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Adewoye, Adeolu Badi, Dimitris Tampakis, Antonia Follenzi, and Alexandra Stolzing. "Multiparameter flow cytometric detection and quantification of senescent cells in vitro." Biogerontology 21, no. 6 (August 10, 2020): 773–86. http://dx.doi.org/10.1007/s10522-020-09893-9.
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Abstract It has been over half a century since cellular senescence was first noted and characterized, and yet no consensus senescent marker has been reliably established. This challenge is compounded by the complexity and heterogenic phenotypes of senescent cells. This necessitates the use of multiple biomarkers to confidently characterise senescent cells. Despite cytochemical staining of senescence associated-beta-galactosidase being a single marker approach, as well as being time and labour-intensive, it remains the most popular detection method. We have developed an alternative flow cytometry-based method that simultaneously quantifies multiple senescence markers at a single-cell resolution. In this study, we applied this assay to the quantification of both replicative and induced senescent primary cells. Using this assay, we were able to quantify the activity level of SA β-galactosidase, the expression level of p16INK4a and γH2AX in these cell populations. Our results show this flow cytometric approach to be sensitive, robust, and consistent in discriminating senescent cells in different cell senescence models. A strong positive correlation between these commonly- used senescence markers was demonstrated. The method described in this paper can easily be scaled up to accommodate high-throughput screening of senescent cells in applications such as therapeutic cell preparation, and in therapy-induced senescence following cancer treatment.
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Bojko, Agnieszka, Joanna Czarnecka-Herok, Agata Charzynska, Michal Dabrowski, and Ewa Sikora. "Diversity of the Senescence Phenotype of Cancer Cells Treated with Chemotherapeutic Agents." Cells 8, no. 12 (November 23, 2019): 1501. http://dx.doi.org/10.3390/cells8121501.
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It is acknowledged that cancer cells are able to undergo senescence in response to clinically used chemotherapeutics. Moreover, recent years have provided evidence that some drugs can selectively remove senescent cells. Therefore, it is essential to properly identify and characterize senescent cells, especially when it comes to cancer. Senescence was induced in various cancer cell lines (A549, SH-SY-5Y, HCT116, MDA-MB-231, and MCF-7) following treatment with doxorubicin, irinotecan, methotrexate, 5-fluorouracil, oxaliplatin, or paclitaxel. Treatment with tested chemotherapeutics resulted in upregulation of p21 and proliferation arrest without cytotoxicity. A comparative analysis with the use of common senescence markers (i.e., morphology, SA-β-galactosidase, granularity, secretory phenotype, and the level of double-stranded DNA damage) revealed a large diversity in response to the chemotherapeutics used. The strongest senescence inducers were doxorubicin, irinotecan, and methotrexate; paclitaxel had an intermediate effect and oxaliplatin and 5-fluorouracil did not induce senescence. In addition, different susceptibility of cancer cells to senescence was observed. A statistical analysis aimed at finding any relationship between the senescence markers applied did not show clear correlations. Moreover, increased SA-β-gal activity coupled with p21 expression proved not to be an unequivocal senescence marker. This points to a need to simultaneously analyze multiple markers, given their individual limitations.
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Verma, Dinesh Kumar, Bo Am Seo, Anurupa Ghosh, Shi-Xun Ma, Karina Hernandez-Quijada, Julie K. Andersen, Han Seok Ko, and Yong-Hwan Kim. "Alpha-Synuclein Preformed Fibrils Induce Cellular Senescence in Parkinson’s Disease Models." Cells 10, no. 7 (July 5, 2021): 1694. http://dx.doi.org/10.3390/cells10071694.
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Emerging evidence indicates that cellular senescence could be a critical inducing factor for aging-associated neurodegenerative disorders. However, the involvement of cellular senescence remains unclear in Parkinson’s disease (PD). To determine this, we assessed the effects of α-synuclein preformed fibrils (α-syn PFF) or 1-methyl-4-phenylpyridinium (MPP+) on changes in cellular senescence markers, employing α-syn PFF treated-dopaminergic N27 cells, primary cortical neurons, astrocytes and microglia and α-syn PFF-injected mouse brain tissues, as well as human PD patient brains. Our results demonstrate that α-syn PFF-induced toxicity reduces the levels of Lamin B1 and HMGB1, both established markers of cellular senescence, in correlation with an increase in the levels of p21, a cell cycle-arrester and senescence marker, in both reactive astrocytes and microglia in mouse brains. Using Western blot and immunohistochemistry, we found these cellular senescence markers in reactive astrocytes as indicated by enlarged cell bodies within GFAP-positive cells and Iba1-positive activated microglia in α-syn PFF injected mouse brains. These results indicate that PFF-induced pathology could lead to astrocyte and/or microglia senescence in PD brains, which may contribute to neuropathology in this model. Targeting senescent cells using senolytics could therefore constitute a viable therapeutic option for the treatment of PD.
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Kim, Seo Rin, Kai Jiang, Christopher M. Ferguson, Hui Tang, Xiaojun Chen, XiangYang Zhu, LaTonya J. Hickson, Tamara Tchkonia, James L. Kirkland, and Lilach O. Lerman. "Transplanted senescent renal scattered tubular-like cells induce injury in the mouse kidney." American Journal of Physiology-Renal Physiology 318, no. 5 (May 1, 2020): F1167—F1176. http://dx.doi.org/10.1152/ajprenal.00535.2019.
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Cellular senescence, a permanent arrest of cell proliferation, is characterized by a senescence-associated secretory phenotype (SASP), which reinforces senescence and exerts noxious effects on adjacent cells. Recent studies have suggested that transplanting small numbers of senescent cells suffices to provoke tissue inflammation. We hypothesized that senescent cells can directly augment renal injury. Primary scattered tubular-like cells (STCs) acquired from pig kidneys were irradiated by 10 Gy of cesium radiation, and 3 wk later cells were characterized for levels of senescence and SASP markers. Control or senescent STCs were then prelabeled and injected (5 × 105 cells) into the aorta of C57BL/6J mice. Four weeks later, renal oxygenation was studied in vivo using 16.4-T magnetic resonance imaging and function by plasma creatinine level. Renal markers of SASP, fibrosis, and microvascular density were evaluated ex vivo. Per flow cytometry, irradiation induced senescence in 80–99% of STCs, which showed increased gene expression of senescence and SASP markers, senescence-associated β-galactosidase staining, and cytokine levels (especially IL-6) secreted in conditioned medium. Four weeks after injection, cells were detected engrafted in the mouse kidneys with no evidence for rejection. Plasma creatinine and renal tissue hypoxia increased in senescent compared with control cells. Senescent kidneys were more fibrotic, with fewer CD31+ endothelial cells, and showed upregulation of IL-6 gene expression. Therefore, exogenously delivered senescent renal STCs directly injure healthy mouse kidneys. Additional studies are needed to determine the role of endogenous cellular senescence in the pathogenesis of kidney injury and evaluate the utility of senolytic therapy.
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Kritsilis, Marios, Sophia V. Rizou, Paraskevi Koutsoudaki, Konstantinos Evangelou, Vassilis Gorgoulis, and Dimitrios Papadopoulos. "Ageing, Cellular Senescence and Neurodegenerative Disease." International Journal of Molecular Sciences 19, no. 10 (September 27, 2018): 2937. http://dx.doi.org/10.3390/ijms19102937.
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Ageing is a major risk factor for developing many neurodegenerative diseases. Cellular senescence is a homeostatic biological process that has a key role in driving ageing. There is evidence that senescent cells accumulate in the nervous system with ageing and neurodegenerative disease and may predispose a person to the appearance of a neurodegenerative condition or may aggravate its course. Research into senescence has long been hindered by its variable and cell-type specific features and the lack of a universal marker to unequivocally detect senescent cells. Recent advances in senescence markers and genetically modified animal models have boosted our knowledge on the role of cellular senescence in ageing and age-related disease. The aim now is to fully elucidate its role in neurodegeneration in order to efficiently and safely exploit cellular senescence as a therapeutic target. Here, we review evidence of cellular senescence in neurons and glial cells and we discuss its putative role in Alzheimer’s disease, Parkinson’s disease and multiple sclerosis and we provide, for the first time, evidence of senescence in neurons and glia in multiple sclerosis, using the novel GL13 lipofuscin stain as a marker of cellular senescence.
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Coates, Philip J. "Markers of senescence?" Journal of Pathology 196, no. 4 (2002): 371–73. http://dx.doi.org/10.1002/path.1073.
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Wagner, Kay-Dietrich, and Nicole Wagner. "The Senescence Markers p16INK4A, p14ARF/p19ARF, and p21 in Organ Development and Homeostasis." Cells 11, no. 12 (June 19, 2022): 1966. http://dx.doi.org/10.3390/cells11121966.
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It is widely accepted that senescent cells accumulate with aging. They are characterized by replicative arrest and the release of a myriad of factors commonly called the senescence-associated secretory phenotype. Despite the replicative cell cycle arrest, these cells are metabolically active and functional. The release of SASP factors is mostly thought to cause tissue dysfunction and to induce senescence in surrounding cells. As major markers for aging and senescence, p16INK4, p14ARF/p19ARF, and p21 are established. Importantly, senescence is also implicated in development, cancer, and tissue homeostasis. While many markers of senescence have been identified, none are able to unambiguously identify all senescent cells. However, increased levels of the cyclin-dependent kinase inhibitors p16INK4A and p21 are often used to identify cells with senescence-associated phenotypes. We review here the knowledge of senescence, p16INK4A, p14ARF/p19ARF, and p21 in embryonic and postnatal development and potential functions in pathophysiology and homeostasis. The establishment of senolytic therapies with the ultimate goal to improve healthy aging requires care and detailed knowledge about the involvement of senescence and senescence-associated proteins in developmental processes and homeostatic mechanism. The review contributes to these topics, summarizes open questions, and provides some directions for future research.
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Kim, Gee-Hye, Yun Kyung Bae, Ji Hye Kwon, Miyeon Kim, Soo Jin Choi, Wonil Oh, Soyoun Um, and Hye Jin Jin. "Positively Correlated CD47 Activation and Autophagy in Umbilical Cord Blood-Derived Mesenchymal Stem Cells during Senescence." Stem Cells International 2021 (April 15, 2021): 1–13. http://dx.doi.org/10.1155/2021/5582792.
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Autophagy plays a critical role in stem cell maintenance and is related to cell growth and cellular senescence. It is important to find a quality-control marker for predicting senescent cells. This study verified that CD47 could be a candidate to select efficient mesenchymal stem cells (MSCs) to enhance the therapeutic effects of stem cell therapy by analyzing the antibody surface array. CD47 expression was significantly decreased during the expansion of MSCs in vitro ( p < 0.01 ), with decreased CD47 expression correlated with accelerated senescence phenotype, which affected cell growth. UCB-MSCs transfected with CD47 siRNA significantly triggered the downregulation of pRB and upregulation of pp38, which are senescence-related markers. Additionally, autophagy-related markers, ATG5, ATG12, Beclin1, and LC3B, revealed significant downregulation with CD47 siRNA transfection. Furthermore, autophagy flux following treatment with an autophagy inducer, rapamycin, has shown that CD47 is a key player in autophagy and senescence to maintain and regulate the growth of MSCs, suggesting that CD47 may be a critical key marker for the selection of effective stem cells in cell therapy.
