Role of Fibrinolytic System in Vein Graft Remodeling

描述(由申请人提供)： 移植到动脉循环中后，静脉在结构上发生反应，或进行重塑，这一过程称为动脉化。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，因为晚期冠状动脉和外周动脉疾病患者使用大隐静脉进行动脉旁路移植术。外科血管重建术的一个主要限制是在搭桥移植物内发生过度的内膜增生，这可能会阻碍血流并产生组织缺血。纤溶酶原激活物抑制物-1(PAI-1)是一种丝氨酸蛋白酶抑制物，是纤溶和细胞迁移的主要调节因子。PAI-1的功能高度依赖于它的辅因子Vitronectin(VN)。我们实验室开发了一种小鼠搭桥模型，包括将一段下腔静脉移植到颈动脉。我们发现PAI-1基因缺陷的小鼠移植静脉内的内膜增生增加。我们推测，PAI-1通过对血浆激活的血管重塑蛋白、细胞迁移和移植静脉壁细胞外基质的结构特性的影响，是移植静脉动脉化的主要调节因子。我们还假设，PAI-1是血流减少对静脉移植物重塑不利影响的下游中介。1.研究PAI-1基因缺陷和转基因过表达对移植静脉中基质金属蛋白酶-2、9、中性粒细胞弹性蛋白酶和凝血酶活性的影响。根据研究结果，采用双基因敲除策略，包括基质金属蛋白酶-2、基质金属蛋白酶-9或中性粒细胞弹性蛋白酶缺陷小鼠，以确定这些下游蛋白在介导纤溶酶原激活物-1‘S对静脉移植物重塑影响中的作用。2.利用重组PAI-1蛋白探讨PAI-1的S抗蛋白酶、VN结合区和低密度脂蛋白受体相关蛋白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PAI-1和VN表达的影响，探讨PAI-1作为血流影响移植静脉重塑的下游介质的作用。4.确定PAI-1和VN表达水平的遗传改变对移植静脉细胞外基质成分和结构的影响。我们将与经验丰富的合作者团队合作，并采用尖端成像和结构功能方法来实现我们的科学目标。我们预计，拟议的实验将对调控搭桥手术后静脉结构重构的分子和细胞机制产生重要的见解。特别是，拟议的研究将增加我们对纤溶系统如何与其他蛋白酶网络相交以调节静脉移植物重塑的理解，并产生关于动脉化过程中静脉移植物壁细胞外基质发生结构变化的新数据。我们期待着 从我们的实验中获得的信息将导致治疗和预防静脉移植物疾病的新策略。

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.