Centrosome Over-duplication and Blood Vessel Function

描述（由申请人提供）：肿瘤血管具有影响肿瘤发病的形态发生缺陷，主要通过启动缺氧相关信号传导。肿瘤血管迂曲、扩张、渗漏。有限的证据表明，肿瘤内皮细胞（ECs）有过多的中心体，这可能解释了其异常表型。然而，导致这些细胞改变的机制在很大程度上是不明确的。我们的研究小组最近证明，在发育中的血管和人类内皮细胞中，过量的VEGF信号会增加中心体过度复制的频率。由于细胞微管组织中心的破坏，不相等的中心体数目（>2）会产生有害的细胞后果。在血管生成发芽过程中，“缺陷”内皮细胞的繁殖和同化可能部分解释了肿瘤组织中常见的弯曲、渗漏和/或耐药血管表型。本课题旨在验证肿瘤血管ec位于中心体数量失调下游的异常假说，这种失调导致肿瘤血管结构和功能异常。因此，我们将在两个目标中检验这一假设。目的1将确定中心体失调对EC行为的机制和后果。遗传扰动将引入人类原代内皮细胞，导致中心体过度复制，独立于多源性促血管生成信号。经过修饰的内皮细胞将在迁移试验和3d血管生成试验中受到挑战，其中它们将在体外增殖、发芽和分支，以确定中心体失调对血管生成的影响。目的2将确定肿瘤进展与肿瘤血管内皮细胞中心体复制之间的联系。使用一种新的转基因方法来标记EC DNA，我将分析小鼠乳腺肿瘤的内皮腔室中与肿瘤分期和侵袭性相关的中心体异常，以确定中心体复制失调对肿瘤进展的影响。此外，肿瘤和正常的ECs将被分离和体外培养，并在目标1中进行相同的功能测定。这项研究的信息将阐明肿瘤血管新生的关键早期事件（中心体复制失调），并显示这些事件如何与异常血管发育和肿瘤进展联系起来。

DESCRIPTION (provided by applicant): Tumor blood vessels have morphogenetic defects that impact tumor pathogenesis, primarily through the initiation of hypoxia-related signaling. Vessels found in tumors are tortuous, dilated and leaky. Limited evidence has indicated that tumor endothelial cells (ECs) have excess centrosomes, which may explain their abnormal phenotype. However, the mechanisms responsible for these cellular alterations are largely undefined. Our group recently demonstrated that excess VEGF signaling increases the frequency of centrosome over-duplication in developing blood vessels and in human ECs. Unequal centrosome numbers (>2) can have deleterious cellular consequences due to disruption of the cells microtubule-organizing center. Propagation and assimilation of "defective" ECs during angiogenic sprouting may explain, in part, the tortuous, leaky and/or chemoresistant blood vessel phenotype commonly observed in tumor tissue. This proposal aims to test the hypothesis that tumor vessel ECs are abnormal downstream of centrosome number dysregulation, and that this dysregulation contributes to the abnormal structure and function of tumor vessels. Accordingly, we will test this hypothesis in two aims. Aim #1 will determine the mechanisms and consequences of centrosome dysregulation on EC behaviors. Genetic perturbations will be introduced into human primary ECs to cause centrosome over-duplication, independent of pleiotropic proangiogenic signaling. Modified ECs will be challenged in a migratory assay and a 3D-angiogenesis assay in which they will proliferate, sprout and branch in vitro to determine the effects of centrosome dysregulation on angiogenesis. Aim #2 will determine the linkage between tumor progression and centrosome duplication in ECs of tumor vessels. Using a novel transgenic approach to mark EC DNA, I will analyze the endothelial compartment in mouse mammary tumors for centrosome abnormalities to be correlated with tumor stage and invasiveness to determine the effects of dysregulation of centrosome duplication on tumor progression. Additionally, tumor and normal ECs will be isolated and cultured ex vivo and challenged to the same functional assays in aim #1. Information from this investigation will illuminate key early events (dysregulation of centrosome duplication) in tumor blood vessel angiogenesis, and show how these events link to abnormal vessel development and tumor progression.