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，可在发育和疾病中发生，其中血管成分响应于各种刺激。许多证据表明，血管生成依赖于蛋白水解活性。活化的血管表达差异表达的生化标志物，其功能重要性刚刚开始被发现。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，有几种是细胞膜相关蛋白酶。通过使用遗传消除和Diochemical抑制，我们发现了阿帕和CD 13/APN在病理性血管生成中尚未认识到的机制作用。我们推测阿帕和CD 13活性参与了重要的调节途径.在这里，我们建议调查的机制，CD 13和阿帕的表达和活性控制的生理和病理增殖的活化内皮细胞形成血管。首先，我们将研究CD 13和阿帕在血管生成过程中的诱导和活性.我们将评估CD 13和阿帕在血管生成血管中的表达。作为细胞表面，在肿瘤血管中上调的膜结合酶，阿帕和CD 13可以通过（i）降解抑制肽，（ii）激活刺激分子，或两者来促进血管生成的起始和/或进展。我们将确定在活化的血管中，已知结合并被阿帕和CD 13降解的肽的水平是否改变。我们的主要重点将是这些酶，血管紧张素III和VI的相互依赖的底物。血管紧张素III已被证明是促血管生成，支持我们的假设。其次，我们将确定的性质，血管生成刺激导致上调和激活的CD 13/APN和阿帕的内皮细胞和周细胞。我们还将研究CD 13/APN和阿帕抑制剂在血管生成中的作用。我们将利用抗-CD 13和抗-APA抑制性抗体和肽来评估这些肽酶是否增强/加速离体和体内血管生成。最后，我们将使用细胞因子、氧和肿瘤诱导的血管生成模型评估CD 13和阿帕缺陷小鼠的血管生成表型。我们假设这些肽酶在血管生成中起互补作用。我们的研究可能建立一个机制的基础上，促血管生成肽酶的作用，在形成和维持新血管，血管生物学的高度相关性的一个方面。本申请中提出的实验也可能导致开发用于具有血管生成成分的疾病（例如癌症和视网膜病变）的新治疗策略。

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.