PROTEASE/ANTIPROTEASE BALANCE IN GINGIVAL DISEASE MODELS

牙龈疾病模型中的蛋白酶/抗蛋白酶平衡

基本信息

批准号：
2654446
负责人：
SANFORD R SIMON
金额：
$ 19.3万
依托单位：
STATE UNIVERSITY NEW YORK STONY BROOK
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-02-01 至 2000-01-31
项目状态：
已结题

项目摘要

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) 介导由招募到细菌位点的中性粒细胞和巨噬细胞释放过度生长。我们建议研究两种最有效的相互作用这些蛋白酶、中性粒细胞弹性蛋白酶 (NE) 和中性粒细胞胶原酶 (MMP- 8) 介导间质结缔组织损伤。我们将研究已用于治疗的四环素（TC）如何控制牙周病，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，而不是牙周炎动物模型。来自培养物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) 用于体外相互作用研究纯化的蛋白酶与天然抗蛋白酶或合成抑制剂，用于测量白细胞介导的结缔组织降解，以及用于观察白细胞侵袭性迁移。与合作石溪分校的 L.M. Golub 博士，我们将评估几个人的能力经化学修饰的 TC，不具有抑制降解的抗生素活性 R22 ECM 通过纯化的蛋白酶或活化的白细胞进行。我们有报道NE与其主要内源性抑制剂α1-的反应抗胰蛋白酶 (alpha1-PI) 转化为 ECM，并保留抗蛋白酶活性。我们提议将这些研究扩展到另一种 SERine 蛋白酶抑制剂 PAI-1 （丝氨酸蛋白酶抑制剂）可抑制巨噬细胞尿激酶，也可结合间质细胞外基质。 ECM 结合失活的很大一部分丝氨酸蛋白酶抑制剂反映白细胞和间质基质金属蛋白酶的蛋白水解作用起源。我们将追求初步结果，证明 TC 可以抑制液相和 ECM 结合的 alpha1-PI 的蛋白水解失活，如以及其他丝氨酸蛋白酶抑制剂，通过刺激白细胞及其 MMP，以及通过 ECM- 相关的 MMP 活性。这种抑制可能有助于保护丝氨酸蛋白酶抑制剂的抗蛋白酶活性，可能是其主要机制 TC 可减少炎症组织损伤。蛋白酶在迁移中的作用白细胞对炎症灶的影响仍存在争议。我们将使用一个我们开发的白细胞侵入性迁移模型 R22 ECM 在多孔塑料膜底细胞培养插入物上的研究 α1-PI、PAI-1 和 TC 的作用，分别和在组合，通过中性粒细胞和巨噬细胞的入侵间质就像牙龈基质的间质，我们将与之相关联抑制ECM降解。