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Rossi, Clara, Marco Venturin, Jakub Gubala, Angelisa Frasca, Alberto Corsini, Cristina Battaglia, and Stefano Bellosta. "PURPL and NEAT1 Long Non-Coding RNAs Are Modulated in Vascular Smooth Muscle Cell Replicative Senescence." Biomedicines 11, no. 12 (December 6, 2023): 3228. http://dx.doi.org/10.3390/biomedicines11123228.
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Cellular senescence is characterized by proliferation and migration exhaustion, senescence-associated secretory phenotype (SASP), and oxidative stress. Senescent vascular smooth muscle cells (VSMCs) contribute to cardiovascular diseases and atherosclerotic plaque instability. Since there are no unanimously agreed senescence markers in human VSMCs, to improve our knowledge, we looked for new possible senescence markers. To this end, we first established and characterized a model of replicative senescence (RS) in human aortic VSMCs. Old cells displayed several established senescence-associated markers. They stained positive for the senescence-associated β-galactosidase, showed a deranged proliferation rate, a dramatically reduced expression of PCNA, an altered migratory activity, increased levels of TP53 and cell-cycle inhibitors p21/p16, and accumulated in the G1 phase. Old cells showed an altered cellular and nuclear morphology, downregulation of the expression of LMNB1 and HMGB1, and increased expression of SASP molecules (IL1β, IL6, IL8, and MMP3). In these senescent VSMCs, among a set of 12 manually selected long non-coding RNAs (lncRNAs), we detected significant upregulation of PURPL and NEAT1. We observed also, for the first time, increased levels of RRAD mRNA. The detection of modulated levels of RRAD, PURPL, and NEAT1 during VSMC senescence could be helpful for future studies on potential anti-aging factors.
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Galvis, Daniel, Darren Walsh, Lorna W. Harries, Eva Latorre, and James Rankin. "A dynamical systems model for the measurement of cellular senescence." Journal of The Royal Society Interface 16, no. 159 (October 9, 2019): 20190311. http://dx.doi.org/10.1098/rsif.2019.0311.
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Senescent cells provide a good in vitro model to study ageing. However, cultures of ‘senescent’ cells consist of a mix of cell subtypes (proliferative, senescent, growth-arrested and apoptotic). Determining the proportion of senescent cells is crucial for studying ageing and developing new anti-degenerative therapies. Commonly used markers such as doubling population, senescence-associated β-galactosidase, Ki-67, γH2AX and TUNEL assays capture diverse and overlapping cellular populations and are not purely specific to senescence. A newly developed dynamical systems model follows the transition of an initial culture to senescence tracking population doubling, and the proportion of cells in proliferating, growth-arrested, apoptotic and senescent states. Our model provides a parsimonious description of transitions between these states accruing towards a predominantly senescent population. Using a genetic algorithm, these model parameters are well constrained by an in vitro human primary fibroblast dataset recording five markers at 16 time points. The computational model accurately fits to the data and translates these joint markers into the first complete description of the proportion of cells in different states over the lifetime. The high temporal resolution of the dataset demonstrates the efficacy of strategies for reconstructing the trajectory towards replicative senescence with a minimal number of experimental recordings.
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Frescas, David, Christelle M. Roux, Semra Aygun-Sunar, Anatoli S. Gleiberman, Peter Krasnov, Oleg V. Kurnasov, Evguenia Strom, et al. "Senescent cells expose and secrete an oxidized form of membrane-bound vimentin as revealed by a natural polyreactive antibody." Proceedings of the National Academy of Sciences 114, no. 9 (February 13, 2017): E1668—E1677. http://dx.doi.org/10.1073/pnas.1614661114.
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Studying the phenomenon of cellular senescence has been hindered by the lack of senescence-specific markers. As such, detection of proteins informally associated with senescence accompanies the use of senescence-associated β-galactosidase as a collection of semiselective markers to monitor the presence of senescent cells. To identify novel biomarkers of senescence, we immunized BALB/c mice with senescent mouse lung fibroblasts and screened for antibodies that recognized senescence-associated cell-surface antigens by FACS analysis and a newly developed cell-based ELISA. The majority of antibodies that we isolated, cloned, and sequenced belonged to the IgM isotype of the innate immune system. In-depth characterization of one of these monoclonal, polyreactive natural antibodies, the IgM clone 9H4, revealed its ability to recognize the intermediate filament vimentin. By using 9H4, we observed that senescent primary human fibroblasts express vimentin on their cell surface, and MS analysis revealed a posttranslational modification on cysteine 328 (C328) by the oxidative adduct malondialdehyde (MDA). Moreover, elevated levels of secreted MDA-modified vimentin were detected in the plasma of aged senescence-accelerated mouse prone 8 mice, which are known to have deregulated reactive oxygen species metabolism and accelerated aging. Based on these findings, we hypothesize that humoral innate immunity may recognize senescent cells by the presence of membrane-bound MDA-vimentin, presumably as part of a senescence eradication mechanism that may become impaired with age and result in senescent cell accumulation.
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Chen, Jian, Jing Wang, Matt Lucas, Haizhen Liu, Christina Wheeler, Kyrie Johnson, Kathryn Woodard, et al. "Abstract B022: Targeting senescence cells in cancer and aging by conditionally active biologic therapeutics." Cancer Research 83, no. 2_Supplement_1 (January 15, 2023): B022. http://dx.doi.org/10.1158/1538-7445.agca22-b022.
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Abstract Cellular senescence is characterized by stable cell cycle arrest and a secretory program that modulates the tissue microenvironment. The aberrant accumulation of senescent cells in aged and cancerous tissue triggers inflammatory signaling through a senescence-associated secretory phenotype (SASP), promoting aging and tumor progression. Pharmacologically, clearing senescent cells has been shown to have promising effects against cancer and age-related pathologies in preclinical models as well as human pilot clinical trials. However, current senescent cell elimination strategies are focused on the rare senescence targets that are under-represented on normal cells. To date, there are no universal senescence cell specific markers. Therefore, the use of senolytics in the clinic is still limited due to their cytotoxicity to normal cells. A more effective senescence cell targeted approach is needed to reduce side effects and increase the efficiency of senescence cell removal. Conditionally Active Biologics (CAB) technology is a proprietary platform that is unique in its ability to selectively activate in the context of diseased tissues, but not normal tissues, taking advantage of the acidic pH conditions in the inflammatory tumor/aged tissue microenvironment. Senescent cells, like tumor cells, are dependent on glycolysis for energy metabolism, and thus generate a glycolytic microenvironment (GME) that resembles that of the tumor microenvironment. Since our CAB technology is already being deployed in cancer therapy by targeting cell surface markers such as AXL, ROR2, and CTLA4 in the clinical stage, we explored whether CAB technology will allow selective removal of senescent cells in SASP-associated microenvironments such as the GME. To identify senescence-specific surface antigens that are broadly and specifically upregulated in senescent cells, we screened our CAB cell surface antigen library in different senescence models including CDK4/6 inhibitor-induced senescent human breast cancer cells, human lung fibroblast cells, as well as in vivo aging mouse tissues and chronic kidney disease mouse model. We have identified several novel senescence markers which show significantly elevated expression in senescent cells. Furthermore, in vitro binding assays demonstrated that CAB antibodies targeting these senescence markers have no or very low binding to the target antigen on senescence cells in alkaline physiological conditions but have strong binding in glycolytic/acidic SASP conditions. Moreover, we performed antibody-dependent cell-mediated cytotoxicity assays for evaluating the functional activities of CAB antibodies against the senescence targets. Our data demonstrated that CAB antibodies were more potent against senescent cells compared to proliferating cells and displayed high selectivity for the glycolytic/acidic microenvironment condition. In conclusion, CAB technology should provide a new generation of biologics with an increased safety margin and therapeutic index in the clinic targeting senescence cells in cancer and age-related diseases. Citation Format: Jian Chen, Jing Wang, Matt Lucas, Haizhen Liu, Christina Wheeler, Kyrie Johnson, Kathryn Woodard, Cathy Chang, Gerhard Frey, William J. Boyle, Jay M. Short. Targeting senescence cells in cancer and aging by conditionally active biologic therapeutics [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B022.
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Bernadotte, Alexandra, Victor M. Mikhelson, and Irina M. Spivak. "Markers of cellular senescence. Telomere shortening as a marker of cellular senescence." Aging 8, no. 1 (January 23, 2016): 3–11. http://dx.doi.org/10.18632/aging.100871.
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Yang, Niuxin, Masato Nakagawa, Aki Nishiura, Masahiro Yamada, Hidetoshi Morikuni, Yoshitomo Honda, and Naoyuki Matsumoto. "Identification of Senescent Cells in Peri-Implantitis and Prevention of Mini-Implant Loss Using Senolytics." International Journal of Molecular Sciences 24, no. 3 (January 28, 2023): 2507. http://dx.doi.org/10.3390/ijms24032507.
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Peri-implantitis is a disease that causes the detachment of orthodontic mini-implants. Recently, stress-induced senescent cells have been reported to be involved in various inflammatory diseases. Senescent cell-eliminating drugs, termed “senolytics”, can improve the symptoms of such diseases. However, the relationship between peri-implantitis and senescent cells remains unclear. In this study, we evaluated the presence of senescent cells in a rat peri-implantitis model developed with a gum ring. The effect on bone resorption and implant loss was also investigated with and without senolytics (Dasatinib and Quercetin). The number of senescence markers (p19, p21, and p16) was found to increase, and implant detachment occurred in 24 days. After the administration of senolytics, the number of senescence markers decreased and implant detachment was inhibited. This study suggests that senescent cells aggravate peri-implantitis and senolytic administration latently reduces implant loss by inhibiting senescence-related mechanisms.
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Kawka, Edyta, Janusz Witowski, Pilar Sandoval, Andras Rudolf, Angela Rynne Vidal, Manuel Lopez Cabrera, and Achim Jörres. "Epithelial-To-Mesenchymal Transition and Migration of Human Peritoneal Mesothelial Cells Undergoing Senescence." Peritoneal Dialysis International: Journal of the International Society for Peritoneal Dialysis 39, no. 1 (January 2019): 35–41. http://dx.doi.org/10.3747/pdi.2017.00244.
