PAI-1 and Vitronectin in Failure of Coronary Revascularization

性状（由申请方提供）：纤溶酶原激活物抑制剂-1（派-1）是组织型和尿型纤溶酶原激活物的主要抑制剂。玻连蛋白（VN）是存在于细胞外基质（ECM）和血浆中的粘附糖蛋白，其稳定派-1，并且其功能受派-1调节。派-1和VN在调节血管平滑肌细胞（VSMC）迁移和内膜增生中发挥重要作用，而后者导致经皮冠状动脉介入治疗（PCI）后再狭窄，是动脉粥样硬化和其他人类血管疾病发展的中心过程。现有的研究表明，派-1和VN可以促进或抑制VSMC迁移和内膜增生，这取决于实验条件。然而，以前的体外研究受到限制的事实是，他们主要涉及二维细胞培养系统和纯化的试剂，这并不足以模拟ECM中的VSMC在体内迁移或评估派-1和VN的功能产生的VSMC本身。我们已经开发了一个实验模型，以研究通过由胶原蛋白，VN和其他ECM分子组成的三维基质的派-1和VN表达的遗传改变的VSMC的迁移。我们的初步数据表明，派-1和VN的化学计量关系在确定派-1的促和抗迁移作用中起着至关重要的作用，派-1通过VSMC调节VN表达。关于派-1和VN在调节动脉重塑中的作用的可用体内数据几乎完全来自敲除小鼠的实验。虽然这些研究提供了丰富的信息，但它们一直存在争议。因此，关于派-1对动脉重塑的净效应缺乏明确的共识，这阻碍了靶向派-1以治疗或预防人类血管疾病的药理学策略的发展。我们推测，阐明派-1在人类血管疾病中的真实功能需要涉及血管损伤的临床相关形式的动物实验，这些动物的血管大小、结构和功能更接近于人类。因此，我们已经开发了一个猪PCI模型和一组特定的，药理学派-1抑制剂，破坏派-1功能的几个不同的机制。我们对这个大型动物模型的初步数据表明，显性阴性形式的派-1抑制PCI后内膜增生。在所提出的实验中，我们将通过采用三维VSMC迁移测定来探测派-1和VN在血管重塑中的功能，研究具有派-1和VN表达水平谱的小鼠中的血管重塑，并确定一组重组派-1蛋白和小分子派-1抑制剂在小鼠血管损伤模型和临床相关的PCI猪模型中的作用。为了实现我们的目标，我们组建了一支多元化的研究团队，他们在派-1和VN生物化学，细胞和分子生物学，药理学以及兽医生物医学科学和临床心血管医学方面具有相当的专业知识。我们预计，本提案中概述的实验将有助于阐明派-1和VN在生理和临床相关条件下的血管功能，并将有助于确定派-1靶向化合物作为预防和治疗人类血管疾病的潜在药物的作用。 公共卫生相关性：每年有数百万美国人接受经皮冠状动脉介入（“支架”）手术以治疗冠状动脉疾病。然而，经皮冠状动脉介入治疗可能由于血管成形术部位的动脉复发性狭窄或支架段内血栓形成而失败。本申请将研究纤溶酶原激活物抑制剂-1和玻连蛋白在经皮冠状动脉介入治疗失败中的作用。

DESCRIPTION (provided by applicant): Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of tissue- and urinary-type plasminogen activators. Vitronectin (VN) is an adhesive glycoprotein present in extracellular matrix (ECM) and plasma that stabilizes PAI-1 and whose function is regulated by PAI-1. PAI-1 and VN play major roles in regulating vascular smooth muscle cell (VSMC) migration and intimal hyperplasia, which cause restenosis after percutaneous coronary intervention (PCI) and are central processes in the development of atherosclerosis and other human vascular diseases. Available studies suggest that PAI-1 and VN can either promote or inhibit VSMC migration and intimal hyperplasia, depending on experimental conditions. However, previous in vitro studies have been limited by the fact that they largely involved 2-dimensional cell culture systems and purified reagents, which do not adequately model the ECM in which VSMC migrate in vivo or assess the functions of PAI-1 and VN produced by VSMC themselves. We have developed an experimental model to study the migration of VSMC with genetic alterations in PAI-1 and VN expression through 3-dimensional matrices composed of collagen, VN, and other ECM molecules. Our preliminary data suggest that the stoichiometric relationship of PAI-1 and VN plays a critical role in determining PAI-1's pro- and anti-migratory effects, and that PAI-1 regulates VN expression by VSMC. Available in vivo data regarding the roles of PAI-1 and VN in regulating arterial remodeling have been derived nearly exclusively from experiments with knockout mice. While these studies have yielded a wealth of information, they have been controversial. Consequently, a clear consensus regarding the net effect of PAI-1 on arterial remodeling is lacking, which has hindered development of pharmacological strategies to target PAI-1 to treat or prevent human vascular disease. We hypothesize that elucidation of the authentic function of PAI-1 in human vascular diseases will require experiments involving clinically relevant forms of vascular injury in animals whose vascular size, structure, and function more closely resembles those of humans. Consequently, we have developed a porcine model of PCI and a panel of specific, pharmacological PAI-1 inhibitors that disrupt PAI-1 function by several distinct mechanisms. Our preliminary data with this large animal model suggest that a dominant-negative form of PAI-1 inhibits intimal hyperplasia after PCI. In the proposed experiments we will probe the functions of PAI-1 and VN in vascular remodeling by employing 3-dimensional VSMC migration assays, studying vascular remodeling in mice with a spectrum of PAI-1 and VN expression levels, and determining the effects of a panel of recombinant PAI-1 proteins and small molecule PAI-1 inhibitors in a murine vascular injury model and a clinically relevant porcine model of PCI. To achieve our objectives we have assembled a diverse team of investigators with considerable expertise in PAI-1 and VN biochemistry, cellular and molecular biology, and pharmacology, as well as in veterinary biomedical sciences and clinical cardiovascular medicine. We anticipate that the experiments outlined in this proposal will help to elucidate the vascular functions of PAI-1 and VN under physiologically and clinically relevant conditions and will help to define the role of PAI-1 targeting compounds as potential agents to prevent and treat human vascular disease. PUBLIC HEALTH RELEVANCE: Each year millions of American undergo percutaneous coronary intervention ("stent") procedures for treatment of coronary artery disease. However, percutaneous coronary interventions can fail due to recurrent narrowing of the artery at the site of angioplasty or formation of a thrombus within the stented segment. This application will study the roles of plasminogen activator inhibitor-1 and vitronectin in the failure of percutaneous coronary interventions.