The Role of Proteinases and Vascular Lesion Formation

蛋白酶和血管病变形成的作用

• 批准号: 6611772
• 负责人: MICHAEL A. REIDY
• 金额: $ 37.9万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6611772
• 关键词: SDS polyacrylamide gel electrophoresis; biological signal transduction; blood vessel disorder; cadherins; cell migration; cell proliferation; collagenase; confocal scanning microscopy; enzyme activity; extracellular matrix; growth factor; immunoelectron microscopy; laboratory mouse; mitogen activated protein kinase; muscle cells; phenotype; phosphatidylinositol 3 kinase; plasmin; plasminogen; protein localization; protein structure function; smooth muscle; tissue inhibitor of metalloproteinases; transfection /expression vector

DESCRIPTION (provided by applicant): This proposal will examine the importance of matrix metalloproteinase-9 (MMP-9) as a regulator of intimal lesion formation. The rationale for this study is that migration of SMC into the new neointima is a critical step in arterial lesion formation. This hypothesis is now supported by our data showing a significant reduction in the size of the neointima in MMP-9 -/- as compared to wild type arteries (FVB background). The experiments of the first aim will validate if this phenotypic change is influenced by the mouse background strain. SMC replication and migration will be analyzed in MMP-9 -/- arteries on a C57BL/6 background. Studies will also determine if direct inhibition of MMP-9 in wild type cells (FVB) will retard migration and replication. Aim 2 will ask if activation of MMP-9 requires plasmin activity. SMC from plaminogen -/- mice will be stimulated to express MMPs and the ratio of inactive and active MMP-9. In a similar manner, the migration of SMC from plaminogen -/- and of wild type arteries will be measured in vitro. A direct role for plasmin activity regulating SMC migration will be tested by transfecting MMP-9 -/- SMC with an adenoviral construct expressing uPA. The next aim will explore how MMP-9 regulates SMC replication. Experiments will determine if MMP-9 regulates the release of arterial growth factors from the extracellular matrix. The activation of ERK and PI3K signal transduction pathways will be measured in injured wild type and MMP-9 -/- arteries. Studies will then document the presence of mitogens in injured wild type and MMP-9 -/- arteries by immuno-electron microscopy. Finally since our preliminary data suggest that MMP-9 may regulate SMC replication by clipping the ectodomain of cadherin, we will evaluate the localization of beta-catenin in SMC isolated from wild type and MMP-9 -/- arteries. We will also measure activation of a beta-catenin/ LEF1 reporter construct in wild type and MMP-9 -/- cells since this is known to upregulate cyclin D1 expression. The final study will be to provide evidence of activation of the beta-catenin/LEF1 site in injured wild type and MMP-9 -/- arteries.

描述（由申请人提供）： 该提案将探讨基质金属蛋白酶 9 (MMP-9) 作为内膜病变形成调节剂的重要性。这项研究的基本原理是，SMC 迁移到新内膜中是动脉病变形成的关键步骤。这一假设现在得到了我们的数据的支持，显示与野生型动脉（FVB 背景）相比，MMP-9 -/- 中的新内膜尺寸显着减小。第一个目标的实验将验证这种表型变化是否受到小鼠背景品系的影响。将在 C57BL/6 背景下分析 MMP-9 -/- 动脉中的 SMC 复制和迁移。研究还将确定直接抑制野生型细胞 (FVB) 中的 MMP-9 是否会阻碍迁移和复制。目标 2 将询问 MMP-9 的激活是否需要纤溶酶活性。来自纤溶酶原-/-小鼠的SMC将被刺激表达MMP以及非活性和活性MMP-9的比率。以类似的方式，将在体外测量来自纤溶酶原-/-和野生型动脉的SMC的迁移。通过用表达 uPA 的腺病毒构建体转染 MMP-9 -/- SMC 来测试纤溶酶活性调节 SMC 迁移的直接作用。下一个目标将探讨 MMP-9 如何调节 SMC 复制。实验将确定 MMP-9 是否调节细胞外基质中动脉生长因子的释放。将在受伤的野生型和 MMP-9 -/- 动脉中测量 ERK 和 PI3K 信号转导途径的激活。然后，研究将通过免疫电子显微镜记录受损野生型和 MMP-9 -/- 动脉中丝裂原的存在。最后，由于我们的初步数据表明 MMP-9 可能通过剪切钙粘蛋白的胞外域来调节 SMC 复制，因此我们将评估从野生型和 MMP-9 -/- 动脉分离的 SMC 中 β-连环蛋白的定位。我们还将测量野生型和 MMP-9 -/- 细胞中 β-连环蛋白/LEF1 报告构建体的激活，因为已知这会上调细胞周期蛋白 D1 的表达。最终研究将提供受损野生型和 MMP-9 -/- 动脉中 β-catenin/LEF1 位点激活的证据。