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Background Epithelial-to-mesenchymal transition (EMT) of human peritoneal mesothelial cells (HPMCs) contributes to fibrotic thickening of the peritoneum that develops in patients on peritoneal dialysis (PD). The process is thought to be largely mediated by transforming growth factor-beta (TGF-β). As TGF-β has also been implicated in senescence of HPMCs, we have performed an exploratory study to examine if senescent HPMCs can undergo EMT. Methods Omentum-derived HPMCs were rendered senescent by repeated passages in culture. Features of EMT were assessed by immunostaining and quantitative polymerase chain reaction (qPCR) at various stages of the HPMC lifespan and after treatment with or without TGF-β. The motility of HPMCs was assessed in a scratch wound migration assay. Results Replicative senescence of HPMCs was associated with a gradual increase in the constitutive expression of EMT markers, including increased production of extracellular matrix proteins. However, senescent HPMCs also retained epithelial cell features such as cytokeratin, calretinin, and E-cadherin and showed decreased, rather than increased, motility. In contrast, exposure to TGF-β resulted in an up-regulation of mesenchymal markers and down-regulation of epithelial markers. Such effects of TGF-β occurred both in young and senescent cells, although they were less pronounced in senescence. Conclusions Senescence of HPMCs is associated with spontaneous development of several EMT features. At the same time, senescent HPMCs preserve epithelial cell-like characteristics and are less prone to develop a full EMT phenotype in response to TGF-β. These observations may support the concept of cellular senescence being antagonistically pleiotropic with regard to EMT.
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Norton, Emma J., Leslie R. Bridges, Lawrence C. Kenyon, Margaret M. Esiri, Dorothy C. Bennett, and Atticus H. Hainsworth. "Cell Senescence and Cerebral Small Vessel Disease in the Brains of People Aged 80 Years and Older." Journal of Neuropathology & Experimental Neurology 78, no. 11 (August 28, 2019): 1066–72. http://dx.doi.org/10.1093/jnen/nlz088.
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Abstract Cerebral small vessel disease (cSVD) in penetrating arteries is a major cause of age-related morbidity. Cellular senescence is a molecular process targeted by novel senolytic drugs. We quantified senescence in penetrating arteries and tested whether myocyte senescence was associated with cSVD. We immunolabeled subcortical white matter of older persons (age 80–96 years, n = 60) with minimal AD, using antibodies to 2 established senescence markers (H3K9me3, γH2AX) and a myocyte marker (hSMM). Within the walls of penetrating arteries (20–300 µm), we quantified senescence-associated heterochromatic foci (SAHF)-positive nuclei, cell density (nuclei/µm2), and sclerotic index (SI). Senescent-appearing mural cells were present in small arteries of all cases. cSVD cases exhibited a lower proportion of senescent-appearing cells and lower area fraction (AF%) of SAHF-positive nuclei compared to controls (p = 0.014, 0.016, respectively). cSVD severity and SI both correlated negatively with AF% (p = 0.013, 0.002, respectively). Mural cell density was lower (p < 0.001) and SI higher (p < 0.001) in cSVD, relative to controls. In conclusion, senescent myocyte-like cells were universal in penetrating arteries of an AD-free cohort aged 80 years and older. Senescent-appearing nuclei were more common in persons aged 80 years and older without cSVD compared to cSVD cases, indicating caution in senolytic drug prescribing. Myocyte senescence and cSVD may represent alternative vessel fates in the aging human brain.
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Choi, Eun-Jeong, In Sup Kil, and Eun-Gyung Cho. "Extracellular Vesicles Derived from Senescent Fibroblasts Attenuate the Dermal Effect on Keratinocyte Differentiation." International Journal of Molecular Sciences 21, no. 3 (February 4, 2020): 1022. http://dx.doi.org/10.3390/ijms21031022.
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The skin is a multilayered and primary defensive organ. Intimate intercellular communication in the skin is necessary to ensure effective surveillance. Extracellular vesicles (EVs) are being explored for their involvement in intercellular skin communication. The aim of this study was to evaluate how human dermal fibroblasts (HDFs) accelerate EV production during senescence and the effects of senescence-associated EVs on epidermal homeostasis. Replicative senescent HDFs were assessed with senescence-associated β-galactosidase staining and the expression of senescence-related markers. Isolated EVs were characterized by dynamic light scattering and EV marker expression. EVs secreted from untreated young or senescent HDFs, or from those treated with a nSMase inhibitor, antioxidant, and lysosomal activity regulators, were determined by sandwich ELISA for CD81. Human epidermal keratinocytes were treated with young- and senescent HDF-derived EVs. Compared to young HDFs, senescent HDFs produced relatively high levels of EVs due to the increased nSMase activity, oxidative stress, and altered lysosomal activity. The nSMase inhibitor, antioxidant, and agents that recovered lysosomal activity reduced EV secretion in senescent HDFs. Relative to young HDF-derived EVs, senescent HDF-derived EVs were less supportive in keratinocyte differentiation and barrier function but increased proinflammatory cytokine IL-6 levels. Our study suggests that dermis-derived EVs may regulate epidermal homeostasis by reflecting cellular status, which provides insight as to how the dermis communicates with the epidermis and influences skin senescence.
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Dewald, Hannah K., Ricardo Iván Martínez-Zamudio, Themistoklis Vasilopoulos, Joycelyn Radeny, Utz Herbig, and Patricia Fitzgerald-Bocarsly. "Senescence-associated β-galactosidase activity and other markers of senescence are present in human peripheral blood CD8+ cells during healthy aging." Journal of Immunology 206, no. 1_Supplement (May 1, 2021): 98.31. http://dx.doi.org/10.4049/jimmunol.206.supp.98.31.
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Abstract Aging is associated with a decline in the immune system, termed “immunosenescence”. Cellular senescence is defined by persistent cellular growth arrest and overall loss of function. If immune cells undergo cellular senescence in vivo, and if it has a role in immunosenesence, remains controversial due to the lack of specific markers to identify these cells. Human CD8+ T cells from younger and older donors were isolated from blood and labeled with a fluorescent substrate for senescence-associated b-galactosidase (SA-βGal). CD8+ T cells were sorted based on SA-βGal and analyzed by qRT-PCR, RNA sequencing, and microscopy. We found a higher percentage of CD8+ T cells with high SA-βGal activity in the older cohort. SA-βGal high CD8+ T cells displayed increased p21, p16INK4a, and inflammatory cytokine transcripts. They also had greater DNA damage response foci and p16INK4a protein and had deficient proliferation. SA-βGal increased as T cells matured, which was consistent regardless of age. Intriguingly, a portion of effector cells did not exhibit a senescent phenotype and maintained low SA-βGal. RNA sequencing of CD8+ T cells revealed cells with low, medium, and high SA-βGal had distinct profiles. In addition, SA-βGal high CD8+ T cells had larger mitochondrial mass than cells with low SA-βGal as measured by MitoID staining. This may indicate differences in metabolism, which may contribute to senescence. The presence of p16INK4a, DNA damage, and inflammatory cytokines and deficient proliferation demonstrate that SA-βGal is a strong indicator of cellular senescence in CD8+. T cells. SA-βGal can be leveraged to further identify senescent immune cells and elucidate cellular senescence’s role in immunosenesence.
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Dewald, Hannah K., Ricardo Iván Martínez-Zamudio, Themistoklis Vasilopoulos, Utz Herbig, and Patricia Fitzgerald-Bocarsly. "Senescence-associated β-galactosidase activity and other markers of senescence are present in human peripheral blood mononuclear cells during healthy aging." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 154.15. http://dx.doi.org/10.4049/jimmunol.204.supp.154.15.
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Abstract Aging is associated with a decline in the immune system, termed “immunosenescence”. Cellular senescence is defined by persistent cellular growth arrest and overall loss of function. If immune cells undergo cellular senescence in vivo and whether cellular senescence has a role in immunosenesence remains controversial due to the lack of specific markers to reliably identify these cells in blood. Freshly isolated human peripheral blood mononuclear cells from younger and older donors were fluorescently labeled for senescence-associated β-galactosidase (SA-βGal). CD8+ T cells were sorted based on SA-βGal and analyzed by qRT-PCR, RNA sequencing, and microscopy. We found a higher percentage of cells with high SA-βGal in the older vs. younger cohort. This increase was apparent in NK cells, CD4+ and CD8+ T cells. CD8+ T cells with high SA-βGal displayed increased transcripts of p21, p16INK4a, and inflammatory cytokines. They also had greater DNA damage response foci and p16INK4a protein. SA-βGal increased as T cells matured, which was consistent regardless of age. Intriguingly, a portion of T effector cells did not exhibit a senescent phenotype and maintained low SA-βGal. RNA sequencing of CD8+ T cells revealed alterations in gene expression were associated with SA-βGal and cells with low, medium, and high SA-βGal shared similar profiles, independent of age. The presence of p16INK4a, DNA damage, and inflammatory cytokines demonstrate that SA-βGal is a strong indicator of cellular senescence. SA-βGal can be leveraged to further identify senescent immune cells and elucidate cellular senescence’s role in immunosenesence. Understanding this connection in healthy aging will further our understanding of premature aging seen chronic diseases.
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Macieira-Coelho, A. "Markers of `cell senescence'." Mechanisms of Ageing and Development 103, no. 1 (June 1998): 105–9. http://dx.doi.org/10.1016/s0047-6374(98)00038-4.
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Martyshkina, Yuliya S., Valeriy P. Tereshchenko, Daria A. Bogdanova, and Stanislav A. Rybtsov. "Reliable Hallmarks and Biomarkers of Senescent Lymphocytes." International Journal of Molecular Sciences 24, no. 21 (October 27, 2023): 15653. http://dx.doi.org/10.3390/ijms242115653.
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The phenomenon of accumulation of senescent adaptive immunity cells in the elderly is attracting attention due to the increasing risk of global epidemics and aging of the global population. Elderly people are predisposed to various infectious and age-related diseases and are at higher risk of vaccination failure. The accumulation of senescent cells increases age-related background inflammation, “Inflammaging”, causing lymphocyte exhaustion and cardiovascular, neurodegenerative, autoimmune and cancer diseases. Here, we present a comprehensive contemporary review of the mechanisms and phenotype of senescence in the adaptive immune system. Although modern research has not yet identified specific markers of aging lymphocytes, several sets of markers facilitate the separation of the aging population based on normal memory and exhausted cells for further genetic and functional analysis. The reasons for the higher predisposition of CD8+ T-lymphocytes to senescence compared to the CD4+ population are also discussed. We point out approaches for senescent-lymphocyte-targeting markers using small molecules (senolytics), antibodies and immunization against senescent cells. The suppression of immune senescence is the most relevant area of research aimed at developing anti-aging and anti-cancer therapy for prolonging the lifespan of the global population.
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Kaur, Gagandeep, Isaac K. Sundar, and Irfan Rahman. "p16-3MR: A Novel Model to Study Cellular Senescence in Cigarette Smoke-Induced Lung Injuries." International Journal of Molecular Sciences 22, no. 9 (May 3, 2021): 4834. http://dx.doi.org/10.3390/ijms22094834.
