Senescence in Health and Disease.

Many cellular stresses activate senescence, a persistent hyporeplicative state characterized in part by expression of the p16INK4a cell-cycle inhibitor. Senescent cell production occurs throughout life and plays beneficial roles in a variety of physiological and pathological processes including embryogenesis, wound healing, host immunity, and tumor suppression. Meanwhile, the steady accumulation of senescent cells with age also has adverse consequences. These non-proliferating cells occupy key cellular niches and elaborate pro-inflammatory cytokines, contributing to aging-related diseases and morbidity. This model suggests that the abundance of senescent cells in vivo predicts "molecular," as opposed to chronologic, age and that senescent cell clearance may mitigate aging-associated pathology.

Suppression of autophagy induces senescence in the heart.

Aging is a critical risk factor for heart disease, including ischemic heart disease and heart failure. Cellular senescence, characterized by DNA damage, resistance to apoptosis and the senescence-associated secretory phenotype (SASP), occurs in many cell types, including cardiomyocytes. Senescence precipitates the aging process in surrounding cells and the organ through paracrine mechanisms. Generalized autophagy, which degrades cytosolic materials in a non-selective manner, is decreased during aging in the heart. This decrease causes deterioration of cellular quality control mechanisms, facilitates aging and negatively affects lifespan in animals, including mice. Although suppression of generalized autophagy could promote senescence, it remains unclear whether the suppression of autophagy directly stimulates senescence in cardiomyocytes, which, in turn, promotes myocardial dysfunction in the heart. We addressed this question using mouse models with a loss of autophagy function. Suppression of general autophagy in cardiac-specific Atg7 knockout (Atg7cKO) mice caused accumulation of senescent cardiomyocytes. Induction of senescence via downregulation of Atg7 was also observed in chimeric Atg7 cardiac-specific KO mice and cultured cardiomyocytes in vitro, suggesting that the effect of autophagy suppression upon induction of senescence is cell autonomous. ABT-263, a senolytic agent, reduced the number of senescent myocytes and improved cardiac function in Atg7cKO mice. Suppression of autophagy and induction of senescence were also observed in doxorubicin-treated hearts, where reactivation of autophagy alleviated senescence in cardiomyocytes and cardiac dysfunction. These results suggest that suppression of general autophagy directly induces senescence in cardiomyocytes, which in turn promotes cardiac dysfunction.

Senescence and senolysis in cancer: The latest findings.

Aging is a life phenomenon that occurs in most living organisms and is a major risk factor for many diseases, including cancer. Cellular senescence is a cellular trait induced by various genomic and epigenetic stresses. Senescent cells are characterized by irreversible cell growth arrest and excessive secretion of inflammatory cytokines (senescence-associated secretory phenotypes, SASP). Chronic tissue microinflammation induced by SASP contributes to the pathogenesis of a variety of age-related diseases, including cancer. Senolysis is a promising new strategy to selectively eliminate senescent cells in order to suppress chronic inflammation, suggesting its potential use as an anticancer therapy. This review summarizes recent findings on the molecular basis of senescence in cancer cells and senolysis.

Network of extracellular vesicles surrounding senescent cells.

Extracellular vesicles (EVs) are small lipid bilayers released from cells that contain cellular components such as proteins, nucleic acids, lipids, and metabolites. Biological information is transmitted between cells via the EV content. Cancer and senescent cells secrete more EVs than normal cells, delivering more information to the surrounding recipient cells. Cellular senescence is a state of irreversible cell cycle arrest caused by the accumulation of DNA damage. Senescent cells secrete various inflammatory proteins known as the senescence-associated secretory phenotype (SASP). Inflammatory SASP factors, including small EVs, induce chronic inflammation and lead to various age-related pathologies. Recently, senolytic drugs that selectively induce cell death in senescent cells have been developed to suppress the pathogenesis of age-related diseases. This review describes the characteristics of senescent cells, the functions of EVs released from senescent cells, and the therapeutic effects of EVs on age-related diseases. Understanding the biology of EVs secreted from senescent cells will provide valuable insights for achieving healthy longevity in an aging society.

Identification of a new human senescent skin cell marker ribonucleoside-diphosphate reductase subunit M2 B.

