A senescent cell bystander effect: senescence-induced senescence

Senescent cells produce and secrete various bioactive molecules including interleukins, growth factors, matrix-degrading enzymes and reactive oxygen species (ROS). Thus, it has been proposed that senescent cells can damage their local environment, and a stimulatory effect on tumour cell growth and invasiveness has been documented. However, it was unknown what effect, if any, senescent cells have on their normal, proliferation-competent counterparts. We show here that senescent cells induce a DNA damage response, characteristic for senescence, in neighbouring cells via gap junction-mediated cell-cell contact and processes involving ROS. Continuous exposure to senescent cells induced cell senescence in intact bystander fibroblasts. Hepatocytes bearing senescence markers clustered together in mice livers. Thus, senescent cells can induce a bystander effect, spreading senescence towards their neighbours in vitro and, possibly, in vivo.     

Analysis of individual cells identifies cell‐to‐cell variability following induction of cellular senescence

Senescent cells play important roles in both physiological and pathological processes, including cancer and aging. In all cases, however, senescent cells comprise only a small fraction of tissues. Senescent phenotypes have been studied largely in relatively homogeneous populations of cultured cells. In vivo, senescent cells are generally identified by a small number of markers, but whether and how these markers vary among individual cells is unknown. We therefore utilized a combination of single‐cell isolation and a nanofluidic PCR platform to determine the contributions of individual cells to the overall gene expression profile of senescent human fibroblast populations. Individual senescent cells were surprisingly heterogeneous in their gene expression signatures. This cell‐to‐cell variability resulted in a loss of correlation among the expression of several senescence‐associated genes. Many genes encoding senescence‐associated secretory phenotype (SASP) factors, a major contributor to the effects of senescent cells in vivo, showed marked variability with a subset of highly induced genes accounting for the increases observed at the population level. Inflammatory genes in clustered genomic loci showed a greater correlation with senescence compared to nonclustered loci, suggesting that these genes are coregulated by genomic location. Together, these data offer new insights into how genes are regulated in senescent cells and suggest that single markers are inadequate to identify senescent cells in vivo.     

Emerging roles of extracellular vesicles in cellular senescence and aging

Cellular senescence is a cellular program that prevents the proliferation of cells at risk of neoplastic transformation. On the other hand, age‐related accumulation of senescent cells promotes aging at least partially due to the senescence‐associated secretory phenotype, whereby cells secrete high levels of inflammatory cytokines, chemokines, and matrix metalloproteinases. Emerging evidence, however, indicates that extracellular vesicles (EVs) are important mediators of the effects of senescent cells on their microenvironment. Senescent cells secrete more EphA2 and DNA via EVs, which can promote cancer cell proliferation and inflammation, respectively. Extracellular vesicles secreted from DNA‐damaged cells can also affect telomere regulation. Furthermore, it has now become clear that EVs actually play important roles in many aspects of aging. This review is intended to summarize these recent progresses, with emphasis on relationships between cellular senescence and EVs.     

Quantitative identification of senescent cells in aging and disease

Senescent cells are present in premalignant lesions and sites of tissue damage and accumulate in tissues with age. In vivo identification, quantification and characterization of senescent cells are challenging tasks that limit our understanding of the role of senescent cells in diseases and aging. Here, we present a new way to precisely quantify and identify senescent cells in tissues on a single‐cell basis. The method combines a senescence‐associated beta‐galactosidase assay with staining of molecular markers for cellular senescence and of cellular identity. By utilizing technology that combines flow cytometry with high‐content image analysis, we were able to quantify senescent cells in tumors, fibrotic tissues, and tissues of aged mice. Our approach also yielded the finding that senescent cells in tissues of aged mice are larger than nonsenescent cells. Thus, this method provides a basis for quantitative assessment of senescent cells and it offers proof of principle for combination of different markers of senescence. It paves the way for screening of senescent cells for identification of new senescence biomarkers, genes that bypass senescence or senolytic compounds that eliminate senescent cells, thus enabling a deeper understanding of the senescent state in vivo.     