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Cellular senescence and lung aging are associated with the pathogenesis of chronic obstructive pulmonary disease (COPD). COPD progresses with aging, and chronic smoking is the key susceptibility factor in lung pathological changes concurrent with mitochondrial dysfunction and biological aging. However, these processes involving cigarette smoke (CS)-mediated lung cellular senescence are difficult to distinguish. One of the impediments to studying cellular senescence in relation to age-related lung pathologies is the lack of a suitable in vivo model. In view of this, we provide evidence that supports the suitability of p16-3MR mice to studying cellular senescence in CS-mediated and age-related lung pathologies. p16-3MR mice have a trimodal reporter fused to the promoter of the p16INK4a gene that enables detection, isolation, and selective elimination of senescent cells, thus making them a suitable model to study cellular senescence. To determine their suitability in CS-mediated lung pathologies, we exposed young (12–14 months) and old (17–20 months) p16-3MR mice to 30 day CS exposure and studied the expression of senescent genes (p16, p21, and p53) and SASP-associated markers (MMP9, MMP12, PAI-1, and FN-1) in air- and CS-exposed mouse lungs. Our results showed that this model could detect cellular senescence using luminescence and isolate cells undergoing senescence with the help of tissue fluorescence in CS-exposed young and old mice. Our results from the expression of senescence markers and SASP-associated genes in CS-exposed young and old p16-3MR mice were comparable with increased lung cellular senescence and SASP in COPD. We further showed alteration in the; (i) tissue luminescence and fluorescence, (ii) mRNA and protein expressions of senescent markers and SASP genes, and (iii) SA-β-gal activity in CS-exposed young and old p16-3MR mice as compared to their air controls. Overall, we showed that p16-3MR is a competent model for studying the cellular senescence in CS-induced pathologies. Hence, the p16-3MR reporter mouse model may be used as a novel tool for understanding the pathobiology of cellular senescence and other underlying mechanisms involved in COPD and fibrosis.
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Chen, Jian, Christina Wheeler, Jing Wang, Matt Lucas, Haizhen Liu, Kyrie Johnson, Kathryn Woodard, et al. "Abstract 4795: Conditionally active biologics eliminates senescence cells in cancer and aging." Cancer Research 83, no. 7_Supplement (April 4, 2023): 4795. http://dx.doi.org/10.1158/1538-7445.am2023-4795.
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Abstract The aberrant accumulation of senescent cells in aged and cancerous tissue triggers inflammatory signaling through a senescence-associated secretory phenotype (SASP), promoting aging and tumor progression. Pharmacologically, clearing senescent cells has been shown to have promising effects against cancer and age-related pathologies in preclinical models as well as early human clinical trials. However, current senescent cell elimination strategies are focused on targets that do not distinguish between hyper-inflamed or SASP senescent cells versus relatively non-inflamed senescent cells. In addition, current senescence targets are predominately intracellular with important functions in normal cell population. Therefore, the use of senolytics in the clinic is still limited due to their cytotoxicity to either normal cells or potentially beneficial senescent cells. A more effective senescence cell targeted approach is needed to reduce side effects and increase the efficiency of senescence cell removal or reduction. Conditionally Active Biologics (CAB) technology is a proprietary platform that is unique in its ability to selectively activate in the context of diseased tissues, but not normal tissues, taking advantage of the acidic pH conditions in the inflammatory tumor/aged tissue microenvironment. Senescent cells, similar to tumor cells, are dependent on glycolysis to support molecule synthesis, for hyperinflammation in the case of senescent cells, and thus generate a glycolytic microenvironment (GME), resembling that of the tumor microenvironment. Since our CAB technology is already being deployed in cancer therapy by targeting cell surface markers such as AXL, ROR2, and CTLA4 in clinical studies, we explored whether CAB technology allows selective removal of senescent cells in SASP-associated microenvironments. To identify senescence-specific surface antigens that are broadly and specifically upregulated in senescent cells, we screened our CAB cell surface antigen library in different senescence in vitro models, in vivo aging mouse tissues and a chronic kidney disease mouse model. We have identified several novel senescence markers. Furthermore, in vitro binding assays demonstrated that CAB antibodies targeting these senescence markers have no or very low binding to the target antigen on senescence cells in physiological, alkaline conditions, but have strong binding in glycolytic, acidic SASP conditions. Moreover, we performed antibody-dependent cell-mediated cytotoxicity assays and demonstrated that CAB antibodies were more potent against senescent cells compared to proliferating cells and displayed high selectivity for the glycolytic, acidic microenvironment condition. In conclusion, CAB technology provides a new generation of biologics with an increased safety margin and therapeutic index targeting SASP, acidic senescence cells in cancer and age-related diseases. Citation Format: Jian Chen, Christina Wheeler, Jing Wang, Matt Lucas, Haizhen Liu, Kyrie Johnson, Kathryn Woodard, Solmarie Joyner, Cathy Chang, William J. Boyle, Jay M. Short. Conditionally active biologics eliminates senescence cells in cancer and aging. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4795.
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Fischer, Bernard M., Jessica K. Wong, Simone Degan, Apparao B. Kummarapurugu, Shuo Zheng, Prashamsha Haridass, and Judith A. Voynow. "Increased expression of senescence markers in cystic fibrosis airways." American Journal of Physiology-Lung Cellular and Molecular Physiology 304, no. 6 (March 15, 2013): L394—L400. http://dx.doi.org/10.1152/ajplung.00091.2012.
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Cystic Fibrosis (CF) is a chronic lung disease characterized by chronic neutrophilic airway inflammation and increased levels of neutrophil elastase (NE) in the airways. We have previously reported that NE treatment triggers cell cycle arrest. Cell cycle arrest can lead to senescence, a complete loss of replicative capacity. Importantly, senescent cells can be proinflammatory and would perpetuate CF chronic inflammation. By immunohistochemistry, we evaluated whether airway sections from CF and control subjects expressed markers of senescence, including p16INK4a(p16), a cyclin-dependent kinase inhibitor, phospho-Histone H2A.X (γH2A.X), and phospho-checkpoint 2 kinase (phospho-Chk2), which are also DNA damage response markers. Compared with airway epithelium from control subjects, CF airway epithelium had increased levels of expression of all three senescence markers. We hypothesized that the high load of NE in the CF airway triggers epithelial senescence by upregulating expression of p16, which inhibits cyclin-dependent kinase 4 (CDK4). Normal human bronchial epithelial (NHBE) cells, cultured in air-liquid interface were treated with NE (0, 200, and 500 nM) to induce visible injury. Total cell lysates were collected and evaluated by Western analysis for p16 protein expression and CDK4 kinase activity. NE significantly increased p16 expression and decreased CDK4 kinase activity in NHBE cells. These results support the concept that NE triggers expression of senescence markers in CF airway epithelial cells.
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Salech, Felipe, Carol D. SanMartín, Jorge Concha-Cerda, Esteban Romero-Hernández, Daniela P. Ponce, Gianella Liabeuf, Nicole K. Rogers, et al. "Senescence Markers in Peripheral Blood Mononuclear Cells in Amnestic Mild Cognitive Impairment and Alzheimer’s Disease." International Journal of Molecular Sciences 23, no. 16 (August 20, 2022): 9387. http://dx.doi.org/10.3390/ijms23169387.
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Recent studies suggest that cellular senescence plays a role in Alzheimer’s Disease (AD) pathogenesis. We hypothesize that cellular senescence markers might be tracked in the peripheral tissues of AD patients. Senescence hallmarks, including altered metabolism, cell-cycle arrest, DNA damage response (DDR) and senescence secretory associated phenotype (SASP), were measured in peripheral blood mononuclear cells (PBMCs) of healthy controls (HC), amnestic mild cognitive impairment (aMCI) and AD patients. Senescence-associated βeta-galactosidase (SA-β-Gal) activity, G0-G1 phase cell-cycle arrest, p16 and p53 were analyzed by flow cytometry, while IL-6 and IL-8 mRNA were analyzed by qPCR, and phosphorylated H2A histone family member X (γH2AX) was analyzed by immunofluorescence. Senescent cells in the brain tissue were determined with lipofuscin staining. An increase in the number of senescent cells was observed in the frontal cortex and hippocampus of advanced AD patients. PBMCs of aMCI patients, but not in AD, showed increased SA-β-Gal compared with HCs. aMCI PBMCs also had increased IL-6 and IL8 mRNA expression and number of cells arrested at G0-G1, which were absent in AD. Instead, AD PBMCs had significantly increased p16 and p53 expression and decreased γH2Ax activity compared with HC. This study reports that several markers of cellular senescence can be measured in PBMCs of aMCI and AD patients.
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Zhou, Lin, Jarin Snyder, Katherine C. Murphy, Kelly D. DeMarco, Sachliv Chana, Karl Simin, Zhong Jiang, and Marcus Ruscetti. "Abstract A028: Dissecting the role of cellular senescence in prostate cancer initiation and immune suppression." Cancer Research 83, no. 11_Supplement (June 2, 2023): A028. http://dx.doi.org/10.1158/1538-7445.prca2023-a028.
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Abstract Prostate cancer is the most common malignancy and the second leading cause of cancer-related death in American men. Although many disease cases remain latent for decades, others progress to malignant prostate cancer with high rates of mortality. The molecular underpinnings driving malignancy are still poorly understood, and there is an urgent need to identify mechanisms and informative biomarkers of disease progression. Using the Pb-Cre+; Ptenfl/fl (CP) mouse model, we and others found that Pten genomic loss, a common occurrence in human prostate cancer, leads to induction of cellular senescence, senescence-associated inflammation, and immune suppression during tumor onset. Similarly, senescence markers have also been identified in benign and early prostate cancer lesions in humans. This suggests that senescence may play a yet uncharacterized functional role in prostate tumorigenesis. Cellular senescence is a damage-induced pathway leading to durable growth arrest and has long been considered a potent tumor suppressive mechanism that limits the ability of pre-malignant cells to proliferate and develop into malignant tumors. However, recent findings suggest that the accumulation of senescent cells can paradoxically lead to chronic inflammation that fosters an environment for eventual tumor development and progression through the induction of growth and inflammatory factors collectively known as the senescence-associated secretory phenotype (SASP). Thus, it remains unclear whether senescence or specific senescent cell populations block or rather promote prostate tumor development. Leveraging the CP prostate cancer mouse model and genetic tools to track senescent cells, we observed that markers of senescence (SA-β-gal, p16, Bcl2) and the inflammatory SASP (NF-𝜅B, IL-1b, CXCL1, CSF-1) were not only present in early prostate intraepithelial neoplasia (PIN) lesions but remained elevated following adenocarcinoma development and progression, and could be found in both epithelial cells as well as other immune and stromal populations. Expression of the canonical senescence marker p16 also correlated with disease progression in human prostate cancer samples. Moreover, senescent cell accumulation was associated with a reduction in cytotoxic lymphocytes and an influx of suppressive myeloid populations that we and others have previously shown contribute to prostate cancer progression. Senolytic approaches to eliminate these senescent cell populations reduced progression to adenocarcinoma and remodeled the immune system to reverse myeloid-mediated immune suppression and activate cytotoxic lymphocyte immunity. Together, our preliminary results suggest that senescence might contribute to prostate cancer initiation and progression, and that removal of senescent cells could delay tumor onset and reverse prostate cancer immune suppression. Citation Format: Lin Zhou, Jarin Snyder, Katherine C. Murphy, Kelly D. DeMarco, Sachliv Chana, Karl Simin, Zhong Jiang, Marcus Ruscetti. Dissecting the role of cellular senescence in prostate cancer initiation and immune suppression [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A028.
