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。 初步观察结果表明，没有引起肌动蛋白细胞骨架的重组，并且它增加了大鼠主动脉平滑肌细胞在原发性培养中的运动性，而不是亚培养物。这些作用与原发性培养物中蛋白质酪氨酸磷酸酶1D（PTP1D）的水平增加有关，而不是亚文化。 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未诱导的运动性。 该项目的总体目的是研究PTP1D在培养和体内无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.目的是处理未刺激的PTP1D表达AI的机制。 A.II。）确定NO是否增加了PTP1D mRNA水平，如果是，则这种作用是否需要CGMP依赖性蛋白激酶（PKG）活性。 如果mRNA水平升高，请确定增加mRNA合成是否可以解释这种影响，或者是否涉及增加mRNA稳定性。 A.III。）确定NO是否增加了PTP1D合成的速率和/或降低PTP1D代谢的速率。 B.目的是处理培养细胞中PKG或PTP1D功能获得或功能丧失的后果。 B.I.）确定缺乏PKG的亚培养细胞中PKG的表达是否会恢复NO增加PTP1D蛋白水平并刺激细胞运动的能力。 b.ii。）确定在缺乏PTP1D的亚培养细胞中强制强制表达野生型PTP1D，但不是催化无效的突变体PTP1D，会增强细胞运动。 b.iii。）确定未引起的肌动蛋白细胞骨架重组和原发性培养的细胞运动增加是否被干扰PTP1D功能的药物阻止，以及PTP1D的过度表达是否模仿NO的影响。 C.与体内PTP1D对PTP1D表达的表达和细胞运动的调节：确定血管损伤是否会增加PTP1D蛋白水平以及PTP1D衰减的显性差异突变体的表达是否会导致血管损伤诱导的细胞运动和NEINTIMA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的动作能力的机制的新信息。