Macrophage Cell-Surface Proteolysis and Inflammation

巨噬细胞表面蛋白水解和炎症

• 批准号: 7140029
• 负责人: Elaine W Raines
• 金额: $ 24.16万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-12-01 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7140029
• 关键词: atherosclerosis; atherosclerotic plaque; cell cell interaction; cell membrane; cell type; endopeptidases; enzyme activity; extracellular; gene expression; inflammation; intracellular; laboratory mouse; lead; lipids; macrophage; macrophage inflammatory proteins; pathologic process; proteolysis; proteomics; scavenger receptor; smooth muscle

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.

许多研究通过人类和动物模型检查动脉粥样硬化病变 确立了巨噬细胞在动脉粥样硬化中的核心作用。尽管有这些观察结果，但仍然 不清楚巨噬细胞的多种促炎和抗炎能力如何在内部平衡 病变。它们调节其功能的一种潜在的重要机制是通过快速调节 通过蛋白水解“脱落”在细胞表面表达的蛋白质库。此外 动态改变细胞表面成分，脱落还导致可溶性胞外域的释放 具有独特的生物学特性。该提案将重点关注已获得认可的 ADAM 蛋白酶家族 被识别为胞外域脱落的主要效应器。 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 造成胆固醇大量积累的原因是它们对动脉粥样硬化的重要性 多基因敲除研究强调了这一点。 Fas 配体 (FasL) 是细胞表面的关键调节因子 巨噬细胞凋亡和激活。清道夫受体和 FasL 均可被蛋白水解裂解 从细胞表面导致细胞表达下调。可溶性清除剂的释放 FasL 受体可以在体外和体内抑制泡沫细胞形成，而 FasL 在体内和体外均通过蛋白水解释放 活动和非活动形式。因此，清道夫受体和 FasL 的蛋白水解脱落可以调节 病变的发生和进展。然而，负责其脱落的酶尚未完全被研究出来。 特点。在目标 1 中，我们将确定参与清道夫受体脱落的蛋白酶 和 FasL，脱落对动脉粥样硬化形成的功能意义将在目标 4 中通过以下方式进行检查： 表达这些底物的不可切割突变体。 除了可能影响病变发生之外，胞外域脱落也可能导致病变 进展和斑块破裂。巨噬细胞活化诱导大量炎症物质脱落 ADAM17 已被证明与其中一些介质的脱落有关。在目标 4 中， 我们将通过基因调节来测试 ADAM17 在病变发生、进展和斑块破裂中的作用 它在巨噬细胞中表达。此外，蛋白质组学方法将用于识别新的底物 ADAM17 可能参与动脉粥样硬化（目标 2），并确定氧化剂在动脉粥样硬化中所起的作用 与项目 4 合作调节该酶的活性（目标 3）。