevant guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). Particular attention will be paid to SGEF, which co-localizes with ICAM-1 in cups. SGEF activates RhoG, a Rho protein which induces dorsal membrane ruffles. We will also investigate the pathways downstream from RhoA and Rac1 that promote junctional disassembly. In the second aim, the hypothesis that endothelial junctions are regulated by Rap1 activity in response to leukocyte adhesion will be examined. We will use mouse models of inflammation to investigate the roles of Rap isoforms in endothelial cells in mice that are null for Rapla or Raplb. In preliminary work, we have shown that leukocyte adhesion stimulates the tyrosine phosphorylation of endothelial junctional components. In the third aim, we will investigate the pathway by which this occurs, whether it is in response to the activation of Rac1 and generation of reactive oxygen species. We will look for the tyrosine kinases and phosphatases involved and determine whether the tyrosine phosphorylation of VE-cadherin leads to its removal from junctions by endocytosis. Several receptor tyrosine phosphatases reside in endothelial junctions. We will test the hypothesis that these may interact with and be inhibited by extravasating leukocytes so as to elevate levels of phosphotyrosine in junctions. Because leukocyte migration across the endothelial barrier lining blood vessels is a critical step in inflammation, the elucidation of signaling pathways that regulate this process may reveal novel targets for the development of therapies to control inflammation and inflammatory diseases.