The consequences of environmental conditions for antagonistic pleiotropic effects of cellular
senescence
van Vliet, Thijmen
DOI:
10.33612/diss.156836397
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van Vliet, T. (2021). The consequences of environmental conditions for antagonistic pleiotropic effects of
cellular senescence. University of Groningen. https://doi.org/10.33612/diss.156836397
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126 (1996).
258. Blagosklonny, M. V. Hypoxia, MTOR and autophagy Converging on senescence or quiescence.
Autophagy 9 260–262 (2013).
259. D’Ignazio, L. & Rocha, S. Hypoxia Induced NF-κB. Cells 2016, 1-8 (2016).
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Chapter 7
General discussion
Thijmen van Vliet
European Research Institute for the Biology of Ageing (ERIBA), University Medical Center Groningen (UMCG), 9713AV Groningen, The Netherlands
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128 Summary
The research presented in this thesis aims to dissect the role of environmental conditions on the antagonistic pleiotropic effects of senescent cells in aging and tissue repair (Chapter 2-6). The exponential increase in life expectancy of the human population pushed the incidence of devastating age-related diseases beyond the sustainability of our healthcare systems, instigating the urgent need to develop anti-aging interventions. The chronic accumulation of senescent cells in essentially all tissues during aging boost age-related diseases thus providing a useful target for the development of anti-aging therapies. However, the transient appearance of senescent cells have essential beneficial functions during wound healing, tissue remodeling and embryonic development and therefore represent a major hurdle to develop senescence targeting therapies with minimal toxicities and adverse effects.
In Chapter 2, we reviewed the current literature indicating that senescence targeting therapies, collectively named senotherapies, influence beneficial functions of senescent cells in wound healing, reprogramming and fibrosis.
The underlying causes of the paradoxical opposing functions of senescent cells remains only partly understood. A new concept emerging in relation to this topic states that both the functions and phenotypes of senescent cells depend on environmental conditions such as oxygen pressure and nutrient availability, creating specific senescence subtypes with distinct functions. In Chapter 3, we analyzed colon tissues from both humans and mice under a caloric restriction (CR) diet for senescence associated markers. In accordance with previous literature we show a reduction of transcriptional senescence markers in the colon of mice. In addition, to our knowledge we are the first to show in human samples that the expression of senescence associated markers are reduced upon CR in aging. Even though the central dogma exist that CR extends health- and lifespan, CR has several side effects which are often overlooked and are relatively understudied. In Chapter 4, we aimed to dissect the role of cellular senescence during delayed wound healing observed after CR. In a murine model for cutaneous wound healing we confirmed that CR delays wound contraction in mice on a CR diet compared to Ad Libitum-fed (AL) counterparts. In addition, after wound closure increased collagen depositions was observed. Our data showed that quantities of transiently present senescent cells in wounds are retarded when mice were fed a CR diet, 2 weeks prior to and during the wound healing process. Furthermore, we show that wounds from caloric restricted animals show higher numbers of TUNNEL-positive nuclei (apoptotic marker), possibly indicating a cell fate switch from senescence to apoptotic cell death explaining the impairment of wound-associated senescent cell burden. We also
129
show that transplantation of senescent cells in wounds of CR mice, was not sufficient to restore impaired wound healing capacity caused by CR.
Oxygen pressure is one of the most varying physiological conditions between and within organs. In Chapter 5, we analyzed the effects of physiological normoxic and hypoxic oxygen conditions on different aspects of the senescent phenotype. First, we show that hypoxic oxygen conditions restrain the expression and secretion of pro-inflammatory SASP without affecting other classical senescence markers such as cell cycle arrest, β-galactosidase staining and cell size. Second, we showed that detrimental paracrine effects of SASP on cancer cell migration are restrained under hypoxia in vitro. Third, we provided in vivo evidence in both human and mouse tissues that lower oxygenated tissues and tissue regions show comparable proportions of senescent cells but express significantly lower levels of pro-inflammatory SASP factors. Fourth, we identified AMPK as a central mediator in the reduction of SASP under hypoxia via het inhibition of mTOR and NF-kB; two major pathways that are known to promote pro-inflammatory SASP. Fifth we discovered that hypoxia mimetic agents act as senostatics by impairing the expression and secretion of SASP in vitro and aged mice.
The data from Chapters 3,4 and 5 indicate that metabolic alterations could influence detrimental effects of senescent cells. In chapter 6, we broaden the scope of this concept beyond senescence and reviewed the currently available literature on how oxygen and oxygen-associated pathways impact on different aspects of aging and physiology. We also discuss the clinical manifestations of dysregulated oxygen pathways and how targeting these pathways can help to improve health.
In line with data presented in this thesis I formulated new research questions. In the next section I discuss how these questions can be addressed in the future.
1. What is the role of cellular senescence in CR mediated effects on health- and lifespan?
CR is one of the most effective and robust non-genetic approaches to increase life- and healthspan in a wide variety of model organisms including non-human primates, rodents, fish, worms and some insects1. Clinical research indicate that CR or fasting mimicking diets, could be utilized in the clinic to
promote health and possibly longevity 2–5. Exactly how CR impact on health and longevity remains open
for debate but can at least in part be explained by the reduction of inflammation and reactive oxygen species (ROS) 1. Senescent cells are important drivers of inflammation and ROS, possibly providing a
mechanistic link between CR and aging 6,7.
In chapter 3, we show that CR reduces the expression of senescence associated markers in both mouse and aged human colon. Cell type specific data on senescence reduction are not well defined and could be further explored by comparative histological analysis of different organs from AL and CR fed
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128 Summary
The research presented in this thesis aims to dissect the role of environmental conditions on the antagonistic pleiotropic effects of senescent cells in aging and tissue repair (Chapter 2-6). The exponential increase in life expectancy of the human population pushed the incidence of devastating age-related diseases beyond the sustainability of our healthcare systems, instigating the urgent need to develop anti-aging interventions. The chronic accumulation of senescent cells in essentially all tissues during aging boost age-related diseases thus providing a useful target for the development of anti-aging therapies. However, the transient appearance of senescent cells have essential beneficial functions during wound healing, tissue remodeling and embryonic development and therefore represent a major hurdle to develop senescence targeting therapies with minimal toxicities and adverse effects.