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Chen, Jian, Jian Wang, Haizheng Liu, Cathy Chang, William J. Boyle, and Jay M. Short. "Abstract 2969: Targeting novel senescence markers by conditionally active biologics eliminates senescence-associated secretory phenotype in in vitro and in vivo models." Cancer Research 84, no. 6_Supplement (March 22, 2024): 2969. http://dx.doi.org/10.1158/1538-7445.am2024-2969.
Full textAbstract:
Abstract Clearing senescent cells has been shown to have promising effects against cancer and age-related pathologies in preclinical models as well as early human clinical trials. However, current senescent cell elimination strategies are focused on targets that do not distinguish between hyper-inflamed or SASP senescent cells versus relatively non-inflamed senescent cells. The use of senolytics in the clinic is still limited due to their cytotoxicity to either normal cells or potentially beneficial senescent cells. A more effective senescence cell targeted approach is needed to reduce side effects and increase the efficiency of senescence cell removal or reduction. Conditionally Active Biologics (CAB) technology is a proprietary platform unique in its ability to be selectively active in the context of diseased tissues, but not normal tissues. The aberrant accumulation of senescent cells in aged and cancerous tissue triggers inflammatory signaling through a senescence-associated secretory phenotype (SASP), promoting aging and tumor progression. Since our CAB technology is currently being tested as a cancer therapy by targeting cell surface markers such as AXL, ROR2, and CTLA4 in clinical studies, we explored whether CAB technology allows selective removal of senescent cells in SASP-associated microenvironments. In our previous report, we demonstrated several novel senescence-specific surface antigens upregulated in senescent cells (1). CAB antibodies targeting these senescence markers have no or very low binding to the target antigen on senescence cells in physiological, alkaline conditions, but have strong binding in glycolytic, acidic SASP conditions in in vitro binding assays. In in vitro antibody-dependent cell-mediated cytotoxicity assays, CAB antibodies were more potent against senescent cells compared to proliferating cells and displayed high selectivity for the glycolytic, acidic condition. To study whether CAB antibody reduces senescent cell occurrence in vivo, we developed a unilateral ureteral obstruction (UUO) mouse model to induce senescent cells, fibrosis, and inflammation in mouse kidney. The mice treated with CAB antibodies had a significant reduction of senescent cells and inflammatory cells infiltrated in the renal cortex, compared to the mice treated with benchmark and isotype antibodies in UUO kidneys. Thus, the CAB antibody-based therapeutic is a promising strategy for senescent cell removal in an inflammatory microenvironment. In conclusion, CAB technology provides a new generation of biologics with an increased safety margin and therapeutic index targeting SASP, acidic senescence cells in cancer and age-related diseases. (1). Chen J, Wang J, Liu H, Chang C, Boyle WJ, and Short JM. Abstract 4795: Conditionally active biologics eliminates senescence cells in cancer and aging. Cancer Res (2023) 83. Citation Format: Jian Chen, Jian Wang, Haizheng Liu, Cathy Chang, William J. Boyle, Jay M. Short. Targeting novel senescence markers by conditionally active biologics eliminates senescence-associated secretory phenotype in in vitro and in vivo models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2969.
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Waters, David W., Michael Schuliga, Prabuddha S. Pathinayake, Lan Wei, Hui-Ying Tan, Kaj E. C. Blokland, Jade Jaffar, et al. "A Senescence Bystander Effect in Human Lung Fibroblasts." Biomedicines 9, no. 9 (September 4, 2021): 1162. http://dx.doi.org/10.3390/biomedicines9091162.
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Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterised by a dense fibrosing of the lung parenchyma. An association between IPF and cellular senescence is well established and several studies now describe a higher abundance of senescent fibroblasts and epithelial cells in the lungs of IPF patients compared with age-matched controls. The cause of this abnormal accumulation of senescent cells is unknown but evidence suggests that, once established, senescence can be transferred from senescent to non-senescent cells. In this study, we investigated whether senescent human lung fibroblasts (LFs) and alveolar epithelial cells (AECs) could induce a senescent-like phenotype in “naïve” non-senescent LFs in vitro. Primary cultures of LFs from adult control donors (Ctrl-LFs) with a low baseline of senescence were exposed to conditioned medium (CM) from: (i) Ctrl-LFs induced to become senescent using H2O2 or etoposide; (ii) LFs derived from IPF patients (IPF-LFs) with a high baseline of senescence; or (iii) senescence-induced A549 cells, an AEC line. Additionally, ratios of non-senescent Ctrl-LFs and senescence-induced Ctrl-LFs (100:0, 0:100, 50:50, 90:10, 99:1) were co-cultured and their effect on induction of senescence measured. We demonstrated that exposure of naïve non-senescent Ctrl-LFs to CM from senescence-induced Ctrl-LFs and AECs and IPF-LFs increased the markers of senescence including nuclear localisation of phosphorylated-H2A histone family member X (H2AXγ) and expression of p21, IL-6 and IL-8 in Ctrl-LFs. Additionally, co-cultures of non-senescent and senescence-induced Ctrl-LFs induced a senescent-like phenotype in the non-senescent cells. These data suggest that the phenomenon of “senescence-induced senescence” can occur in vitro in primary cultures of human LFs, and provides a possible explanation for the abnormal abundance of senescent cells in the lungs of IPF patients.
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Herrmann, Jaqueline, Milen Babic, Markus Tölle, Kai-Uwe Eckardt, Markus van der Giet, and Mirjam Schuchardt. "A Novel Protocol for Detection of Senescence and Calcification Markers by Fluorescence Microscopy." International Journal of Molecular Sciences 21, no. 10 (May 14, 2020): 3475. http://dx.doi.org/10.3390/ijms21103475.
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Vascular calcification and stiffening of the arterial wall is a systemic phenomenon that is associated with aging and it can be increased by several risk factors. The underlying mechanisms, especially the pathways of cellular senescence, are under current investigation. Easily manageable in vitro settings help to study the signaling pathways. The experimental setting presented here is based on an in vitro model using rat vascular smooth muscle cells and the detection of senescence and osteoblastic markers via immunofluorescence and RNAscope™. Co-staining of the senescence marker p21, the osteoblastic marker osteopontin, detection of senescence-associated heterochromatin foci, and senescence-associated β-galactosidase is possible within one test approach requiring fewer cells. The protocol is a fast and reliable evaluation method for multiplexing of calcifying and senescence markers with fluorescence microscopy detection. The experimental setting enables analysis on single cell basis and allows detection of intra-individual variances of cultured cells.
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Riordan, Ruben. "Brain Cellular Senescence in Mouse Models of Alzheimer's Disease." Innovation in Aging 5, Supplement_1 (December 1, 2021): 929. http://dx.doi.org/10.1093/geroni/igab046.3363.
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Abstract Abstract The accumulation of senescent cells contributes to aging pathologies, including neurodegenerative diseases, and its selective removal improves physiological and cognitive function in wild type mice as well as in Alzheimer’s disease (AD) models. AD models recapitulate some, but not all components of disease and do so at different rates. Whether brain cellular senescence is recapitulated in some or all AD models, and whether the emergence of cellular senescence in AD mouse models occurs before or after the expected onset of AD-like cognitive deficits in these models is not yet known. The goal of this study was to identify mouse models of AD and AD-related dementias that develop measurable markers of cellular senescence in brain and thus may be useful to study the role of cellular senescence in these conditions. We measured levels of cellular senescence markers in brains of P301S(PS19), P301L, hTau, and 3xTg-AD mice that model amyloidopathy and/or tauopathy in AD and related dementias, and in wild type, age-matched control mice for each strain. Expression of cellular senescence markers in brains of transgenic P301L and 3xTg-AD mice was largely indistinguishable from that in WT control age-matched mice. In contrast, markers of cellular senescence were significantly increased in brains of transgenic P301S and hTau mice as compared to WT control mice at the expected time of onset of AD-like cognitive deficits. Taken together, our data suggest that P301S(PS19) and hTau mice may be useful for the study of brain cellular senescence in tauopathies including, but not limited to, AD.
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Wicher, Sarah A., Benjamin B. Roos, Jacob J. Teske, Yun Hua Fang, Christina Pabelick, and Y. S. Prakash. "Aging increases senescence, calcium signaling, and extracellular matrix deposition in human airway smooth muscle." PLOS ONE 16, no. 7 (July 29, 2021): e0254710. http://dx.doi.org/10.1371/journal.pone.0254710.
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Lung function declines as people age and their lungs become stiffer. With an increasing elderly population, understanding mechanisms that contribute to these structural and functional changes in the aging lung is important. Part of the aging process is characterized by thicker, more fibrotic airways, and senile emphysema caused by changes in lung parenchyma. There is also senescence, which occurs throughout the body with aging. Here, using human airway smooth muscle (ASM) cells from patients in different age groups, we explored senescence pathways and changes in intracellular calcium signaling and extracellular matrix (ECM) deposition to elucidate potential mechanisms by which aging leads to thicker and stiffer lungs. Senescent markers p21, γH2AX, and β-gal, and some senescence-associated secretory proteins (SASP) increased with aging, as shown by staining and biochemical analyses. Agonist-induced intracellular Ca2+ responses, measured using fura-2 loaded cells and fluorescence imaging, increased with age. However, biochemical analysis showed that expression of the following markers decreased with age: M3 muscarinic receptor, TRPC3, Orai1, STIM1, SERCA2, MMP2 and MMP9. In contrast, collagen III, and fibronectin deposition increased with age. These data show that senescence increases in the aging airways that is associated with a stiffer but surprisingly greater intracellular calcium signaling as a marker for contractility. ASM senescence may enhance fibrosis in a feed forward loop promoting remodeling and altered calcium storage and buffering.
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Chadebech, Philippe, Gwellaouen Bodivit, Gaétana Di Liberto, Alicia Jouard, Corinne Vasseur, France Pirenne, and Pablo Bartolucci. "Ex Vivo Activation of Red Blood Cell Senescence by Plasma from Sickle-Cell Disease Patients: Correlation between Markers and Adhesion Consequences during Acute Disease Events." Biomolecules 11, no. 7 (June 30, 2021): 963. http://dx.doi.org/10.3390/biom11070963.
