Role of Fibrinolytic System in Vein Graft Remodeling

描述（由申请人提供）： 静脉在被移植到动脉循环中后，在结构上做出反应或重塑，这一过程被称为动脉化。对这一现象的研究有可能对血压和血流对血管结构的影响产生重大的科学见解。此外，静脉动脉化的过程具有重要的临床意义，因为隐静脉用于在患有晚期冠状动脉和外周动脉疾病的患者中进行动脉旁路移植术。手术血运重建的一个主要限制是旁路移植物内过度内膜增生的发展，这会阻碍血流并产生组织缺血。 纤溶酶原激活物抑制剂-1（派-1）是一种丝氨酸蛋白酶抑制剂，是纤维蛋白溶解和细胞迁移的主要调节剂。派-1的功能高度依赖于其辅因子玻连蛋白（VN）。 我们的实验室已经建立了一个小鼠模型的旁路移植术，包括移植一段下腔静脉到颈动脉。我们已经发现派-1缺陷小鼠静脉移植物内内膜增生增加。我们假设派-1是通过其对血浆激活的血管重塑蛋白酶、细胞迁移和静脉移植物壁的细胞外基质的结构特性的影响来调节静脉移植物动脉化的主要调节剂。我们还假设派-1是血流减少对静脉移植物重塑的不利影响的下游介质。我们提出以下具体目标来检验我们的假设：1。确定派-1基因缺陷和转基因过表达对静脉移植物内基质金属蛋白酶-2（MMP-2）、MMP-9、中性粒细胞弹性蛋白酶和凝血酶活性的影响。基于结果，使用涉及MMP-2-、MMP-9-或嗜酸性弹性蛋白酶缺陷小鼠的双敲除策略来确定这些下游蛋白酶在介导派-1对静脉移植物重塑的作用中的作用。 2.使用重组派-1蛋白来探测派-1的抗蛋白酶、VN结合和LDL受体相关蛋白（LRP）结合域在静脉移植物重塑中的作用。 3.研究血流量减少对移植静脉中派-1和VN表达的影响，并检测派-1作为血流量影响移植静脉重塑的下游介质的作用。 4.确定派-1和VN表达水平的遗传改变对细胞凋亡的影响。 静脉移植物细胞外基质的组成和结构。 我们将与经验丰富的合作者团队合作，采用尖端的成像和结构功能方法来实现我们的科学目标。我们预计，所提出的实验将产生重要的见解的分子和细胞机制，调节结构重建的静脉搭桥手术后。特别是，拟议的研究将增加我们的理解，纤溶系统如何与其他蛋白酶网络交叉，以调节静脉移植物重塑，并产生新的数据，发生在动脉化过程中的静脉移植物壁的细胞外基质的结构变化。我们预计 从我们的实验中获得的信息将导致治疗和预防静脉移植疾病的新策略。

DESCRIPTION (provided by applicant): Vein responds structurally, or remodel, after being transplanted into the arterial circulation, a process referred to as arterialization. A study of this phenomenon has the potential to yield major scientific insights into the effects of blood pressure and flow on vascular structure. In addition, the process of venous arterialization has major clinical significance, as saphenous veins are used to perform arterial bypass grafting in patients with advanced coronary artery and peripheral artery disease. A major limitation of surgical revascularization is the development of excessive intimal hyperplasia within bypass grafts, which can obstruct blood flow and produce tissue ischemia. Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor and a major regulator of fibrinolysis and cell migration. PAI-1 function is highly dependent on its cofactor, vitronectin (VN). Our laboratory has developed a murine model of bypass grafting that involves transplantation of a segment of the inferior vena cava into the carotid artery. We have shown that PAI-1-deficient mice develop increased intimal hyperplasia within vein grafts. We hypothesize that PAI-1 is a major regulator of vein graft arterialization via its effects on plasmi-activated vascular remodeling proteases, cell migration, and the structural properties of the extracellular matrix of the vein graft wall. We also hypothesize that PAI-1 is a downstream mediator of the adverse effects of reduced blood flow on vein graft remodeling. We propose the following specific aims to test our hypotheses: 1. Determine the effects of genetic deficiency and transgenic over-expression of PAI-1 on the activities of matrix metalloproteinase-2 (MMP-2), MMP-9, neutrophil elastase, and thrombin within vein grafts. Based on results, use a double-knock-out strategy involving MMP-2-, MMP-9-, or neutrophil-elastase-deficient mice to determine the roles of these downstream proteases in mediating PAI-1's effects on vein graft remodeling. 2. Use recombinant PAI-1 proteins to probe the roles of PAI-1's anti-protease, VN-binding, and LDL receptor-related protein (LRP)-binding domains in vein graft remodeling. 3. Study the effects of reduced blood flow on the expression of PAI-1 and VN in vein grafts and examine the role of PAI-1 as a downstream mediator of blood flow's effects on vein graft remodeling. 4. Determine the impact of genetic alterations in expression levels of PAI-1 and VN on the composition and structure of the extracellular matrix of vein grafts. We will work with a experienced team of collaborators and employ cutting-edge imaging and structure-function approaches to achieve our scientific objectives. We anticipate that the proposed experiments will yield important insights into the molecular and cellular mechanisms that regulate the structural remodeling of veins after bypass surgery. In particular, the proposed studies will increase our understanding of how the fibrinolytic system intersects with other protease networks to regulate vein graft remodeling and yield novel data regarding the structural changes that occur in the extracellular matrix of the vein graft wall during arterialization. We anticipate that the information gained from our experiments will lead to new strategies to treat and prevent vein graft disease.