In skin aging, it has been hypothesized that aging fibroblasts accumulate within the epidermal basal layer, dermis, and subcutaneous fat, causing abnormal tissue remodeling and extracellular matrix dysfunction, thereby inducing an aging-related secretory phenotype (SASP). A new treatment for skin aging involves the specific elimination of senescent skin cells, especially fibroblasts within the dermis and keratinocytes in the basal layer. This requires the identification of specific protein markers of senescent cells, such as ribonucleoside-diphosphate reductase subunit M2 B (RRM2B), which is upregulated in various malignancies in response to DNA stress damage. However, the behavior and role of RRM2B in skin aging remain unclear. Therefore, we examined whether RRM2B functions as a senescence marker using a human dermal fibroblast model of aging. In a model of cellular senescence induced by replicative aging and exposure to ionizing radiation or UVB, RRM2B was upregulated at the gene and protein levels. This was correlated with decreased uptake of the senescence-associated ß-galactosidase activity and proliferation marker bromodeoxyuridine. RRM2B upregulation was concurrent with the increased expression of SASP factor genes. Furthermore, using fluorescence flow cytometry, RRM2B-positive cells were recovered more frequently in the aging cell population. In aging human skin, RRM2B was also found to be more abundant in the dermis and epidermal basal layer than other proteins. Therefore, RRM2B may serve as a clinical marker to identify senescent skin cells.

Cellular senescence and kidney aging.

Life expectancy is increasing worldwide, and by 2050 the proportion of the world's population over 65 years of age is estimated to surpass 1.5 billion. Kidney aging is associated with molecular and physiological changes that cause a loss of renal function and of regenerative potential. As the aging population grows, it is crucial to understand the mechanisms underlying these changes, as they increase the susceptibility to developing acute kidney injury (AKI) and chronic kidney disease (CKD). Various cellular processes and molecular pathways take part in the complex process of kidney aging. In this review, we will focus on the phenomenon of cellular senescence as one of the involved mechanisms at the crossroad of kidney aging, age-related disease, and CKD. We will highlight experimental and clinical findings about the role of cellular senescence in kidney aging and CKD. In addition, we will review challenges in senescence research and emerging therapeutic aspects. We will highlight the great potential of senolytic strategies for the elimination of harmful senescent cells to promote healthy kidney aging and to avoid age-related disease and CKD. This review aims to give insight into recent discoveries and future developments, providing a comprehensive overview of current knowledge on cellular senescence and anti-senescent therapies in the kidney field.

Exploration of New Therapies for Heart Failure Targeting Age-Related Mechanisms.

Evidence indicates a role of cellular senescence and systemic insulin resistance (hyperinsulinemia) in the pathogenesis of age-related cardiovascular-metabolic disorders, including heart failure, atherosclerotic diseases, obesity, and diabetes. "Metabolic remodeling" is one of the keywords for aging research, and studies with brown adipose tissue have shown that maintaining the homeostasis of this organ is crucial to suppressing the progression of pathologies in obesity and heart failure. The mechanisms contributing to the synchronization of aging (sync-aging) are mysterious and interesting. "Senometabolite" or "senoprotein" are defined as circulating molecules that have causal roles in sync-aging, which requires the establishment of new concepts: age-related fibrotic disorders (A-FiDs), and senometabolite-related disorders (SRDs). Globally, researchers are active in comprehensive and conclusive studies targeting age-related circulating molecules. Recently, the senolytic approach opened a new avenue for aging research. Senolysis, mediated through a genetic/pharmacologic/vaccination approach, reversed aging and pathologies in age-related diseases. Suppression of prosenescent molecules (senocules) and senolysis, the specific depletion of senescent cells, will become next-generation therapies for cardiovascular diseases.

New Senolysis Approach via Antibody-Drug Conjugate Targeting of the Senescent Cell Marker Apolipoprotein D for Skin Rejuvenation.

Senescent cells accumulate in aging skin, causing age-related changes and a decline in functional efficiency. Therefore, senolysis, a treatment that specifically removes senescent cells and rejuvenates the skin, should be explored. We targeted apolipoprotein D (ApoD), a previously identified marker expressed on senescent dermal fibroblasts, and investigated a novel senolysis approach using a monoclonal antibody against this antigen and a secondary antibody conjugated with the cytotoxic drug pyrrolobenzodiazepine. Observations using fluorescently labeled antibodies revealed that ApoD functions as a surface marker of senescent cells and that the antibody is taken up and internalized only by such cells. The concurrent administration of the antibody with the PBD-conjugated secondary antibody specifically eliminated only senescent cells without harming young cells. The antibody-drug conjugate treatment of aging mice combined with the administration of antibodies reduced the number of senescent cells in the dermis of mice and improved the senescent skin phenotype. These results provide a proof-of-principle evaluation of a novel approach to specifically eliminate senescent cells using antibody-drug conjugates against senescent cell marker proteins. This approach is a potential candidate for clinical applications to treat pathological skin aging and related diseases via the removal of senescent cells.