In Chapter 2, we reviewed the current literature indicating that senescence targeting therapies, collectively named senotherapies, influence beneficial functions of senescent cells in wound healing, reprogramming and fibrosis.
The underlying causes of the paradoxical opposing functions of senescent cells remains only partly understood. A new concept emerging in relation to this topic states that both the functions and phenotypes of senescent cells depend on environmental conditions such as oxygen pressure and nutrient availability, creating specific senescence subtypes with distinct functions. In Chapter 3, we analyzed colon tissues from both humans and mice under a caloric restriction (CR) diet for senescence associated markers. In accordance with previous literature we show a reduction of transcriptional senescence markers in the colon of mice. In addition, to our knowledge we are the first to show in human samples that the expression of senescence associated markers are reduced upon CR in aging. Even though the central dogma exist that CR extends health- and lifespan, CR has several side effects which are often overlooked and are relatively understudied. In Chapter 4, we aimed to dissect the role of cellular senescence during delayed wound healing observed after CR. In a murine model for cutaneous wound healing we confirmed that CR delays wound contraction in mice on a CR diet compared to Ad Libitum-fed (AL) counterparts. In addition, after wound closure increased collagen depositions was observed. Our data showed that quantities of transiently present senescent cells in wounds are retarded when mice were fed a CR diet, 2 weeks prior to and during the wound healing process. Furthermore, we show that wounds from caloric restricted animals show higher numbers of TUNNEL-positive nuclei (apoptotic marker), possibly indicating a cell fate switch from senescence to apoptotic cell death explaining the impairment of wound-associated senescent cell burden. We also
129
show that transplantation of senescent cells in wounds of CR mice, was not sufficient to restore impaired wound healing capacity caused by CR.
Oxygen pressure is one of the most varying physiological conditions between and within organs. In Chapter 5, we analyzed the effects of physiological normoxic and hypoxic oxygen conditions on different aspects of the senescent phenotype. First, we show that hypoxic oxygen conditions restrain the expression and secretion of pro-inflammatory SASP without affecting other classical senescence markers such as cell cycle arrest, β-galactosidase staining and cell size. Second, we showed that detrimental paracrine effects of SASP on cancer cell migration are restrained under hypoxia in vitro. Third, we provided in vivo evidence in both human and mouse tissues that lower oxygenated tissues and tissue regions show comparable proportions of senescent cells but express significantly lower levels of pro-inflammatory SASP factors. Fourth, we identified AMPK as a central mediator in the reduction of SASP under hypoxia via het inhibition of mTOR and NF-kB; two major pathways that are known to promote pro-inflammatory SASP. Fifth we discovered that hypoxia mimetic agents act as senostatics by impairing the expression and secretion of SASP in vitro and aged mice.
The data from Chapters 3,4 and 5 indicate that metabolic alterations could influence detrimental effects of senescent cells. In chapter 6, we broaden the scope of this concept beyond senescence and reviewed the currently available literature on how oxygen and oxygen-associated pathways impact on different aspects of aging and physiology. We also discuss the clinical manifestations of dysregulated oxygen pathways and how targeting these pathways can help to improve health.
In line with data presented in this thesis I formulated new research questions. In the next section I discuss how these questions can be addressed in the future.
1. What is the role of cellular senescence in CR mediated effects on health- and lifespan?
CR is one of the most effective and robust non-genetic approaches to increase life- and healthspan in a wide variety of model organisms including non-human primates, rodents, fish, worms and some insects1. Clinical research indicate that CR or fasting mimicking diets, could be utilized in the clinic to
promote health and possibly longevity 2–5. Exactly how CR impact on health and longevity remains open
for debate but can at least in part be explained by the reduction of inflammation and reactive oxygen species (ROS) 1. Senescent cells are important drivers of inflammation and ROS, possibly providing a
mechanistic link between CR and aging 6,7.
In chapter 3, we show that CR reduces the expression of senescence associated markers in both mouse and aged human colon. Cell type specific data on senescence reduction are not well defined and could be further explored by comparative histological analysis of different organs from AL and CR fed
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animals. In addition, it remains elusive if observed reduction in inflammation is attributed to a reduction in senescence burden on its own or if CR utilizes direct effects on distinct inflammation drivers such as immune cells. It is likely that both pathways play a role and could be dissected further. Recent evidences indicate that not caloric intake per se, but the composition and quality of macronutrients in the diet can regulate aging and longevity independent from total caloric intake8.
Reduction of components in diets without reducing total caloric intake (isocaloric diet) could therefore be an interesting approach to dissect which macronutrients impact on senescent cell burden and associated phenotypes such as SASP expression.
In recent years, a sizeable fraction of beneficial effects of CR on longevity and health span have been linked to reduced intake of proteins or certain amino acids9. For example, reducing protein content
extends lifespan of mice compared to isocaloric fed counterparts and reduces the onset of age-related diseases including cardiovascular disease8. Furthermore, diets restricted in methionine extend mouse
lifespan and retards age-associated deteriations including cataracts and T-cell related immunosesncence10. Interestingly, a recent report shows that methionine restriction delays the
induction of senescence and dampens the SASP 11. It is therefore tempting to speculate that isocaloric
diets with varying protein and/or amino acid compositions have the capacity to modulate senescence induction and/or senescence associated phenotypes and could be an interesting direction for future research.
Recent advances in the field of CR and aging indicate that other discrepancies about CR mediated effects on life- and healthspan dependent on gender, genetic background, meal frequency and time of feeding 1. The samples from mice and humans characterized in Chapter 3 are biased towards the male
gender and CR diet in mice is achieved by providing one meal per day always at the same timepoint. Differences between males and females related to senescence induction and extension of data to other mouse strains, different feeding times and possibly different model organisms could be performed to investigate whether these parameters play a role.