Senescent vs. non-senescent cells in the human annulus in vivo: Cell harvest with laser capture microdissection and gene expression studies with microarray analysis

Background  

Senescent cells are well-recognized in the aging/degenerating human disc. Senescent cells are viable, cannot divide, remain metabolically active and accumulate within the disc over time. Molecular analysis of senescent cells in tissue offers a special challenge since there are no cell surface markers for senescence which would let one use fluorescence-activated cell sorting as a method for separating out senescent cells.     

To clear,or not to clear (senescent cells)? That is the question

Cellular senescence is an anti‐proliferative program that restricts the propagation of cells subjected to different kinds of stress. Cellular senescence was initially described as a cell‐autonomous tumor suppressor mechanism that triggers an irreversible cell cycle arrest that prevents the proliferation of damaged cells at risk of neoplastic transformation. However, discoveries during the last decade have established that senescent cells can also impact the surrounding tissue microenvironment and the neighboring cells in a non‐cell‐autonomous manner. These non‐cell‐autonomous activities are, in part, mediated by the selective secretion of extracellular matrix degrading enzymes, cytokines, chemokines and immune modulators, which collectively constitute the senescence‐associated secretory phenotype. One of the key functions of the senescence‐associated secretory phenotype is to attract immune cells, which in turn can orchestrate the elimination of senescent cells. Interestingly, the clearance of senescent cells seems to be critical to dictate the net effects of cellular senescence. As a general rule, the successful elimination of senescent cells takes place in processes that are considered beneficial, such as tumor suppression, tissue remodeling and embryonic development, while the chronic accumulation of senescent cells leads to more detrimental consequences, namely, cancer and aging. Nevertheless, exceptions to this rule may exist. Now that cellular senescence is in the spotlight for both anti‐cancer and anti‐aging therapies, understanding the precise underpinnings of senescent cell removal will be essential to exploit cellular senescence to its full potential.     

清除衰老细胞在衰老与老龄化相关疾病中的研究进展

衰老是一个新兴的重要研究领域,随着该领域相关知识的积累和技术的进步,人们逐渐意识到衰老本身可以被针对性地干预,实现延长寿命并且延缓衰老相关疾病的发生发展,具有重要的科学和现实意义.引起个体衰老的众多因素中,衰老细胞的积累被认为是导致器官衰老发生退行性变,最终引起衰老相关疾病的重要原因.近年来,多项研究表明,清除体内衰老细胞可以延缓多种衰老相关疾病的发生,直接证明了衰老细胞是导致衰老相关疾病的重要原因之一,为治疗衰老相关疾病提供了新靶点.细胞衰老是由于损伤积累诱发了细胞周期抑制通路的激活,细胞永久地退出细胞增殖周期.衰老细胞会发生细胞形态、转录谱、蛋白质稳态、表观遗传以及代谢等系列特征的改变,同时衰老细胞对凋亡发生抵抗从而在体内多器官组织积累.衰老细胞会激活炎症因子分泌通路,导致组织局部非感染性炎症微环境,进而导致器官退行性变及多种衰老相关疾病的发生发展.因此针对衰老细胞对凋亡抵抗的特性,多个研究小组通过筛选小分子化合物库,发现某些化合物能够选择性清除衰老细胞,这些小分子化合物被称为"senolytics",意为"衰老细胞杀伤性化合物".衰老细胞杀伤性化合物在多种衰老相关疾病动物模型中能够延缓疾病的发展并延长哺乳动物寿命.因此,靶向杀伤衰老细胞对多种衰老相关疾病的治疗从而提高健康寿命具有重要的临床应用前景.除靶向杀伤衰老细胞策略以外,干细胞移植、基因编辑、异体共生等策略在抗衰老研究发展中也具有重要意义,具有启发性.本文通过汇总近期衰老细胞清除领域的重要进展和多种抗衰老策略,将细胞衰老研究发展史做简要梳理,就细胞衰老与衰老相关疾病的关系作一综述,重点讨论衰老细胞在多种衰老相关疾病中作为治疗靶点的应用潜力,并就其局限性和进一步的研究方向进行探讨.     

