What Is Aging?

Cellular senescence prevents the proliferation of cells at risk for neoplastic transformation. Nonetheless, the senescence response is thought to be antagonistically pleiotropic and thus contribute to aging phenotypes, including, ironically, late life cancers. The cancer-promoting activity of senescent cells is likely due to secreted molecules, the identity of which remains largely unknown. Here, we have shown that senescent fibroblasts, much more than presenescent fibroblasts, stimulate tumor vascularization in mice. Weakly malignant epithelial cells co-injected with senescent fibroblasts had larger and greater numbers of blood vessels compared with controls. Accordingly, increased vascular endothelial growth factor (VEGF) expression was a frequent characteristic of senescent human and mouse fibroblasts in culture. Importantly, conditioned medium from senescent fibroblasts, more than medium from presenescent cells, stimulates cultured human umbilical vein endothelial cells to invade a basement membrane, a hallmark of angiogenesis. Increased VEGF expression was specific to the senescent phenotype and increased whether senescence was induced by replicative exhaustion, overexpression of p16(Ink4a), or overexpression of oncogenic RAS. The senescence-dependent increase in VEGF production was accompanied by very little increase in hypoxic-inducible (transcription) factor 1 alpha protein levels, and hypoxia further induced VEGF in senescent cells. This result suggests the rise in VEGF expression at senescence is not a hypoxic response. Our findings may in part explain why senescent cells stimulate tumorigenesis in vivo and support the idea that senescent cells may facilitate age-associated cancer development by secreting factors that promote malignant progression.

Aging is a progressive degenerative process involving a chronic low-grade inflammation and the accumulation of senescent cells. One major issue is to reveal the mechanisms which promote the deposition of pro-inflammatory senescent cells within tissues. The accumulation involves mechanisms which increase cellular senescence as well as those inhibiting the clearance of senescent cells from tissues. It is known that a persistent inflammatory state evokes a compensatory immunosuppression which inhibits pro-inflammatory processes by impairing the functions of effector immune cells, e.g., macrophages, T cells and natural killer (NK) cells. Unfortunately, these cells are indispensable for immune surveillance and the subsequent clearance of senescent cells, i.e., the inflammation-induced counteracting immunosuppression prevents the cleansing of host tissues. Moreover, senescent cells can also repress their own clearance by expressing inhibitors of immune surveillance and releasing the ligands of NKG2D receptors which impair their surveillance by NK and cytotoxic CD8+ T cells. It seems that cellular senescence and immunosuppression establish a feed-forward process which promotes the aging process and age-related diseases. I will examine in detail the immunosuppressive mechanisms which impair the surveillance and clearance of pro-inflammatory senescent cells with aging. In addition, I will discuss several therapeutic strategies to halt the degenerative feed-forward circuit associated with the aging process and age-related diseases.

Amyloid β and Tumorigenesis: Amyloid β-Induced Telomere Dysfunction in Tumor Cells

Cellular senescence, defined as an irreversible cell cycle arrest, exacerbates the tissue microenvironment. Our previous study demonstrated that mouse uterine senescent cells were physiologically increased according to gestational days and that their abnormal accumulation was linked to the onset of preterm delivery. We hypothesized that there is a mechanism for removal of senescent cells after parturition to maintain uterine function. In the current study, we noted abundant uterine senescent cells and their gradual disappearance in wild-type postpartum mice. F4/80+ macrophages were present specifically around the area rich in senescent cells. Depletion of macrophages in the postpartum mice using anti-F4/80 antibody enlarged the area of senescent cells in the uterus. We also found excessive uterine senescent cells and decreased second pregnancy success rate in a preterm birth model using uterine p53-deleted mice. Furthermore, a decrease in F4/80+ cells and an increase in CD11b+ cells with a senescence-associated inflammatory microenvironment were observed in the p53-deleted uterus, suggesting that uterine p53 deficiency affects distribution of the macrophage subpopulation, interferes with senescence clearance, and promotes senescence-induced inflammation. These findings indicate that the macrophage is a key player in the clearance of uterine senescent cells to maintain postpartum uterine function.