2. What contributes to the difference in wound associated senescent cell burden between ad libitum-fed and caloric restricted mice?
Senescent cells that transiently appear in cutaneous wounds are regarded as beneficial 12. In Chapter
4, we demonstrate in a murine model for cutaneous wound healing that CR prior and during wound healing is associated with delayed wound closure and coincides with reduced numbers of senescent cells. Molecular mechanisms underlying the reduction in senescent cells in wounds of caloric restricted animals remains to be further explored.
131
One hypothesis that we have postulates that senescence induction is prevented during wound healing by the interference with senescence promoting events in wounds. Even though it is not exactly clear what the main inducer of cellular senescence in cutaneous wounds is, CCN1 has been suggested to induce p16 mediated senescence in wounds via the generation of ROS13. CR is well known to reduce
ROS formation which may explain lower senescence burden in wounds of caloric restricted mice. This hypothesis can be tested by measurement and modulation of ROS levels in wound of CR fed mice and assessment of the consequent effects on senescence induction during wound healing.
Another hypothesis we generated, postulates that the reduction in senescent cells could be explained by CR mediated altered cell fate choice towards apoptotic cell death instead of senescence in wounds. In chapter 4, preliminary data shows that the number of TUNEL-positive cells in wounds of caloric restricted animals is significantly higher compared to wounds from AL fed mice supporting this hypothesis. Future research should aim at the discovery of molecular pathways involved in cell fate decision between apoptosis and senescence during wound healing. A pathway that can be investigated related to cell fate decision in wound healing is mTOR. mTOR has been shown to promote geroconversion: the process that converts a temporary growth arrest (termed quiescence), into an irreversible growth arrest normally seen in senescent cells14. It is well documented that mTOR is
inhibited by CR. In addition, rapamycin (and other pharmacological inhibitors of mTOR) delay wound healing as described in chapter 1. In vitro and In vivo experiments can be performed using pharmacological mTOR activators under CR conditions to test whether the interference with this pathway is sufficient to restore wound healing speed and wound associated senescence.
CR is believed to, at least in part, execute its health benefits via the reduction of the IGF-1 and insulin signaling pathways. These pathways have a common downstream effector protein called protein kinase B, also known as AKT, which is important for cell fate decision in response to cellular stresses. AKT is a robust positive regulator of cell survival by inhibition of pro-apoptotic proteins in senescent cells and might play a role as a mediator of apoptosis induction during CR in cutaneous wounds15. In
line with this, reports show that Incubation of human epithelial cells with serum from IGF-1 deficient individuals before providing oxidative stress, leads to less DNA damage but increased apoptotic cell death16. In addition, treatment of AKT deficient cells with hydrogen peroxide induced oxidative stress,
increased apoptotic cell death compared to cells with intact AKT16. This implies that impairment of
IGF-1 signaling and downstream effector protein AKT lowers the threshold to execute apoptotic cell death in response to stress.
Future research could start by measuring AKT activity in skin and wounds of CR mice in comparison to AL fed counterparts. In addition, following apoptotic cell death during the process of wound healing in
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animals. In addition, it remains elusive if observed reduction in inflammation is attributed to a reduction in senescence burden on its own or if CR utilizes direct effects on distinct inflammation drivers such as immune cells. It is likely that both pathways play a role and could be dissected further. Recent evidences indicate that not caloric intake per se, but the composition and quality of macronutrients in the diet can regulate aging and longevity independent from total caloric intake8.
Reduction of components in diets without reducing total caloric intake (isocaloric diet) could therefore be an interesting approach to dissect which macronutrients impact on senescent cell burden and associated phenotypes such as SASP expression.
In recent years, a sizeable fraction of beneficial effects of CR on longevity and health span have been linked to reduced intake of proteins or certain amino acids9. For example, reducing protein content
extends lifespan of mice compared to isocaloric fed counterparts and reduces the onset of age-related diseases including cardiovascular disease8. Furthermore, diets restricted in methionine extend mouse
lifespan and retards age-associated deteriations including cataracts and T-cell related immunosesncence10. Interestingly, a recent report shows that methionine restriction delays the
induction of senescence and dampens the SASP 11. It is therefore tempting to speculate that isocaloric
diets with varying protein and/or amino acid compositions have the capacity to modulate senescence induction and/or senescence associated phenotypes and could be an interesting direction for future research.
Recent advances in the field of CR and aging indicate that other discrepancies about CR mediated effects on life- and healthspan dependent on gender, genetic background, meal frequency and time of feeding 1. The samples from mice and humans characterized in Chapter 3 are biased towards the male
gender and CR diet in mice is achieved by providing one meal per day always at the same timepoint. Differences between males and females related to senescence induction and extension of data to other mouse strains, different feeding times and possibly different model organisms could be performed to investigate whether these parameters play a role.
2. What contributes to the difference in wound associated senescent cell burden between ad libitum-fed and caloric restricted mice?
Senescent cells that transiently appear in cutaneous wounds are regarded as beneficial 12. In Chapter
4, we demonstrate in a murine model for cutaneous wound healing that CR prior and during wound healing is associated with delayed wound closure and coincides with reduced numbers of senescent cells. Molecular mechanisms underlying the reduction in senescent cells in wounds of caloric restricted animals remains to be further explored.
131
One hypothesis that we have postulates that senescence induction is prevented during wound healing by the interference with senescence promoting events in wounds. Even though it is not exactly clear what the main inducer of cellular senescence in cutaneous wounds is, CCN1 has been suggested to induce p16 mediated senescence in wounds via the generation of ROS13. CR is well known to reduce
ROS formation which may explain lower senescence burden in wounds of caloric restricted mice. This hypothesis can be tested by measurement and modulation of ROS levels in wound of CR fed mice and assessment of the consequent effects on senescence induction during wound healing.
