TNF receptor-2 signaling in arteriogenesis/angiogenesis

描述(申请人提供)：有两种不同的肿瘤坏死因子特异性质膜受体，I型55 kDa TNFR(TNFR1)和II型75 kDa TNFR(TNFR2)。TNFR1广泛表达，而TNFR2受到严格调控，主要分布于内皮细胞(EC)。肿瘤坏死因子通过肿瘤坏死因子受体1和相关的接头分子引起广泛的生物学效应，包括增殖、分化和凋亡。关于TNFR2招募到的蛋白以及TNFR2在EC中的功能，人们知之甚少。针对TNFR1和TNFR2的抗肿瘤坏死因子治疗在治疗类风湿性关节炎方面取得了成功，但在心血管疾病的治疗中失败了。我们假设，TNFR2信号在心血管系统中发挥有益的作用，如缺血启动的动脉生成/血管生成和重塑。我们发现，TNFR2的基因缺失，而不是TNFR1的基因缺失，会损害缺血启动的血流恢复，从而导致严重的肢体缺血。这可能通过受损的动脉生成、血管生成或内皮祖细胞动员而发生。我们已经建立了各种检测方法来剖析TNFR2-KO小鼠的这些缺陷。同时，我们还努力确定了TNFR2血管生成信号通路中的关键下游效应因子。我们发现，TRAF2是目前已知的唯一一种与TNFR2相互作用的蛋白，它调节TNFR2的抗凋亡功能；我们已经鉴定出第一个TNFR2特异的激酶ETK，它对肿瘤坏死因子诱导的EC迁移和管状形成至关重要。因此，在动脉形成/血管形成过程中，TNFR2的肿瘤坏死因子激活导致EC存活、迁移和管状形成的不同但协同的下游信号。成功培育出具有TNFR1、TNFR2、TRAF2和ETK基因缺陷的小鼠，从这些小鼠中分离出小鼠肌肉微血管内皮细胞，并建立体内血管生成/动脉生成实验，将使我们能够剖析TNFR2的信号转导机制。我们提出以下具体目标：1)利用TNFR2-KO小鼠，明确TNFR2介导血管生成/动脉生成的机制。2)剖析了TNFR2在EC中的信号转导机制。3)利用转基因小鼠模型，明确EC表达的TNFR2在炎性血管生成中的作用。这一建议将为TNFR2及其下游效应因子TRAF2和ETK在炎性血管生成/动脉生成中的作用提供新的信息，并促进控制血管生成/动脉生成依赖型心血管疾病的新治疗方法的开发。血管的生长和成熟对缺血的心、脑和四肢的修复至关重要。我们将研究膜受体TNFR2在这一过程中的作用。我们的工作可能会带来更好的缺血性疾病治疗方法。

DESCRIPTION (provided by applicant): There are two distinct TNF-specific plasma membrane-localized receptors, type I 55 kDa TNFR (TNFR1) and type II 75 kDa TNFR (TNFR2). TNFR1 is expressed ubiquitously, whereas TNFR2 is tightly regulated and found predominantly on endothelial cells (EC). TNF via TNFR1 and associated adaptor molecules elicits a broad spectrum of biological effects including proliferation, differentiation and apoptosis. Little is known regarding the proteins recruited to TNFR2 and the function of TNFR2 in EC. Anti-TNF therapy targeting both TNFR1 and TNFR2 has been successful in treating rheumatoid arthritis but failed in the treatment of cardiovascular diseases. We hypothesize that TNFR2 signaling plays beneficial roles in the cardiovascular system such as ischemia-initiated arteriogenesis/angiogenesis and remodeling. We show that genetic loss of TNFR2, but not of TNFR1, impairs ischemia initiated blood flow recovery resulting in critical limb ischemia. This may occur via impaired arteriogenesis, angiogenesis or mobilization of endothelial progenitor cells. We have established various assays in order to dissect these defects in TNFR2-KO mice. Meanwhile, we have made efforts to define the critical downstream effectors in TNFR2 angiogenic signaling. We show that TRAF2, the only previously known TNFR2-interacting protein, regulates the anti-apoptotic function of TNFR2; we have identified the first TNFR2-specific kinase, Etk, critical for TNF-induced EC migration and tube formation. Thus TNF activation of TNFR2 leads to distinct but cooperative downstream signaling of EC survival, migration and tube formation during arteriogenesis/angiogenesis. The successful breeding of genetically-deficient mice for TNFR1, TNFR2, TRAF2 and Etk, isolation of mouse muscle microvessel EC from these mice and establishment of in vivo angiogenesis/arteriogenesis assays will allow us to dissect the signaling by TNFR2. We propose the following specific aims: 1) Define the mechanism by which TNFR2 mediates angiogenesis/arteriogenesis using TNFR2-KO mice. 2) Dissect TNFR2 signaling in EC. 3) Define the role of EC-expressed TNFR2 in inflammatory angiogenesis using transgenic mouse models. This proposal should provide novel information on the role of TNFR2 and its downstream effectors TRAF2 and Etk in inflammatory angiogenesis/arteriogenesis, and facilitate development of new therapeutic approaches to control angiogenesis/arteriogenesis-dependent cardiovascular diseases. Growth and maturation of blood vessels are critical for the repair of ischemic heart, brain and extremity. We will study the role of a membrane receptor TNFR2 in the process. Our work may lead to better treatments for ischemic diseases.