PROTEASE/ANTIPROTEASE BALANCE IN GINGIVAL DISEASE MODELS

牙龈疾病模型中的蛋白酶/抗蛋白酶平衡

• 批准号: 2132005
• 负责人: SANFORD R SIMON
• 金额: $ 17.16万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-02-01 至 1999-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2132005
• 关键词: cell migration; cellular pathology; chemical association; collagenase; elastase inhibitor; elastases; enzyme activity; enzyme mechanism; extracellular matrix proteins; gingivitis; human subject; intermolecular interaction; interstitial; macrophage; neutrophil; periodontitis; plasminogen activator inhibitors; protease inhibitor; protein degradation; serine proteinases; stromelysin; tetracyclines; tissue /cell culture; tissue inhibitor of metalloproteinases; trypsin inhibitors

Inflammatory injury to the gingival stroma in periodontal disease is mediated in part by serine proteases and matrix metalloproteases (MMPs) released by neutrophils and macrophages recruited to sites of bacterial overgrowth. We propose to study interactions of two of the most potent of these proteases, neutrophil elastase (NE), and neutrophil collagenase (MMP- 8), in mediating injury to interstitial connective tissue. We will investigate how tetracycline (TCs), which have been used therapeutically in controlling periodontal disease, might affect these interacting proteases. Instead of animal models of periodontitis, we will employ a complete biosynthetically labeled interstitial extracellular matrix from cultured R22 rat smooth muscle cells (R22 ECM) for in vitro studies on interactions of purified proteases with natural antiproteases or synthetic inhibitors, for measurements of leukocyte-mediated connective tissue degradation, and for observations of leukocyte invasive migration. In collaboration with Dr. L.M. Golub at Stony Brook, we will evaluate the capacity of several chemically modified TCs with no antibiotic activity to inhibit degradation of R22 ECM by purified proteases or by activated leukocytes. We have reported that reaction of NE with its major endogenous inhibitor alpha1- antitrypsin (alpha1-PI) to ECM with retention of antiprotease activity. We propose to extend these studies to PAI-1, another SERine Protease inhibitor (serpin) which inhibits macrophage urokinase and can also bind to interstitial ECM. A significant portion of the inactivation of ECM-bound serpins reflects proteolytic cleavage by MMPs of leukocyte and interstitial origin. We will pursue initial results which show that TCs can inhibit proteolytic inactivation of both fluid phase and ECM-bound alpha1-PI, as well as other serpins, by stimulated leukocytes and their MMPs, and by ECM- associated MMP activity. Such inhibition may help to preserve the antiprotease activity of the serpins, and may be a major mechanism by which TCs reduce inflammatory tissue damage. The role of proteases in migration of leukocytes to inflammatory foci is still controversial. We will use a model we have developed for invasive migration of leukocytes through the R22 ECM on porous plastic membrane-bottomed cell culture inserts to study the effects of alpha1-PI, PAI-1, and the TCs, separately and in combination, on invasion of neutrophils and macrophages through an interstitium like that of the gingival stroma, which we will correlate with inhibition of ECM degradation.

牙周疾病中牙龈基质的炎症损伤是 部分由丝氨酸蛋白酶和基质金属蛋白酶（MMP）介导 由中性粒细胞和巨噬细胞释放到细菌部位 过度生长。 我们建议研究两个最有效的相互作用 这些蛋白酶，中性粒细胞弹性酶（NE）和中性粒细胞胶原酶（MMP- 8），在介导间质结缔组织的损伤中。 我们将 研究如何在治疗中使用的四环素（TCS） 控制牙周疾病可能会影响这些相互作用的蛋白酶。 我们将采用完整的牙周炎动物模型 从培养的生物合成标记的间质细胞外基质 R22大鼠平滑肌细胞（R22 ECM）用于相互作用的体外研究 纯化蛋白酶，具有天然抗细菌或合成抑制剂的蛋白酶， 用于测量白细胞介导的结缔组织降解和 观察白细胞侵入性迁移。 与 Stony Brook的L.M. Golub博士，我们将评估几个的能力 化学修饰的TCs没有抗生素活性以抑制降解 通过纯化蛋白酶或活化的白细胞通过R22 ECM的of。 我们有 报道了NE与其主要的内源抑制剂α1-的反应 抗抗蛋白酶活性的抗胰蛋白酶（α1-P​​I）。 我们 建议将这些研究扩展到PAI-1，另一种丝氨酸蛋白酶抑制剂 （SERPIN）抑制巨噬细胞尿激酶，也可以结合 间质ECM。 ECM结合的大部分 Serpins反映了白细胞和间质的MMP的蛋白水解裂解 起源。 我们将追求初始结果，这表明TC可以抑制 流体相和ECM结合α1-PI的蛋白水解灭活，AS 以及其他Serpins，通过刺激的白细胞及其MMP，以及ECM- 相关的MMP活性。 这种抑制可能有助于保存 丝丁蛋白的抗蛋白酶活性，可能是一种主要机制 TCS减少炎症组织损伤。 蛋白酶在迁移中的作用 炎症灶的白细胞仍然存在争议。 我们将使用一个 我们已经开发了用于通过白细胞侵入性迁移的模型 R22 ECM在多孔塑料膜底部细胞培养物插入物上进行研究 alpha1-pi，pai-1和tcs的影响，分别 结合，通过浸入中性粒细胞和巨噬细胞的结合 像牙龈基质一样的间质，我们将与之相关 抑制ECM降解。