VASCULAR SMOOTH MUSCLE GROWTH AND FIBRONECTIN MATRIX

血管平滑肌细胞（VSMCs）的增殖受到控制， 通过生长因子和细胞与细胞外 矩阵（ECM）。 我们的目标是了解纤维连接蛋白的作用 基质在控制VSMC增殖中的作用。我们发现， 重组纤连蛋白片段（蛋白III 1-C）抑制VSMC 纤连蛋白基质组装并且还抑制VSMC增殖。 此外，III 1-C阻断生长因子诱导的和粘附诱导的 VSMC中的MAP激酶活化。 为了确定是否抑制 纤连蛋白基质组装阻断MAP激酶活化，我们将测试 是否其它抑制纤连蛋白基质组装的方法也 抑制VSMC增殖和MAP激酶信号传导。 可以 III 1-C抑制VSMC增殖的活性除了 纤连蛋白基质组装的抑制。 因此，我们将测试 III 1-C是否与纤维连接蛋白以外的蛋白质相互作用， VSMCs表面。 这将通过几种方法来实现， 包括标记的III 1-C与细胞表面蛋白的交联， III 1-C亲和层析分离和鉴定细胞表面 与III 1-C相互作用的蛋白质。 的分子机制 III 1-C阻断MAP激酶信号传导将通过系统分析 测试III 1-C对已知成员的活性的影响， Ras/MAP激酶通路，从而使我们能够确定III 1- C破坏了这个信号通路。 我们还将测试 III 1-C在再狭窄模型中对VSMC增殖的体内作用。III 1-C将 插入腺病毒表达构建体中，所述腺病毒表达构建体包括 最近发现的SMC特异性启动子。 这将使高水平的 VSMCs中III 1-C的表达和分泌。 将在大鼠颈动脉中测试III 1-C-腺病毒载体 球囊损伤模型 如果III 1-C对VSMC具有相同的作用， 在体内增殖，因为它在细胞培养中，那么III 1-C- 腺病毒载体应抑制新生内膜的形成，在这个大鼠颈动脉 动脉再狭窄模型。 这项拨款中提出的工作将有助于 确定ECM的重要性，特别是 纤连蛋白基质在培养和细胞增殖中对VSMC生长的调节作用 vivo. 通过了解ECM在病理过程中的作用 例如再狭窄和动脉粥样硬化，我们可以开始设计更多 有效治疗这些疾病。

The proliferation of vascular smooth muscle cells (VSMCs) is controlled by growth factors and by the interaction of cells with the extracellular matrix (ECM). Our goal is to understand the role of the fibronectin matrix in controlling VSMC proliferation. We have found that a recombinant fragment of fibronectin (protein III1-C) inhibits VSMC fibronectin matrix assembly and also inhibits VSMC proliferation. Moreover, III1-C blocks both growth factor-induced and adhesion-induced MAP kinase activation in VSMCs. To determine whether inhibition of fibronectin matrix assembly blocks MAP kinase activation, we will test whether other methods of inhibiting fibronectin matrix assembly also inhibit VSMC proliferation and MAP kinase signaling. It is possible that III1-C inhibits VSMC proliferation by some activity other than inhibition of fibronectin matrix assembly. We will therefore test whether III1-C interacts with proteins other than fibronectin on the surface of VSMCs. This will be accomplished by several methods, including crosslinking of labeled III1-C to cell surface proteins and III1-C affinity chromatography to isolate and identify cell surface proteins that interact with III1-C. The molecular mechanism by which III1-C blocks MAP kinase signaling will be analyzed by systematically testing the effects of III1-C on the activities of the known members of the Ras/MAP kinase pathway, thereby allowing us to determine where III1- C disrupts this signaling pathway. We will also test the effect of III1-C on VSMC proliferation in vivo in a restenosis model. III1-C will be inserted into an adenovirus expression construct that includes a recently discovered SMC-specific promoter. This will allow high levels of expression and secretion of III1-C specifically in VSMCs in vivo. The III1-C-adenovirus vector will be tested in the rat carotid artery balloon injury model. If III1-C has the same effect on VSMC proliferation in vivo as it does in cell culture, then the III1-C- adenovirus vector should inhibit neointima formation in this rat carotid artery restenosis model. The work proposed in this grant will help establish the importance of the ECM, and in particular of the fibronectin matrix in regulating VSMC growth both in culture and in vivo. By understanding the role of the ECM in pathological processes such as restenosis and atherosclerosis we may begin to design more effective treatments for these conditions.