NO-INDUCED VASCULAR SMOOTH MUSCLE CELL MOTILITY

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、新生内膜形成和组织重塑至关重要。 初步观察表明，一氧化氮能促进肌动蛋白细胞骨架的重组，并能增加大鼠主动脉平滑肌细胞原代培养时的运动能力，但对传代培养无影响。这些效应与原代培养中蛋白酪氨酸磷酸酶1D（PTP 1D）水平升高有关，但与传代培养无关。 我们还发现用反义寡脱氧核苷酸（ODN）降低PTP 1D蛋白水平减弱了原代培养物中NO诱导的运动性。 本项目的总体目的是研究PTP 1D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.目的处理NO刺激的PTP 1D表达的机制A.i.）确定NO是否增加PTP 1D酶活性，同时增加蛋白质水平。A.二.）确定NO是否增加PTP 1D mRNA水平，如果是，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，确定mRNA合成增加是否可以解释这种效应，或者是否涉及mRNA稳定性增加。A. iii.）确定NO是否增加PTP 1D合成速率和/或降低PTP 1D代谢速率。 B。目的是处理培养细胞中PKG或PTP 1D功能获得或功能丧失的后果。B. i.） 确定在PKG缺陷的继代培养细胞中PKG的强制表达是否恢复NO增加PTP 1D蛋白水平和刺激细胞运动的能力。B. ii.） 确定在PTP 1D缺陷的传代培养细胞中，野生型PTP 1D而非催化失活突变型PTP 1D的强制表达是否增强细胞运动性。B. iii.） 确定原代培养中NO诱导的肌动蛋白细胞骨架重组和增加的细胞运动性是否被干扰PTP 1D功能的试剂阻断，以及PTP 1D的过表达是否模拟NO. C的作用。目的研究PTP 1D在体内的表达及PTP 1D对细胞运动的调节：确定血管损伤是否增加PTP 1D蛋白水平，以及PTP 1D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。 我们预计，该项目将提供新的信息机制，可能解释的motogenic能力的一氧化氮在主动脉平滑肌细胞和体内。

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.