Integrating cellular senescence with the concept of damage accumulation in aging: Relevance for clearance of senescent cells

Understanding the aging process and ways to manipulate it is of major importance for biology and medicine. Among the many aging theories advanced over the years, the concept most consistent with experimental evidence posits the buildup of numerous forms of molecular damage as a foundation of the aging process. Here, we discuss that this concept integrates well with recent findings on cellular senescence, offering a novel view on the role of senescence in aging and age‐related disease. Cellular senescence has a well‐established role in cellular aging, but its impact on the rate of organismal aging is less defined. One of the most prominent features of cellular senescence is its association with macromolecular damage. The relationship between cell senescence and damage concerns both damage as a molecular signal of senescence induction and accelerated accumulation of damage in senescent cells. We describe the origin, regulatory mechanisms, and relevance of various damage forms in senescent cells. This view on senescent cells as carriers and inducers of damage puts new light on senescence, considering it as a significant contributor to the rise in organismal damage. Applying these ideas, we critically examine current evidence for a role of cellular senescence in aging and age‐related diseases. We also discuss the differential impact of longevity interventions on senescence burden and other types of age‐related damage. Finally, we propose a model on the role of aging‐related damage accumulation and the rate of aging observed upon senescent cell clearance.     

Galactose‐modified duocarmycin prodrugs as senolytics

Senescence is a stable growth arrest that impairs the replication of damaged, old or preneoplastic cells, therefore contributing to tissue homeostasis. Senescent cells accumulate during ageing and are associated with cancer, fibrosis and many age‐related pathologies. Recent evidence suggests that the selective elimination of senescent cells can be effective on the treatment of many of these senescence‐associated diseases. A universal characteristic of senescent cells is that they display elevated activity of the lysosomal β‐galactosidase, and this has been exploited as a marker for senescence (senescence‐associated β‐galactosidase activity). Consequently, we hypothesized that galactose‐modified cytotoxic prodrugs will be preferentially processed by senescent cells, resulting in their selective killing. Here, we show that different galactose‐modified duocarmycin (GMD) derivatives preferentially kill senescent cells. GMD prodrugs induce selective apoptosis of senescent cells in a lysosomal β‐galactosidase (GLB1)‐dependent manner. GMD prodrugs can eliminate a broad range of senescent cells in culture, and treatment with a GMD prodrug enhances the elimination of bystander senescent cells that accumulate upon whole‐body irradiation treatment of mice. Moreover, taking advantage of a mouse model of adamantinomatous craniopharyngioma (ACP), we show that treatment with a GMD prodrug selectively reduced the number of β‐catenin‐positive preneoplastic senescent cells. In summary, the above results make a case for testing the potential of galactose‐modified duocarmycin prodrugs to treat senescence‐related pathologies.     

Replicative aging down-regulates the myogenic regulatory factors in human myoblasts

Background information. Aging of human skeletal muscle results in a decline in muscle mass and force, and excessive turnover of muscle fibres, such as in muscular dystrophies, further increases this decline. Although it has been shown in rodents, by cross‐age transplantation of whole muscles, that the environment plays an important role in this process, the implication of proliferating aging of the muscle progenitors has been poorly investigated, particularly in humans, since the regulation of cell proliferation differs between rodents and humans. The myogenic differentiation of human myoblasts is regulated by the muscle‐specific regulatory factors. Cross‐talk between the muscle‐specific regulatory factors and the cell cycle regulators is essential for differentiation. The aim of the present study was to determine the effects of replicative senescence on the myogenic programme of human myoblasts. Results. We showed that senescent myoblasts, which could not re‐enter the cell cycle, are still able to differentiate and form multinucleated myotubes. However, these myotubes are significantly smaller. The expression of muscle‐specific regulatory factors and cell cycle regulators was analysed in proliferating myoblasts and compared with senescent cells. We have observed a delay and a decrease in the muscle‐specific regulatory factors and the cyclin‐dependent kinase inhibitor p57 during the early step of differentiation in senescent myoblasts, as well as an increase in the fibroblastic markers. Conclusions. Our results demonstrate that replicative senescence alters the expression of the factors triggering muscle differentiation in human myoblasts and could play a role in the regenerative defects observed in muscular diseases and during normal skeletal‐muscle aging.     

