COBRE: LSU HSC: P3: GROWTH FACTOR & INTEGRIN SIGNALING IN VSMC

COBRE：路易斯安那州立大学 HSC：P3：增长因子

• 批准号: 7382065
• 负责人: ANDREW D CATLING
• 金额: $ 20.56万
• 依托单位: LSU HEALTH SCIENCES CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7382065
• 关键词: COBRE; LSU; HSC; P3; GROWTH

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Proliferation and migration of vascular smooth muscle cells (VSMC) occur rapidly after vessel injury concomitant with switching of VSMC from a contractile to a synthetic phenotype. These changes in VSMC behavior contribute to intimal expansion and vascular remodeling seen in atherosclerotic plaques. Phenotypic switching is associated with changes in the local extracellular matrix that function to suppress or enhance VSMC proliferation, migration or contractility. The MAP kinase pathway governs each of these endpoints, and is regulated by stimulatory and inhibitory adhesion signals. Three Specific Aims were proposed to dissect how adhesion signals regulate ERK signaling in VSMC. The progress towards completion of Aims I - III made during the second budget period is summarized below: Aims I/II. Determine the matrix-specific modulation of MAP kinase activation by growth factor stimulation in VSMC. We have visualized phospho-ERK in femoral artery sections from balloon catheter-injured rats. Intimal expansion is apparent 3-7 days after injury (3 rats per time point). Furthermore, co-staining with anti phospho-ERK and anti smooth muscle actin antisera reveals that ERK is activated in smooth muscle cells at the injury site at these time points following injury. In collaboration with Dr. Park we have generated lentiviral constructs for the expression of epitope-tagged MEK mutants and other signaling molecules in vascular endothelial and smooth muscle cells in situ. We will express phosphorylation-defective and phosphorylation-mimetic MEK1 in situ to test the hypothesis that MEK1 phosphorylation controls ERK activation and VSMC function during restenosis. Aim III. Identify matrix-specific MAP kinase substrates following growth factor stimulation in VSMC. Initially we proposed to identify and characterize matrix-dependent ERK substrates in VSMC in culture before asking whether these targets are important in vivo. However, difficulties encountered in our tissue culture model have led us to attempt to identify putative ERK substrates regulated in concert with VSMC phenotypic switching in situ in balloon-injured rabbit vessel sections. Our strategy is to use lentiviral reagents to manipulate MEK and ERK signaling in situ, and phosphoprotein-enrichment combined with mass spectrometry to identify putative ERK substrates in vessel tissue. We have made the necessary viral constructs, and "proof of principle" experiments have identified phosphorylation sites in multiple VSMC proteins.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。血管损伤后，血管平滑肌细胞（VSMC）的增殖和迁移迅速发生，伴随着VSMC从收缩型向合成型的转变。VSMC行为的这些变化有助于动脉粥样硬化斑块中可见的内膜扩张和血管重塑。表型转换与局部细胞外基质的变化有关，其功能是抑制或增强VSMC增殖、迁移或收缩性。MAP激酶途径支配这些终点中的每一个，并且由刺激性和抑制性粘附信号调节。本论文提出了三个具体的目的来研究粘附信号对VSMC ERK信号的调节作用。第二个预算期间在完成目标一至三方面取得的进展概述如下：确定基质特异性调节MAP激酶激活的生长因子刺激VSMC。我们已经可视化磷酸化ERK在股动脉节从球囊导管损伤大鼠。损伤后3-7天内膜扩张明显（每个时间点3只大鼠）。此外，用抗磷酸化ERK和抗平滑肌肌动蛋白抗血清的共染色揭示，在损伤后的这些时间点，ERK在损伤部位的平滑肌细胞中被激活。 与Park博士合作，我们已经产生了用于在血管内皮细胞和平滑肌细胞中原位表达表位标记的MEK突变体和其他信号分子的慢病毒构建体。我们将在原位表达磷酸化缺陷和磷酸化模拟MEK 1，以检验再狭窄期间MEK 1磷酸化控制ERK激活和VSMC功能的假设。 Aim III.在VSMC中生长因子刺激后识别基质特异性MAP激酶底物。最初，我们提出了确定和表征基质依赖性ERK底物在培养VSMC之前，问这些目标是否是重要的在体内。然而，在我们的组织培养模型中遇到的困难，使我们试图确定假定的ERK底物调节一致的VSMC表型转换原位球囊损伤的兔血管节。我们的策略是使用慢病毒试剂来原位操纵MEK和ERK信号传导，并将磷蛋白富集与质谱法相结合来识别血管组织中推定的ERK底物。我们已经制造了必要的病毒结构，并且“原理证明”实验已经确定了多种VSMC蛋白中的磷酸化位点。