PAI-1 and Vitronectin in Failure of Coronary Revascularization

说明(申请人提供)：纤溶酶原激活物抑制物-1(PAI-1)是组织和尿型纤溶酶原激活物的主要抑制物。Vitronectin(VN)是一种存在于细胞外基质(ECM)和血浆中的粘附性糖蛋白，稳定PAI-1，其功能受PAI-1调节。PAI-1和VN在调节血管平滑肌细胞(VSMC)迁移和内膜增生中起重要作用，可导致经皮冠状动脉介入术后再狭窄，是动脉粥样硬化和其他人类血管疾病发生发展的中心过程。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，以往的体外研究主要涉及二维细胞培养系统和纯化试剂，不能很好地模拟VSMC在体内迁移的ECM，也不能评估VSMC本身产生的PAI-1和VN的功能。我们建立了一个实验模型来研究PAI-1和VN表达发生遗传变化的VSMC通过由胶原、VN和其他ECM分子组成的三维基质的迁移。我们的初步数据表明，PAI-1和VN的化学计量关系在决定PAI-1的促迁移和抗迁移作用中起着关键作用，并且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活体数据几乎完全来自于基因敲除小鼠的实验。虽然这些研究提供了丰富的信息，但它们也存在争议。因此，对于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物策略的发展。我们假设，要阐明PAI-1在人类血管疾病中的真实功能，需要对血管大小、结构和功能与人类更接近的动物进行临床相关形式的血管损伤实验。因此，我们开发了一种猪的经皮冠状动脉介入治疗模型和一组特定的、药理上的PAI-1抑制剂，它们通过几种不同的机制破坏PAI-1的功能。我们在这个大型动物模型上的初步数据表明，显性-阴性形式的PAI-1抑制了经皮冠状动脉介入治疗后的内膜增生。在拟议的实验中，我们将通过三维VSMC迁移分析来探讨PAI-1和VN在血管重塑中的作用，研究PAI-1和VN表达水平的小鼠血管重塑，并确定一组重组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冠状动脉介入治疗模型中的作用。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拥有丰富的专业知识。我们期望这项建议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靶向化合物作为潜在的药物来预防和治疗人类血管疾病的作用。 与公共卫生相关：每年有数百万美国人接受经皮冠状动脉介入治疗(“支架”)治疗冠状动脉疾病。然而，由于血管成形术部位的动脉反复狭窄或支架节段内形成血栓，经皮冠状动脉介入治疗可能失败。这项应用将研究纤溶酶原激活物抑制物-1和玻璃体连结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.