Effect of clonal senescence on low density lipoprotein-receptor activity of bovine arterial endothelial cells

Senescent and young bovine arterial endothelial cells derived from the same parental cell clone were compared to test the effect of in vitro endothelial cell senescence on low density lipoprotein (LDL) and modified LDL-receptor activities. Low density lipoprotein binding and degradation were both increased in cells that underwent a larger number of population doublings, whereas acetyl LDL binding and degradation were unchanged. The increased LDL-receptor activity associated with endothelial cell senescence remained significant after variation of cell number among senescent and young clones was taken into account. Thus, aging endothelial cells seem capable of continuing to process LDL and modified LDL, which could play a role in the arterial wall changes that occur with age in vivo.     

Fat tissue,aging, and cellular senescence

Fat tissue, frequently the largest organ in humans, is at the nexus of mechanisms involved in longevity and age‐related metabolic dysfunction. Fat distribution and function change dramatically throughout life. Obesity is associated with accelerated onset of diseases common in old age, while fat ablation and certain mutations affecting fat increase life span. Fat cells turn over throughout the life span. Fat cell progenitors, preadipocytes, are abundant, closely related to macrophages, and dysdifferentiate in old age, switching into a pro‐inflammatory, tissue‐remodeling, senescent‐like state. Other mesenchymal progenitors also can acquire a pro‐inflammatory, adipocyte‐like phenotype with aging. We propose a hypothetical model in which cellular stress and preadipocyte overutilization with aging induce cellular senescence, leading to impaired adipogenesis, failure to sequester lipotoxic fatty acids, inflammatory cytokine and chemokine generation, and innate and adaptive immune response activation. These pro‐inflammatory processes may amplify each other and have systemic consequences. This model is consistent with recent concepts about cellular senescence as a stress‐responsive, adaptive phenotype that develops through multiple stages, including major metabolic and secretory readjustments, which can spread from cell to cell and can occur at any point during life. Senescence could be an alternative cell fate that develops in response to injury or metabolic dysfunction and might occur in nondividing as well as dividing cells. Consistent with this, a senescent‐like state can develop in preadipocytes and fat cells from young obese individuals. Senescent, pro‐inflammatory cells in fat could have profound clinical consequences because of the large size of the fat organ and its central metabolic role.     

Senescence chips for ultrahigh‐throughput isolation and removal of senescent cells

Cellular senescence plays an important role in organismal aging and age‐related diseases. However, it is challenging to isolate low numbers of senescent cells from small volumes of biofluids for downstream analysis. Furthermore, there is no technology that could selectively remove senescent cells in a high‐throughput manner. In this work, we developed a novel microfluidic chip platform, termed senescence chip, for ultrahigh‐throughput isolation and removal of senescent cells. The core component of our senescence chip is a slanted and tunable 3D micropillar array with a variety of shutters in the vertical direction for rapid cell sieving, taking advantage of the characteristic cell size increase during cellular senescence. The 3D configuration achieves high throughput, high recovery rate, and device robustness with minimum clogging. We demonstrated proof‐of‐principle applications in isolation and enumeration of senescent mesenchymal stem cells (MSCs) from undiluted human whole blood, and senescent cells from mouse bone marrow after total body irradiation, with the single‐cell resolution. After scale‐up to a multilayer and multichannel structure, our senescence chip achieved ultrahigh‐throughput removal of senescent cells from human whole blood with an efficiency of over 70% at a flow rate of 300 ml/hr. Sensitivity and specificity of our senescence chips could be augmented with implementation of multiscale size separation, and identification of background white blood cells using their cell surface markers such as CD45. With the advantages of high throughput, robustness, and simplicity, our senescence chips may find wide applications and contribute to diagnosis and therapeutic targeting of cellular senescence.     