Normal and cancer cells facing their demise following exposure to radio-chemotherapy can actively participate in choosing their subsequent fate. These programmed cell fate decisions include true cell death (apoptosis-necroptosis) and therapy-induced cellular senescence (TIS), a permanent "proliferative arrest" commonly portrayed as premature cellular aging. Despite a permanent loss of proliferative potential, senescent cells remain viable and are highly bioactive at the microenvironment level, resulting in a prolonged impact on tissue architecture and functions. Cellular senescence is primarily documented as a tumor suppression mechanism that prevents cellular transformation. In the context of normal tissues, cellular senescence also plays important roles in tissue repair, but contributes to age-associated tissue dysfunction when senescent cells accumulate. Theoretically, in multi-step cancer progression models, cancer cells have already bypassed cellular senescence during their immortalization step (see hallmarks of cancer). It is then perhaps surprising to find that cancer cells often retain the ability to undergo TIS, or premature aging. This occurs because cellular senescence results from multiple signalling pathways, some retained in cancer cells, aiming to prevent cell cycle progression in damaged cells. Since senescent cancer cells persist after therapy and secrete an array of cytokines and growth factors that can modulate the tumor microenvironment, these cells may have beneficial and detrimental effects regarding immune modulation and survival of remaining proliferation-competent cancer cells. Similarly, while normal cells undergoing senescence are believed to remain indefinitely growth arrested, whether this is true for senescent cancer cells remains unclear, raising the possibility that these cells may represent a reservoir for cancer recurrence after treatment. This review discusses our current knowledge on cancer cell senescence and highlight questions that must be addressed to fully understand the beneficial and detrimental impacts of cellular senescence during cancer therapy.

Myofibroblasts play an important role in fibrogenesis. Hepatic stellate cells are the main precursors of myofibroblasts. Cellular senescence is the terminal cell fate in which proliferating cells undergo irreversible cell cycle arrest. Senescent hepatic stellate cells were identified in liver fibrosis. Senescent hepatic stellate cells display decreased collagen production and proliferation. Therefore, induction of senescence could be a protective mechanism against progression of liver fibrosis and the concept of therapy-induced senescence has been proposed to treat liver fibrosis. In this review, characteristics of senescent hepatic stellate cells and the essential signaling pathways involved in senescence are reviewed. Furthermore, the potential impact of senescent hepatic stellate cells on other liver cell types are discussed. Senescent cells are cleared by the immune system. The persistence of senescent cells can remodel the microenvironment and interact with inflammatory cells to induce aging-related dysfunction. Therefore, senolytics, a class of compounds that selectively induce death of senescent cells, were introduced as treatment to remove senescent cells and consequently decrease the disadvantageous effects of persisting senescent cells. The effects of senescent hepatic stellate cells in liver fibrosis need further investigation.

The aging process is associated with a low-grade chronic inflammation and the accumulation of senescent cells into tissues. Diverse stresses can trigger cellular senescence, a cell fate characterized by cell-cycle arrest and flat morphology. Oncogenic signaling can also induce cellular senescence which has been termed oncogene-induced senescence (OIS). Senescent cells display a pro-inflammatory phenotype which has been called the senescence-associated secretory phenotype (SASP). The secretomes associated with SASP contain colony-stimulating factors and chemokines which stimulate the generation of myeloid-derived suppressor cells (MDSC) by enhancing myelopoiesis in bone marrow and spleen. Enhanced myelopoiesis and increased level of MDSCs have been observed in bone marrow, spleen, and blood in both tumor-bearing and aged mice. Immunosuppressive MDSCs are recruited via chemotaxis into inflamed tissues where they proliferate and consequently suppress acute inflammatory reactions by inhibiting the functions of distinct components of innate and adaptive immunity. For instance, MDSCs stimulate the activity of immunosuppressive regulatory T-cells (Tregs). They also increase the expression of amino acid catabolizing enzymes and the secretion of anti-inflammatory cytokines, e.g. IL-10 and TGF-β, and reactive oxygen species (ROS). On the other hand, the accumulation of MDSCs into tissues exerts harmful effects in chronic pathological disorders, e.g. tumors and many age-related diseases, since the immunosuppression induced by MDSCs impairs the clearance of senescent and cancer cells and also disturbs the maintenance of energy metabolism and tissue proteostasis. The co-operation between senescent cells and immunosuppressive MDSCs regulates not only tumorigenesis and chronic inflammatory disorders but it also might promote inflammaging during the aging process.

