Regulatory Roles for Vascular Peptidases in Angiogenesis

描述(申请人提供)：血管生成是一个复杂的多步骤过程，可以发生在发育和疾病中，血管成分对各种刺激做出反应。许多证据表明，血管生成依赖于蛋白水解酶活性。激活的血管表达差异表达的生化标记物，其功能重要性刚刚开始被发现。尽管到目前为止只有几个与血管新生血管相关的标记物被报道，但值得注意的是，有几个是细胞膜相关的蛋白酶。通过基因消除和二次化学抑制，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。我们假设APA和CD13的活性对重要的调控途径有贡献。在此，我们拟探讨CD13和APA的表达和活性控制血管内皮细胞的生理性和病理性增殖的机制。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在血管新生血管中的表达。作为细胞表面，在肿瘤血管、APA和CD13中上调的膜结合酶可能通过(I)降解抑制性多肽，(Ii)激活刺激分子，或两者兼而有之，从而促进血管生成的启动和/或进展。我们将确定已知的APA和CD13结合和降解的多肽水平在激活的血管中是否发生变化。我们将主要关注这些酶的相互依赖的底物，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抑制抗体和多肽来评估这些多肽酶是否在体外和体内增强/加速血管生成。最后，我们将使用细胞因子、氧气和肿瘤诱导的血管生成模型来评估CD13和APA缺陷小鼠的血管生成表型。我们的假设是，这些肽酶在血管生成中起互补作用。我们的研究可能为促血管生成多肽酶在新生血管的形成和维持中的作用建立一个机制基础，这是血管生物学中高度相关的一个方面。这一应用中拟议的实验还可能导致开发新的治疗策略，用于癌症和视网膜病变等具有血管生成成分的疾病。

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.