PLASMIN & GELATINASE & ECM CATABOLISM BY MESANGIAL CELLS

纤溶酶

• 批准号: 3246939
• 负责人: WILLIAM H BARICOS
• 金额: $ 13.76万
• 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 1995-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3246939
• 关键词: collagen; collagenase; enzyme activity; extracellular matrix proteins; fibronectins; heparan sulfate; interleukin 1; laminin; mesangium; plasmin; plasminogen; plasminogen activator; protease inhibitor; protein degradation; radiotracer; superoxides; tissue /cell culture; transforming growth factors; tumor necrosis factor alpha

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（改编自申请人的摘要）：负责的机制 系膜细胞对系膜基质的降解是 未知。该应用程序的总体目标是描绘 肾小球系膜细胞降解的生化机制 系膜基质，包括特定蛋白酶的鉴定 所涉及的因素和相互作用，调节其活动和 表达。 系膜细胞产生多种可能参与的物质 基质降解，包括纤溶酶原激活剂，潜伏 明胶酶和明胶酶抑制剂 TIMP-1 和 TIMP-2（组织 金属蛋白酶抑制剂）。 系膜细胞也产生并做出反应 多肽生长因子和活性氧代谢物、制剂 在其他组织中调节基质降解的活性 蛋白酶及其抑制剂。 申请人的初步研究 表明培养的系膜细胞会降解细胞外基质 涉及纤溶酶（产生于 纤溶酶原（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 由基质降解蛋白酶（明胶酶、 纤溶酶）、其抑制剂（TIMP 和 α-2-抗纤溶酶）以及特异性 调节剂（多肽生长因子、活性氧代谢物） 由 MC 和/或白细胞产生。 使用薄层培养的系膜细胞 放射性标记 ECM（基质胶）或纯化基质成分（类型 IV 胶原蛋白、层粘连蛋白、纤连蛋白、硫酸乙酰肝素蛋白聚糖），他将： 1. 确认并扩展他的初步观察结果，即 ECM 降解 培养的系膜细胞是纤溶酶（原）依赖性的，但也涉及 系膜细胞明胶酶。 2. 确定相对贡献 通过检查纤溶酶和系膜细胞明胶酶对 ECM 的降解：a) 纤溶酶原浓度增加的影响； b) 的影响 外源添加明胶酶特异性抑制剂（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) ECM降解和明胶酶之间的相关性， 纤溶酶、TIMP 和系膜细胞纤溶酶原激活剂活性。 3. 通过以下方法确定活性氧代谢物在 ECM 降解中的作用 通过检查活性氧的影响培养系膜细胞 代谢物清除剂（包括 SOD、过氧化氢酶、DMTU、蛋氨酸和 去铁胺）对培养的系膜细胞 ECM 降解的影响。 4. 检查 明胶酶、纤溶酶、TIMP、α-2- 之间的潜在相互作用 抗纤溶酶和活性氧代谢物参与 ECM 降解 培养的系膜细胞包括 a) 纤溶酶和反应性的能力 氧代谢物激活潜在的系膜细胞明胶酶和 灭活 TIMP； b) 系膜细胞明胶酶的能力 灭活 α-2 抗纤溶酶； 5.检查TGFbeta的作用， TNFα 和 IL-1（已知每种都会改变 ECM 降解的表达） 蛋白酶和/或其抑制剂）对：a）完整 ECM 的降解 并通过培养系膜细胞纯化ECM成分；和 b) 培养基和纤溶酶原中纤溶酶、明胶酶和 TIMP 的活性 系膜细胞的激活剂。