Another hypothesis we generated, postulates that the reduction in senescent cells could be explained by CR mediated altered cell fate choice towards apoptotic cell death instead of senescence in wounds. In chapter 4, preliminary data shows that the number of TUNEL-positive cells in wounds of caloric restricted animals is significantly higher compared to wounds from AL fed mice supporting this hypothesis. Future research should aim at the discovery of molecular pathways involved in cell fate decision between apoptosis and senescence during wound healing. A pathway that can be investigated related to cell fate decision in wound healing is mTOR. mTOR has been shown to promote geroconversion: the process that converts a temporary growth arrest (termed quiescence), into an irreversible growth arrest normally seen in senescent cells14. It is well documented that mTOR is
inhibited by CR. In addition, rapamycin (and other pharmacological inhibitors of mTOR) delay wound healing as described in chapter 1. In vitro and In vivo experiments can be performed using pharmacological mTOR activators under CR conditions to test whether the interference with this pathway is sufficient to restore wound healing speed and wound associated senescence.
CR is believed to, at least in part, execute its health benefits via the reduction of the IGF-1 and insulin signaling pathways. These pathways have a common downstream effector protein called protein kinase B, also known as AKT, which is important for cell fate decision in response to cellular stresses. AKT is a robust positive regulator of cell survival by inhibition of pro-apoptotic proteins in senescent cells and might play a role as a mediator of apoptosis induction during CR in cutaneous wounds15. In
line with this, reports show that Incubation of human epithelial cells with serum from IGF-1 deficient individuals before providing oxidative stress, leads to less DNA damage but increased apoptotic cell death16. In addition, treatment of AKT deficient cells with hydrogen peroxide induced oxidative stress,
increased apoptotic cell death compared to cells with intact AKT16. This implies that impairment of
IGF-1 signaling and downstream effector protein AKT lowers the threshold to execute apoptotic cell death in response to stress.
Future research could start by measuring AKT activity in skin and wounds of CR mice in comparison to AL fed counterparts. In addition, following apoptotic cell death during the process of wound healing in
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comparative studies of AL fed and CR fed mice that are pharmacologically or genetically modified for the IGF-1- and insulin signaling axis might provide useful insights. Senescence and apoptosis induction by CCN1 and oxidative stress can be investigated in vitro, in the presence of serum from AL and CR fed mice to further investigate cell fate decision upon exposure to these stimuli. The development of reporter mouse models that allow for simultaneous tracking of senescence and apoptosis by taking advantage of live imaging could offer a valid research tool in this context.
3. What is the role of HIF-1 in SASP regulation?
In chapter 5, we identified that the effects of hypoxia on the expression of pro-inflammatory SASP are independent from Hypoxia inducible factor-1 (HIF). In accordance with previous literature that uncovered pro-inflammatory functions of HIF and the activation of HIF in senescent cells, we showed that genetic ablation of HIF under normoxia, impairs pro-inflammatory SASP expression. The underlying molecular mechanisms could be further developed.
In the past, it has been established that HIF can transcriptionally regulate pro-inflammatory factors directly via the activation of pro-inflammatory transcription factor NF-kB 17. Transcriptomics analysis
of senescent cells, modified for HIF can help to identify the full scope of HIF dependent SASP factors. In addition, HIF physically interacts and regulates a variety of proteins that include well established SASP and senescence regulators such as: p53, mTOR and p16 18. These proteins could be further
investigated in relation to different senescence phenotypes such as SASP and cell cycle arrest. The involvement of these proteins could be tested by co-Immunoprecipitation experiments of HIF and interactors in senescent cells.
Extracellular vesicles are a recently emerged arm of the secretory program of senescent cells which function as conveyors of senescence associated paracrine effects 19,20. HIF has been shown to play a
major role in the development of extracellular vesicles released by cancer cells under hypoxia21.
Detailed analysis of extracellular vesicles secreted by senescent cells that are modified for HIF might help to further develop the understanding about the involvement of HIF this arm of the SASP. Because of the pro-tumorigenic role of HIF in cancer progression, a wide variety of pharmacological HIF inhibitors have been developed. An interesting future direction could be to test these compounds for their potential to modulate senescence phenotypes and battle the detrimental effects of senescent cells.
4. How do hypoxia mimetic agents mechanistically restrain the SASP?
In Chapter 5, we show that relatively short treatments (2 weeks) with hypoxia mimetics, Roxadustat or Dihydroxy benzoic acid (DHB), retrain the expression and secretion of pro-inflammatory SASP
133
factors in RAS induced senescent cells and aged mice. In addition, we show that treatment of senescent cells with hypoxia mimetics retards the transcriptional activity of NF-kB, a master regulator of pro-inflammatory SASP expression. We still lack information about upstream regulators of NF-kb that are involved in SASP suppression by hypoxia mimetics.
Future experiments might investigate whether SASP inhibition by hypoxia mimetics requires HIF activity and which isoform of the HIF family and propyl hydroxylase family are involved. A transcriptome and proteome analysis of hypoxia mimetics treated senescent cells, genetically modified for HIF and PHD isoforms, will help to address this point.
Another hypothesis that we generated postulates that SASP inhibition by hypoxia mimetics is mediated by HIF-independent functions of propyl hydroxylases. From the literature it is clear that PHD inhibition is different from solely HIF activation and PHD isoforms have a number of distinct substrates22. This
supposition is supported by reports showing that neuroprotective effects of hypoxia mimetics (including DHB) are independent from HIF-signaling but rather depend on different pathways orchestrated by inhibition of PHD123. Other reports show that PHD inhibition by amino acid deprivation
or hypoxia mimetic treatment (by a compound called DMOG), leads to mTORC1 inhibition which does not require HIF-1α24. In chapter 5, we also identified that SASP inhibition by hypoxia is dependent on
mTORC1 but independent from HIF-1α. It would be of interest to investigate if mTORC1 inhibition is achieved upon treatment with hypoxia mimetics in vitro and in vivo. In addition, reporter assays for the different PHD isoforms and testing whether SASP inhibition by hypoxia mimetics could be reversed by knockdown of different PHD isoforms will provide insights about primary molecular targets of hypoxia mimetics.