Induction of ferroptosis selectively eliminates senescent tubular cells.

The accumulation of senescent cells is an important contributor to kidney aging, chronic renal disease, and poor outcome after kidney transplantation. Approaches to eliminate senescent cells with senolytic compounds have been proposed as novel strategies to improve marginal organs. While most existing senolytics induce senescent cell clearance by apoptosis, we observed that ferroptosis, an iron-catalyzed subtype of regulated necrosis, might serve as an alternative way to ablate senescent cells. We found that murine kidney tubular epithelial cells became sensitized to ferroptosis when turning senescent. This was linked to increased expression of pro-ferroptotic lipoxygenase-5 and reduced expression of anti-ferroptotic glutathione peroxidase 4 (GPX4). In tissue slice cultures from aged kidneys low dose application of the ferroptosis-inducer RSL3 selectively eliminated senescent cells while leaving healthy tubular cells unaffected. Similar results were seen in a transplantation model, in which RSL3 reduced the senescent cell burden of aged donor kidneys and caused a reduction of damage and inflammatory cell infiltration during the early post-transplantation period. In summary, these data reveal an increased susceptibility of senescent tubular cells to ferroptosis with the potential to be exploited for selective reduction of renal senescence in aged kidney transplants.

Dietary restriction in senolysis and prevention and treatment of disease.

Aging represents a key risk factor for a plethora of diseases. Targeting detrimental processes which occur during aging, especially before onset of age-related disease, could provide drastic improvements in healthspan. There is increasing evidence that dietary restriction (DR), including caloric restriction, fasting, or fasting-mimicking diets, extend both lifespan and healthspan. This has sparked interest in the use of dietary regimens as a non-pharmacological means to slow aging and prevent disease. Here, we review the current evidence on the molecular mechanisms underlying DR-induced health improvements, including removal of senescent cells, metabolic reprogramming, and epigenetic rejuvenation.

PALVEN: phase Ib trial of palbociclib, letrozole and venetoclax in estrogen receptor- and BCL2-positive advanced breast cancer.

The addition of a CDK4/6 inhibitor to endocrine therapy improves progression-free and overall survival in women with metastatic estrogen receptor-positive breast cancer. In that setting, CDK4/6 inhibitors induce a potent cell-cycle arrest (which may be accompanied by tumor senescence) but fail to induce apoptotic cell death. Venetoclax is a potent inhibitor of BCL2, a pro-survival protein overexpressed in the majority of estrogen receptor-positive cancers. Pre-clinical findings indicate that venetoclax augments tumor response to the CDK4/6 inhibitor palbociclib by triggering apoptosis, including in senescent cells. The PALVEN phase Ib trial will further examine this finding. The primary objective is to identify the maximum tolerated dose and determine the recommended phase II dose for palbociclib, letrozole and venetoclax combination therapy. Clinical Trial Registration: NCT03900884 (ClinicalTrials.gov).

The current 'gold standard' treatment for estrogen receptor-positive, HER2-negative metastatic breast cancer is endocrine therapy with a CDK4/6 inhibitor. This combination improves tumor response and patient outcomes, primarily by reducing tumor cell growth. Paradoxically, less killing of tumor cells is observed in the presence of a CDK4/6 inhibitor. The authors hypothesize that co-treatment with venetoclax, an inhibitor of the BCL2 survival protein, will help trigger tumor death, thereby further improving tumor responses and patient outcomes. As a first step, combination therapy comprising letrozole, palbociclib and venetoclax will be tested in a phase I trial to identify the recommended doses for subsequent studies.

Apoptosis resistance of senescent cells is an intrinsic barrier for senolysis induced by cardiac glycosides.

Targeted elimination of senescent cells, senolysis, is one of the core trends in the anti-aging therapy. Cardiac glycosides were recently proved to be a broad-spectrum senolytics. Here we tested senolytic properties of cardiac glycosides towards human mesenchymal stem cells (hMSCs). Cardiac glycosides had no senolytic ability towards senescent hMSCs of various origins. Using biological and bioinformatic approaches we compared senescence development in 'cardiac glycosides-sensitive' A549 and '-insensitive' hMSCs. The absence of senolysis was found to be mediated by the effective potassium import and increased apoptosis resistance in senescent hMSCs. Weakening "antiapoptotic defense" predisposes hMSCs to senolysis. We revealed that apoptosis resistance, previously recognized as a common characteristic of senescence, in fact, is not a general feature of senescent cells. Moreover, only apoptosis-prone senescent cells are sensitive to cardiac glycosides-induced senolysis. Thus, we can speculate that the effectiveness of senolysis might depend on whether senescent cells indeed become apoptosis-resistant as compared to their proliferating counterparts.

