HEPARIN AND AORTIC SMOOTH MUSCLE CELL PROLIFERATION

新生内膜平滑肌细胞增殖是导致 大多数血管成形术后再狭窄。 可以理解， 因此对开发平滑肌细胞抑制剂有很大兴趣 增殖 最有前途的代理商是非抗凝部分 肝素是一种有效的平滑肌细胞抑制剂， 扩散和迁移。 尽管肝素的作用机制 最近的证据表明， 肝素的抑制活性是由于它能够阻断 血小板反应蛋白的细胞表面表达。 这个大（450 kD）， 同源三聚体、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，可以作为重点， 蛋白酶（纤溶酶）活性。 有证据表明血小板反应蛋白是 与主动脉平滑肌细胞增殖密切相关的包括：（a） 血小板反应蛋白mRNA和蛋白的快速诱导 主动脉平滑肌细胞与血小板衍生生长因子，一种有效的 促有丝分裂原;（B）主动脉平滑肌细胞的促有丝分裂反应增强 表皮生长因子的作用， 纯化的血小板反应蛋白;（c）抑制平滑肌细胞之间的相关性。 肝素对肌细胞生长的影响以及肝素阻断 血小板反应蛋白掺入细胞表面的能力;（d） 单克隆抗体，其识别沿着所述免疫球蛋白的沿着的不同表位， 抑制主动脉平滑肌增殖的血小板反应蛋白分子 培养物中的细胞;（e）血小板反应蛋白的强烈新生内膜沉积 动脉球囊导管损伤。 在本提案中，我们描述了 抑制血小板反应蛋白表达和平滑的替代方法 肌肉增殖和迁移。 这些方法包括开发 血小板反应蛋白细胞表面表达的竞争性抑制剂， 重组肝素结合结构域和肝素结合肽及其用途 抑制血小板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 血小板反应蛋白的结构域是一种亲和基质，以富集血小板反应蛋白的部分， 抑制平滑肌细胞增殖肝素。 因此，具体目标是：具体目标1。 使用重组 肝素结合结构域、合成的肝素结合肽和载体 表达反义转录物以抑制血小板反应蛋白的生物合成， 平滑肌细胞增殖、迁移和细胞表面蛋白酶 一代 具体目标2。 重组肝素结合结构域的用途 作为亲和基质，富集肝素抑制剂级分， 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.