5. What is the therapeutic potential of hypoxia mimetics treatment and environmental oxygen oscillations to alleviate aging and age-related diseases?
Due to the capacity of hypoxia mimetics to restrain SASP, perhaps these drugs carry the capacity to fight detrimental effects of senescent cells via interference with various senescence features. Given the direct anti-inflammatory effects on senescent cells, hypoxia mimetics could be used to alleviate diseases and age-related functional decline, which are both associated to senescence mediated chronic inflammation. It would be of interest to investigate functional performance in aged mice after the treatment with hypoxia mimetics, for example muscle strength, endurance and spontaneous activity. In addition, broadening the scope of organs in which SASP is reduced upon hypoxia mimetics treatments might help to predict functional parameters and disease states that potentially benefit from hypoxia mimetic treatment.
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comparative studies of AL fed and CR fed mice that are pharmacologically or genetically modified for the IGF-1- and insulin signaling axis might provide useful insights. Senescence and apoptosis induction by CCN1 and oxidative stress can be investigated in vitro, in the presence of serum from AL and CR fed mice to further investigate cell fate decision upon exposure to these stimuli. The development of reporter mouse models that allow for simultaneous tracking of senescence and apoptosis by taking advantage of live imaging could offer a valid research tool in this context.
3. What is the role of HIF-1 in SASP regulation?
In chapter 5, we identified that the effects of hypoxia on the expression of pro-inflammatory SASP are independent from Hypoxia inducible factor-1 (HIF). In accordance with previous literature that uncovered pro-inflammatory functions of HIF and the activation of HIF in senescent cells, we showed that genetic ablation of HIF under normoxia, impairs pro-inflammatory SASP expression. The underlying molecular mechanisms could be further developed.
In the past, it has been established that HIF can transcriptionally regulate pro-inflammatory factors directly via the activation of pro-inflammatory transcription factor NF-kB 17. Transcriptomics analysis
of senescent cells, modified for HIF can help to identify the full scope of HIF dependent SASP factors. In addition, HIF physically interacts and regulates a variety of proteins that include well established SASP and senescence regulators such as: p53, mTOR and p16 18. These proteins could be further
investigated in relation to different senescence phenotypes such as SASP and cell cycle arrest. The involvement of these proteins could be tested by co-Immunoprecipitation experiments of HIF and interactors in senescent cells.
Extracellular vesicles are a recently emerged arm of the secretory program of senescent cells which function as conveyors of senescence associated paracrine effects 19,20. HIF has been shown to play a
major role in the development of extracellular vesicles released by cancer cells under hypoxia21.
Detailed analysis of extracellular vesicles secreted by senescent cells that are modified for HIF might help to further develop the understanding about the involvement of HIF this arm of the SASP. Because of the pro-tumorigenic role of HIF in cancer progression, a wide variety of pharmacological HIF inhibitors have been developed. An interesting future direction could be to test these compounds for their potential to modulate senescence phenotypes and battle the detrimental effects of senescent cells.
4. How do hypoxia mimetic agents mechanistically restrain the SASP?
In Chapter 5, we show that relatively short treatments (2 weeks) with hypoxia mimetics, Roxadustat or Dihydroxy benzoic acid (DHB), retrain the expression and secretion of pro-inflammatory SASP
133
factors in RAS induced senescent cells and aged mice. In addition, we show that treatment of senescent cells with hypoxia mimetics retards the transcriptional activity of NF-kB, a master regulator of pro-inflammatory SASP expression. We still lack information about upstream regulators of NF-kb that are involved in SASP suppression by hypoxia mimetics.
Future experiments might investigate whether SASP inhibition by hypoxia mimetics requires HIF activity and which isoform of the HIF family and propyl hydroxylase family are involved. A transcriptome and proteome analysis of hypoxia mimetics treated senescent cells, genetically modified for HIF and PHD isoforms, will help to address this point.
Another hypothesis that we generated postulates that SASP inhibition by hypoxia mimetics is mediated by HIF-independent functions of propyl hydroxylases. From the literature it is clear that PHD inhibition is different from solely HIF activation and PHD isoforms have a number of distinct substrates22. This
supposition is supported by reports showing that neuroprotective effects of hypoxia mimetics (including DHB) are independent from HIF-signaling but rather depend on different pathways orchestrated by inhibition of PHD123. Other reports show that PHD inhibition by amino acid deprivation
or hypoxia mimetic treatment (by a compound called DMOG), leads to mTORC1 inhibition which does not require HIF-1α24. In chapter 5, we also identified that SASP inhibition by hypoxia is dependent on
mTORC1 but independent from HIF-1α. It would be of interest to investigate if mTORC1 inhibition is achieved upon treatment with hypoxia mimetics in vitro and in vivo. In addition, reporter assays for the different PHD isoforms and testing whether SASP inhibition by hypoxia mimetics could be reversed by knockdown of different PHD isoforms will provide insights about primary molecular targets of hypoxia mimetics.
5. What is the therapeutic potential of hypoxia mimetics treatment and environmental oxygen oscillations to alleviate aging and age-related diseases?
Due to the capacity of hypoxia mimetics to restrain SASP, perhaps these drugs carry the capacity to fight detrimental effects of senescent cells via interference with various senescence features. Given the direct anti-inflammatory effects on senescent cells, hypoxia mimetics could be used to alleviate diseases and age-related functional decline, which are both associated to senescence mediated chronic inflammation. It would be of interest to investigate functional performance in aged mice after the treatment with hypoxia mimetics, for example muscle strength, endurance and spontaneous activity. In addition, broadening the scope of organs in which SASP is reduced upon hypoxia mimetics treatments might help to predict functional parameters and disease states that potentially benefit from hypoxia mimetic treatment.
