Neointimal smooth muscle cell proliferation is responsible for the vast majority of restenosis following angioplasty. Understandably, there has thus been great interest in developing inhibitors of smooth muscle cell proliferation. The most promising agent is the non-anticoagulant fraction of heparin which is a potent inhibitor of both smooth muscle cell proliferation and migration. Although the mechanisms by which heparin accomplishes this have been poorly understood, recent evidence suggests that heparin's inhibitory activities are due to its ability to block the cell surface expression of thrombospondin. This large (450 kD), homotrimeric, multidomain, cell surface/matrix molecule binds to cell surfaces via its heparin binding domain and can serve as a focus for protease (plasmin) activity. Evidence suggesting that thrombospondin is intimately linked to aortic smooth muscle cell proliferation includes: (a) the rapid induction of thrombospondin mRNA and protein after stimulation of aortic smooth muscle cells with platelet derived growth factor, a potent mitogen; (b) the enhanced mitogenic response of aortic smooth muscle cells to epidermal growth factor following prior exposure of the cells to purified thrombospondin; (c) the correlation between inhibition of smooth muscle cell growth by heparin and the ability of heparin to block thrombospondin incorporation on the cell surface; (d) the ability of monoclonal antibodies which recognize distinct epitopes along the thrombospondin molecule to inhibit proliferation of aortic smooth muscle cells in culture; (e) intense neointimal deposition of thrombospondin following arterial balloon catheter injury. In this proposal we describe alternative methods of inhibiting thrombospondin expression and smooth muscle proliferation and migration. These methods include developing competitive inhibitors of thrombospondin cell surface expression such as recombinant heparin binding domain and heparin binding peptides and using antisense vectors or oligonucleotides to inhibit thrombospondin biosynthesis. In addition, we will use the recombinant heparin binding domain of thrombospondin s an affinity matrix to enrich for the fraction of heparin that is inhibitory to smooth muscle cell proliferation. Accordingly, the specific aims are:
Specific Aim 1. To use recombinant heparin binding domain, synthetic heparin binding peptides and vectors expressing antisense transcript to inhibit thrombospondin biosynthesis, smooth muscle cell proliferation, migration and cell surface protease generation.
Specific Aim 2. Use of the recombinant heparin binding domain as an affinity matrix to enrich for the fraction of heparin inhibitory to smooth muscle cell proliferation.