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BACKGROUND: Blood transfusion remains a key treatment for managing occlusive episodes and painful crises in sickle-cell disease (SCD). In that clinical context, red blood cells (RBCs) from donors and transfused to patients, may be affected by plasma components in the recipients’ blood. Senescence lesion markers appear on the red cells after transfusion, shortening the RBC lifespan in circulation. In the specific context of SCD, senescence signals can also trigger the occlusive painful events, typical of the disease. This work follows through our previous data that described a RBC senescence process, rapidly detected after challenge with SCD pathological plasmas. In this clinical context, we wanted here to further explore the characteristics and physiologic consequences of AA RBC lesions associated with senescence, as lesions caused by RBCs after transfusion may have adverse consequences for SCD patients. METHODS: Plasma samples from SCD patients, with acute symptoms (n = 20) or steady-state disease (n = 34) were co-incubated with donor AA RBCs from blood units for 24 to 48 h. Specific markers signing RBC senescence were quantified after the incubation with SCD plasma samples. The physiologic in-flow adhesion was investigated on senescent RBCs, an in vitro technic into biochips that mimic adherence of RBCs during the occlusive events of SCD. RESULTS: Senescence markers on AA RBCs, together with their in-flow adhesion to the plasma-bridging protein thrombospondin, were associated with the clinical status of the SCD patients from whom plasma was obtained. In these experiments, the highest values were obtained for SCD acute plasma samples. Adhesion of senescent RBCs into biochips, which is not reversed by a pre-treatment with recombinant Annexin V, can be reproduced with the use of chemical agents acting on RBC membrane channels that regulate either Ca2+ entry or modulating RBC hydration. CONCLUSION: We found that markers on red cells are correlated, and that the senescence induced by SCD plasma provokes the adhesion of RBCs to the vessel wall protein thrombospondin. In-flow adhesion of senescent red cells after plasma co-incubations can be reproduced with the use of modulators of RBC membrane channels; activating the Piezo1 Ca2+ mechanosensitive channel provokes RBC adhesion of normal (non-senescent) RBCs, while blocking the Ca2+-dependent K+ Gardos channel, can reverse it. Clinically modulating the RBC adhesion to vascular wall proteins might be a promising avenue for the treatment of painful occlusive events in SCD.
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Casella, Gabriel, Rachel Munk, Kyoung Mi Kim, Yulan Piao, Supriyo De, Kotb Abdelmohsen, and Myriam Gorospe. "Transcriptome signature of cellular senescence." Nucleic Acids Research 47, no. 14 (June 28, 2019): 7294–305. http://dx.doi.org/10.1093/nar/gkz555.
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Abstract Cellular senescence, an integral component of aging and cancer, arises in response to diverse triggers, including telomere attrition, macromolecular damage and signaling from activated oncogenes. At present, senescent cells are identified by the combined presence of multiple traits, such as senescence-associated protein expression and secretion, DNA damage and β-galactosidase activity; unfortunately, these traits are neither exclusively nor universally present in senescent cells. To identify robust shared markers of senescence, we have performed RNA-sequencing analysis across eight diverse models of senescence triggered in human diploid fibroblasts (WI-38, IMR-90) and endothelial cells (HUVEC, HAEC) by replicative exhaustion, exposure to ionizing radiation or doxorubicin, and expression of the oncogene HRASG12V. The intersection of the altered transcriptomes revealed 50 RNAs consistently elevated and 18 RNAs consistently reduced across all senescence models, including many protein-coding mRNAs and some non-coding RNAs. We propose that these shared transcriptome profiles will enable the identification of senescent cells in vivo, the investigation of their roles in aging and malignancy and the development of strategies to target senescent cells therapeutically.
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Abdul-Aziz, Amina, Raymond D. Devine, Justin M. Lyberger, Hsiaochi Chang, Amy Kovacs, James R. Lerma, Andrew M. Rogers, John C. Byrd, Erin Hertlein, and Gregory K. Behbehani. "Mass Cytometry as a Tool for Investigating Senescence in Multiple Model Systems." Cells 12, no. 16 (August 11, 2023): 2045. http://dx.doi.org/10.3390/cells12162045.
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Cellular senescence is a durable cell cycle arrest as a result of the finite proliferative capacity of cells. Senescence responds to both intrinsic and extrinsic cellular stresses, such as aging, mitochondrial dysfunction, irradiation, and chemotherapy. Here, we report on the use of mass cytometry (MC) to analyze multiple model systems and demonstrate MC as a platform for senescence analysis at the single-cell level. We demonstrate changes to p16 expression, cell cycling fraction, and histone tail modifications in several established senescent model systems and using isolated human T cells. In bone marrow mesenchymal stromal cells (BMSCs), we show increased p16 expression with subsequent passage as well as a reduction in cycling cells and open chromatin marks. In WI-38 cells, we demonstrate increased p16 expression with both culture-induced senescence and oxidative stress-induced senescence (OSIS). We also use Wanderlust, a trajectory analysis tool, to demonstrate how p16 expression changes with histone tail modifications and cell cycle proteins. Finally, we demonstrate that repetitive stimulation of human T cells with CD3/CD28 beads induces an exhausted phenotype with increased p16 expression. This p16-expressing population exhibited higher expression of exhaustion markers such as EOMES and TOX. This work demonstrates that MC is a useful platform for studying senescence at a single-cell protein level, and is capable of measuring multiple markers of senescence at once with high confidence, thereby improving our understanding of senescent pathways.
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Cherif, Hosni, Daniel Bisson, Peter Jarzem, Michael Weber, Jean Ouellet, and Lisbet Haglund. "Curcumin and o-Vanillin Exhibit Evidence of Senolytic Activity in Human IVD Cells In Vitro." Journal of Clinical Medicine 8, no. 4 (March 29, 2019): 433. http://dx.doi.org/10.3390/jcm8040433.
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Curcumin and o-Vanillin cleared senescent intervertebral disc (IVD) cells and reduced the senescence-associated secretory phenotype (SASP) associated with inflammation and back pain. Cells from degenerate and non-mildly-degenerate human IVD were obtained from organ donors and from patients undergoing surgery for low back pain. Gene expression of senescence and SASP markers was evaluated by RT-qPCR in isolated cells, and protein expression of senescence, proliferation, and apoptotic markers was evaluated by immunocytochemistry (ICC). The expression levels of SASP factors were evaluated by enzyme-linked immunosorbent assay (ELISA). Matrix synthesis was verified with safranin-O staining and the Dimethyl-Methylene Blue Assay for proteoglycan content. Western blotting and ICC were used to determine the molecular pathways targeted by the drugs. We found a 40% higher level of senescent cells in degenerate compared to non-mildly-degenerate discs from unrelated individuals and a 10% higher level in degenerate compared to non-mildly-degenerate discs from the same individual. Higher levels of senescence were associated with increased SASP. Both drugs cleared senescent cells, and treatment increased the number of proliferating as well as apoptotic cells in cultures from degenerate IVDs. The expression of SASP factors was decreased, and matrix synthesis increased following treatment. These effects were mediated through the Nrf2 and NFkB pathways.
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Teti, Gabriella, Eleonora Mazzotti, Valentina Gatta, Francesca Chiarini, Maria Laura Alfieri, and Mirella Falconi. "Implication of Cellular Senescence in Osteoarthritis: A Study on Equine Synovial Fluid Mesenchymal Stromal Cells." International Journal of Molecular Sciences 24, no. 4 (February 4, 2023): 3109. http://dx.doi.org/10.3390/ijms24043109.
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Osteoarthritis (OA) is described as a chronic degenerative disease characterized by the loss of articular cartilage. Senescence is a natural cellular response to stressors. Beneficial in certain conditions, the accumulation of senescent cells has been implicated in the pathophysiology of many diseases associated with aging. Recently, it has been demonstrated that mesenchymal stem/stromal cells isolated from OA patients contain many senescent cells that inhibit cartilage regeneration. However, the link between cellular senescence in MSCs and OA progression is still debated. In this study, we aim to characterize and compare synovial fluid MSCs (sf-MSCs), isolated from OA joints, with healthy sf-MSCs, investigating the senescence hallmarks and how this state could affect cartilage repair. Sf-MSCs were isolated from tibiotarsal joints of healthy and diseased horses with an established diagnosis of OA with an age ranging from 8 to 14 years. Cells were cultured in vitro and characterized for cell proliferation assay, cell cycle analysis, ROS detection assay, ultrastructure analysis, and the expression of senescent markers. To evaluate the influence of senescence on chondrogenic differentiation, OA sf-MSCs were stimulated in vitro for up to 21 days with chondrogenic factors, and the expression of chondrogenic markers was compared with healthy sf-MSCs. Our findings demonstrated the presence of senescent sf-MSCs in OA joints with impaired chondrogenic differentiation abilities, which could have a potential influence on OA progression.
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Schwartz, Rachael E., and Irina M. Conboy. "Non-Intrinsic, Systemic Mechanisms of Cellular Senescence." Cells 12, no. 24 (December 5, 2023): 2769. http://dx.doi.org/10.3390/cells12242769.
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Cellular senescence is believed to contribute to aging and disease through the activity of secreted factors that promote inflammation, remodel the extracellular matrix, and adversely modify the behavior of non-senescent cells. While the markers and properties of senescent cells are still under investigation, it is postulated that cellular senescence manifests in vivo as the consequence of cellular damage that accumulates and becomes exacerbated with time. Yet, the notions that senescence has a solely intrinsic and time-dependent nature are questioned by the rapid induction of senescence in young mice and young cells in vitro by exposure to blood from aged animals. Here, we review some of the research on the systemically present factors that increase with age and may contribute to extrinsically induced senescence or “bystander senescence”. These include proteins, reactive oxygen species, lipids, and nucleic acids, which may be present in individual soluble form, in vesicles, and in non-membranous multi-component macromolecules.
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LaFougere, Christian, Brigitte Gueckel, Helmut Dittmann, Nils Trautwein, Martina Hinterleitner, Jonathan Cotton, Gerald Reischl, et al. "Abstract CT095: Update: a phase 1/2, open-label study to assess safety, tolerability, biodistribution, radiation dosimetry and PET imaging characteristics of [18F]FPyGal in comparison to in-vitro diagnostic for the assessment of senescence in oncological patients (NCT04536454)." Cancer Research 83, no. 8_Supplement (April 14, 2023): CT095. http://dx.doi.org/10.1158/1538-7445.am2023-ct095.