Due to both internal and external factors, our bodies are constantly exposed to tissue damage. Effective tissue regeneration is relatively rare in nature, and we are no different. While some degree of self-repair occurs, the damaged tissue is usually replaced with fibrotic material, which does not restore the original function of what was lost. The limited regenerative potential of our body also progressively declines as we age, and is accompanied by the wide-spread accumulation of irreparable, non-proliferating cells: senescent cells. Over the last few years, an association has been observed between senescent cells and stem cells. It has been hypothesized that these two cell types may influence each other in the context of tissue damage and repair, and that this interaction might play an important role in how well, or how poorly, the tissue regenerates. To be able to develop better methods to improve tissue repair, it is therefore desirable to understand how senescence and stemness are causally connected. When it comes to regeneration after damage, the liver is perhaps one of the most relevant organs in the body, since it is capable of remarkable recovery after insult. However, when this recovery is dysfunctional, chronic liver damage usually leads to the development of serious pathologies, such as liver fibrosis, cirrhosis, and cancer. Both the liver and kidney are known to present an accumulation of senescent and stem cells after damaging insults. Models for these two tissues are therefore particularly relevant to causally assess how cellular senescence may drive features of stemness and decline in tissue function. In this thesis we studied the manner through which senescent cells are able to influence the differentiation of neighboring cells, both in a liver and kidney context, and how this influence might affect how the organ responds to damage. We explored these aspects through the use of both in vitro and in vivo models. In Chapter 2 we showcase how iPSC-derived multicellular liver structures can be used to model various types of liver damage, thus offering a valuable intermediate model to bridge in vitro and in vivo studies. We also showcase how these models can be used to test the efficacy of anti-fibrotic drugs. In Chapter 3 we show that senescent cells are able to induce de-differentiation of human hepatocytes, and that this process is mediated by IL-1β and IL-6, through the activation of NF-κB in the hepatocytes, with CD44 potentially playing a key causative role. We also show that senescent cells prolong the liver’s normal damage response, by locking liver cells into a poorly differentiated state. Finally, we show that hepatocyte de-differentiation can be reversed, leading to better hepatocyte differentiation, and improved in vivo homeostasis recovery, through the selective removal of senescent cells. In Chapter 4 we showcase iPSC-derived multicellular kidney structures as a valuable model for studying the influence of senescent cells on kidney differentiation. In Chapter 5 we offer a general discussion of the results, and the implications of our findings for the development of future therapeutic strategies.

