Macrophage Cell-Surface Proteolysis and Inflammation

从人类和动物模型中检测动脉粥样硬化病变的大量研究 确定了巨噬细胞在动脉粥样硬化中的中心作用。尽管有这些观察，但它仍然是 不清楚巨噬细胞的多种促炎和抗炎能力是如何在 损伤。它们调节其功能的一个潜在的重要机制是通过快速调节 通过蛋白水解性“脱落”在细胞表面表达的蛋白质。除了……之外 动态改变细胞表面成分，脱落也会导致可溶性胞外结构域的释放 具有独特的生物学特性。这项提案将重点放在亚当家族的蛋白水解酶上 识别为胞外区域脱落的主要效应者。 动脉粥样硬化的早期病变以充满脂质的巨噬细胞为特征。清道夫受体 对胆固醇的大量积累负有责任，而它们对动脉粥样硬化形成的意义是 多项基因敲除研究突出了这一点。Fas配体(FasL)是一种关键的细胞表面调节因子 巨噬细胞的凋亡和激活。清道夫受体和FasL都可以被蛋白水解性切割 从细胞表面分离，导致细胞表达下调。可溶性清道夫的释放 受体在体外和体内均能抑制泡沫细胞的形成，而FasL在体内和体外都能被蛋白分解释放 活动和非活动表单。因此，清道夫受体和FasL的蛋白水解性脱落可以调节 病变的发生和发展。然而，负责它们脱落的酶还没有完全分解。 特色化的。在目标1中，我们将确定参与清道夫受体脱落的蛋白水解酶。 和FasL，脱落在动脉粥样硬化形成中的功能意义将在目标4中通过 表达这些底物的不可切割突变体。 除了对病变起始的可能影响外，胞外结构域脱落也可能对病变起作用。 进展和斑块破裂。巨噬细胞活化诱导大量炎性细胞的分泌 调解人，而反兴奋剂机构17已被证明对其中几个问题的减少负有责任。在目标4中， 我们将通过基因调控来测试ADAM17在病变的发生、发展和斑块破裂中的作用 它在巨噬细胞中的表达。此外，还将利用蛋白质组学方法来鉴定新的底物。 可能参与动脉粥样硬化的ADAM17(目标2)，并确定氧化剂在 与项目4合作，调节这种酶的活性(目标3)。

Numerous studies examining atherosclerotic lesions from human and animal models have established the central role of the macrophage in atherosclerosis. Despite these observations, it is still unclear how the multiple pro- and anti-inflammatory capabilities of the macrophage are balanced within lesions. One potentially important mechanism for them to regulate their function is by the rapid modulation of the repertoire of proteins expressed on their cell surface through proteolytic "shedding". In addition to dynamically altering the cell surface constituents, shedding also leads to the release of soluble ectodomains with distinct biological properties. This proposal will focus on the ADAM family of proteases that have gained recognition as primary effectors of ectodomain shedding. Early lesions of atherosclerosis are characterized by lipid-filled macrophages. Scavenger receptors are responsible for this massive accumulation of cholesterol, and their significance for atherogenesis is highlighted by multiple gene knockout studies. Fas ligand (FasL) is a key cell surface regulator of macrophage apoptosis and activation. Both scavenger receptors and FasL can be proteolytically cleaved from the cell surface resulting in down-regulated cellular expression. The release of soluble scavenger receptor can inhibit foam cell formation in vitro and in vivo, while FasL is proteolytically released in both active and inactive forms. Thus, proteolytic shedding of scavenger receptors and FasL could modulate lesion initiation and progression. However, the enzymes responsible for their shedding have not been fully characterized. In Aim 1, we will determine the proteases involved in the shedding of scavenger receptors and FasL, and the functional significance of shedding on atherogenesis will be examined in Aim 4 by expressing uncleavable mutants of these substrates. In addition to possible effects on lesion initiation, ectodomain shedding may also contribute to lesion progression and plaque rupture. Macrophage activation induces the shedding a multitude of inflammatory mediators, and ADAM17 has been shown to be responsible for the shedding of several of these. In Aim 4, we will test the role of ADAM17 in lesion initiation, progression and plaque rupture by genetically modulating its expression in macrophages. In addition, proteomic approaches will be utilized to identify novel substrates for ADAM17 potentially involved in atherosclerosis (Aim 2), and to determine the role played by oxidants in regulating the activity of this enzyme (Aim 3) in collaboration with Project 4.