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Abstract It is well established that senescence of cancer cells can be induced by treatment with classical cytotoxic therapies but also by molecularly targeted therapies or immunotherapies, a phenomenon referred to as therapy induced senescence (TIS). Occurrence of intratumoral senescence harbors broad therapeutic implications and may limit the prognosis of cancer patients, as senescent cells, via their senescence associated secretory phenotype (SASP), can suppress anti-tumor immune responses and may increase the metastatic potential of non-senescent cancer cells. Preclinical data suggest that an eradication of senescent cells (senolytic therapies) or an application of SASP modulating therapies might improve the outcome of systemic cancer therapies, however, as a prerequisite to guide such approaches, robust and non-invasive modalities to visualize and quantify intratumoral senescence are needed. We recently reported on the safety evaluation of the novel Positron-Emission-Tomography (PET) tracer [18F]FPyGal, a radiolabeled substrate of senescence-associated ꞵ-galactosidase in healthy volunteers (AACR 2022, #7958). In summary, our data showed that the use of [18F]FPyGal is safe and well-tolerated in the described human study population. In the second stage of our trial, we are now investigating the concordance of histopathological senescence markers with [18F]FPyGal senescence signals in i) rectum cancer-, ii) non-small cell lung cancer- and iii) adenocarcinoma of the esophagogastric junction (AEG) patients, who are undergoing neoadjuvant therapy. Here, we report on patients from the rectum cancer cohort of the trial. [18F]FPyGal PET imaging was done before and after administration of neoadjuvant radiochemotherapy (RCTx). [18F]FPyGal PET signal was correlated with immunohistochemical senescence markers (p16, p21, p53) and quantification of SA-ß-gal (Senescence-associated beta-galactosidase) positive cells in biopsy materials (pre-therapy) and surgical resection materials (post-therapy). Expectedly, senescent cells were only detected at very low frequency in therapy naïve tumors and the lack of histological detection of senescence correlated with negativity in [18F]FPyGal imaging. In stark contrast, homogenous senescence, quantified with the indicated senescence markers, was detected in the majority of cancer cells after RCTx. Interestingly, in one rectum carcinoma, which underwent a full remission after RCTx, senescence of stroma cells in the area of the former tumor bed was detected by PET imaging and histologically. Collectively, our data indicate the utility of [18F]FPyGal PET imaging to non-invasively detect senescence. Future trials using [18F]FPyGal to guide oncological therapy, e.g. to apply senolytic drugs in a personalized manner, are warranted. Citation Format: Christian LaFougere, Brigitte Gueckel, Helmut Dittmann, Nils Trautwein, Martina Hinterleitner, Jonathan Cotton, Gerald Reischl, Gabriele Kienzle, Stephan Singer, Cihan Gani, Ulrich Lauer, Bernd Pichler, Lars Zender. Update: a phase 1/2, open-label study to assess safety, tolerability, biodistribution, radiation dosimetry and PET imaging characteristics of [18F]FPyGal in comparison to in-vitro diagnostic for the assessment of senescence in oncological patients (NCT04536454) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT095.
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Wang, Zijia, Ying Han, Ying Peng, Shuhui Shao, Huanquan Nie, Kun Xia, Haofeng Xiong, and Tong Su. "Senescent epithelial cells remodel the microenvironment for the progression of oral submucous fibrosis through secreting TGF-β1." PeerJ 11 (April 19, 2023): e15158. http://dx.doi.org/10.7717/peerj.15158.
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Objectives Cellular senescence is strongly associated with fibrosis and tumorigenesis. However, whether the epithelium of oral submucous fibrosis (OSF) undergoes premature senescence remains unclear. This study investigates the roles of senescent epithelial cells in OSF. Methods The immunohistochemistry and Sudan black B staining were performed to identify epithelium senescence in OSF tissues. Arecoline was used to induce human oral keratinocytes (HOKs) senescence. The cell morphology, senescence-associated β galactosidase activity, cell counting Kit 8, immunofluorescence, quantitative real-time PCR, and western blot assay were used to identification of senescent HOKs. The enzyme-linked immunosorbent assay was exerted to evaluate the levels of transforming growth factor β1 (TGF-β1) in the supernatants of HOKs treated with or without arecoline. Results The senescence-associated markers, p16 and p21, were overexpressed in OSF epithelium. These expressions were correlated with alpha-smooth actin (α-SMA) positively and proliferating cell nuclear antigen (PCNA) negatively. Moreover, Sudan black staining showed that there was more lipofuscin in OSF epithelium. In vitro, HOKs treated with arecoline showed senescence-associated characteristics including enlarged and flattened morphology, senescence-associated β galactosidase staining, cell growth arrest, γH2A.X foci, upregulation of p53, p21, and TGF-β1 protein levels. Moreover, senescent HOKs secreted more TGF-β1. Conclusions Senescent epithelial cells are involved in OSF progression and may become a promising target for OSF treatment.
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Herman, Allison B., Carlos Anerillas, Sophia C. Harris, Rachel Munk, Jennifer L. Martindale, Xiaoling Yang, Krystyna Mazan-Mamczarz, et al. "Reduction of lamin B receptor levels by miR-340-5p disrupts chromatin, promotes cell senescence and enhances senolysis." Nucleic Acids Research 49, no. 13 (June 28, 2021): 7389–405. http://dx.doi.org/10.1093/nar/gkab538.
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Abstract A major stress response influenced by microRNAs (miRNAs) is senescence, a state of indefinite growth arrest triggered by sublethal cell damage. Here, through bioinformatic analysis and experimental validation, we identified miR-340-5p as a novel miRNA that foments cellular senescence. miR-340-5p was highly abundant in diverse senescence models, and miR-340-5p overexpression in proliferating cells rendered them senescent. Among the target mRNAs, miR-340-5p prominently reduced the levels of LBR mRNA, encoding lamin B receptor (LBR). Loss of LBR by ectopic overexpression of miR-340-5p derepressed heterochromatin in lamina-associated domains, promoting the expression of DNA repetitive elements characteristic of senescence. Importantly, overexpressing miR-340-5p enhanced cellular sensitivity to senolytic compounds, while antagonization of miR-340-5p reduced senescent cell markers and engendered resistance to senolytic-induced cell death. We propose that miR-340-5p can be exploited for removing senescent cells to restore tissue homeostasis and mitigate damage by senescent cells in pathologies of human aging.
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Baboota, Ritesh K., Aidin Rawshani, Laurianne Bonnet, Xiangyu Li, Hong Yang, Adil Mardinoglu, Tamar Tchkonia, et al. "BMP4 and Gremlin 1 regulate hepatic cell senescence during clinical progression of NAFLD/NASH." Nature Metabolism 4, no. 8 (August 22, 2022): 1007–21. http://dx.doi.org/10.1038/s42255-022-00620-x.
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AbstractThe role of hepatic cell senescence in human non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) is not well understood. To examine this, we performed liver biopsies and extensive characterization of 58 individuals with or without NAFLD/NASH. Here, we show that hepatic cell senescence is strongly related to NAFLD/NASH severity, and machine learning analysis identified senescence markers, the BMP4 inhibitor Gremlin 1 in liver and visceral fat, and the amount of visceral adipose tissue as strong predictors. Studies in liver cell spheroids made from human stellate and hepatocyte cells show BMP4 to be anti-senescent, anti-steatotic, anti-inflammatory and anti-fibrotic, whereas Gremlin 1, which is particularly highly expressed in visceral fat in humans, is pro-senescent and antagonistic to BMP4. Both senescence and anti-senescence factors target the YAP/TAZ pathway, making this a likely regulator of senescence and its effects. We conclude that senescence is an important driver of human NAFLD/NASH and that BMP4 and Gremlin 1 are novel therapeutic targets.
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Piccinato, Carla A., Andrea L. Sertie, Natália Torres, Mario Ferretti, and Eliane Antonioli. "HighOCT4and Lowp16INK4AExpressions DetermineIn VitroLifespan of Mesenchymal Stem Cells." Stem Cells International 2015 (2015): 1–11. http://dx.doi.org/10.1155/2015/369828.
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After long-term culture, mesenchymal stem cells alter their biological properties and enter into a state of replicative senescence. Although several classical biomarkers have been used for quantitative assessment of cellular senescence, no hallmark has been proven completely unique to the senescent state in cells. We used bone marrow-derived MSCs (BM-MSCs) from different healthy young donors and anin vitromodel with well-defined senescence end points to identify a set of robust markers that could potentially predict the expansion capacity of MSCs preparations before reaching senescence. For each early passage BM-MSC sample (5th or 6th passages), the normalized protein expression levels of senescence-associated markersp16INK4A,p21WAF1, SOD2, andrpS6S240/244; the concentration of IL6 and IL8 in cell culture supernatants; and the normalized gene expression levels of pluripotency markersOCT4,NANOG, andSOX2were correlated with final population doubling (PD) number. We revealed that the low expression ofp16INK4Aprotein and a highOCT4gene expression, rather than other evaluated markers, might be potential hallmarks and predictors of greaterin vitrolifespan and growth potential, factors that can impact the successful therapeutic use of MSCs preparations.
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Prlic, Martin, Jilian Sacks, and Michael Bevan. "Dissociating markers of senescence and protective ability in memory T cells (110.16)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 110.16. http://dx.doi.org/10.4049/jimmunol.188.supp.110.16.
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Abstract A set of surface markers is commonly used to predict the potential of CD8 effector T cells to differentiate into memory cells. Similarly, these surface markers together with TNF family member CD27 are frequently used to predict a memory T cell’s ability to mount a recall response. Expression of these markers changes every time a memory cell is stimulated and repeated stimulation can lead to T cell senescence and loss of memory T cell responsiveness. This is a concern for prime-boost vaccine strategies which repeatedly stimulate T cells to increase memory T cell frequency. The molecular cues that cause senescence are still unknown, but cell division history is likely to play a major role. We sought to dissect the roles of inflammation and cell division history in developing T cell senescence and their impact on the expression pattern of commonly used markers of senescence. We developed a system that allows priming of CD8 T cells with minimal inflammation and without acquisition of maximal effector function, such as granzyme expression, but a cell division history similar to priming with systemic inflammation. Memory cells derived from minimal effector T cells are fully functional upon rechallenge, have full access to non-lymphoid tissue and appear to be less senescent by phenotype upon rechallenge. However, these currently used biomarkers to measure senescence do not predict proliferative potential or protective ability, but merely reflect initial priming conditions.
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Mao, Genxiang, and Xiaogang Xu. "Exosomes Derived From Senescent Cells Promote Cellular Senescence." Innovation in Aging 4, Supplement_1 (December 1, 2020): 132–33. http://dx.doi.org/10.1093/geroni/igaa057.435.
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Abstract Exosomes are one type of small-cell extracellular vesicles (sEVs), which together with the senescence-associated secretory phenotype (SASP) mainly constitute the senescent microenvironment and perform remotely intercellular communication. However, the effects of senescence on exosomes biosynthesis and secretion and its role in the cell senescence are still obscure. Here, we used human fetal lung diploid fibroblasts (2BS) passaged to PD50 to construct the senescent cells model in vitro, which were confirmed by senescence-related β-galactosidase staining, cell cycle distribution, and intracellular ROS levels. PD30 2BS was used as young control. We evaluated the exosomes derived from senescence and young control group respectively and investigated their regulation of senescence. We found that exosomes released from 2BS had typical sizes and cup-shapes morphology and their surface presented typical exosome-associated proteins. The number of exosomes secreted by senescent cells was significantly higher than that of young cells. Moreover, exosomal markers Alix, TSG101, and CD63 were all more expressed than young cells. Furthermore, we treat young cells with exosomes secreted by senescent cells, which can induce senescence-like changes in young cells, including increased SA-β-Gal activity, up-regulated p16 protein expression, and activation of the Notch signaling pathway. The above results imply that exosomes derived from senescent cells can promote cell senescence. The findings expand the current knowledge on exosomes-mediated aging and provide a novel understanding of the relationship between SASP and senescence. This study is supported by National Natural Science Foundation of China (No. 81771520 and 31702144).