Increased insulin resistance and impaired insulin secretion are significant characteristics manifested by patients with type 2 diabetes mellitus (T2DM). The degree and extent of these two features in T2DM vary among races and individuals. Insulin resistance is accelerated by obesity and is accompanied by accumulation of dysfunctional adipose tissues. In addition, dysfunction of pancreatic β-cells impairs insulin secretion. T2DM is significantly affected by aging, as the β-cell mass diminishes with age. Moreover, both obesity and hyperglycemia-related metabolic changes in developing diabetes are associated with accumulation of senescent cells in multiple organs, that is, organismal aging. Cellular senescence is defined as a state of irreversible cell cycle arrest with concomitant functional decline. It is caused by telomere shortening or senescence-inducing stress. Senescent cells secrete proinflammatory cytokines and chemokines, which is designated as the senescence-associated secretory phenotype (SASP), and this has a negative impact on adipose tissues and pancreatic β-cells. Recent advances in aging research have suggested that senolysis, the removal of senescent cells, can be a promising therapeutic approach to prevent or improve aging-related diseases, including diabetes. The attenuation of a SASP may be beneficial, although the pathophysiological involvement of cellular senescence in diabetes is not fully understood. In the clinical application of senotherapy, tissue-context-dependent senescent cells are increasingly being recognized as an issue to be solved. Recent studies have observed highly heterogenic and complex senescent cell populations that serve distinct roles among tissues, various stages of disease, and different ages. For example, in high-fat-diet induced diabetes with obesity, mouse adipose tissues display accumulation of p21 Cip1-highly-expressing (p21 high) cells in the early stage, followed by increases in both p21 high and p16 INK4a-highly-expressing (p16 high) cells in the late stage. Interestingly, elimination of p21 high cells in visceral adipose tissue can prevent or improve insulin resistance in mice with obesity, while p16 high cell clearance is less effective in alleviating insulin resistance. Importantly, in immune-deficient mice transplanted with fat from obese patients, dasatinib plus quercetin, a senolytic cocktail that reduces the number of both p21 high and p16 high cells, improves both glucose tolerance and insulin resistance. On the other hand, in pancreatic β cells, p16 high cells become increasingly predominant with age and development of diabetes. Consistently, elimination of p16 high cells in mice improves both glucose tolerance and glucose-induced insulin secretion. Moreover, a senolytic compound, the anti-Bcl-2 inhibitor ABT263 reduces p16 INK4a expression in islets and restores glucose tolerance in mice when combined with insulin receptor antagonist S961 treatment. In addition, efficacy of senotherapy in targeting mouse pancreatic β cells has been validated not only in T2DM, but also in type 1 diabetes mellitus. Indeed, in non-obese diabetic mice, treatment with anti-Bcl-2 inhibitors, such as ABT199, eliminates senescent pancreatic β cells, resulting in prevention of diabetes mellitus. These findings clearly indicate that features of diabetes are partly determined by which or where senescent cells reside in vivo, as adipose tissues and pancreatic β cells are responsible for insulin resistance and insulin secretion, respectively. In this review, we summarize recent advances in understanding cellular senescence in adipose tissues and pancreatic β cells in diabetes. We review the different potential molecular targets and distinctive senotherapeutic strategies in adipose tissues and pancreatic β cells. We propose the novel concept of a dual-target tailored approach in senotherapy against diabetes.

Molecular Modeling of the FOXO4-TP53 Interaction to Design Senolytic Peptides for the Elimination of Senescent Cancer Cells

Radiation Induced and FACS-Sorted Senescent tdTOMp16+ Cells Upregulate Profibrotic Gene Expression in Mesenchymal Stem Cells (Stromal Cells)

Why biological age is a major risk factor for many of the most important human diseases remains mysterious. We know that as organisms age, stem cell pools are exhausted while senescent cells progressively accumulate. Independently, induction of pluripotency via expression of Yamanaka factors (Oct4, Klf4, Sox2, c-Myc; OKSM) and clearance of senescent cells have each been shown to ameliorate cellular and physiological aspects of aging, suggesting that both processes are drivers of organismal aging. But stem cell exhaustion and cellular senescence likely interact in the etiology and progression of age-dependent diseases because both undermine tissue and organ homeostasis in different if not complementary ways. Here, we combine transient cellular reprogramming (stem cell rejuvenation) with targeted removal of senescent cells to test the hypothesis that simultaneously targeting both cell-fate based aging mechanisms will maximize life and health span benefits. We find that OKSM extends lifespan and show that both interventions protect the intestinal stem cell pool, lower inflammation, activate pro-stem cell signaling pathways, and synergistically improve health and lifespan. Our findings suggest that a combination therapy, simultaneously replacing lost stem cells and removing senescent cells, shows synergistic potential for anti-aging treatments. Our finding that transient expression of both is the most effective suggests that drug-based treatments in non-genetically tractable organisms will likely be the most translatable.

