VASCULAR SMOOTH MUSCLE MEMBRANE PROTEOGLYCANS

VSM细胞受制于一系列调控信号 它们的生长、分化和功能活动。了解以下内容 这些监管信号是如何发挥作用的，将为 了解并最终控制VSM细胞的活动 病理情况。这个项目的长期目标是 阐明膜相关蛋白多糖(MPGs)在血管内皮细胞生长中的作用 血管平滑肌(VSM)细胞的功能和病理生物学 尽管MPG是质膜的普遍成分，并且具有 与控制细胞生长和分化有关，它们 具体功能仍不清楚。最近的研究结果表明， MPG参与了细胞与重要调控因子的相互作用。 分子，特别是细胞外基质(ECM)黏附蛋白 和多肽生长因子。生化与分子生物学 我们实验室的研究已经确定了四个结构上不同的 由大鼠主动脉VSM细胞合成的MPG。其中包括3个 基因家族中不同但结构上相关的成员 跨膜蛋白多糖和脂质锚定的mpg。一位少校 拟议的研究背后的问题是，什么是功能性的 这种结构多样性的意义。的三个具体方面 VSM mpg生物学在提案中涉及：1)模式 VSM细胞在大鼠体内发育过程中合成这些mpg的研究 作为对伤害的反应，这些研究将检查mpg的水平。 Northern印迹分析和核糖核酸酶保护实验 免疫印迹和免疫细胞化学法测定MPG水平；2)分析 这些MPG与ECM分子的结合及其在VSM中的作用 细胞-细胞外基质相互作用；在这些实验中，mpg将被提纯 常规和免疫亲和培养的大鼠主动脉VSM细胞 层析并检测它们与一组 纯化的ECM分子；我们还将使用抗MPG抗体， 反义抑制和mpg过表达直接检测其 VSM细胞在ECM上的附着、扩散和迁移中的作用； 特异性mpgs在生长因子刺激的VSM中的作用分析 细胞增殖；抗mpg抗体的作用，反义 抑制mpg合成和特异性mpg过表达对速率 对VSM细胞增殖和c-fos基因表达的影响 将测量生长因子，以及绑定到 高亲和力细胞表面受体。

VSM cells are subject to an array of regulatory signals that control their growth, differentiation and functional activity. Knowledge of how these regulatory signals work would provide important insights into understanding and ultimately controlling VSM cell activity in pathological conditions. The long range goal of this project is to elucidate the roles of membrane associated proteoglycans (mPGs) in the function and pathobiology of vascular smooth muscle (VSM) cells. Although mPGs are ubiquitous components of plasma membranes, and have been implicated in control of cell growth and differentiation, their specific functions are still unknown. Recent findings suggest that mPGs are involved in interactions of cells with important regulatory molecules, in particular extracellular matrix (ECM) adhesive proteins and polypeptide growth factors. Biochemical and molecular biological studies from our laboratory have identified 4 structurally distinct mPGs that are synthesized by rat aortic VSM cells. These include 3 different but structurally related members of a gene family of transmembrane proteoglycans, and a lipid-anchored mPG. A major question underlying the proposed studies is what is the functional significance of this structural diversity. Three specific aspects of VSM mPG biology are addressed in the proposal: 1) the patterns of synthesis of these mPGs by VSM cells in vivo in rats during development and in response to injury; these studies will examine levels of mPG mRNA by Northern blot analysis and ribonuclease protection assay and mPG levels by immunoblotting and immunocytochemistry; 2) analysis of the binding of these mPGs to ECM molecules and their roles in VSM cell-ECM interactions; for these experiments the mPGs will be purified from rat aortic VSM cell cultures by conventional and immunoaffinity chromatography and assayed for their ability to bind to a panel of purified ECM molecules; we will also use anti-mPG antibodies, anti-sense inhibition, and mPG overexpression to test directly their role in VSM cell attachment, spreading and migration on ECM; and 3) analysis of the role of specific mPGs in growth factor stimulated VSM cell proliferation; effects of anti-mPG antibodies, anti-sense inhibition of mPG synthesis, and specific mPG overexpression on rates of VSM cell proliferation and c-fos mRNA induction in response to growth factors will be measured, as well as growth factor binding to high affinity cell surface receptors.