A Senescence-Centric View of Aging:Implications for Longevity and Disease

Cellular senescence is a state of stable cell cycle arrest associated with macromolecular alterations and secretion of proinflammatory cytokines and molecules. From their initial discovery in the 1960s, senescent cells have been hypothesized as potential contributors to the age-associated loss of regenerative potential. Here, we discuss recent evidence that implicates cellular senescence as a central regulatory mechanism of the aging process. We provide a comprehensive overview of age-associated pathologies in which cellular senescence has been implicated. We describe mechanisms by which senescent cells drive aging and diseases, and we discuss updates on exploiting these mechanisms as therapeutic targets. Finally, we critically analyze the use of senotherapeutics and their translation to the clinic, highlighting limitations and suggesting ideas for future applications and developments

Algorithmic assessment of cellular senescence in experimental and clinical specimens

The development of genetic tools allowed for the validation of the pro-aging and pro-disease functions of senescent cells in vivo. These discoveries prompted the development of senotherapies—pharmaceutical interventions aimed at interfering with the detrimental effect of senescent cells—that are now entering the clinical stage. However, unequivocal identification and examination of cellular senescence remains highly difficult because of the lack of universal and specific markers. Here, to overcome the limitation of measuring individual markers, we describe a detailed two-phase algorithmic assessment to quantify various senescence-associated parameters in the same specimen. In the first phase, we combine the measurement of lysosomal and proliferative features with the expression of general senescence-associated genes to validate the presence of senescent cells. In the second phase we measure the levels of pro-inflammatory markers for specification of the type of senescence. The protocol can help graduate-level basic scientists to improve the characterization of senescence-associated phenotypes and the identification of specific senescent subtypes. Moreover, it can serve as an important tool for the clinical validation of the role of senescent cells and the effectiveness of anti-senescence therapies

Pharmacological CDK4/6 inhibition reveals a p53-dependent senescent state with restricted toxicity

Cellular senescence is a state of stable growth arrest and a desired outcome of tumor suppressive interventions. Treatment with many anti‐cancer drugs can cause premature senescence of non‐malignant cells. These therapy‐induced senescent cells can have pro‐tumorigenic and pro‐disease functions via activation of an inflammatory secretory phenotype (SASP). Inhibitors of cyclin‐dependent kinases 4/6 (CDK4/6i) have recently proven to restrain tumor growth by activating a senescence‐like program in cancer cells. However, the physiological consequence of exposing the whole organism to pharmacological CDK4/6i remains poorly characterized. Here, we show that exposure to CDK4/6i induces non‐malignant cells to enter a premature state of senescence dependent on p53. We observe in mice and breast cancer patients that the CDK4/6i‐induced senescent program activates only a partial SASP enriched in p53 targets but lacking pro‐inflammatory and NF‐κB‐driven components. We find that CDK4/6i‐induced senescent cells do not acquire pro‐tumorigenic and detrimental properties but retain the ability to promote paracrine senescence and undergo clearance. Our results demonstrate that SASP composition is exquisitely stress‐dependent and a predictor for the biological functions of different senescence subsets

Physiological hypoxia restrains the senescence-associated secretory phenotype via AMPK-mediated mTOR suppression

Cellular senescence is a state of stable proliferative arrest triggered by damaging signals. Senescent cells persist during aging and promote age-related pathologies via the pro-inflammatory senescence-associated secretory phenotype (SASP), whose regulation depends on environmental factors. In vivo, a major environmental variable is oxygenation, which varies among and within tissues. Here, we demonstrate that senescent cells express lower levels of detrimental pro-inflammatory SASP factors in physiologically hypoxic environments, as measured in culture and in tissues. Mechanistically, exposure of senescent cells to low-oxygen conditions leads to AMPK activation and AMPK-mediated suppression of the mTOR-NF-kappa B signaling loop. Finally, we demonstrate that treatment with hypoxia-mimetic compounds reduces SASP in cells and tissues and improves strength in chemotherapy-treated and aged mice. Our findings highlight the importance of oxygen as a determinant for pro-inflammatory SASP expression and offer a potential new strategy to reduce detrimental paracrine effects of senescent cells

Physiological hypoxia restrains the senescence-associated secretory phenotype via AMPK-mediated mTOR suppression

