Mechanisms by which MAGP-2 Promotes Angiogenesis

描述（申请人提供）：肿瘤生长和转移需要血管生成。在肿瘤形成过程中发生的关键事件之一是血管静止微环境向促血管生成微环境的重塑。这种重塑为血管内皮细胞（ECs）提供了血管生成前的通信，这对正常和病理血管生成至关重要。因此，干扰内皮细胞与其微环境之间的通信的治疗干预是抑制血管生成和癌症的潜在方法。不幸的是，关于调节血管生成的微环境分子和这些分子发挥作用的分子机制，还有很多需要了解的。为了解决这个问题，我们最近对体外血管生成的内皮细胞进行了基于微阵列的转录组分析。我们发现了39种以前与血管生成无关的分泌蛋白。我们对该数据集的首次分析是通过在内皮细胞中使用逆转录病毒过表达策略进行的。总的来说，该方法证实了七个研究蛋白中的两个（即MAGP-2和lumican）在体外和体内都控制血管生成。本项修订申请的总体目标是描述MAGP-2促进血管生成的分子机制。我们在这个项目上取得了重大进展，并确定了MAGP-2通过与Notch信号通路相互作用来控制血管生成。正在进行的实验正在剖析MAGP-2操纵Notch信号的分子细节，以及内皮特异性含有颗粒的MAGP-2在血管生成调节中的作用。虽然这个项目将帮助我们了解血管生成调节的基本方面，但它并不寻求在原始微阵列数据集中寻找血管生成的其他调节因子。这是一个错失的机会，因为很有可能在我们原始的微阵列数据中仍有其他新的血管生成调节因子有待发现。本修订应用程序的目标是发现和表征在我们的微阵列结果的初始分析中未研究的血管生成的其他基质和细胞膜结合调节剂。这是一个重要而重点突出的项目，基于先前的成功，有很大的可能性发现新的血管生成调节因子。重要的是，根据R15资助机制和ARRA，拟议的项目将为学生研究人员提供优秀的培训机会，将对一个没有得到NIH大量资助的机构进行强有力的投资，因此将有助于推动印第安纳州农村地区的经济复苏。为了实现我们的目标，我们提出了两个具体目标。在目标1中，我们将确定血管生成的其他基质和细胞膜结合调节剂。为了实现这一目标，我们将采用基于morpholino的斑马鱼胚胎候选基因敲低方法来确定这些基因是否是血管生成的重要调节因子。基因敲除的效果将通过对fl1 - gfp + GATA-1 RFP双转基因斑马鱼品系的微血管造影分析来监测。这种方法将使我们能够快速有效地筛选原始微阵列数据中剩余的推定血管生成调节因子。由于其他的血管生成调节因子已经从这个数据集中分离出来，我们相信这个项目将发现更多的新的血管生成调节因子。事实上，正如我们的初步结果所示，我们已经通过这种方法确定了另外4种可能的血管生成调节因子。在目标2中，这些新的假定的血管生成调节因子以及在目标1中确定的其他基因将被监测其促血管生成或抗血管生成活性。为了实现这一目标，候选基因将在哺乳动物内皮细胞中过度表达和/或敲低，并与对照基因进行比较，以改变血管生成活性，包括细胞侵袭、增殖、对血管生成生长因子的敏感性和毛细血管样结构的形成。

DESCRIPTION (provided by applicant): Angiogenesis is required for the growth and metastasis of cancers. One of the critical events that occur during tumor formation is the remodeling of the angiostatic vascular microenvironment to a pro-angiogenic microenvironment. This remodeling provides pro- angiogenic communications to vascular endothelial cells (ECs) that are critical for normal and pathological angiogenesis. Therefore, therapeutic interventions that disrupt communications between ECs and their microenvironment represent a potential approach for inhibiting angiogenesis and cancer. Unfortunately, there remains a great deal to learn about the microenvironment molecules that regulate angiogenesis and the molecular mechanisms by which these molecules functions. To address this problem, we recently conducted a microarray based transcriptome analysis of ECs undergoing angiogenesis in vitro. We identified 39 secreted proteins that were not previously associated with angiogenesis. Our first analysis of this data set was performed by employing a retroviral overexpression strategy in endothelial cells. Overall, this approach confirmed that two out of seven investigate proteins (i.e. MAGP-2 and lumican) control angiogenesis both in vitro and in vivo. The overall goal of the parent grant to which this revision application is amended is to characterize the molecular mechanisms by which MAGP-2 promotes angiogenesis. We have made significant progress on this project and have determined that MAGP-2 controls angiogenesis by interacting with Notch signaling pathways. Ongoing experimentation in the parent grant is dissecting the molecular details by which MAGP-2 manipulates Notch signaling and the role of endothelial specific MAGP-2 containing granules in angiogenesis regulation. While this project will help us to understand basic aspects of angiogenesis regulation, it does not seek to pursue additional regulators of angiogenesis present within the original microarray data set. This is a missed opportunity since it is highly likely that additional novel regulators of angiogenesis remain to be discovered in our original microarray data. The goal of this revision application is to discover and characterize additional stromal and cell membrane bound regulators of angiogenesis that were not investigated in the initial analysis of our microarray results. This is an important and focused project that based on prior success, has a high probability to uncover novel regulators of angiogenesis. Importantly, in accord with the R15 funding mechanism and the ARRA, the proposed projects will provide outstanding training opportunities for student researchers, will make a strong investment in an institution that has not received significant NIH funding, and therefore will help drive economic recovery in rural Indiana. We propose two specific aims to achieve our goals. In aim 1, we will identify additional stromal and cell membrane bound regulators of angiogenesis. To accomplish this, we will employ a morpholino based knockdown approach of candidate genes in zebrafish embryos to determine if these genes are important regulators of angiogenesis. The effect of gene knockdown will be monitored by microangiogram analysis of Fli1-GFP + GATA-1 RFP double transgenic zebrafish lines. This approach will allow us to rapidly and efficiently screen through the remaining putative regulators of angiogenesis in our original microarray data. Since other regulators of angiogenesis have already been isolated from this dataset, we are confident this project will uncover additional novel angiogenesis regulators. Indeed, as shown in our preliminary results, we have already identified 4 additional putative regulators of angiogenesis using this procedure. In aim 2 these new putative regulators of angiogenesis plus additional genes identified in aim 1 will be monitored for either pro- or anti-angiogenic activities. To accomplish this, candidate genes will be overexpressed and/or knocked down in mammalian endothelial cells and compared to their control counterparts for altered angiogenic activities including cell invasion, proliferation, sensitivity to angiogenic growth factors, and formation of capillary-like structures. PUBLIC HEALTH RELEVANCE: Cancer continues to be a leading cause of death and suffering in the United States. Since all solid tumors depend on the infiltration of new blood vessels (i.e. angiogenesis), strategies to block angiogenesis may provide therapeutic opportunities to treat cancer. To address this medical need, this proposal seeks to characterize new molecules and mechanisms by which angiogenesis is controlled so that we can one day use this information to block angiogenesis and combat cancer.