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Senescent cells are more resistant to cell death compared to proliferating and quiescent cells, via the activation of anti-apoptotic pathways25. SASP factors have been shown to orchestrate important
pro-survival networks in established senescent cells via paracrine and autocrine manners leading to accumulation and prolonged persistence of senescent cells25. In addition, the SASP promotes paracrine
senescence and prolonged SASP signaling is postulated to fuel the aberrant accumulation of senescent cells associated with tissue disfunction and pathology. An interesting future direction could be to test whether treatment with hypoxia mimetics impair (SASP) signaling associated to survival pathways in senescent cells. In addition, testing whether hypoxia mimetics treatment renders susceptibility to senolytics (compounds that selectively eliminate senescent cells) that target anti-apoptotic signaling pathways might be of interest.
In Chapter 5, we showed that conditioned medium from senescent cells treated with hypoxia mimetics failed to promote robust cancer cell migration in vitro. It would be interesting to investigate whether, co-injection of hypoxia mimetics treated senescent cells or senescent cells that are genetically ablated for molecular targets of hypoxia mimetics, with cancer cells in mice show a retarded capacity to promote tumor progression such as growth and metastasis.
In Chapter 5, we also show that the exposure of cells to hypoxic oxygen conditions restrain the expression and secretion of pro-inflammatory SASP in vitro, raising the possibility to use natural oxygen oscillations, for example by residence at high altitude, to influence senescence phenotypes such as SASP. In Chapter 6, we described that individuals residing at medium but not extreme high altitude regions show reduced likelihood to develop age-related diseases such as cardiovascular disease compared to individuals living close to sea level altitude. However, a clear link with senescence has never been established. In future research, tissue material from high and low residing individuals could be analyzed for senescence and inflammation markers. Aging studies with mice that are maintained in oxygen conditions that resemble pressures at different altitudes could be performed. In dept analysis of tissue from mice exposed to different oxygen pressures for senescence markers will help to clarify direct effects on senescence accumulation and secretion of inflammatory SASP factors. In addition, aging parameters such as physical activity, muscle strength, endurance and intrinsic aging phenotypes such as tissue damage and age-related pathologies could be explored.
The converse question could also be asked, namely, would hyperoxic oxygen, which is used in therapeutic strategies against lung diseases, lead to increased senescence associated detrimental effects? In Chapter 6, we stated that several studies report adverse effects after prolonged treatment with hyperbaric oxygen such as difficulty in middle ear equalization, paranasal sinuses pressure sensation, dental barotrauma and emphysematous bulla, likely due to induction of oxidative stress. In
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addition, different studies indicate that senescent cells act as drivers of chronic lung disease progression for example as a mediator of inflammation, which is possibly affected by high oxygen conditions after hyperbaric oxygen therapies 26. Future lines of research could investigate senescence
induction and inflammation in lungs of mice that are exposure to hyperbaric oxygen conditions and explore the potential of senolytics or genetic ablation of senescent cells to alleviate adverse effects of hyperbaric oxygen treatment.
One risk with the use of hypoxic oxygen conditions or hypoxia mimetics on the long term, is the occurrence of natural (metabolic) adaptation responses that potentially, over time, diminish the effects initiated by short term hypoxia mimetics treatment or acute hypoxia. Monitoring SASP expression after prolonged treatment with hypoxia mimetics or hypoxia will indicate whether the effects on senescent cells are overshadowed by adaptive responses, possibly leading to a re-establishment of SASP. In addition, data not presented in this thesis, indicate that observed SASP limiting effects are reversible upon re-exposure of hypoxic senescent cells to normoxic oxygen conditions or removal of hypoxia mimetics. In the future, research should aim at formulating optimal and sufficient therapeutic windows for hypoxia mimetic administration or hypoxia exposure to achieve SASP inhibition leading to reduction of senescence mediated detrimental effects.
References
1. Fontana, L. & Partridge, L. Promoting health and longevity through diet: from model organisms to humans. Cell 161, 106–118 (2015).
2. Fontana, L., Meyer, T. E., Klein, S. & Holloszy, J. O. Long-term calorie restriction is highly effective in reducing the risk for atherosclerosis in humans. PNAS 101, 6659–6663 (2004). 3. Redman, L. M. et al. Metabolic Slowing and Reduced Oxidative Damage with Sustained Caloric
Restriction Support the Rate of Living and Oxidative Damage Theories of Aging. Cell Metab. 27, 805-815 (2018).
4. Il’yasova, D. et al. Effects of 2 years of caloric restriction on oxidative status assessed by urinary F2-isoprostanes: The CALERIE 2 randomized clinical trial. Aging Cell 17, 1–9 (2018).
5. Brandhorst, S. et al. A Periodic Diet that Mimics Fasting Promotes Multi-System Regeneration, Enhanced Cognitive Performance, and Healthspan. Cell Metab. 22, 86–99 (2015).
6. Coppé, J. P. et al. Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol. 6, e301 (2008).
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Senescent cells are more resistant to cell death compared to proliferating and quiescent cells, via the activation of anti-apoptotic pathways25. SASP factors have been shown to orchestrate important
pro-survival networks in established senescent cells via paracrine and autocrine manners leading to accumulation and prolonged persistence of senescent cells25. In addition, the SASP promotes paracrine
senescence and prolonged SASP signaling is postulated to fuel the aberrant accumulation of senescent cells associated with tissue disfunction and pathology. An interesting future direction could be to test whether treatment with hypoxia mimetics impair (SASP) signaling associated to survival pathways in senescent cells. In addition, testing whether hypoxia mimetics treatment renders susceptibility to senolytics (compounds that selectively eliminate senescent cells) that target anti-apoptotic signaling pathways might be of interest.
In Chapter 5, we showed that conditioned medium from senescent cells treated with hypoxia mimetics failed to promote robust cancer cell migration in vitro. It would be interesting to investigate whether, co-injection of hypoxia mimetics treated senescent cells or senescent cells that are genetically ablated for molecular targets of hypoxia mimetics, with cancer cells in mice show a retarded capacity to promote tumor progression such as growth and metastasis.
