Intimal thickening is a complex, common response to many forms of vascular injury. It occurs during native atherosclerosis as well as after angioplasty or vein graft arterialization, and is mediated in part by growth factor stimulated smooth muscle cell (SMC) proliferation, migration, and matrix accumulation. The molecular and cellular events controlling these responses are poorly understood; discovery of novel mechanisms of regulation may enable new therapeutic modalities. Our laboratory has studied the regulation of plasminogen activators (PA) by vascular cells. PAs, which are secreted by SMC after vascular injury, may bind to specific cellular receptors, thereby generating plasmin locally and facilitating the movement of SMC through tissue barriers and extracellular matrix. Three recent findings have suggested additional mechanisms of PA regulation of SMC function: l) tissue-type PA (t-PA) activity directly stimulates SMC proliferation; 2) low density lipoprotein receptor related protein (LRP) can internalize and degrade PAs and PA/PA inhibitor complexes; and 3) urokinase-type PA receptor (u-PAR) directly modulates monocyte migration independent of u-PA activity. We have hypothesized that: a) t-PA, possibly in the presence of a cofactor, can proteolytically cleave SMC thrombin receptors, inducing proliferation; b) interaction of PAs with one of several possible receptors will alter migration and matrix invasion; c) intimal thickening in diseased vessels and arterialized vein grafts is associated with an altered expression of plasminogen activator system components in the vessel wall; and d) genetic deletion of u-PA or t-PA but not PAI-1 in mice will limit the vascular injury response in vivo. Using radioligand binding studies and ELISAs with monoclonal antibodies to different portions of the human thrombin receptor, we will determine if t-PA binds to and cleaves the SMC thrombin receptor. We will study the effect of alterations in the plasminogen activator system in vitro on vascular smooth muscle cell proliferation, migration, and invasion, and preliminarily determine which domains of the molecules are involved. We will determine the extent and location of protein and mRNA expression for plasminogen activator system components in normal and diseased human vascular tissue and in rabbit jugular veins before and after arterialization. We will use a carotid injury model in mice genetically deficient in u-PA, t-PA, or PAI-1 to determine the effect of deletion of these specific PA system components on the vascular response to injury. Finally, to identify genes that are upregulated early after vascular injury, we will use differential display and RNA isolated from normal rabbit jugular vein and arterialized vein grafts. Candidate amplified cDNAs will be labeled for use in Northern blotting to confirm altered expression and then sequenced. At least two full length cDNA clones will be obtained from cDNA libraries for apparently novel sequences. Vascular cell expression will be evaluated by in situ hybridization. In summary, a greater understanding of the basic pathobiologic processes underlying intimal thickening following vascular injury should allow better development of pharmacologic agents to inhibit these processes.
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