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抑制物，特别是 基质金属蛋白酶及其抑制物TIMPs (金属蛋白酶组织抑制物)。基质降解是一种 是血管生成、肿瘤形成和转移的重要前提。 这一建议是基于这样一个假设，即蛋白质水解物的变化 有利于MMP或TIMP的平衡可能具有重要的 对血管生成调控的临床相关作用。非常 最近，基质金属蛋白酶被认为与血管生成的过程有关。 前体/母体蛋白转化为其生物活性成分。在 具体目标1的背景下，我们将研究在 MMPs和TIMPs之间的蛋白分解平衡可能在 血管生成抑制物的加工是更大的， 失活蛋白，特别是血管抑素和内皮抑素，最近的两个 发现了抑制剂。这将通过使用 底物FEL电泳法、放射性基质金属蛋白酶测定法和Western 和Northern杂交分析，并辅之以一系列正义和 相关MMPs和TIMPs的反义转染研究。 这些研究将随后进行体内生物测定，以评估 这些基因修改的表型后果。 新的和有趣的功能归因于TIMP，因为 井。除了它们对基质金属蛋白酶的抑制活性外，其中一些 抑制剂还具有对接血管生成的活性。虽然 关于TIMP结构域的鉴定已知很多 对于基质金属蛋白酶的抑制是重要的，相对来说什么都不知道 关于TIMP域(S)对它们的影响 血管生成。在特定目标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。然后这些区域将在一系列血管生成中进行测试 体外和体内的检测，以确定结构 TIMP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.