PA/PLASMIN/GELATINASE CASCADE IN DIABETIC NEPHROPATHY

糖尿病肾病中的 PA/纤溶酶/明胶酶级联

• 批准号: 6176549
• 负责人: WILLIAM H BARICOS
• 金额: $ 20.8万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6176549
• 关键词: albumins; animal tissue; collagenase; diabetic nephropathy; enzyme activity; extracellular matrix; extracellular matrix proteins; gene expression; gene targeting; genetically modified animals; glucose; human tissue; laboratory mouse; laboratory rat; matrigel; mesangium; plasmids; plasmin; plasminogen activator; protein degradation; renal glomerulus; tissue /cell culture; transfection; transforming growth factors; transposon /insertion element

DESCRIPTION (Adapted from Applicant's Abstract):The mechanisms responsible for the degradation of the mesangial matrix by mesangial cells are unknown. The overall objective of this application is to delineate the biochemical mechanisms by which glomerular mesangial cells degrade mesangial matrix, including identification of the specific proteinases involved and the factors and interactions that regulate their activity and expression. Mesangial cells produce several agents potentially involved in matrix degradation including plasminogen activator(s), latent gelatinase and the gelatinase inhibitors, TIMP-1 and TIMP-2 (tissue inhibitor of metalloproteinase). Mesangial cells also produce and respond to polypeptide growth factors and reactive oxygen metabolites, agents which in other tissues modulate the activities of matrix degrading proteinases and their inhibitors. The applicant's preliminary studies indicate that extracellular matrix degradation by cultured mesangial cells involves cooperative interactions between plasmin (generated from plasminogen by MC plasminogen activator) and mesangial cell gelatinase. His hypothesis is that in vivo, degradation of the mesangial matrix is controlled by interactions among matrix degrading proteinases (gelatinase, plasmin), their inhibitors (TIMPs and alpha-2-antiplasmin), and specific modulators (polypeptide growth factors, reactive oxygen metabolites) produced by MC and/or leukocytes. Using mesangial cells cultured on thin films of radiolabeled ECM (Matrigel) or purified matrix components (type IV collagen, laminin, fibronectin, heparan sulfate proteoglycan), he will: 1. Confirm and extend his preliminary observations that ECM degradation by cultured mesangial cells is plasmin(ogen) dependent but also involves mesangial cell gelatinase. 2. Determine the relative contribution of plasmin and mesangial cell gelatinase to ECM degradation by examining: a) the effect of increasing concentrations of plasminogen; b) the effect of exogenously added specific inhibitors of gelatinase (eg TIMP, antigelatinase antibodies) and plasmin (eg alpha-2-antiplasmin, aprotinin); c) the correlations among ECM degradation and gelatinase, plasmin, TIMP, and mesangial cell plasminogen activator activities. 3. Determine the role of reactive oxygen metabolites in ECM degradation by cultured mesangial cells by examining the effects of reactive oxygen metabolite scavengers (including SOD, catalase, DMTU, methionine, and deferroxamine) on ECM degradation by cultured mesangial cells. 4. Examine potential interactions among gelatinase, plasmin, TIMPs, alpha-2- antiplasmin, and reactive oxygen metabolites in ECM degradation by cultured mesangial cells including a) the ability of plasmin and reactive oxygen metabolites to activate latent mesangial cell gelatinase and inactivate TIMPs; and b) the ability of mesangial cell gelatinase to inactivate alpha-2 antiplasmin; 5. Examine the effects of TGFbeta, TNFalpha, and IL-1 (each known to alter the expression of ECM degrading proteinases and/or their inhibitors) on: a) the degradation of intact ECM and purified ECM components by cultured mesangial cells; and b) the activities of plasmin, gelatinase, and TIMPs in medium and plasminogen activator in mesangial cells.

