HEPARIN AND AORTIC SMOOTH MUSCLE CELL PROLIFERATION

新生血管内膜平滑肌细胞的大量增殖 血管成形术后的大部分再狭窄。可以理解的是，有 因此，开发平滑肌细胞抑制剂引起了人们的极大兴趣 扩散。最有希望的药剂是非抗凝剂部分 肝素是一种有效的平滑肌细胞抑制物 扩散和迁徙。尽管肝素的作用机制 最近的证据表明，实现这一目标的人知之甚少 肝素的抑制活性是由于它能阻止 细胞表面凝血酶原蛋白的表达。这个大的(450 KD)， 均三聚体、多结构域、细胞表面/基质分子与细胞结合 表面通过其肝素结合域，并可作为焦点 蛋白酶(纤溶酶)活性。有证据表明凝血酶原蛋白是 与主动脉平滑肌细胞增殖密切相关的因素包括：(A) 凝血酶原激活剂刺激后凝血酶反应蛋白mRNA和蛋白的快速诱导 含血小板衍化生长因子的主动脉平滑肌细胞 促有丝分裂原；(B)增强的主动脉平滑肌细胞的有丝分裂反应 对表皮生长因子的影响 纯化的凝血酶原蛋白；(C)平滑抑制之间的相关性 肝素对肌细胞生长及阻断能力的影响 凝血酶原蛋白在细胞表面的掺入；(D) 识别沿线不同表位的单抗 凝血酶敏感蛋白分子抑制血管平滑肌增殖的研究 培养中的细胞；(E)血栓反应蛋白强烈的新生内膜沉积 动脉球囊导管损伤后。在这份提案中，我们描述了 抑制凝血酶反应蛋白表达和平滑的替代方法 肌肉的增殖和迁移。这些方法包括开发 血栓反应蛋白细胞表面表达的竞争性抑制物，如 重组肝素结合区及肝素结合肽及其用途 抑制血栓反应蛋白的反义载体或寡核苷酸 生物合成。此外，我们将使用重组肝素结合 凝血酶原蛋白S结构域的亲和基质为富集组分 肝素对血管细胞增殖有抑制作用。 相应地，具体目标是：具体目标1。使用重组 肝素结合域、合成肝素结合肽和载体 表达反义转录物以抑制凝血酶敏感蛋白的生物合成， 平滑肌细胞增殖、迁移与细胞表面蛋白水解酶 一代。特定目的2.重组肝素结合域的用途 作为亲和基质富集肝素抑制组分 平滑肌细胞增殖。

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.