Mural thrombus formation after balloon angioplasty (PTCA) may result in acute closure, leading to myocardial infarction and sudden death, and may participate in the development of intimal hyperplasia, the pathologic hallmark of restenosis after PTCA. Initiation of the clotting cascade in vivo occurs, predominantly via the tissue factor (TF) pathway. We have recently demonstrated that balloon dilatation of the rat aorta rapidly induces the expression of TF mRNA and activity in arterial smooth muscle. In cultured rat aortic vascular smooth muscle cells (VSMC), TF is markedly induced by thrombin, a central regulatory molecule of the hemostatic system. The central tenets of this proposal are (i) that the induction of TF plays a major role in the generation of a mural thrombus after vessel wall injury, and (ii) that this induction results in part from the regulatory effects that thrombin exerts on TF gene expression in vivo. To test these hypotheses, we propose to study the effects of inhibiting the induced expression of TF mRNA on thrombosis and intimal hyperplasia after balloon injury. To specifically and locally inhibit arterial TF expression in- vivo, a porous balloon catheter will be used to deliver antisense oligonucleotides that have been demonstrated to block TF induction in culture. In addition, ribozymes (short catalytic RNAs possessing specific endoribonuclease activity), will be designed against TF. Ribozymes will be linked to transferrin to enhance transfection efficiency through receptor- mediated endocytosis, and then used to inhibit TF in culture and in vivo. To investigate the mechanisms through which thrombin regulates TF expression, the rat TF promoter will be analyzed in culture to identify a cis-acting thrombin response element. Once identified, promoter constructs in which the thrombin response element has been deleted or mutated will be transfected into the vessel wall. These vessels will then be subjected to balloon injury to determine whether the thrombin response element defined in cell culture is necessary for the response of the TF gene to balloon injury in vivo. The proposal involves a variety of techniques which are new for the applicant, such as ribozyme synthesis and promoter analysis. Both the sponsors and the institution are ideally suited to train the applicant in the areas of vascular molecular biology and thrombosis. The information gained from the proposed studies will provide insights into the relationship between vessel injury, coagulation, and gene regulation that may lead to novel anti-thrombotic therapies.
| Cavusoglu, Erdal; Chen, Irene; Rappaport, Jay et al. (2002) Inhibition of tissue factor gene induction and activity using a hairpin ribozyme. Circulation 105:2282-7 |
| Dangas, G; Badimon, J J; Coller, B S et al. (1998) Administration of abciximab during percutaneous coronary intervention reduces both ex vivo platelet thrombus formation and fibrin deposition: implications for a potential anticoagulant effect of abciximab. Arterioscler Thromb Vasc Biol 18:1342-9 |
| Marmur, J D; Thiruvikraman, S V; Fyfe, B S et al. (1996) Identification of active tissue factor in human coronary atheroma. Circulation 94:1226-32 |
