Plasticity of the Vascular Smooth Muscle Cytoskeleton

增殖细胞，如成纤维细胞，众所周知具有非常"可塑性"的细胞骨架。与此相反， 已知横纹肌细胞具有刚性细胞骨架。分化的血管平滑肌细胞 （dVSMC），基于有限的现有文献和我们的初步数据，似乎在某些地方发挥作用， 这两种细胞类型之间的联系。本项目的长期目标是确定在何种程度上以及通过何种方式 机制"非肌肉"细胞骨架在激动剂-， 其结构的流体动力学和疾病引起的重塑。今后五年的具体目标是： 1.为了验证这一假设，根据初步数据，肌动蛋白丝重塑在激动剂刺激 并在dVSMC中伸展，以确定这是否会改变收缩性。2.为了验证这个假设， 特异性成核/延伸和稳定蛋白参与dSMC的细胞骨架重塑， 首先关注于profilin、N-WASP和VASP（成核/延伸），以及CaD、Tm和CaP（稳定化）。 我们还将检验平滑肌假定的丝稳定蛋白的分布， CaD、Tm和CaP在确定较不稳定的肌动蛋白丝的定位中起作用。3.测试 假设致密斑块和/或致密体和/或它们与肌动蛋白的连接的重塑 纤维发生在dVSMC中，并且，非受体酪氨酸激酶FAK和Src调节 dVSMC中的细胞骨架/斑块重塑。技术将包括差速离心和体内 标记以监测F和G肌动蛋白群体，双色免疫印迹，质谱，细胞内光亲和性 交联和FRET，细胞渗透性诱饵肽，显性负性蛋白片段，反义 技术和标准细胞、生物化学和分子方法学。该项目的成果将 进一步我们的理解，但不清楚的机制ofee维护dVSMC和将 增加我们对非肌肉细胞骨架在控制血管生成中作用的基本理解， 功能 总结：目前治疗心血管疾病的药物的局限性部分是由于我们的有限性 了解血管中的肌肉细胞如何发挥作用。这个项目将扩大我们对 的VSMC，并有可能确定全新的药物开发的目标。

Proliferative cells, such as fibroblasts, are well known to have a very "plastic" cytoskeleton. In contrast, striated muscle cells are known to have a rigid cytoskeleton. Differentiated vascular smooth muscle cells (dVSMCs), based on limited existing literature and our preliminary data, appear to function somewhere in between these two cell types. The long-term goal of this project is to determine to what degree and by what mechanisms the "non-muscle" cytoskeleton modulates smooth muscle contractility during agonist-, hydrodynamic and disease-induced remodeling of its structure. The Specific Aims over the next 5 years are: 1. To test the hypothesis, based on preliminary data, that actin filaments remodel during agonist stimulation and stretch in dVSMCs and to determine whether this alters contractility. 2. To test the hypothesis that specific nucleation/elongation and stabilizing proteins are involved in cytoskeletal remodeling of dSMC, focusing first on profilin, N-WASP, and VASP (nucleating/elongation), and CaD, Tm, and CaP (stabilizing). We will also test the hypothesis that the distribution of smooth muscle putative filament stabilizer proteins, CaD, Tm, and CaP, play a role in defining the localizationof the less labile actin filaments. 3. To test the hypothesis that remodeling of the dense plaques and/or dense bodies and/or their attachements to actin filaments occurs in dVSMCs, and, that the non-receptor tyrosine kinases FAK and Src regulate cytoskeleton/plaque remodeling in dVSMC. Techniques will include differential centrifugation, and in vivo labeling to monitor F and G actin populations, 2-color immunoblot, mass spectrometry, in-cell photoaffinity cross-linking and FRET, cell permeant decoy peptides, dominant negative protein fragments, antisense technology and standard cellular, biochemical and molecular methodologies. The results from this project will further our understanding of the, yet unclear mechanisms offeree maintenance in the dVSMC and will increase our fundamental understanding of the role of the nonmuscle cytoskeleton in the control of vascular function. LAY SUMMARY: The limitations of current drugs to treat cardiovascular disease is due, in part, to our limited understanding of how the muscle cells of blood vessels function. This project will expand our understanding of the VSMC and has the potential of identifying entirely novel targets for drug development.