Cellular senescence is a state of stable proliferative arrest triggered by damaging signals. Senescent cells persist during aging and promote age-related pathologies via the pro-inflammatory senescence-associated secretory phenotype (SASP), whose regulation depends on environmental factors. In vivo, a major environmental variable is oxygenation, which varies among and within tissues. Here, we demonstrate that senescent cells express lower levels of detrimental pro-inflammatory SASP factors in physiologically hypoxic environments, as measured in culture and in tissues. Mechanistically, exposure of senescent cells to low-oxygen conditions leads to AMPK activation and AMPK-mediated suppression of the mTOR-NF-κB signaling loop. Finally, we demonstrate that treatment with hypoxia-mimetic compounds reduces SASP in cells and tissues and improves strength in chemotherapy-treated and aged mice. Our findings highlight the importance of oxygen as a determinant for pro-inflammatory SASP expression and offer a potential new strategy to reduce detrimental paracrine effects of senescent cells

Physiological hypoxia restrains the senescence-associated secretory phenotype via AMPK-mediated mTOR suppression

Cellular senescence is a state of stable proliferative arrest triggered by damaging signals. Senescent cells persist during aging and promote age-related pathologies via the pro-inflammatory senescence-associated secretory phenotype (SASP), whose regulation depends on environmental factors. In vivo, a major environmental variable is oxygenation, which varies among and within tissues. Here, we demonstrate that senescent cells express lower levels of detrimental pro-inflammatory SASP factors in physiologically hypoxic environments, as measured in culture and in tissues. Mechanistically, exposure of senescent cells to low-oxygen conditions leads to AMPK activation and AMPK-mediated suppression of the mTOR-NF-κB signaling loop. Finally, we demonstrate that treatment with hypoxia-mimetic compounds reduces SASP in cells and tissues and improves strength in chemotherapy-treated and aged mice. Our findings highlight the importance of oxygen as a determinant for pro-inflammatory SASP expression and offer a potential new strategy to reduce detrimental paracrine effects of senescent cells. © 2021 Elsevier Inc

Algorithmic assessment of cellular senescence in experimental and clinical specimens

The development of genetic tools allowed for the validation of the pro-aging and pro-disease functions of senescent cells in vivo. These discoveries prompted the development of senotherapies—pharmaceutical interventions aimed at interfering with the detrimental effect of senescent cells—that are now entering the clinical stage. However, unequivocal identification and examination of cellular senescence remains highly difficult because of the lack of universal and specific markers. Here, to overcome the limitation of measuring individual markers, we describe a detailed two-phase algorithmic assessment to quantify various senescence-associated parameters in the same specimen. In the first phase, we combine the measurement of lysosomal and proliferative features with the expression of general senescence-associated genes to validate the presence of senescent cells. In the second phase we measure the levels of pro-inflammatory markers for specification of the type of senescence. The protocol can help graduate-level basic scientists to improve the characterization of senescence-associated phenotypes and the identification of specific senescent subtypes. Moreover, it can serve as an important tool for the clinical validation of the role of senescent cells and the effectiveness of anti-senescence therapies. © 2021, The Author(s), under exclusive licence to Springer Nature Limited

Connexin43-positive exosomes from osteoarthritic chondrocytes spread senescence and inflammatory mediators to nearby synovial and bone cells