Accumulation of apoptosis‐insensitive human bone marrow‐mesenchymal stromal cells after long‐term expansion

Cells undergo replicative senescence during in vitro expansion, which is induced by the accumulation of cellular damage caused by excessive reactive oxygen species. In this study, we investigated whether long‐term‐cultured human bone marrow mesenchymal stromal cells (MSCs) are insensitive to apoptotic stimulation. To examine this, we established replicative senescent cells from long‐term cultures of human bone marrow MSCs. Senescent cells were identified based on declining population doublings, increased expression of senescence markers p16 and p53 and increased senescence‐associated β‐gal activity. In cell viability assays, replicative senescent MSCs in late passages (i.e. 15–19 passages) resisted damage induced by oxidative stress more than those in early passages did (i.e. 7–10 passages). This resistance occurred via caspase‐9 and caspase‐3 rather than via caspase‐8. The senescent cells are gradually accumulated during long‐term expansion. The oxidative stress‐sensitive proteins ataxia‐telangiectasia mutated and p53 were phosphorylated, and the expression of apoptosis molecules Bax increased, and Bcl‐2 decreased in early passage MSCs; however, the expression of the apoptotic molecules did less change in response to apoptotic stimulation in late‐passage MSCs, suggesting that the intrinsic apoptotic signalling pathway was not induced by oxidative stress in long‐term‐cultured MSCs. Based on these results, we propose that some replicative senescent cells may avoid apoptosis signalling via impairment of signalling molecules and accumulation during long‐term expansion. Copyright © 2016 John Wiley & Sons, Ltd.     

Elimination of p19ARF‐expressing cells protects against pulmonary emphysema in mice

Senescent cells accumulate in tissues during aging and are considered to underlie several aging‐associated phenotypes and diseases. We recently reported that the elimination of p19^ARF‐expressing senescent cells from lung tissue restored tissue function and gene expression in middle‐aged (12‐month‐old) mice. The aging of lung tissue increases the risk of pulmonary diseases such as emphysema, and cellular senescence is accelerated in emphysema patients. However, there is currently no direct evidence to show that cellular senescence promotes the pathology of emphysema, and the involvement of senescence in the development of this disease has yet to be clarified. We herein demonstrated that p19^ARF facilitated the development of pulmonary emphysema in mice. The elimination of p19^ARF‐expressing cells prevented lung tissue from elastase‐induced lung dysfunction. These effects appeared to depend on reduced pulmonary inflammation, which is enhanced after elastase stimulation. Furthermore, the administration of a senolytic drug that selectively kills senescent cells attenuated emphysema‐associated pathologies. These results strongly suggest the potential of senescent cells as therapeutic/preventive targets for pulmonary emphysema.     

Amelioration of age‐related brain function decline by Bruton's tyrosine kinase inhibition