Targeting Multiple Homeostasis-Maintaining Systems by Ionophore Nigericin Is a Novel Approach for Senolysis.

Within the present study we proposed a novel approach for senolysis based on the simultaneous disturbance of the several homeostasis-maintaining systems in senescent cells including intracellular ionic balance, energy production and intracellular utilization of damaged products. Of note, we could not induce senolysis by applying ouabain, amiloride, valinomycin or NH4Cl-compounds that modify each of these systems solely. However, we found that ionophore nigericin can disturb plasma membrane potential, intracellular pH, mitochondrial membrane potential and autophagy at once. By affecting all of the tested homeostasis-maintaining systems, nigericin induced senolytic action towards stromal and epithelial senescent cells of different origins. Moreover, the senolytic effect of nigericin was independent of the senescence-inducing stimuli. We uncovered that K+ efflux caused by nigericin initiated pyroptosis in senescent cells. According to our data, the higher sensitivity of senescent cells compared to the control ones towards nigericin-induced death was partially mediated by the lower intracellular K+ content in senescent cells and by their predisposition towards pyroptosis. Finally, we proposed an interval dosing strategy to minimize the negative effects of nigericin on the control cells and to achieve maximal senolytic effect. Hence, our data suggest ionophore nigericin as a new senotherapeutic compound for testing against age-related diseases.

Senescence of Tumor Cells in Anticancer Therapy-Beneficial and Detrimental Effects.

Cellular senescence process results in stable cell cycle arrest, which prevents cell proliferation. It can be induced by a variety of stimuli including metabolic stress, DNA damage, telomeres shortening, and oncogenes activation. Senescence is generally considered as a process of tumor suppression, both by preventing cancer cells proliferation and inhibiting cancer progression. It can also be a key effector mechanism for many types of anticancer therapies such as chemotherapy and radiotherapy, both directly and through bioactive molecules released by senescent cells that can stimulate an immune response. Senescence is characterized by a senescence-associated secretory phenotype (SASP) that can have both beneficial and detrimental impact on cancer progression. Despite the negatives, attempts are still being made to use senescence to fight cancer, especially when it comes to senolytics. There is a possibility that a combination of prosenescence therapy-which targets tumor cells and causes their senescence-with senotherapy-which targets senescent cells, can be promising in cancer treatment. This review provides information on cellular senescence, its connection with carcinogenesis and therapeutic possibilities linked to this process.

Selective Elimination of Senescent Fibroblasts by Targeting the Cell Surface Protein ACKR3.

The accumulation of senescent cells in aging tissues is associated with age-related diseases and functional decline. Thus, senolysis, a therapy aimed at rejuvenation by removing senescent cells from the body, is being developed. However, this therapy requires the identification of membrane surface antigens that are specifically expressed on senescent cells for their selective elimination. We showed that atypical chemokine receptor 3 (ACKR3), a receptor of the CXC motif chemokine 12 (CXCL12) implicated in cancer, inflammation, and cardiovascular disorders, is selectively expressed on the surface of senescent human fibroblasts but not on proliferating cells. Importantly, the differential presence of ACKR3 enabled the isolation of senescent cells by flow cytometry using anti-ACKR3 antibodies. Furthermore, antibody-dependent cellular cytotoxicity assays revealed that cell surface ACKR3 preferentially sensitizes senescent but not dividing fibroblasts to cell injury by natural killer cells. Conclusively, the selective expression of ACKR3 on the surface of senescent cells allows the preferential elimination of senescent cells. These results might contribute to the future development of novel senolysis approaches.

Senolytic treatment modulates decidualization in human endometrial stromal cells.

Decidualization - the differentiation of endometrial stromal cells (ESCs) into decidual cells - is a crucial step for successful embryo implantation and placentation that is initiated in the secretory phase of the menstrual cycle. During decidualization, ESCs undergo proliferation arrest and secrete inflammatory mediators, including senescence-associated secretory phenotype (SASP). Although several senolytic agents improve age-related diseases, their effects on cellular senescence in decidualizing ESCs has not been explored. To do this, we treated decidualized ESCs with the senolytic agents Quercetin (Que), Dasatinib (Das), and BPTES. Que decreased the number of senescence-associated ß-galactosidase (SA-ß-Gal) positive cells and expression of senescence markers in ESCs treated with the decidual stimulus (dibutyryl-cAMP plus progesterone: DP). Concomitantly, Que markedly increased the expression of the decidualization markers IGFBP1, PRL, and FOXO1, in decidualizing ESCs. Similar to Que, Das also stimulated decidualization. Treatment with a combination of Que and Das synergistically increased the expression of decidualization markers and senescence markers compared with treatment with Que or Das alone. However, BPTES did not enhance the expression of decidualization markers. These results imply that treatment with Que and/or Das can remove senescent decidual cells and enhance the decidualization of the rest of ESCs. Thus, senolytic modulation of abnormal ESC decidualization could alleviate infertility caused by dysfunctions of endometrial receptivity and embryo implantation.

