Role of plasminogen activator inhibitor-1 in mediating age-related fibrosis

描述(由申请人提供)：心血管老化导致心肌梗死(MI)后心脏纤维化、舒张期功能障碍和伤口愈合减弱。虽然许多研究表明纤溶酶原激活物抑制物-1(PAI-1)的增加与衰老有关，但还没有人直接研究PAI-1介导的胶原转化抑制的可能机制。这项拟议的研究试图确定PAI-1在调节与年龄相关的心脏纤维化中的作用。PAI-1的年龄依赖性增加与基质金属蛋白酶(MMP)活性降低、胶原沉积增加和钝化炎症反应相关。作为ECM降解和成纤维细胞迁移受损的结果，基质金属蛋白酶活性的降低可能通过增加心肌梗死后的胶原沉积和减轻炎症反应而导致心肌纤维化。分离的大鼠心脏成纤维细胞(CFs)将被用来研究PAI-1的释放与胶原沉积和CF迁移的潜在影响之间的关系。这项研究的第一个目的是询问CFS产生和释放的PAI-1是否通过抑制基质金属蛋白酶的激活而增加胶原沉积。CFS中的总MMPs和活性MMPs将通过蛋白质印迹、酶谱和ELISA法检测。第二个目的是比较PAI-1在年轻和老年大鼠分离的CFs中的表达，以检查其表达是否随着年龄的增长而增加，并与胶原蛋白的增加有关。推测PAI-1随增龄增加可减弱基质金属蛋白酶的活性。这反过来会减少胶原的降解，也可能会抑制CF的迁移。最后，这项研究的第三个目标将使用体外大鼠缺血再灌注模型。这提供了一个系统，以确定PAI-1在老年心脏中的表达是否在基础上增加，以及在缺血应激后，PAI-1是否在生理相关的浓度下上调。PAI-1对基质金属蛋白酶活性的急性抑制可能解释了老年人心肌梗死后炎症反应和伤口愈合反应减弱的原因。总之，这些发现可能确定一种新的PAI-1依赖机制，在这种机制中，老年心脏在缺血损伤后更容易出现纤维化和伤口愈合减弱。

DESCRIPTION (provided by applicant): Cardiovascular aging results in cardiac fibrosis, diastolic dysfunction, and attenuated wound healing post-myocardial infarction (MI). While numerous studies have correlated increases in plasminogen activator inhibitor-1 (PAI-1) with aging, none have directly examined possible mechanisms of PAI-1-mediated inhibition of collagen turnover. The proposed study seeks to identify a role of PAI-1 in regulating age-related cardiac fibrosis. Age-dependent increases of PAI-1 correlate with decreased matrix metalloprotease (MMP) activity, increased collagen deposition, and blunted inflammatory response. As a consequence of impaired ECM degradation and fibroblast migration, the reduction in MMP activity may cause ventricular fibrosis by increasing collagen deposition and diminished inflammatory responses post-MI. Isolated rat cardiac fibroblasts (CFs) will be used to investigate the relationship between PAI-1 release and potential effects on collagen deposition and CF migration. The first aim of this study asks whether PAI-1 produced and released by CFs increases collagen deposition by inhibiting MMP activation. Total and active MMPs will be assayed in CFs by Western blotting, zymography, and ELISA. The second aim compares PAI-1 expression in CFs isolated from young and aged rats to examine whether expression increases with age and is responsible for increased collagen accumulation. The increase of PAI-1 with age is hypothesized to attenuate MMP activity. This in turn decreases collagen degradation and may also inhibit CF migration. Finally, the third aim of the study will use an ex vivo rat ischemia-reperfusion model. This provides a system to ascertain whether PAI-1 expression is increased basally in aged hearts and whether PAI-1 is up-regulated in physiologically relevant concentrations following ischemic stress. Acute inhibition of MMP activity by PAI-1 may explain the attenuated inflammatory and wound healing responses post-MI seen in aged individuals. Together, these findings may identify a novel PAI-1 dependent mechanism in which aged hearts are more prone to both fibrosis and attenuated wound healing following ischemic damage.