Regulatory Roles for Vascular Peptidases in Angiogenesis

描述（由申请人提供）：血管生成是一个复杂的多步骤过程，可发生在发育和疾病中，具有血管成分，以响应各种刺激。许多证据表明血管生成依赖于蛋白水解活性。激活血管表达的生化标记物是差异表达的，其功能重要性刚刚被发现。虽然迄今为止只有少数与血管生成血管相关的标志物被报道，但值得注意的是，其中一些是细胞膜相关蛋白酶。通过基因消除和双化学抑制，我们发现了APA和CD13/APN在病理性血管生成中的一个尚未被认识的机制作用。我们假设APA和cd13的活性参与了重要的调控途径。本研究旨在探讨cd13和APA的表达和活性如何调控血管内皮细胞的生理性和病理性增殖。首先，我们将研究cd13和APA在血管生成过程中的诱导和活性。我们将评估cd13和APA在血管生成系统中的表达。作为肿瘤血管中细胞表面、膜结合酶的上调，APA和cd13可能通过(i)降解抑制肽，（ii）激活刺激分子，或两者兼而有之，促进血管生成的开始和/或进展。我们将确定在激活的血管中，已知与APA和cd13结合并被其降解的肽的水平是否发生改变。我们的主要重点将放在这些酶的相互依赖的底物上，血管紧张素III和血管紧张素VI。血管紧张素III已被证明是促进血管生成的，支持我们的假设。其次，我们将确定导致内皮细胞和周细胞中CD13/APN和APA上调和激活的血管生成刺激的性质。我们还将研究CD13/APN和APA抑制剂在血管生成中的作用。我们将利用抗cd13和抗apa抑制抗体和肽来评估这些肽酶是否在体内和体外促进/加速血管生成。最后，我们将利用细胞因子、氧和肿瘤诱导的血管生成模型，评估CD13和APA缺陷小鼠在血管生成方面的表型。我们的假设是这些肽酶在血管生成中起互补作用。我们的研究很可能为促血管生成肽酶在新生血管形成和维持中的作用建立一个机制基础，这是血管生物学中一个高度相关的方面。在这个应用中提出的实验也可能导致新的治疗策略的发展与血管生成成分的疾病，如癌症和视网膜病变。

DESCRIPTION (provided by applicant): Angiogenesis is a complex multi-step process that can occur in development and diseases with a vascular component in response to various stimuli. Many lines of evidence indicate that angiogenesis depends on proteolytic activity. Activated blood vessels express biochemical markers that are differentially expressed and whose functional importance tias just begun to be uncovered. While only a few markers associated with angiogenic blood vessels have thus far been reported, it is remarkable that several are cell membrane-associated proteinases. By using genetic elimination and Diochemical inhibition, we have uncovered an as yet unrecognized mechanistic role for APA and CD13/APN in pathological angiogenesis. We hypothesize that APA and CD 13 activity contribute to important regulatory pathways. Here, we propose to investigate the mechanisms by which CD 13 and APA expression and activity control the physiological and pathological proliferation of activated endothelial cells forming blood vessels. First, we will study the induction and activity of CD 13 and APA during angiogenesis. We will evaluate the expression of CD 13 and of APA in angiogenic vasculature. As cell surface, membrane-bound enzymes upregulated in tumor vessels, APA and CD 13 may contribute to the initiation and/or progression of angiogenesis by (i) degrading inhibitory peptides, (ii) activating stimulatory molecules, or both. We will establish whether or not the levels of peptides known to bind and be degraded by APA and CD 13 are altered in activated blood vessels. Our main focus will be on the interdependent substrates for these enzymes, angiotensin III and VI. Angiotensin III has been shown to be pro-angiogenic, supporting our hypothesis. Second, we will determine the nature of the angiogenic stimuli leading to upregulation and activation of CD13/APN and APA in endothelial cells and pericytes. We will also examine the effects of inhibitors of CD13/APN and APA in angiogenesis. We will utilize anti-CD 13 and anti-APA inhibitory antibodies and peptides to evaluate if these peptidases enhance/accelerate angiogenesis ex-vivo and in vivo. Finally, we will evaluate the phenotype of CD13 and APA deficient mice with respect to angiogenesis using cytokine-, oxygen-and tumor-induced angiogenesis models. It is our hypothesis that these peptidases play complementary roles in angiogenesis. Our studies are likely to establish a mechanistic basis for the role of pro-angiogenic peptidases in the formation and maintenance of neovasculature, an aspect of high relevance in vascular biology. The proposed experiments in this application may also lead to development of new therapeutic strategies for diseases with an angiogenic component such as cancer and retinopathies.