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Chen, Yu-Hsiu, Xin Zhang, Kuei-Yueh Ko, Ming-Feng Hsueh, and Virginia Byers Kraus. "CBX4 Regulates Replicative Senescence of WI-38 Fibroblasts." Oxidative Medicine and Cellular Longevity 2022 (February 23, 2022): 1–15. http://dx.doi.org/10.1155/2022/5503575.
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Cellular senescence is characterized by cell cycle arrest and senescence-associated secretory phenotypes. Cellular senescence can be caused by various stress stimuli such as DNA damage, oxidative stress, and telomere attrition and is related to several chronic diseases, including atherosclerosis, Alzheimer’s disease, and osteoarthritis. Chromobox homolog 4 (CBX4) has been shown to alleviate cellular senescence in human mesenchymal stem cells and is considered a possible target for senomorphic treatment. Here, we explored whether CBX4 expression is associated with replicative senescence in WI-38 fibroblasts, a classic human senescence model system. We also examined whether and how regulation of CBX4 modifies the senescence phenotype and functions as an antisenescence target in WI-38. During the serial culture of the WI-38 primary fibroblast cell line to a senescent state, we found increased expression of senescence markers, including senescence β-galactosidase (SA-βgal) activity, protein expression of p16, p21, and DPP4, and decreased proliferation marker EdU; moreover, CBX4 protein expression declined. With knockdown of CBX4, SA-βgal activity and p16 protein expression increased, and EdU decreased. With the activation of CBX4, SA-βgal activity, p16, and DPP4 protein decreased. In addition, CBX4 knockdown increased, while CBX4 activation decreased, gene expression of both CDKN2A (encoding the p16 protein) and DPP4. Genes related to DNA damage and cell cycle pathways were regulated by CBX4. These results demonstrate that CBX4 can regulate replicative senescence in a manner consistent with a senomorphic agent.
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Jo, Hye-Ram, and Jae-Hoon Jeong. "MicroRNA-Mediated Downregulation of HMGB2 Contributes to Cellular Senescence in Microvascular Endothelial Cells." Cells 11, no. 3 (February 8, 2022): 584. http://dx.doi.org/10.3390/cells11030584.
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High mobility group box 2 (HMGB2) is a non-histone chromosomal protein involved in various biological processes, including cellular senescence. However, its role in cellular senescence has not been evaluated extensively. To determine the regulatory role and mechanism of HMGB2 in cellular senescence, we performed gene expression analysis, senescence staining, and tube formation assays using young and senescent microvascular endothelial cells (MVECs) after small RNA treatment or HMGB2 overexpression. HMGB2 expression decreased with age and was regulated at the transcriptional level. siRNA-mediated downregulation inhibited cell proliferation and accelerated cellular senescence. In contrast, ectopic overexpression delayed senescence and maintained relatively higher tube-forming activity. To determine the HMGB2 downregulation mechanism, we screened miRNAs that were significantly upregulated in senescent MVECs and selected HMGB2-targeting miRNAs. Six miRNAs, miR-23a-3p, 23b-3p, -181a-5p, -181b-5p, -221-3p, and -222-3p, were overexpressed in senescent MVECs. Ectopic introduction of miR-23a-3p, -23b-3p, -181a-5p, -181b-5p, and -221-3p, with the exception of miR-222-3p, led to the downregulation of HMGB2, upregulation of senescence-associated markers, and decreased tube formation activity. Inhibition of miR-23a-3p, -181a-5p, -181b-5p, and -221-3p delayed cellular senescence. Restoration of HMGB2 expression using miRNA inhibitors represents a potential strategy to overcome the detrimental effects of cellular senescence in endothelial cells.
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Romashkan, Sergei, Henry Chang, and Evan C. Hadley. "National Institute on Aging Workshop: Repurposing Drugs or Dietary Supplements for Their Senolytic or Senomorphic Effects: Considerations for Clinical Trials." Journals of Gerontology: Series A 76, no. 6 (February 2, 2021): 1144–52. http://dx.doi.org/10.1093/gerona/glab028.
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Abstract Background Cell senescence is implicated in numerous age-related conditions. Drugs and nutritional supplements developed for a variety of purposes kill senescent cells (senolytics) or suppress their secretions (senomorphics). There is interest in repurposing such drugs to treat or prevent age-related diseases. To date, only small-scale preliminary trials have been conducted. Method At a workshop convened by the National Institute on Aging in August 2019, academic, industry, and government scientists reviewed issues for phase II trials of potentially repurposable drugs, or dietary supplements, to assess benefits and risks of their senolytic (killing senescent cells) or senomorphic (altering senescent cells’ phenotypes) effects in treating or preventing age-related conditions. Results Participants reviewed mechanisms and effects of cellular senescence, senolytics, and senomorphics of several classes and their potential role in treating or preventing disease, modulators of the senescence-associated secretory phenotype, needs for senescence markers, data and specimen resources, infrastructure for planning trials, and potential effects on outcomes in older patients with multimorbidity and polypharmacy. Conclusions Participants noted the importance of considering potential effects of candidate drugs on multiple aging outcomes. It is important to assess drugs’ specificity for killing senescent cells and the balance between senolytic and cytotoxic effects. Markers of specific senescent cell types are needed to assess intervention responses. There are potential interactions with coexisting diseases and their treatments in older persons. Standardized measures could enhance comparisons and pooling of data. Additional characterization of human cell senescent phenotypes is needed for developing better and more specific senolytics and senomorphics.
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Xu, Linshan, Yuyang Wang, Jianping Wang, Jianglong Zhai, Li Ren, and Guoying Zhu. "Radiation-Induced Osteocyte Senescence Alters Bone Marrow Mesenchymal Stem Cell Differentiation Potential via Paracrine Signaling." International Journal of Molecular Sciences 22, no. 17 (August 28, 2021): 9323. http://dx.doi.org/10.3390/ijms22179323.
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Cellular senescence and its senescence-associated secretory phenotype (SASP) are widely regarded as promising therapeutic targets for aging-related diseases, such as osteoporosis. However, the expression pattern of cellular senescence and multiple SASP secretion remains unclear, thus leaving a large gap in the knowledge for a desirable intervention targeting cellular senescence. Therefore, there is a critical need to understand the molecular mechanism of SASP secretion in the bone microenvironment that can ameliorate aging-related degenerative pathologies including osteoporosis. In this study, osteocyte-like cells (MLO-Y4) were induced to cellular senescence by 2 Gy γ-rays; then, senescence phenotype changes and adverse effects of SASP on bone marrow mesenchymal stem cell (BMSC) differentiation potential were investigated. The results revealed that 2 Gy irradiation could hinder cell viability, shorten cell dendrites, and induce cellular senescence, as evidenced by the higher expression of senescence markers p16 and p21 and the elevated formation of senescence-associated heterochromatin foci (SAHF), which was accompanied by the enhanced secretion of SASP markers such as IL-1α, IL-6, MMP-3, IGFBP-6, resistin, and adiponectin. When 0.8 μM JAK1 inhibitors were added to block SASP secretion, the higher expression of SASP was blunted, but the inhibition in osteogenic and adipogenic differentiation potential of BMSCs co-cultured with irradiated MLO-Y4 cell conditioned medium (CM- 2 Gy) was alleviated. These results suggest that senescent osteocytes can perturb BMSCs’ differential potential via the paracrine signaling of SASP, which was also demonstrated by in vivo experiments. In conclusion, we identified the SASP factor partially responsible for the degenerative differentiation of BMSCs, which allowed us to hypothesize that senescent osteocytes and their SASPs may contribute to radiation-induced bone loss.
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Moimas, Silvia, Francesco Salton, Beata Kosmider, Nadja Ring, Maria C. Volpe, Karim Bahmed, Luca Braga, et al. "miR-200 family members reduce senescence and restore idiopathic pulmonary fibrosis type II alveolar epithelial cell transdifferentiation." ERJ Open Research 5, no. 4 (October 2019): 00138–2019. http://dx.doi.org/10.1183/23120541.00138-2019.
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RationaleAlveolar type II (ATII) cells act as adult stem cells contributing to alveolar type I (ATI) cell renewal and play a major role in idiopathic pulmonary fibrosis (IPF), as supported by familial cases harbouring mutations in genes specifically expressed by these cells. During IPF, ATII cells lose their regenerative potential and aberrantly express pathways contributing to epithelial–mesenchymal transition (EMT). The microRNA miR-200 family is downregulated in IPF, but its effect on human IPF ATII cells remains unproven. We wanted to 1) evaluate the characteristics and transdifferentiating ability of IPF ATII cells, and 2) test whether miR-200 family members can rescue the regenerative potential of fibrotic ATII cells.MethodsATII cells were isolated from control or IPF lungs and cultured in conditions promoting their transdifferentiation into ATI cells. Cells were either phenotypically monitored over time or transfected with miR-200 family members to evaluate the microRNA effect on the expression of transdifferentiation, senescence and EMT markers.ResultsIPF ATII cells show a senescent phenotype (p16 and p21), overexpression of EMT (ZEB1/2) and impaired expression of ATI cell markers (AQP5 and HOPX) after 6 days of culture in differentiating medium. Transfection with certain miR-200 family members (particularly miR-200b-3p and miR-200c-3p) reduced senescence marker expression and restored the ability to transdifferentiate into ATI cells.ConclusionsWe demonstrated that ATII cells from IPF patients express senescence and EMT markers, and display a reduced ability to transdifferentiate into ATI cells. Transfection with certain miR-200 family members rescues this phenotype, reducing senescence and restoring transdifferentiation marker expression.
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Mullani, Nowsheen, Yevheniia Porozhan, Adèle Mangelinck, Christophe Rachez, Mickael Costallat, Eric Batsché, Michele Goodhardt, Giovanni Cenci, Carl Mann, and Christian Muchardt. "Reduced RNA turnover as a driver of cellular senescence." Life Science Alliance 4, no. 3 (January 14, 2021): e202000809. http://dx.doi.org/10.26508/lsa.202000809.
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Accumulation of senescent cells is an important contributor to chronic inflammation upon aging. The inflammatory phenotype of senescent cells was previously shown to be driven by cytoplasmic DNA. Here, we propose that cytoplasmic double-stranded RNA has a similar effect. We find that several cell types driven into senescence by different routes share an accumulation of long promoter RNAs and 3′ gene extensions rich in retrotransposon sequences. Accordingly, these cells display increased expression of genes involved in response to double stranded RNA of viral origin downstream of the interferon pathway. The RNA accumulation is associated with evidence of reduced RNA turnover, including in some cases, reduced expression of RNA exosome subunits. Reciprocally, depletion of RNA exosome subunit EXOSC3 accelerated expression of multiple senescence markers. A senescence-like RNA accumulation was also observed in cells exposed to oxidative stress, an important trigger of cellular senescence. Altogether, we propose that in a subset of senescent cells, repeat-containing transcripts stabilized by oxidative stress or reduced RNA exosome activity participate in driving and maintaining the permanent inflammatory state characterizing cellular senescence.