Purpose: Chondrocytes in articular cartilage undergo phenotypic changes and senescence, restricting cartilage regeneration and favoring osteoarthritis (OA) progression, characterized by degradation of articular cartilage, physical disability and pain. Like other wound healing disorders, chondrocytes and synovial cells from OA patients show a chronic increase in the transmembrane channel protein connexin43 (Cx43), which through the exchange of ions and molecule or binding of signaling factors to the membrane regulates signal transduction. Extracellular vesicles (EVs), including exosomes, have been show to play important roles in many biological functions and harbour connexin channels that allow the formation of gap junctions between the exosome and the target cell. However the role of these vesicles and exosomal-Cx43 in OA progression has not been studied yet. The main objective of this study was to investigate the role of exosomes released by osteoarthritic chondrocytes in cellular plasticity and senescence of surrounding tissues. Methods: Chondrocytes, bone and synovial cells were isolated from human tissues from healthy and OA donors. Extracellular vesicles (EVs), including exosomes, were obtained by differential ultracentrifugation and their protein content was analysed by LC-MS/MS using 6600 triple TOF. Protein levels were evaluated by western blot, immunofluorescence and flow cytometry. RNA expression was evaluated by RT-qPCR. Gap junction intercellular communication was studied by scrape loading assay and flow cytometry. Results: OA chondrocytes (OACs) showed increased levels of Cx43 within their EVs in comparison to the EVs isolated from healthy donors. Furthermore, overexpression of Cx43 in chondrocytes with a vector or by treatment with the mitochondrial inhibitor oligomycin increased senescence and the total content of Cx43 in the EVs. Interestingly, the treatment of chondrocytes, bone cells and synoviocytes (target cells) with EVs containing Cx43, released by OACs, led to a significant increase in both Cx43 mRNA and protein levels in the recipient cells. The increase of Cx43 in target cells acted as a positive regulator of the reversion of the recipient cells to a less differentiated state via EMT by activation of Twist-1, associated with increased levels of the mesenchymal markers CD105 and CD166, as well as N-cadherin, Slug, Vimentin and Smad2/3. The phenotypic changes detected in OACs lead to a decrease in collagen II and aggrecan expression in chondrocytes, and increased the levels of cellular senescence and the senescence-associated secretory phenotype (SASP) in target cells via p53/p16 and NF-kß. These results were corroborated by analysing the protein cargo of these Cx43 positive EVs by LC-MS/MS, where we found an enrichment in proteins related with the catabolic, senescence and wound-healing pathways together with factors of the complement system and innate immunity. Conclusions: Our results indicate that Cx43-positive exosomes and extracellular vesicles released by osteoarthritic chondrocytes may be involved in the spread of cellular senescence and inflammatory and reprogramming factors involved in wound healing failure to neighbouring tissues in the joint, spreading OA among cartilage, synovium and subchondral bone. The results suggest that further understanding of the role of exosomal Cx43 in OA will help to halt the disease spread and progression

Spread of senescence and joint inflammation via connexin43-positive exosomes released by osteoarthritic chondrocytes

Background: Chondrocytes (CHs) in articular cartilage undergo phenotypic changes and senescence, restricting cartilage regeneration and favoring osteoarthritis (OA) progression. CHs and synovial cells from OA patients show a chronic increase in the channel protein connexin43 (Cx43), which regulates signal transduction. Extracellular vesicles (EVs), including exosomes, have been shown to play important roles in many biological functions and harbour Cx channels that allow the formation of gap junctions (GJs) between the exosome and the target cell, but the role of these EVs and exosomal-Cx43 in OA progression has not been studied yet. Objectives: The objective of this study was to investigate the role of EVs released by OA chondrocytes (OACs) in cellular plasticity, inflammation and senescence of surrounding joint tissues. Methods: CHs, bone and synovial cells were isolated from healthy and OA donors. EVs were obtained by ultracentrifugation and their protein content was analysed by LC-MS/MS. Protein levels were evaluated by western blot, immunofluorescence and flow cytometry. RNA expression was evaluated by RT-qPCR. Senescence and GJ intercellular communication was studied by flow cytometry and scrape loading assay, respectively. Results: OACs showed increased levels of Cx43 within their EVs in comparison to the EVs isolated from healthy donors. Overexpression of Cx43 in CHs increased senescence and exosomal Cx43 levels. Interestingly, the treatment of CHs, bone cells and synoviocytes (target cells) with Cx43-EVs released by OACs, led to a significant increase in both Cx43 mRNA and protein levels in the recipient cells. The increase of Cx43 in target cells acted as a positive regulator of the reversion to a less differentiated state via EMT by activation of Twist-1, associated with increased levels of the mesenchymal markers, as CD105/CD166. The phenotypic changes detected in OACs lead to a decrease in Col2A1 and aggrecan expression in CHs, and increased the levels of cellular senescence and the senescence-associated secretory phenotype (SASP) in the target cells in target cells via p53/p16 and NF-kß. These results were corroborated by analysing the protein cargo of these Cx43 positive EVs by LC-MS/MS, finding enrichment in proteins related with catabolic, senescence and wound-healing pathways. Conclusion: Our results indicate that Cx43-positive exosomes released by OACs may be involved in the spread of cellular senescence and inflammation involved in wound healing failure. Further understanding of the role of exosomal Cx43 in OA will help to halt the disease spread and progression