PAI-1 and Vitronectin in Failure of Coronary Revascularization

描述（由申请人提供）：纤溶酶原激活剂抑制剂-1 （PAI-1）是组织型和尿型纤溶酶原激活剂的主要抑制剂。玻璃体连接蛋白（VN）是一种存在于细胞外基质（ECM）和血浆中的黏附糖蛋白，其作用是稳定PAI-1，其功能受PAI-1的调节。PAI-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，而血管平滑肌细胞迁移和内膜增生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生的中心过程。现有研究表明，PAI-1和VN可促进或抑制VSMC迁移和内膜增生，具体取决于实验条件。然而，先前的体外研究受到限制，因为它们主要涉及二维细胞培养系统和纯化试剂，无法充分模拟VSMC在体内迁移的ECM，也无法评估VSMC本身产生的PAI-1和VN的功能。我们建立了一个实验模型，通过胶原、VN和其他ECM分子组成的三维基质，研究PAI-1和VN表达的遗传改变对VSMC的迁移。我们的初步数据表明，PAI-1与VN的化学计量关系在决定PAI-1的亲迁移和抗迁移作用中起关键作用，PAI-1通过VSMC调节VN的表达。关于PAI-1和VN在调节动脉重塑中的作用的体内数据几乎完全来自基因敲除小鼠的实验。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PAI-1对动脉重塑的净作用缺乏明确的共识，这阻碍了针对PAI-1治疗或预防人类血管疾病的药理学策略的发展。我们假设，阐明PAI-1在人类血管疾病中的真实功能，将需要在血管大小、结构和功能更接近人类的动物中进行涉及临床相关血管损伤形式的实验。因此，我们开发了猪PCI模型和一组特异性的药理学PAI-1抑制剂，通过几种不同的机制破坏PAI-1的功能。我们这个大型动物模型的初步数据表明，PCI术后PAI-1的显性阴性形式抑制内膜增生。在我们的实验中，我们将通过三维VSMC迁移实验来探讨PAI-1和VN在血管重构中的功能，研究具有PAI-1和VN表达水平谱的小鼠血管重构，并确定重组PAI-1蛋白和小分子PAI-1抑制剂在小鼠血管损伤模型和临床相关的猪PCI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PAI-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PAI-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PAI-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。

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.