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 (MMP) 及其抑制剂 TIMP （金属蛋白酶的组织抑制剂）。 基质降解是 血管生成、肿瘤发生和转移的重要先决条件。 该提议基于蛋白水解酶发生变化的假设 有利于 MMP 或 TIMP 的平衡可能具有重要且 对血管生成调节的临床相关影响。 非常 最近，MMP 被卷入了 前体/母体蛋白质转化为其生物活性成分。 内 在具体目标 1 的背景下，我们将研究在 MMP 和 TIMP 之间的蛋白水解平衡可能影响 血管生成抑制剂的加工，这些抑制剂是较大的片段， 无活性蛋白质，特别是血管抑制素和内皮抑素，最近有两种 发现了抑制剂。 这将通过使用来完成 底物 fel 电泳、放射性 MMP 测定和 Western 和 Northern 印迹分析，并辅以一系列有义和 相关 MMP 和 TIMP 的反义转染研究。 随后将进行体内生物测定以评估研究结果 这些基因修饰的表型后果。 新的、有趣的功能被归因于 TIMP： 好吧。 除了 MMP 抑制活性外，其中一些 抑制剂还具有血管生成调节活性。 虽然 关于 TIMP 域的识别，我们已经了解很多 对于 MMP 抑制很重要，相对来说还一无所知 关于 TIMP 结构域对它们的影响 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。 然后这些域将在一系列血管生成中进行测试 体外和体内测定，以确定结构 TIMPs 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.