NO-INDUCED VASCULAR SMOOTH MUSCLE CELL MOTILITY

血管内皮细胞的迁移对血管的发育、新生血管的形成以及血管损伤后的组织重塑起着至关重要的作用。初步观察表明，NO可引起肌动蛋白细胞骨架的重组，并能增加原代培养的大鼠主动脉平滑肌细胞的运动能力，但不能增加传代培养的细胞活力。这些影响与原代培养中蛋白酪氨酸磷酸酶1D(PTP1D)水平的增加有关，但与传代培养无关。我们还发现，在原代培养中，用反义寡核苷酸(ODN)降低PTP1D蛋白水平可以减弱NO诱导的运动。本项目的总体目的是研究PTP1D在培养和体内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.旨在研究NO刺激PTP1D表达的机制。确定一氧化氮是否增加了PTP1D酶的活性，同时也增加了蛋白质水平。A.ii.)确定NO是否增加PTP1D的mRNA水平，如果是，这种作用是否需要cGMP依赖的蛋白激酶(PKG)活性。如果信使核糖核酸水平升高，确定信使核糖核酸合成增加是否可以解释这种效应，或者是否可能与信使核糖核酸稳定性增强有关。A.III.)确定NO是否增加PTP1D合成和/或降低PTP1D代谢率。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PKG缺陷的传代细胞中强制表达PKG是否恢复了NO增加PTP1D蛋白水平和刺激细胞运动的能力。B.ii.)确定在PTP1D缺陷的传代细胞中强制表达野生型PTP1D而不是催化失活突变体PTP1D是否能增强细胞的运动能力。B.III.)确定原代培养中NO诱导的肌动蛋白细胞骨架重组和细胞活力增加是否被干扰PTP1D功能的药物阻断，以及PTP1D的过度表达是否模仿NO的影响。目的研究PTP1D在体内的表达及PTP1D对细胞运动的调节：确定血管损伤是否增加PTP1D蛋白水平，以及PTP1D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生内膜形成。我们期望这个项目能为解释NO在主动脉平滑肌细胞和体内的运动生成能力的机制提供新的信息。

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.