Role of Anthrax Toxin Receptors in Angiogenesis and Endothelium

描述(申请人提供)：允许炭疽与宿主相互作用的细胞受体被认为是血管生成受体。了解它们的功能对于设计干扰炭疽毒素作用的策略可能至关重要。例如，阻断炭疽-受体相互作用的试剂可能会干扰天然配体-受体相互作用，从而影响血管完整性、血管生成和表皮细胞。这一建议定义了与炭疽保护性抗原(PA)亲和力最高的受体的血管生成功能，即ANTXR2/CMG2。ANTXR2/CMG2的功能将在炭疽暴露的组织部位进行研究，特别是皮肤。我们的初步研究表明，ANTXR2/CMG2对毛细血管样管的形成、内皮细胞的增殖和迁移具有重要作用。我们描述了与炭疽暴露相关的ANTXR2/CMG2表达的位置，并证明了CMG2胞外区的一种可溶形式抑制了皮肤中由血管内皮生长因子驱动的新生血管。我们的重点将放在ANTXR2/CMG2在真皮血管生成、角质形成细胞的功能以及炭疽保护性抗原(PA)与这些细胞的结合上。我们的总体目标是了解ANTXR2/CMG2在皮肤细胞中的功能，以研究炭疽毒素/受体相互作用抑制对宿主细胞功能的影响。为了实现这一目标，在目标I中，我们使用基因敲除和受体拮抗剂来干预ANTXR2/CMG2在真皮微血管内皮细胞中的功能。我们假设ANTXR2/CMG2促进真皮血管生成的关键步骤。这一假说也将在AIMS II和III中得到评估，这两个项目的重点是在体内评估ANTXR2/CMG2在血管发育、血管内皮生长因子诱导的真皮血管生成和伤口修复过程中的功能。在AIM III中，我们将有条件地去除ANTXR2/CMG2的表达。我们的初步结果显示，ANTRX2/CMG2在不同的细胞类型中表达，因此我们将有条件地去除内皮细胞或角质形成细胞中的基因功能，这两个潜在的炭疽相互作用部位。最后，在第四个目标中，我们通过在内皮特异性表达缺失的内皮细胞和突变小鼠中评估PA-受体的相互作用来检验ANTXR2/CMG2作为内皮中主要的炭疽受体的假设。允许炭疽与宿主相互作用的细胞受体被认为参与新血管的生长，因此了解它们的功能可能对于设计干扰炭疽毒素作用的策略至关重要。例如，阻断炭疽毒素的试剂可能会干扰正常的受体功能，从而影响皮肤、肺和肠道的血管和其他细胞。这项研究将确定使用旨在阻止炭疽病的新试剂后的潜在不良影响，从而帮助确保这种策略是安全的。

DESCRIPTION (provided by applicant): The cellular receptors that allow Anthrax interaction with host are proposed to be angiogenic receptors. Understanding their function may be critical in designing strategies to interfere with Anthrax toxin action. For instance, reagents that block anthrax-receptor interaction may interfere with native ligand-receptor interactions thus affecting vessel integrity, angiogenesis and epidermal cells. This proposal defines the angiogenic functions of the receptor with highest affinity for the anthrax protective antigen (PA), that is ANTXR2/CMG2. The function of ANTXR2/CMG2 will be studied in tissues sites of anthrax exposure, specifically skin. Our preliminary studies shows that ANTXR2/CMG2 is important for capillary-like tube formation, proliferation and migration of endothelial cells. We describe sites of ANTXR2/CMG2 expression of relevance to Anthrax exposure and we demonstrate that a soluble form of the extracellular domain of CMG2 inhibits VEGF-driven neovascularization in skin. Our emphasis here will be on ANTXR2/CMG2 function in dermal angiogenesis, in keratinocyte function and on anthrax protective antigen (PA) binding to such cells. Our overall objective is to understand ANTXR2/CMG2 function in cells of the skin, in order to study the consequence of inhibition of anthrax toxin/receptor interaction on host cell function. To achieve this objective, in Aim I we evaluate ANTXR2/CMG2 function in dermal microvascular endothelial cells, using knockdown and receptor antagonists to interfere with function. We hypothesize that ANTXR2/CMG2 promotes key steps in dermal angiogenesis. This hypothesis will also be evaluated in Aims II and III that focus on in vivo assessment of ANTXR2/CMG2 function during vascular development, dermal angiogenesis induced by VEGF and during wound repair. In Aim III we will conditionally ablate ANTXR2/CMG2 expression. Our preliminary results show that ANTRX2/CMG2 is expressed in diverse cell types thus we will conditionally remove gene function in endothelial cells or keratinocytes, both potential sites of anthrax interaction. Finally, in Aim IV we test the hypothesis that ANTXR2/CMG2 serves as the major anthrax receptor in endothelium by evaluating PA-receptor interaction in knockdown endothelial cells and mutant mice with endothelial-specific loss of receptor expression.The cellular receptors that allow Anthrax interaction with host are thought to be involved in the growth of new blood vessels thus understanding their function may be critical in designing strategies to interfere with Anthrax toxin action. For instance, reagents that block anthrax toxin may interfere with the normal receptor function thus affecting blood vessels and other cells in skin, lung, and gut. This study will define the potential adverse effects after use of new agents designed to block anthrax and thus help assure that such strategies are safe.