ENDOTHELIAL CELL SENESCENCE GENES

描述(改编自调查人员摘要)：调查人员 人脐静脉内皮细胞衰老表型的研究 (HUVEC)，以了解其非复制性的机制 组件。他们实验室之前的研究有力地证明了 细胞因子白介素1α作为血管紧张素转换酶抑制剂的重要作用 成纤维细胞生长因子-1诱导的人脐静脉内皮细胞及其诱导剂 体外衰老。最近的研究表明，细胞内 IL-1α是成纤维细胞生长因子受体(R)信号转导的解偶联分子 事实上，衰老的HUVEC群体不能诱导酪氨酸 FGFR底物Src的磷酸化，即使外源成纤维细胞生长因子-1 能够上调即刻-早期和早期的稳定状态mRNA水平 中晚期成纤维细胞生长因子反应基因。因为HUVEC的种群非常 难以稳定地转染/转导，一直难以推广 这些观察和这种顽固的行为取得的进展有限。 然而，他们最近获得了稳定的前体IL-1α 使用ECV细胞的转染体，一种自发的HUVEC转化子，和数据 从这一系统中得出的结论是，前体IL-1α而不是成熟的 形式的IL-1α能够减弱细胞的增殖和迁移 体外培养ECV细胞的潜能不依赖于外源性 IL-1受体拮抗剂(IRAP)。这些数据表明，前兆 IL-1α作为ECV细胞迁移的细胞内抑制因子 成长。有趣的是，前体IL-1α而不是成熟的IL-1α ECV细胞转染者表现出明显的F-肌动蛋白染色， 与体外衰老的人脐静脉内皮细胞表型相似。因此，为了阐明 细胞内IL-1α前体作用机制的研究进展 建议鉴定和鉴定前体IL-1α相关蛋白 试图阐明细胞内前体IL-1α的途径 沟通。他们还建议利用观察到的 FGFR和IL-1R系统不限制细胞内流量或 外源重组嵌合配体的信号转导能力， 成纤维细胞生长因子-1：β-半乳糖苷酶(GAL)和前体IL-1α：β-GAL。因此，作为 一种替代HUVEC低效转导能力的方法 群体中，他们建议构建和表达一种重组 成纤维细胞生长因子-1：β-半乳糖：前体IL-1α嵌合体。这种双配体嵌合体应该 使他们能够通过FGFR贩运将前体IL-1α内化 IRAP处理的人脐静脉内皮细胞中的通路。这一新方法 不仅应该使他们能够建立细胞内机制， 前体IL-1α作为人脐静脉内皮细胞衰老和静止的修饰物，但可能 也被证明是一种有价值的进口其他蛋白质和 它们的突变体变成了顽固的人类细胞。因此，调查人员 建议对ECV使用这种和更传统的基因转移方法 细胞了解抑制Src酪氨酸的分子基础 衰老人脐静脉内皮细胞表型中的磷酸化。这些研究应该 更好地了解成纤维细胞生长因子-1和前体IL-1α信号转导 HUVEC群体，并牢固地建立前体IL-1α作为 HUVEC衰老。

DESCRIPTION (Adapted from Investigator's Abstract): The investigators have studied the senescent phenotype of the human umbilical vein endothelial cell (HUVEC) in order to understand the mechanism of its non-replicative component. Prior studies from their laboratory have strongly implicated a prominent role for the cytokine, interleukin (IL)-1alpha as an inhibitor of fibroblast growth factor (FGF)-1-induced HUVEC and an inducer of HUVEC senescence in vitro. More recent studies have suggested that intracellular IL-1alpha act as an uncoupler of FGF receptor (R) signal transduction and indeed senescent HUVEC populations are not able to induce the tyrosine phosphorylation of the FGFR substrate Src even though exogenous FGF-1 is able to upregulate the steady-state mRNA levels of both immediate-early and mid-to-late FGF response genes. Because HUVEC populations are extremely difficult to stably transfect/transduce, it has been difficult to extend these observations and this recalcitrant behavior has limited progress. However, they have recently obtained stable precursor IL-1alpha transfectants using the ECV cell, a spontaneous HUVEC transformant, and data obtained from this system argues that precursor IL-1alpha but not the mature form of IL-1alpha is able to attenuate the proliferative and migratory potential of the ECV cell in vitro independent of the presence of exogenous IL-1 receptor antagonist (IRAP). These data demonstrate that precursor IL-1alpha functions as a intracellular repressor of ECV cell migration and growth. Interestingly, the precursor IL-1alpha but not the mature IL-1alpha ECV cell transfectants exhibit a prominent F-actin staining that strongly resembles the senescent HUVEC phenotype in vitro. Thus, to elucidate the mechanism of intracellular IL-1alpha precursor function the investigators propose to identify and characterize precursor IL-1alpha associated proteins in an attempt to elucidate the pathway of intracellular precursor IL-1alpha communication. They also propose to take advantage of the observation that the FGFR and IL-1R systems do not restrict the intracellular traffic or signaling capacities of the exogenous recombinant chimeric ligands, FGF-1:beta-galactosidase (gal) and precursor IL-1alpha:beta-gal. Thus, as an alternative to the inefficient transfection/transduction ability of HUVEC populations, they propose to construct and express a recombinant FGF-1:beta-gal:precursor IL-1alpha chimera. This dual ligand chimera should enable them to internalize precursor IL-1alpha via the FGFR trafficking pathway in HUVEC populations of pretreated with IRAP. This novel approach should not only enable them to establish the intracellular mechanism of precursor IL-1alpha as a modifier of HUVEC senescence and quiescence but may also prove valuable as a general method of importing other proteins and their mutants into recalcitrant human cells. Thus, the investigators propose to use this and more conventional gene transfer methods with ECV cells to understand the molecular basis for the repression of Src tyrosine phosphorylation in the senescent HUVEC phenotype. These studies should yield a better understanding of FGF-1 and precursor IL-1alpha signaling in HUVEC populations and firmly establish precursor IL-1alpha as a modifier of HUVEC senescence.