Plasticity of the Vascular Smooth Muscle Cytoskeleton

众所周知，增殖性细胞，如成纤维细胞，具有非常“可塑性”的细胞骨架。相比之下， 众所周知，横纹肌细胞具有坚硬的细胞骨架。分化的血管平滑肌细胞 (DVSMCs)，基于有限的现有文献和我们的初步数据，似乎在 在这两种细胞类型之间。这个项目的长期目标是确定在什么程度上和在什么程度上 “非肌肉”细胞骨架在激动剂中调节平滑肌收缩能力的机制-- 流体力学和疾病诱导的结构重塑。今后5年的具体目标是： 1.基于初步数据，检验肌动蛋白细丝在激动剂刺激过程中重塑的假设 并在dVSMCs中拉伸，以确定这是否改变收缩能力。2.检验假设 特定的核/伸长和稳定蛋白参与了细胞骨架的重塑， 首先关注Profilin、N-WASP和Vasp(成核/伸长)，以及CAD、TM和CAP(稳定)。 我们还将检验这样一种假设，即平滑肌假定的细丝稳定剂蛋白的分布， CAD、TM和CAP在定义不太稳定的肌动蛋白细丝的定位方面发挥了作用。3.测试 假设致密斑块和/或致密小体和/或它们与肌动蛋白的附着发生重塑 细丝存在于dVSMCs中，非受体酪氨酸激酶FAK和Src调节 DVSMC细胞骨架/斑块重构。技术将包括差速离心法和在体 用于监测F和G肌动蛋白群体的标记、双色免疫印迹、质谱学、细胞内光亲和力 交联与FRET、细胞诱骗多肽、显性负性蛋白片段、反义 技术和标准的细胞、生物化学和分子方法。这个项目的结果将是 促进我们对dVSMC中提供维护的尚不清楚的机制的理解，并将 加深我们对非肌肉细胞骨架在血管控制中的作用的基本了解 功能。 总结：目前治疗心血管疾病的药物的局限性在一定程度上是由于我们有限的 了解血管的肌肉细胞如何运作。这个项目将扩大我们的理解 并有可能为药物开发确定全新的靶点。

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.