Guaraná (Paullinia cupana) improves the proliferation and oxidative metabolism of senescent adipocyte stem cells derived from human lipoaspirates

Introduction: Senescent cells have emerged as bona fide drivers of ageing and age-related cardiovascular disease, with senescent cells accumulating in the aged heart and following damage/injury. We have shown that the removal of senescent cells using senolytics can rejuvenate the regenerative capacity of the aged heart. Aim: To investigate the effects of cell senescence and the action of the senolytics, Dasatinib (D) and Quercetin (Q) on human iPSC-derived cardiomyocyte survival and cell cycle, and endothelial cell survival, cell cycle, migration and tube formation in vitro. Methods and Results: We developed a transwell insert co-culture stress-induced premature senescence human cell model system to test the effects of senolytics D+Q in vitro. Co-culture of iPSC-derived cardiomyocytes (iPSC-CMs) with senescent cardiac stromal progenitor cells (senCPCs) led to decreased number and DNA-synthesising activity of iPSC-CMs. Treatment with senolytics D+Q led to the elimination of senCPCs in the co-culture and the rescue of iPSC-CM number and DNA synthesis. Treatment of HUVECs with senCPC conditioned media decreased HUVEC number, cell cycle activity, migration, and tube formation. Treatment of HUVECs with D+Q conditioned media rescued HUVEC number, migration and tube formation. Next, we investigated the effects of co-culture of senescent HUVECs (senHUVECs) with HUVECs and showed decreased HUVEC number and DNA synthesis. Treatment with senolytics D+Q led to the elimination of senHUVECs in the co-culture and ameliorated HUVEC number, but not DNA synthesis. Treatment of HUVECs with conditioned media from senHUVECs led to decreased HUVEC migration and tube formation. Treatment of HUVECs with D+Q conditioned media improved HUVEC tube formation but not migration. Luminex analysis of the conditioned media from iPSC-CM and HUVEC co-cultures revealed upregulation of senescence-associated secretory phenotype (SASP) factors, but the level of SASP factors was reduced with the application of D+Q. Conclusion: Senescent cell removal by senolytics D+Q shows therapeutic potential in rejuvenating the reparative activity of human cardiomyocytes and endothelial cells. These results open the path to further studies on using senolytic therapy in age-related cardiac deterioration and rejuvenation. Potential impact of the findings: Senescent cells and their SASP present a promising therapeutic target to rejuvenate the heart’s reparative potential. Clinical trials using senolytics D+Q are already underway and thus far have shown promising results. Further pre-clinical studies are warranted for evidence-based clinical trials using senolytics in age-related cardiovascular diseases.

Simple SummaryAging is a progressive decline of an organism over time. In contrast, senescence occurs throughout an organism’s lifespan. It is a cell-cycle arrest preventing the proliferation of damaged cells. Cellular and molecular senescence timing is crucial for the pace of aging and disease development and progression. The accumulation of senescent cells during a lifespan leads to organismal senescence. Senescent multinucleated giant cells are present in many age-related diseases and cancer. Although senescence was assumed to be irreversible, studies now show that senescent multinucleated giant cells overcome senescence in various cancers, becoming the source of highly aggressive mononucleated stem-like cells, which divide and initiate tumor development and progression.This review introduces the subject of senescence, aging, and the formation of senescent multinucleated giant cells. We define senescence and aging and describe how molecular and cellular senescence leads to organismal senescence. We review the latest information on senescent cells’ cellular and molecular phenotypes. We describe molecular and cellular features of aging and senescence and the role of multinucleated giant cells in aging-related conditions and cancer. We explain how multinucleated giant cells form and their role in aging arteries and gonads. We also describe how multinucleated giant cells and the reversibility of senescence initiate cancer and lead to cancer progression and metastasis. We also describe molecules and pathways regulating aging and senescence in model systems and their applicability to clinical therapies in age-related diseases.