NO-INDUCED VASCULAR SMOOTH MUSCLE CELL MOTILITY

无诱导血管平滑肌细胞运动

• 批准号: 6390572
• 负责人: AVIV HASSID
• 金额: $ 24.85万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6390572
• 关键词: actins; cGMP dependent protein kinase; cell motility; enzyme activity; enzyme biosynthesis; laboratory rat; nitric oxide; nitric oxide synthase; protein tyrosine phosphatase; tissue /cell culture; vascular smooth muscle; vasomotion; western blottings

Migration of smooth muscle cells is of critical importance to vascular development, angiogenesis, neointima formation and tissue remodeling that occurs after vascular injury. Preliminary observations indicate that NO elicits reorganization of the actin cytoskeleton and that it increases the motility of rat aortic smooth muscle cells in primary culture, but not subculture. These effects are associated with increased levels of protein tyrosine phosphatase 1D (PTP1D) in primary culture but not subculture. We have also found that reduction of PTP1D protein levels with antisense oligodeoxynucleotide (ODN) attenuates NO- induced motility in primary cultures. The overall purpose of this project is to investigate the role of PTP1D in NO-induced cell motility in culture and in vivo. The proposal is divided into three interrelated parts. A. Aims dealing with mechanisms of NO-stimulated PTP1D expression A.i.) Determine whether NO increases PTP1D enzyme activity, concomitantly with increased protein levels. A.ii.) Determine whether NO increases PTP1D mRNA levels and if so, whether this effect requires cGMP-dependent protein kinase (PKG) activity. If mRNA levels are increased, determine whether increased mRNA synthesis can explain this effect or whether increased mRNA stability may be involved. A.iii.) Determine whether NO increases the rate of PTP1D synthesis and/or decreases the rate of PTP1D metabolism. B. Aims dealing with consequences of gain-of-function or loss-of-function of PKG or PTP1D in cultured cells. B.i.) Determine whether enforced expression of PKG in subcultured cells deficient in PKG restores the capacity of NO to increase PTP1D proteins levels and stimulate cell motility. B.ii.) Determine whether enforced expression of wild-type PTP1D, but not catalytically inactive mutant PTP1D, in subcultured cells deficient in PTP1D enhances cell motility. B.iii.) Determine whether NO-elicited actin cytoskeletal reorganization and increased cell motility in primary culture are blocked by agents that interfere with PTP1D function and whether overexpression of PTP1D mimics the effects of NO. C. Aim dealing with expression of PTP1D and modulation of cell motility by PTP1D in vivo: Determine whether vascular injury increases PTP1D protein levels and whether expression of dominant-interfering mutant of PTP1D attenuates vascular injury- induced cell motility and neointima formation. We anticipate that this project will provide new information on mechanisms that are likely to explain the motogenic capacity of NO in aortic smooth muscle cells and in vivo.

平滑肌细胞的迁移对于血管损伤后发生的血管发育、血管生成、新内膜形成和组织重塑至关重要。 初步观察表明，NO 会引起肌动蛋白细胞骨架的重组，并增加原代培养中大鼠主动脉平滑肌细胞的运动性，但在传代培养中则不然。这些效应与原代培养物中蛋白酪氨酸磷酸酶 1D (PTP1D) 水平升高有关，但与传代培养物中无关。 我们还发现，用反义寡脱氧核苷酸 (ODN) 降低 PTP1D 蛋白水平会减弱原代培养物中 NO 诱导的运动性。 该项目的总体目的是研究 PTP1D 在培养物和体内 NO 诱导的细胞运动中的作用。 该提案分为三个相互关联的部分。 A. 目的是研究 NO 刺激 PTP1D 表达的机制 A.i.) 确定 NO 是否会增加 PTP1D 酶活性，同时增加蛋白质水平。 A.ii.) 确定 NO 是否会增加 PTP1D mRNA 水平，如果是的话，这种作用是否需要 cGMP 依赖性蛋白激酶 (PKG) 活性。 如果 mRNA 水平增加，请确定 mRNA 合成增加是否可以解释这种效应，或者是否可能涉及 mRNA 稳定性增加。 A.iii.) 确定 NO 是否会增加 PTP1D 合成速率和/或降低 PTP1D 代谢速率。 B. 旨在处理培养细胞中 PKG 或 PTP1D 功能获得或功能丧失的后果。 B.i.) 确定在缺乏 PKG 的传代细胞中强制表达 PKG 是否可以恢复 NO 增加 PTP1D 蛋白水平和刺激细胞运动的能力。 B.ii.) 确定在 PTP1D 缺陷的传代培养细胞中强制表达野生型 PTP1D（而非催化失活突变体 PTP1D）是否会增强细胞运动性。 B.iii.) 确定原代培养物中 NO 引发的肌动蛋白细胞骨架重组和细胞运动性增加是否被干扰 PTP1D 功能的试剂阻断，以及 PTP1D 的过度表达是否模仿 NO 的作用。 C.处理体内PTP1D的表达和PTP1D对细胞运动的调节的目的：确定血管损伤是否增加PTP1D蛋白水平以及PTP1D的显性干扰突变体的表达是否减弱血管损伤诱导的细胞运动和新内膜形成。 我们预计该项目将提供有关可能解释主动脉平滑肌细胞和体内 NO 运动能力的机制的新信息。