Mechanisms by which MAGP-2 Promotes Angiogenesis

MAGP-2 促进血管生成的机制

• 批准号: 7811528
• 负责人: Allan R Albig
• 金额: $ 12.31万
• 依托单位: INDIANA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7811528
• 关键词: Address; Binding; Blood Vessels; Blood capillaries; Candidate Disease Gene; Cause of Death; Cell membrane; Cells; Communication; Cytoplasmic Granules; Data; Data Set; Economics; Embryo; Endothelial Cells; Event; Funding; Funding Mechanisms; Gene Expression Profile; Genes; Goals; Growth; Growth Factor; In Vitro; Indiana; Infiltration; Institution; Investments; Learning; Malignant Neoplasms; Medical; Molecular; Monitor; Neoplasm Metastasis; Notch Signaling Pathway; Pathologic Neovascularization; Probability; Procedures; Proteins; Recovery; Regulation; Research Personnel; Role; Rural; Signal Transduction; Solid Neoplasm; Stromal Cells; Structure; Students; Therapeutic; Therapeutic Intervention; Training; Transgenic Organisms; United States; United States National Institutes of Health; Vascular Endothelial Cell; Zebrafish; angiogenesis; base; capillary; combat; human GATA1 protein; human microfibrillar-associated protein 2; in vivo; knock-down; lumican; notch protein; novel; overexpression; parent grant; public health relevance; success; tumor

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.

描述（由申请人提供）：血管生成是癌症生长和转移所必需的。在肿瘤形成期间发生的关键事件之一是将血管生成抑制的血管微环境重塑为促血管生成的微环境。这种重塑为血管内皮细胞（EC）提供了促血管生成的通讯，这对正常和病理性血管生成至关重要。因此，破坏EC与其微环境之间的通信的治疗干预代表了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以及这些分子发挥作用的分子机制，仍然有很多东西需要了解。为了解决这个问题，我们最近进行了一个基于微阵列的转录组分析的内皮细胞进行血管生成在体外。我们确定了39个分泌蛋白，以前没有与血管生成。我们的第一个分析数据集进行了采用逆转录病毒过表达策略在内皮细胞。总的来说，这种方法证实了七种研究蛋白中的两种（即MAGP-2和lumican）在体外和体内都控制血管生成。本修订申请的母基金的总体目标是表征MAGP-2促进血管生成的分子机制。我们已经在这个项目上取得了重大进展，并确定MAGP-2通过与Notch信号通路相互作用来控制血管生成。在母基金中正在进行的实验是剖析MAGP-2操纵Notch信号传导的分子细节以及含有内皮特异性MAGP-2的颗粒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控的基本方面，但它并不寻求在原始微阵列数据集中寻找其他血管生成调控因子。这是一个错失的机会，因为它是非常有可能的，其他新的调节血管生成仍然被发现在我们的原始微阵列数据。本修订申请的目的是发现和表征在我们的微阵列结果的初始分析中没有研究的血管生成的额外基质和细胞膜结合调节剂。这是一个重要的和集中的项目，基于先前的成功，有很高的概率发现新的血管生成调节因子。重要的是，在与R15资助机制和ARRA的雅阁中，拟议的项目将为学生研究人员提供出色的培训机会，将对一个尚未获得NIH重大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的经济复苏。 我们提出两个具体目标来实现我们的目标。在目标1中，我们将确定额外的基质和细胞膜结合的血管生成调节因子。为了实现这一目标，我们将采用基于吗啉的斑马鱼胚胎候选基因敲除方法，以确定这些基因是否是血管生成的重要调节因子。通过Fli1-GFP+加塔-1 RFP双转基因斑马鱼系的微血管造影分析来监测基因敲减的效果。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假定血管生成调节因子。由于已经从该数据集中分离出了其他血管生成调节因子，我们相信该项目将发现更多新的血管生成调节因子。事实上，正如我们的初步结果所示，我们已经确定了4个额外的假定的调节血管生成使用这个程序。在目标2中，这些新的假定的血管生成调节因子加上目标1中鉴定的其他基因将被监测促血管生成或抗血管生成活性。为了实现这一点，候选基因将在哺乳动物内皮细胞中过表达和/或敲低，并与它们的对照对应物比较改变的血管生成活性，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。 公共卫生相关性：癌症仍然是美国死亡和痛苦的主要原因。由于所有实体瘤都依赖于新血管的浸润（即血管生成），因此阻断血管生成的策略可以提供治疗癌症的治疗机会。为了满足这一医疗需求，该提案旨在描述控制血管生成的新分子和机制，以便我们有一天可以使用这些信息来阻止血管生成并对抗癌症。