PLASMIN & GELATINASE & ECM CATABOLISM BY MESANGIAL CELLS

纤溶酶

• 批准号: 3246938
• 负责人: WILLIAM H BARICOS
• 金额: $ 12.5万
• 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/3246938
• 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）： 系膜细胞对系膜基质的降解是 未知本申请的总体目标是描绘 肾小球系膜细胞降解的生化机制 系膜基质，包括鉴定特异性蛋白酶 参与和因素和相互作用，调节他们的活动， 表情 肾小球系膜细胞产生几种可能涉及 在基质降解中，包括纤溶酶原激活剂，潜在的 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）。 肾小球系膜细胞也会产生和应答 涉及多肽生长因子和活性氧代谢物， 在其他组织中，其调节基质降解活性， 蛋白酶及其抑制剂。 申请人的初步研究 表明培养系膜细胞降解细胞外基质 涉及纤溶酶之间的协同相互作用（由 通过MC纤溶酶原激活剂的纤溶酶原）和系膜细胞明胶酶。 他的假设是，在体内，系膜基质的降解， 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。 利用薄基质上培养的系膜细胞， 放射性标记的ECM（Matrigel）或纯化的基质组分（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.确认并扩展了他的初步观察结果，即ECM降解是由 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 通过检测纤溶酶和系膜细胞明胶酶对ECM的降解： 增加纤溶酶原浓度的作用; B） 外源性加入明胶酶的特异性抑制剂（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物活性。 3. 确定活性氧代谢物在ECM降解中的作用， 通过检测活性氧对培养的系膜细胞的影响， 代谢物清除剂（包括SOD、过氧化氢酶、DMTU、甲硫氨酸和 去铁胺）对培养的系膜细胞降解ECM的影响。 4.审查 明胶酶、纤溶酶、TIMPs、α-2- 抗纤溶酶和活性氧代谢产物在ECM降解中的作用 培养的系膜细胞，包括a）纤溶酶和反应的能力， 氧代谢物以激活潜在的系膜细胞明胶酶， B）肾小球系膜细胞明胶酶的能力， α-2抗纤溶酶; 5.检查TGF β的作用， TNF α和IL-1（每一种都已知改变ECM降解的表达， 蛋白酶和/或其抑制剂）对：a）完整ECM的降解 和纯化的ECM组分;和B）所述ECM组分 培养基和纤溶酶原中纤溶酶、明胶酶和TIMP的活性 系膜细胞中的激活剂。