Leik C E, Su E J, Nambi P, Crandall D L, Lawrence D A
Department of Cardiovascular and Metabolic Diseases Research, Wyeth Research, Collegeville, PA, USA.
J Thromb Haemost. 2006 Dec;4(12):2710-5. doi: 10.1111/j.1538-7836.2006.02244.x. Epub 2006 Oct 2.
Plasminogen activator inhibitor-1 (PAI-1) is integrally involved in tumorigenesis by impacting on both proteolytic activity and cell migration during angiogenesis.
We hypothesized that an orally active small molecule inhibitor of PAI-1 (PAI-039; tiplaxtinin) could affect smooth muscle cell (SMC) attachment and migration in vitro on a vitronectin matrix, and exhibit antiangiogenic activity in vivo.
In vitro assays were used to assess the mechanism of inhibition of PAI-1 by PAI-039 using wild-type PAI-1 in the presence or absence of vitronectin and wild-type PAI-1 and specific PAI-1 mutants in SMC adhesion and migration assays. An in vivo tumor angiogenesis model was used to assess the effect of PAI-039 administration on neovascularization in a Matrigel implant.
PAI-039 dose-dependently inhibited soluble, but not vitronectin-bound, PAI-1. Cell adhesion assays using PAI-1 mutants unable to bind vitronectin (PAI-1K) or inactivate proteases (PAI-1R) further suggested that PAI-039 inactivated PAI-1 by binding near its vitronectin domain. In a tumor angiogenesis model, PAI-039 treatment of wild-type mice dose-dependently decreased hemoglobin concentration and endothelial cell staining within the Matrigel implant, indicating reduced angiogenesis, but exhibited no in vivo efficacy in PAI-1 null mice.
Administration of an orally active PAI-1 inhibitor prevented angiogenesis in a Matrigel implant. The lack of activity of PAI-039 against wild-type PAI-1 bound to vitronectin and PAI-1K suggests PAI-039 binding near the vitronectin-binding site. Our studies further substantiate a role for PAI-1 in cellular motility and tumor angiogenesis, and suggest for the first time that these effects can be modulated pharmacologically.
纤溶酶原激活物抑制剂-1（PAI-1）通过在血管生成过程中影响蛋白水解活性和细胞迁移，全面参与肿瘤发生。
我们推测一种口服活性的PAI-1小分子抑制剂（PAI-039；替普拉西丁）可能会影响体外玻连蛋白基质上平滑肌细胞（SMC）的黏附与迁移，并在体内表现出抗血管生成活性。
体外试验用于在有或无玻连蛋白的情况下，使用野生型PAI-1评估PAI-039对PAI-1的抑制机制，以及在SMC黏附与迁移试验中使用野生型PAI-1和特定PAI-1突变体进行评估。体内肿瘤血管生成模型用于评估给予PAI-039对基质胶植入物中新血管形成的影响。
PAI-039剂量依赖性地抑制可溶性PAI-1，但不抑制与玻连蛋白结合的PAI-1。使用无法结合玻连蛋白的PAI-1突变体（PAI-1K）或使蛋白酶失活的PAI-1突变体（PAI-1R）进行的细胞黏附试验进一步表明，PAI-039通过在其玻连蛋白结构域附近结合使PAI-1失活。在肿瘤血管生成模型中，用PAI-039治疗野生型小鼠可剂量依赖性地降低基质胶植入物中的血红蛋白浓度和内皮细胞染色，表明血管生成减少，但在PAI-1基因敲除小鼠中未表现出体内疗效。
给予口服活性PAI-1抑制剂可防止基质胶植入物中的血管生成。PAI-039对与玻连蛋白结合的野生型PAI-1和PAI-1K缺乏活性，提示PAI-039在玻连蛋白结合位点附近结合。我们的研究进一步证实了PAI-1在细胞运动和肿瘤血管生成中的作用，并首次表明这些作用可通过药理学方法调节。