描述(改编自申请者摘要)：负责的机制 系膜细胞对系膜基质的降解作用 未知。这个应用程序的总体目标是描述 肾小球系膜细胞降解的生化机制 系膜基质，包括特定蛋白水解酶的鉴定 以及调节它们活动和相互作用的因素和相互作用 表情。系膜细胞产生几种可能参与其中的因子 在基质降解中包括纤溶酶原激活剂(S)，潜伏期 明胶酶和明胶酶抑制剂TIMP-1和TIMP-2(组织 金属蛋白酶抑制物)。肾小球系膜细胞也产生和反应 多肽生长因子和活性氧代谢物、试剂 它在其他组织中调节基质降解的活动 蛋白水解酶及其抑制剂。申请人的初步研究 表明培养的肾小球系膜细胞降解细胞外基质 涉及纤溶酶之间的协同作用(产生于 MC纤溶酶原激活剂)和系膜细胞明胶酶。 他的假设是，在体内，系膜基质的降解是 由基质降解蛋白水解酶(明胶酶， 纤溶酶)、其抑制物(TIMPs和α-2-抗纤溶酶)和特异性 调节剂(多肽生长因子、活性氧代谢物) 由MC和/或白细胞产生。利用薄层培养的肾小球系膜细胞 放射性标记ECM(Matrigel)或提纯基质成分(类型)薄膜 IV胶原蛋白、层粘连蛋白、纤维连接蛋白、硫酸乙酰肝素蛋白多糖)，他将： 1.确认并推广他的初步观察结果，即ECM的降解 培养的系膜细胞是纤溶酶(原)依赖的，但也参与 系膜细胞明胶酶。2.确定相对贡献率 纤溶酶和肾小球系膜细胞明胶酶对细胞外基质降解的影响 增加纤溶酶原浓度的影响；b)影响 外源添加特定的明胶酶抑制剂(例如TIMP， 抗明胶酶抗体)和纤溶酶(例如α-2-抗纤溶酶， 抑肽酶)；c)细胞外基质降解和明胶酶之间的相关性， 纤溶酶、组织型纤溶酶原激活物和系膜细胞纤溶酶原激活物活性。3. 通过以下方法确定活性氧代谢产物在ECM降解中的作用 体外培养肾小球系膜细胞检测活性氧的影响 代谢物清除剂(包括超氧化物歧化酶、过氧化氢酶、dmtu、蛋氨酸和 去铁胺)对培养的肾小球系膜细胞ECM降解的影响。4.审查 明胶酶、纤溶酶、TIMPs、α-2-之间的潜在相互作用 抗纤溶酶和活性氧代谢产物在ECM降解中的作用 培养的肾小球系膜细胞包括a)纤溶酶和反应能力 氧代谢产物激活潜在的系膜细胞明胶酶和 失活TIMPs；以及b)系膜细胞明胶酶 灭活α-2抗纤溶酶；5.检测转化生长因子β的作用， 肿瘤坏死因子α和IL-1(每个已知改变ECM降解的表达 蛋白水解酶和/或其抑制物)对：a)完整细胞外基质的降解 以及培养的肾小球系膜细胞纯化的ECM成分； 纤溶酶、明胶酶和TIMPs在培养液和纤溶酶原中的活性 系膜细胞中的激活剂。

项目成果

Chronic renal disease: do metalloproteinase inhibitors have a demonstrable role in extracellular matrix accumulation?

慢性肾病：金属蛋白酶抑制剂在细胞外基质积累中是否具有明显作用？

• 发表时间: 1995
• 期刊: Current opinion in nephrology and hypertension.
• 作者: Baricos,WH

Plasminogen activators augment endothelial cell organization in vitro by two distinct pathways.

纤溶酶原激活剂通过两种不同的途径增强体外内皮细胞组织。

• DOI: 10.1002/jcp.1041650114
• 发表时间: 1995
• 期刊: Journal of cellular physiology
• 影响因子: 5.6
• 作者: Schnaper,HW;Barnathan,ES;Mazar,A;Maheshwari,S;Ellis,S;Cortez,SL;Baricos,WH;Kleinman,HK
• 通讯作者: Kleinman,HK