The Role of Proteinases and Vascular Lesion Formation

描述(由申请人提供)： 这项建议将研究基质金属蛋白酶-9(MMP-9)作为内膜病变形成的调节因子的重要性。这项研究的基本原理是，SMC迁移到新的新生内膜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，该数据显示，与野生型动脉相比，基质金属蛋白酶-9-/-的新生内膜的大小显著减少(FVB背景)。第一个目的的实验将验证这种表型变化是否受小鼠背景品系的影响。SMC的复制和迁移将在C57BL/6背景下分析MMP-9/-动脉中的SMC复制和迁移。研究还将确定在野生型细胞(FVB)中直接抑制基质金属蛋白酶-9是否会延缓迁移和复制。目标2将询问是否激活基质金属蛋白酶-9需要纤溶酶活性。纤溶酶原基因缺失的小鼠的SMC将被刺激表达MMPs和失活/活化的基质金属蛋白酶-9的比例。以类似的方式，将在体外测量SMC从纤溶酶原-/-和野生型动脉中的迁移。纤溶酶活性调节SMC迁移的直接作用将通过将表达uPA的腺病毒载体导入MMP9-/-SMC来检验。下一个目标将探索基质金属蛋白酶-9如何调节SMC复制。实验将确定基质金属蛋白酶-9是否调节细胞外基质中动脉生长因子的释放。ERK和PI3K信号转导通路的激活将在受损的野生型和基质金属蛋白酶-9-/-动脉中被检测。然后，研究将通过免疫电子显微镜证明受损的野生型和基质金属蛋白酶-9/-动脉中有丝分裂原的存在。最后，由于我们的初步数据表明，基质金属蛋白酶-9可能通过截断钙粘附素的胞外结构域来调节SMC的复制，我们将评估β-连环素在野生型和基质金属蛋白酶-9-/-动脉分离的SMC中的定位。我们还将测量野生型和MMP-9-/-细胞中β-连环蛋白/LEF1报告结构的激活，因为这是已知的上调细胞周期蛋白D1的表达。最后的研究将提供在受损的野生型和基质金属蛋白酶-9/-动脉中β-连环蛋白/LEF1位点激活的证据。

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.