Mechanisms by which MAGP-2 Promotes Angiogenesis

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。这种重塑对于在血管内皮细胞（EC）和血管微环境之间提供适当的通信是必不可少的，而血管微环境对于适当的血管生成是至关重要的。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个分泌的蛋白质，以前没有与血管生成和表征的血管生成活性的几个这些蛋白质。特别是，MAGP-2的表达在活化的EC中增加，并被发现促进体外血管生成活性和体内血管生成。我们的结论是，激活的EC分泌MAGP-2进入血管微环境，以促进血管生成。然而，这些研究没有解决MAGP-2促进血管生成的分子机制。我们的初步结果表明，MAGP-2通过阻断内皮细胞中的Notch信号传导促进血管生成。巧合的是，我们还发现MAGP-2在非内皮细胞类型中促进Notch信号传导。同时，我们还发现MAGP-2特异性定位于内皮细胞胞浆颗粒。基于这些关键观察，我们假设MAGP-2通过干扰EC特异性血管生成抑制Notch受体/配体相互作用来促进血管生成，并且MAGP-2响应于特异性促血管生成化学刺激而从EC颗粒分泌。我们将以三个具体目标来讨论这一假设。具体目标1将利用我们的观察结果，即MAGP-2抑制EC中的Notch信号传导，但促进其他细胞类型中的Notch信号传导。使用该系统，我们将确定哪些Notch受体和配体由这些细胞类型表达，表征MAGP-2如何与这些受体，配体和受体/配体对相互作用，并最终检查MAGP-2与各种Notch受体/配体对相互作用的血管生成后果。在具体目标2中，我们将首先确定MAGP-2是否储存在常见的EC特异性储存颗粒（韦伯-帕拉德体）或替代储存颗粒中。然后，我们将继续确定MAGP-2序列是必要的直接颗粒存储，和化学信号，刺激MAGP-2胞吐从EC。通过研究MAGP-2可能促进血管生成的这些分子机制，我们将了解有关血管微环境重塑如何促进血管生成和肿瘤发生的有价值的信息。由于Notch和整合素抑制剂的发展目前正在开发作为抗血管生成治疗剂，并且假设调节的EC胞吐作用在肿瘤发生期间发挥重要作用，这些研究将对理解人类健康和我们如何治疗人类疾病做出直接贡献。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血管生成（即，新血管的浸润）的策略来阻断它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。

DESCRIPTION (provided by applicant): Angiogenesis is required for the growth and metastasis of cancers. One of the critical events that occurs during tumor formation, is the remodeling of the angiostatic vascular microenvironment to a pro-angiogenic microenvironment. This remodeling is essential to provide the proper communications between vascular endothelial cells (ECs) and the vascular microenvironment that is critical for proper angiogenesis. Therefore, therapeutic interventions that disrupt communications between ECs and their microenvironment represents 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 and characterized the angiogenic activity of several of these proteins. In particular, MAGP-2 expression was increased in activated ECs and was found to promote angiogenic activities in vitro and angiogenesis in vivo. We concluded that activated ECs secrete MAGP-2 into the vascular microenvironment to promote angiogenesis. These investigations however did not address the molecular mechanism(s) by which MAGP-2 promotes angiogenesis. Our preliminary results now demonstrate that MAGP-2 promotes angiogenesis by blocking Notch signaling in endothelial cells. Paradoxically, we also find that MAGP-2 promotes Notch signaling in non-endothelial cell types. Simultaneously, we have also found that MAGP-2 is specifically localized to endothelial cytoplasmic granules. Based on these key observations, we hypothesize that MAGP-2 promotes angiogenesis by interfering with EC specific angiostatic Notch receptor/ligand interactions, and that MAGP-2 is secreted from EC granules in response to specific pro-angiogenic chemical stimuli. We will address this hypothesis with three specific aims. Specific aim 1 will take advantage of our observations that MAGP-2 inhibits Notch signaling in ECs but promotes Notch signaling in other cell types. Using this system, we will identify which Notch receptors and ligands are expressed by these cell types, characterize how MAGP-2 interacts with these receptors, ligands, and receptor/ligand pairs, and finally examine the angiogenic consequences of MAGP-2 interaction with various Notch receptor/ligand pairs. In specific aim 2, we will first determine if MAGP-2 is stored in the common EC specific storage granules (Weibel-Palade bodies) or alternative storage granules. We will then proceed to identify MAGP-2 sequences that are necessary to direct granule storage, and the chemical signals that stimulate MAGP-2 exocytosis from ECs. By investigating these molecular mechanisms by which MAGP-2 may promote angiogenesis, we will learn valuable information about how remodeling of vascular microenvironments contributes to angiogenesis and tumorigenesis. Since the development of Notch and integrin inhibitors are currently being developed as anti-angiogenesis therapeutics, and regulated EC exocytosis is hypothesized to play an important role during tumorigenesis, these investigations will make direct contributions to understanding human health and how we treat human disease. PUBLIC HEALTH RELEVANCE: Cancer continues to be a leading cause of death and suffering in the United States. Since all solid tumors depend on angiogenesis (i.e., infiltration of new blood vessels) strategies to block it 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.