MACROPHAGE CELL-SURFACE PROTEOLYSIS IN ATHEROGENESIS

描述(申请人提供)：大量研究从人类和动物模型中检测动脉粥样硬化病变已经确定巨噬细胞在动脉粥样硬化中的中心作用。尽管有这些观察，但仍不清楚巨噬细胞的多种促炎和抗炎能力是如何在病变内平衡的。它们调节其功能的一个潜在的重要机制是通过蛋白水解性“脱落”来快速调节其细胞表面表达的蛋白质。除了动态改变细胞表面成分外，脱落还会导致具有不同生物学特性的可溶性胞外结构域的释放。这项建议将重点放在已经被公认为胞外结构域脱落的主要效应者的ADAM蛋白家族上。动脉粥样硬化的早期病变以充满脂质的巨噬细胞为特征。清道夫受体是胆固醇大量积累的原因，多项基因敲除研究突显了清道夫受体在动脉粥样硬化形成中的重要性。Fas配体(FasL)是巨噬细胞凋亡和激活的关键调节因子。清道夫受体和FasL都可以被蛋白水解性地从细胞表面切割，它们的蛋白水解性脱落可以调节病变的进展。然而，负责它们脱落的酶还没有完全确定。在目标1中，我们将确定参与清道夫受体和FasL脱落的蛋白酶，并将通过在目标3中表达这些底物的不可切割突变来检测这些底物在动脉粥样硬化形成中的功能意义。巨噬细胞在病变发展的所有阶段都被观察到激活，并诱导大量炎性蛋白的脱落。ADAM17被证明是导致大量炎症介质脱落的原因，但激活ADAM17的机制尚不清楚。在目标2中，将研究氧化剂在调节ADAM17活性中所起的作用。最后，在目标3中，我们将通过基因调控ADAM17在巨噬细胞中的表达来测试ADAM17在病变起始、进展和斑块破裂中的作用。

DESCRIPTION (provided by applicant): 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 regulator of macrophage apoptosis and activation. Both scavenger receptors and FasL can be proteolytically cleaved from the cell surface, and their proteolytic shedding could modulate lesion 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 by expressing uncleavable mutants of these substrates in Aim 3. Macrophage activation is observed at all stages of lesion development, and induces the shedding of a multitude of inflammatory proteins. ADAM17 has been shown to be responsible for the shedding of a large number of inflammatory mediators, but the mechanisms that underlie the activation of ADAM17 are poorly understood. In Aim 2, the role played by oxidants in regulating the activity of ADAM17 will be investigated. Finally in Aim 3, we will test the role of ADAM17 in lesion initiation, progression and plaque rupture by genetically modulating its expression in macrophages.