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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。血管平滑肌细胞（VSMC）的增殖和迁移在血管损伤后迅速发生，并伴有血管平滑肌细胞从收缩表型向合成表型的转变。这些VSMC行为的改变有助于动脉粥样硬化斑块中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的增殖、迁移或收缩。MAP激酶通路控制着这些端点，并受刺激和抑制粘附信号的调节。提出了三个特定的目的来剖析粘附信号如何调节VSMC中的ERK信号。在第二个预算期间为完成目标一至三所取得的进展摘要如下：目标一/二。确定生长因子刺激VSMC对MAP激酶激活的基质特异性调节。我们在球囊导管损伤大鼠的股动脉切片中观察到磷酸化erk。损伤后3 ~ 7 d，内膜明显扩张（每个时间点3只）。此外，抗磷酸化ERK和抗平滑肌肌动蛋白抗血清共染色显示，在损伤后的这些时间点，损伤部位的平滑肌细胞中的ERK被激活。在与Park博士的合作中，我们在血管内皮细胞和平滑肌细胞中原位生成了用于表位标记的MEK突变体和其他信号分子表达的慢病毒构建体。我们将原位表达磷酸化缺陷和磷酸化模拟MEK1，以验证MEK1磷酸化在再狭窄期间控制ERK激活和VSMC功能的假设。第三目标。确定基质特异性MAP激酶底物后生长因子刺激VSMC。最初，我们提出在询问这些靶点在体内是否重要之前，在培养的VSMC中鉴定和表征基质依赖的ERK底物。然而，在我们的组织培养模型中遇到的困难使我们试图在气球损伤的兔血管切片中确定与VSMC表型转换相一致的推定ERK底物。我们的策略是使用慢病毒试剂来原位操纵MEK和ERK信号，并将磷酸化蛋白富集与质谱相结合来鉴定血管组织中可能的ERK底物。我们已经构建了必要的病毒结构，并且“原理证明”实验已经确定了多个VSMC蛋白的磷酸化位点。

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.