Centrosome Over-duplication and Blood Vessel Function

描述(申请人提供)：肿瘤血管具有影响肿瘤发病机制的形态发生缺陷，主要是通过启动与缺氧相关的信号。在肿瘤中发现的血管是弯曲的、扩张的和漏水的。有限的证据表明，肿瘤内皮细胞(ECs)具有过量的中心体，这可能是其异常表型的原因。然而，导致这些细胞变化的机制在很大程度上还不清楚。我们的研究小组最近证实，过量的血管内皮生长因子信号会增加发育中的血管和人内皮细胞中中心体过度复制的频率。不相等的中心体数目(&gt；2)可能会由于细胞微管组织中心的破坏而产生有害的细胞后果。血管新生萌发过程中“缺陷”内皮细胞的增殖和同化可能部分解释了肿瘤组织中常见的曲折、渗漏和/或化疗耐药的血管表型。这一建议旨在验证一种假说，即肿瘤血管内皮细胞位于中心体数量失调的下游，这种失调导致了肿瘤血管结构和功能的异常。因此，我们将在两个目标上检验这一假设。目的#1将确定中心体失调对EC行为的机制和后果。基因扰动将被引入到人类原代内皮细胞中，导致中心体过度复制，而不依赖于多效性促血管生成信号。改良的内皮细胞将在迁移试验和3D血管生成试验中受到挑战，在这些试验中，它们将在体外增殖、发芽和分枝，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。利用一种新的转基因方法标记EC DNA，我将分析小鼠乳腺肿瘤中的内皮室，寻找与肿瘤分期和侵袭性相关的中心体异常，以确定中心体复制异常对肿瘤进展的影响。此外，肿瘤和正常内皮细胞将被分离和体外培养，并在AIM#1中接受相同的功能测试。这项研究的信息将阐明肿瘤血管生成中的关键早期事件(中心体复制失调)，并展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.