Identification of molecular mechanisms of carbon nanoparticle-induced senescence and aging ex vivo and in vivo: Role of reactive oxygen species

所有燃烧衍生的碳纳米颗粒（CDNP）的共同原理是具有小于100 nm的初级直径的碳质核。多项研究表明，纯碳纳米颗粒的作用可以解释吸入CDNP引起的肺和心血管系统的几种不良健康结果。流行病学数据表明，慢性暴露于CDNP导致肺过早老化和年龄相关的心血管疾病的特征。在衰老过程中，器官的功能下降，导致多器官衰竭和死亡。衰老的一个标志是细胞衰老的发生。几项研究表明，衰老细胞存在于体内，并且比非衰老细胞功能更差。Haendeler和Unfried团队首次共同证明了纯碳纳米颗粒诱导内皮细胞和肺上皮细胞的细胞衰老特征。这种暴露也增加了活性氧（ROS），据信与衰老和年龄相关疾病有关。因此，本研究提案的目的是确定迄今为止未知的导致原代人肺上皮细胞和血管内皮细胞体外碳纳米颗粒诱导衰老的潜在分子机制，并确定这些机制是否也与体内相关。首先，我们将调查是否ROS是因果关系参与衰老诱导的同时处理不同的抗氧化剂和随后的衰老参数的测量。此外，我们将确定碳纳米颗粒是否改变氧化和/或抗氧化系统的表达和/或活性。如果是这样的话，相应的蛋白质将通过慢病毒方法永久地重新表达或下调。由于线粒体在衰老过程和衰老中起着重要作用，因此将在碳纳米颗粒处理后测量这些细胞器的功能。一种与线粒体功能、老化和衰老相关的蛋白质是线粒体端粒酶逆转录酶（TERT），其被碳纳米颗粒还原。因此，我们将确定是否重新表达肺局部的TERT拯救肺上皮细胞和血管内皮细胞从碳纳米颗粒诱导的衰老。肺和血管功能下降的另一个重要特征是细胞间通讯受损。因此，我们将确定碳纳米颗粒对细胞间通讯的影响，特别关注连接蛋白43。为了进一步了解目前未知的途径，我们将进行转录组和蛋白质组分析。最后，我们将研究非炎症剂量的碳纳米颗粒是否也会诱导体内衰老。为了测试特定的途径，我们将使用两种小鼠模型，一种是在内皮细胞中特异性过表达NADPH氧化酶4，另一种是在所有组织的线粒体中仅含有TERT。

The common principle of all combustion-derived carbon nanoparticles (CDNP) is the carbonaceous core with a primary diameter below 100 nm. Multiple studies have demonstrated that the effects of pure carbon nanoparticles can explain several adverse health outcomes to the lung and the cardiovascular system induced by inhaled CDNP. Epidemiologic data indicate that chronic exposure to CDNP leads to features of premature lung aging and age-related cardiovascular diseases. During the process of aging a functional decline of organs occurs resulting to multiple organ failure and death. One hallmark of aging is the occurrence of cellular senescence. Several studies demonstrated that senescent cells are present in vivo and are less functional than non-senescent cells. The Haendeler and Unfried groups together demonstrated for the first time that pure carbon nanoparticles induce features of cellular senescence in endothelial cells and lung epithelial cells. This exposure also increased reactive oxygen species (ROS), believed to be involved in aging and age-associated diseases. Therefore, the aims of this research proposal are to identify the so far unknown underlying molecular mechanisms leading to carbon nanoparticle-induced senescence in primary human lung epithelial cells and vessel endothelial cells ex vivo and to determine whether these mechanisms are also relevant in vivo. First, we will investigate whether ROS are causally involved in senescence induction by concurrent treatment with different antioxidants and subsequent measurement of senescence parameters. Furthermore, we will determine whether carbon nanoparticles change the expression and/or activity of oxidative and/or antioxidative systems. If so, the respective proteins will be permanently re-expressed or downregulated by a lentiviral approach. As mitochondria play an important role in aging processes and senescence, the functionality of these organelles will be measured after carbon nanoparticle treatment. One relevant protein for mitochondrial functions, aging and senescence is mitochondrial Telomerase Reverse Transcriptase (TERT), which is reduced by carbon nanoparticles. Thus, we will determine whether re-expression of mitochondrially localized TERT rescues lung epithelial cells and vessel endothelial cells from carbon nanoparticle-induced senescence. Another important feature of declined lung and vessel functions is impaired intercellular communication. Thus, we will determine the effect of carbon nanoparticles on intercellular communication with a specific focus on Connexin 43. To get further insights in so far unknown pathways, we will perform transcriptome and proteome analyses. Finally, we will investigate whether carbon nanoparticles in non-inflammatory doses also induce senescence in vivo. To test specific pathways we will use two mouse models, one overexpressing NADPH oxidase 4 specifically in the endothelium and one containing TERT exclusively in the mitochondria of all tissues.