In Chapter 5, we also show that the exposure of cells to hypoxic oxygen conditions restrain the expression and secretion of pro-inflammatory SASP in vitro, raising the possibility to use natural oxygen oscillations, for example by residence at high altitude, to influence senescence phenotypes such as SASP. In Chapter 6, we described that individuals residing at medium but not extreme high altitude regions show reduced likelihood to develop age-related diseases such as cardiovascular disease compared to individuals living close to sea level altitude. However, a clear link with senescence has never been established. In future research, tissue material from high and low residing individuals could be analyzed for senescence and inflammation markers. Aging studies with mice that are maintained in oxygen conditions that resemble pressures at different altitudes could be performed. In dept analysis of tissue from mice exposed to different oxygen pressures for senescence markers will help to clarify direct effects on senescence accumulation and secretion of inflammatory SASP factors. In addition, aging parameters such as physical activity, muscle strength, endurance and intrinsic aging phenotypes such as tissue damage and age-related pathologies could be explored.
The converse question could also be asked, namely, would hyperoxic oxygen, which is used in therapeutic strategies against lung diseases, lead to increased senescence associated detrimental effects? In Chapter 6, we stated that several studies report adverse effects after prolonged treatment with hyperbaric oxygen such as difficulty in middle ear equalization, paranasal sinuses pressure sensation, dental barotrauma and emphysematous bulla, likely due to induction of oxidative stress. In
135
addition, different studies indicate that senescent cells act as drivers of chronic lung disease progression for example as a mediator of inflammation, which is possibly affected by high oxygen conditions after hyperbaric oxygen therapies 26. Future lines of research could investigate senescence
induction and inflammation in lungs of mice that are exposure to hyperbaric oxygen conditions and explore the potential of senolytics or genetic ablation of senescent cells to alleviate adverse effects of hyperbaric oxygen treatment.
One risk with the use of hypoxic oxygen conditions or hypoxia mimetics on the long term, is the occurrence of natural (metabolic) adaptation responses that potentially, over time, diminish the effects initiated by short term hypoxia mimetics treatment or acute hypoxia. Monitoring SASP expression after prolonged treatment with hypoxia mimetics or hypoxia will indicate whether the effects on senescent cells are overshadowed by adaptive responses, possibly leading to a re-establishment of SASP. In addition, data not presented in this thesis, indicate that observed SASP limiting effects are reversible upon re-exposure of hypoxic senescent cells to normoxic oxygen conditions or removal of hypoxia mimetics. In the future, research should aim at formulating optimal and sufficient therapeutic windows for hypoxia mimetic administration or hypoxia exposure to achieve SASP inhibition leading to reduction of senescence mediated detrimental effects.
References
1. Fontana, L. & Partridge, L. Promoting health and longevity through diet: from model organisms to humans. Cell 161, 106–118 (2015).
2. Fontana, L., Meyer, T. E., Klein, S. & Holloszy, J. O. Long-term calorie restriction is highly effective in reducing the risk for atherosclerosis in humans. PNAS 101, 6659–6663 (2004). 3. Redman, L. M. et al. Metabolic Slowing and Reduced Oxidative Damage with Sustained Caloric
Restriction Support the Rate of Living and Oxidative Damage Theories of Aging. Cell Metab. 27, 805-815 (2018).
4. Il’yasova, D. et al. Effects of 2 years of caloric restriction on oxidative status assessed by urinary F2-isoprostanes: The CALERIE 2 randomized clinical trial. Aging Cell 17, 1–9 (2018).
5. Brandhorst, S. et al. A Periodic Diet that Mimics Fasting Promotes Multi-System Regeneration, Enhanced Cognitive Performance, and Healthspan. Cell Metab. 22, 86–99 (2015).
6. Coppé, J. P. et al. Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol. 6, e301 (2008).
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effect is caused by ROS-activated NF-κB signalling. Mech. Ageing Dev. 170, 30–36 (2018). 8. Solon-Biet, S. M. et al. The ratio of macronutrients, not caloric intake, dictates cardiometabolic
health, aging, and longevity in ad libitum-fed mice. Cell Metab. 19, 418–430 (2014).
9. Mirzaei, H., Suarez, J. A. & Longo, V. D. Protein and amino acid restriction, aging and disease: From yeast to humans. Trends Endocrinol. Metab. 25, 558–566 (2014).
10. Miller, R. A. et al. Methionine-deficient diet extends mouse lifespan, slows immune and lens aging, alters glucose, T4, IGF-I and insulin levels, and increases hepatocyte MIF levels and stress resistance. Aging Cell 4, 119–125 (2005).
11. Wang, S. Y. et al. Methionine restriction delays senescence and suppresses the senescence-associated secretory phenotype in the kidney through endogenous hydrogen sulfide. Cell Cycle 18, 1573–1587 (2019).
12. Demaria, M. et al. An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA. Dev. Cell 31, 722–733 (2014).
13. Jun, J. Il & Lau, L. F. The matricellular protein CCN1 induces fibroblast senescence and restricts fibrosis in cutaneous wound healing. Nat. Cell Biol. 12, 676–685 (2010).
14. Leontieva, O.G., et al. Hypoxia suppresses conversion from proliferative arrest to cellular senescence. PNAS 109, 13314–13318 (2012).
15. Nogueira, V. et al. Akt Determines Replicative Senescence and Oxidative or Oncogenic Premature Senescence and Sensitizes Cells to Oxidative Apoptosis. Cancer Cell 14, 458–470 (2008).
16. Guevara-Aguirre, J. et al. Growth hormone receptor deficiency is associated with a major reduction in pro-aging signaling, cancer, and diabetes in humans. Sci. Transl. Med. 3, 20–23 (2011).
17. Scortegagna, M. et al. HIF-1α regulates epithelial inflammation by cell autonomous NFκB activation and paracrine stromal remodeling. Blood 111, 3343–3354 (2008).
