Intimal hyperplasia (IH) is a common response to many forms of vascular injury such as angioplasty and is thought to be mediated in part by a variety of growth factors acting to stimulate vascular smooth muscle cell (VSMC) proliferation and migration from the media into the intima. Plasminogen activators (PA) are a class of fibrinolytic molecules that also facilitate the movement of cells through tissue barriers and extracellular matrix. Cellular receptors for tissue-type (t-PA) and urokinase-type plasminogen activators (u-PA) have also been described which modulate and direct the facility of cellular movement through tissue barriers while plasminogen activator inhibitor type 1 (PAI-1) may inhibit this process. Recent data in vascular injury models have demonstrated increased expression of u-PA and t-PA in temporal association with VSMC proliferation and migration, respectively. Our major objective is to characterize the pathobiology of the plasminogen activator system in VSMC following vascular injury. We will functionally and biochemically characterize u-PA and t-PA receptors expressed by VSMC in culture. We will study the regulation of PAs, PAI-1 and PA receptors in these cells and define which growth factors, cytokines, or pharmacologic agents have the greatest effects on their expression at the protein level using radioligand binding studies and ELISAs and at the mRNA level using Northern blot analysis. We will design antisense oligonucleotides to optimize our ability to inhibit expression of key control proteins in this system and quantify any resulting effect of VSMC proliferation and migration. We will utilize an in vitro invasion assay to test the effects of various PAs or endothelial cell conditioned medium on VSMC migration and attempt to identify positive and negative regulators of this process. Using immunohistochemistry and in situ hybridization, we will study the distribution of PA, PAI-1, and PA receptor protein and mRNAs respectively, in both normal and diseased vascular tissue and correlate their expression with the extent of IH. Finally, we will utilize a rabbit angioplasty restenosis model to study the in vivo expression of the fibrinolytic system and correlate changes in it with the severity of restenosis and test the efficacy of several novel orally active heparin-like molecules (cyclodextrins) on their ability to inhibit IH. A greater understanding of the basic pathobiologic processes underlying intimal hyperplasia following vascular injury will hopefully allow better development of pharmacologic agents to inhibit this process.
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