One of the hallmarks of aging is the progressive accumulation of senescent cells in organisms, which has been proposed to be a contributing factor to age‐dependent organ dysfunction. We recently reported that Bruton's tyrosine kinase (BTK) is an upstream component of the p53 responses to DNA damage. BTK binds to and phosphorylates p53 and MDM2, which results in increased p53 activity. Consistent with this, blocking BTK impairs p53‐induced senescence. This suggests that sustained BTK inhibition could have an effect on organismal aging by reducing the presence of senescent cells in tissues. Here, we show that ibrutinib, a clinically approved covalent inhibitor of BTK, prolonged the maximum lifespan of a Zmpste24^?/? progeroid mice, which also showed a reduction in general age‐related fitness loss. Importantly, we found that certain brain functions were preserved, as seen by reduced anxiety‐like behaviour and better long‐term spatial memory. This was concomitant to a decrease in the expression of specific markers of senescence in the brain, which confirms a lower accumulation of senescent cells after BTK inhibition. Our data show that blocking BTK has a modest increase in lifespan in Zmpste24^?/? mice and protects them from a decline in brain performance. This suggests that specific inhibitors could be used in humans to treat progeroid syndromes and prevent the age‐related degeneration of organs such as the brain.     

Positive crosstalk between arginase‐II and S6K1 in vascular endothelial inflammation and aging

Augmented activities of both arginase and S6K1 are involved in endothelial dysfunction in aging. This study was to investigate whether or not there is a crosstalk between arginase and S6K1 in endothelial inflammation and aging in senescent human umbilical vein endothelial cells and in aging mouse models. We show increased arginase‐II (Arg‐II) expression/activity in senescent endothelial cells. Silencing Arg‐II in senescent cells suppresses eNOS‐uncoupling, several senescence markers such as senescence‐associated‐β‐galactosidase activity, p53‐S15, p21, and expression of vascular adhesion molecule‐1 (VCAM1) and intercellular adhesion molecule‐1 (ICAM1). Conversely, overexpressing Arg‐II in nonsenescent cells promotes eNOS‐uncoupling, endothelial senescence, and enhances VCAM1/ICAM1 levels and monocyte adhesion, which are inhibited by co‐expressing superoxide dismutase‐1. Moreover, overexpressing S6K1 in nonsenescent cells increases, whereas silencing S6K1 in senescent cells decreases Arg‐II gene expression/activity through regulation of Arg‐II mRNA stability. Furthermore, S6K1 overexpression exerts the same effects as Arg‐II on endothelial senescence and inflammation responses, which are prevented by silencing Arg‐II, demonstrating a role of Arg‐II as the mediator of S6K1‐induced endothelial aging. Interestingly, mice that are deficient in Arg‐II gene (Arg‐II^?/?) are not only protected from age‐associated increase in Arg‐II, VCAM1/ICAM1, aging markers, and eNOS‐uncoupling in the aortas but also reveal a decrease in S6K1 activity. Similarly, silencing Arg‐II in senescent cells decreases S6K1 activity, demonstrating that Arg‐II also stimulates S6K1 in aging. Our study reveals a novel mechanism of mutual positive regulation between S6K1 and Arg‐II in endothelial inflammation and aging. Targeting S6K1 and/or Arg‐II may decelerate vascular aging and age‐associated cardiovascular disease development.     

Proteostasis failure and cellular senescence in long‐term cultured postmitotic rat neurons

Cellular senescence, a stress‐induced irreversible cell cycle arrest, has been defined for mitotic cells and is implicated in aging of replicative tissues. Age‐related functional decline in the brain is often attributed to a failure of protein homeostasis (proteostasis), largely in postmitotic neurons, which accordingly is a process distinct by definition from senescence. It is nevertheless possible that proteostasis failure and cellular senescence have overlapping molecular mechanisms. Here, we identify postmitotic cellular senescence as an adaptive stress response to proteostasis failure. Primary rat hippocampal neurons in long‐term cultures show molecular changes indicative of both senescence (senescence‐associated β‐galactosidase, p16, and loss of lamin B1) and proteostasis failure relevant to Alzheimer's disease. In addition, we demonstrate that the senescent neurons exhibit resistance to stress. Importantly, treatment of the cultures with an mTOR antagonist, protein synthesis inhibitor, or chemical compound that reduces the amount of protein aggregates relieved the proteotoxic stresses as well as the appearance of senescence markers. Our data propose mechanistic insights into the pathophysiological brain aging by establishing senescence as a primary cell‐autonomous neuroprotective response.