PROTEASE/ANTIPROTEASE BALANCE--ROLE IN ANGIOGENESIS

在体内，净基质降解是平衡活性的函数， 蛋白水解酶及其内源性抑制剂，特别是 基质金属蛋白酶（MMPs）及其抑制剂， （金属蛋白酶的组织抑制剂）。 基质降解是一种 是血管生成、肿瘤发生和转移的重要先决条件。 这个建议是基于假设，在蛋白水解的变化， 有利于MMP或TIMP的平衡，可能具有重要的， 对血管生成调节的临床相关作用。 非常 最近，MMPs参与了 前体/母体蛋白质转化为它们的生物活性组分。 内 在具体目标1的背景下，我们将研究 MMPs和TIMPs之间的蛋白水解平衡可能在 处理血管生成抑制剂， 非活性蛋白，特别是血管抑素和内皮抑素，最近 发现了抑制剂。 这将通过使用 底物FEL电泳、放射性MMP测定和Western印迹分析。 和北方印迹分析，并辅以一系列正义和 相关MMP和TIMP的反义转染研究。 这些研究之后将进行后续的体内生物测定，以评估 这些遗传修饰的表型后果。 新的和有趣的功能被归因于TIMPs， 好. 除了它们的MMP抑制活性之外，其中一些 抑制剂还具有血管生成-诱导活性。 虽然 关于TIMP结构域的鉴定， 对于MMP抑制很重要， 关于TIMP结构域负责其对 血管生成 在具体目标2的背景下，我们将使用酵母 表达TIMPs-1不同结构域的表达系统 和-2。 这些结构域将在一系列血管生成试验中进行测试。 在体外和体内进行测定，以鉴定结构 TIMP的血管生成调节活性的决定因素。

In vivo, net matrix degradation is a function of the balanced activity of proteolytic enzymes and their endogenous inhibitors, in particular the matrix metalloproteinases (MMPs) and their inhibitors, the TIMPs (Tissue Inhibitors of MetalloProteinases). Matrix degradation is an impotant prerequisite of angiogenesis, tunorigenesis and metastasis. This proposal is based on th hypothesis that shifts in the proteolytic balance favoring either MMPs or TIMPs, may have important and clinically relevant effects on the regulation of angiogenesis. Very recently, MMPs have been implicated in the processing of precursor/parent proteins into their bioactive components. Within the context of Specific Aim 1, we will study the role that shifts in the proteolytic balance between MMPs and TIMPs may have in the processing of angiogenesis inhibitors which are fragments of larger, inactive proteins, in particular angiostatin and endostatin, two recently discovered inhibitors. This will be accomplished through the use of substrate fel electrophoresis, radiometric MMP assays, and Western and Northern blot analyses complemented by a series of sense and antisense transfection studies of the relevant MMPs and TIMPs. There studies will be followed by subsequent in vivo bioassays to assess the phenotypic consequences of these genetic modifications. New and interesting functions are being attributed to the TIMPs as well. In addition to their MMP inhibitory activity, some of these inhibitors also possess angiogenesis-doculating activities. Although much is known with respect to the identification of the TIMP domains that are important for MMP inhibition, relatively nothing is known about the TIMP domain(s) responsible for their effects on angiogenesis. Within the context of Specific Aim 2, we will use a yeast expression system to express different structural domains of TIMPs-1 and -2. These domains will then be tested in a series of angiogenesis assays, both in vitro and in vivo, in order to identify the structural determinants of the angiogenesis-modulating activities of the TIMPs.