18. Semenza, G. L. A compendium of proteins that interact with HIF-1α. EXp. Cell. Res. 356, 128– 135 (2017).
19. Takahashi, A. et al. Exosomes maintain cellular homeostasis by excreting harmful DNA from cells. Nat. Commun. 8, 1-14 (2017).
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20. Borghesan, M. et al. Small Extracellular Vesicles Are Key Regulators of Non-cell Autonomous Intercellular Communication in Senescence via the Interferon Protein IFITM3. Cell Rep. 27, 3956-3971 (2019).
21. Patton, M. C., Zubair, H., Khan, M. A., Singh, S. & Singh, A. P. Hypoxia alters the release and size distribution of extracellular vesicles in pancreatic cancer cells to support their adaptive survival.
J. Cell. Biochem. 121, 828–839 (2020).
22. Chen, R. L. et al. Roles of individual prolyl-4-hydroxylase isoforms in the first 24 hours following transient focal cerebral ischaemia: Insights from genetically modified mice. J. Physiol. 590, 4079–4091 (2012).
23. Siddiq, A. et al. Selective inhibition of hypoxia-inducible factor (HIF) prolyl-hydroxylase 1 mediates neuroprotection against normoxic oxidative death via HIF- and CREB-independent pathways. J. Neurosci. 29, 8828–8838 (2009).
24. Durán, R. V. et al. HIF-independent role of prolyl hydroxylases in the cellular response to amino acids. Oncogene 32, 4549–4556 (2013).
25. Soto-Gamez, A., Quax, W. J. & Demaria, M. Regulation of Survival Networks in Senescent Cells: From Mechanisms to Interventions. J. Mol. Biol. 431, 2629–2643 (2019).
26. Barnes, P. J., Baker, J. & Donnelly, L. E. Cellular senescence as a mechanism and target in chronic lung diseases. Am. J. Respir. Crit. Care Med. 200, 556–564 (2019).
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effect is caused by ROS-activated NF-κB signalling. Mech. Ageing Dev. 170, 30–36 (2018). 8. Solon-Biet, S. M. et al. The ratio of macronutrients, not caloric intake, dictates cardiometabolic
health, aging, and longevity in ad libitum-fed mice. Cell Metab. 19, 418–430 (2014).
9. Mirzaei, H., Suarez, J. A. & Longo, V. D. Protein and amino acid restriction, aging and disease: From yeast to humans. Trends Endocrinol. Metab. 25, 558–566 (2014).
10. Miller, R. A. et al. Methionine-deficient diet extends mouse lifespan, slows immune and lens aging, alters glucose, T4, IGF-I and insulin levels, and increases hepatocyte MIF levels and stress resistance. Aging Cell 4, 119–125 (2005).
11. Wang, S. Y. et al. Methionine restriction delays senescence and suppresses the senescence-associated secretory phenotype in the kidney through endogenous hydrogen sulfide. Cell Cycle 18, 1573–1587 (2019).
12. Demaria, M. et al. An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA. Dev. Cell 31, 722–733 (2014).
13. Jun, J. Il & Lau, L. F. The matricellular protein CCN1 induces fibroblast senescence and restricts fibrosis in cutaneous wound healing. Nat. Cell Biol. 12, 676–685 (2010).
14. Leontieva, O.G., et al. Hypoxia suppresses conversion from proliferative arrest to cellular senescence. PNAS 109, 13314–13318 (2012).
15. Nogueira, V. et al. Akt Determines Replicative Senescence and Oxidative or Oncogenic Premature Senescence and Sensitizes Cells to Oxidative Apoptosis. Cancer Cell 14, 458–470 (2008).
16. Guevara-Aguirre, J. et al. Growth hormone receptor deficiency is associated with a major reduction in pro-aging signaling, cancer, and diabetes in humans. Sci. Transl. Med. 3, 20–23 (2011).
17. Scortegagna, M. et al. HIF-1α regulates epithelial inflammation by cell autonomous NFκB activation and paracrine stromal remodeling. Blood 111, 3343–3354 (2008).
18. Semenza, G. L. A compendium of proteins that interact with HIF-1α. EXp. Cell. Res. 356, 128– 135 (2017).
19. Takahashi, A. et al. Exosomes maintain cellular homeostasis by excreting harmful DNA from cells. Nat. Commun. 8, 1-14 (2017).
137
20. Borghesan, M. et al. Small Extracellular Vesicles Are Key Regulators of Non-cell Autonomous Intercellular Communication in Senescence via the Interferon Protein IFITM3. Cell Rep. 27, 3956-3971 (2019).
21. Patton, M. C., Zubair, H., Khan, M. A., Singh, S. & Singh, A. P. Hypoxia alters the release and size distribution of extracellular vesicles in pancreatic cancer cells to support their adaptive survival.
J. Cell. Biochem. 121, 828–839 (2020).
22. Chen, R. L. et al. Roles of individual prolyl-4-hydroxylase isoforms in the first 24 hours following transient focal cerebral ischaemia: Insights from genetically modified mice. J. Physiol. 590, 4079–4091 (2012).
23. Siddiq, A. et al. Selective inhibition of hypoxia-inducible factor (HIF) prolyl-hydroxylase 1 mediates neuroprotection against normoxic oxidative death via HIF- and CREB-independent pathways. J. Neurosci. 29, 8828–8838 (2009).
24. Durán, R. V. et al. HIF-independent role of prolyl hydroxylases in the cellular response to amino acids. Oncogene 32, 4549–4556 (2013).
25. Soto-Gamez, A., Quax, W. J. & Demaria, M. Regulation of Survival Networks in Senescent Cells: From Mechanisms to Interventions. J. Mol. Biol. 431, 2629–2643 (2019).
26. Barnes, P. J., Baker, J. & Donnelly, L. E. Cellular senescence as a mechanism and target in chronic lung diseases. Am. J. Respir. Crit. Care Med. 200, 556–564 (2019).
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Dutch summary
Thijmen van Vliet
European Research Institute for the Biology of Ageing (ERIBA), University Medical Center Groningen (UMCG), 9713AV Groningen, The Netherlands