Functional changes in beta cells during ageing and senescence.

Insulin secretion from beta cells is crucial for maintaining euglycaemia and preventing type 2 diabetes, a disease correlated with ageing. Therefore, understanding the functional changes that beta cell function undergoes with age can reveal new therapeutic targets and strategies to delay or revert the disease. Herein, a systematic review of the literature agrees that, as humans age, their beta cell function declines, independently of peripheral insulin resistance, BMI and waist circumference. Rodent studies reveal that, with age, basal insulin secretion increases with either no change or an increase in stimulated insulin secretion, but the biological significance of this is unclear. The accumulation of senescent beta cells could explain some of these functional changes: transcriptional analysis of senescent and aged beta cells revealed parallel downregulation of several steps along the pathway linking glucose stimulation and insulin secretion. Moreover, specific deletion of senescent cells (senolysis) improved residual beta cell function, gene expression profile and blood glucose levels. In conclusion, cellular senescence could underlie the functional decline of beta cells during ageing and could represent a novel and promising approach for recovering insulin secretion. Graphical abstract.

Converging Paths of Pulmonary Arterial Hypertension and Cellular Senescence.

Cellular senescence is recognized as a crucial contributor to the pathobiology of various degenerative and cardiovascular diseases, such as idiopathic pulmonary fibrosis and atherosclerosis. We describe the potential link between cellular senescence and the degenerative character of neointimal pulmonary vascular disease in pulmonary arterial hypertension (PAH). Senescence markers have been described in remodeled pulmonary arteries, and PAH and senescence share common triggers and pathogenic pathways, such as transforming growth factor-ß/bone morphogenetic protein and TNF-α. In addition, interventions that target a senescence phenotype also target pulmonary vascular remodeling in vivo. These data provide a basis for further exploration of the role of senescence in the pathobiology of PAH and for preclinical trials with a senolytic class of drugs.

Senotherapy: growing old and staying young?

Cellular senescence, which has been linked to age-related diseases, occurs during normal aging or as a result of pathological cell stress. Due to their incapacity to proliferate, senescent cells cannot contribute to normal tissue maintenance and tissue repair. Instead, senescent cells disturb the microenvironment by secreting a plethora of bioactive factors that may lead to inflammation, regenerative dysfunction and tumor progression. Recent understanding of stimuli and pathways that induce and maintain cellular senescence offers the possibility to selectively eliminate senescent cells. This novel strategy, which so far has not been tested in humans, has been coined senotherapy or senolysis. In mice, senotherapy proofed to be effective in models of accelerated aging and also during normal chronological aging. Senotherapy prolonged lifespan, rejuvenated the function of bone marrow, muscle and skin progenitor cells, improved vasomotor function and slowed down atherosclerosis progression. While initial studies used genetic approaches for the killing of senescent cells, recent approaches showed similar effects with senolytic drugs. These observations open up exciting possibilities with a great potential for clinical development. However, before the integration of senotherapy into patient care can be considered, we need further research to improve our insight into the safety and efficacy of this strategy during short- and long-term use.

Therapeutic Senolysis of Axitinib-Induced Senescent Human Lung Cancer Cells.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) inhibit receptor-mediated signals in cells. Axitinib is a TKI with high specificity for vascular endothelial growth factor receptors (VEGFRs). AIM: We determined whether axitinib could induce senescence in human cancer cells and be lysed by the senolytic drug ABT-263. METHODS: Human lung and breast adenocarcinoma cell lines were used. These cells were cultured with axitinib or a multi-target TKI lenvatinib. The expression of ß-galactosidase, VEGFRs, Ki-67, reactive oxygen species (ROS) of cancer cells, and their BrdU uptake were evaluated by flow cytometry. The mRNA expression of p21 and IL-8 was examined by quantitative PCR. The effects of TKIs on phosphorylation of Akt and Erk1/2, as downstream molecules of VEGFR signaling, were examined by immunoblot. The in vivo anti-cancer effect was examined using a xenograft mice model. RESULTS: Axitinib, but not lenvatinib, induced cellular senescence (increased cell size and enhanced expression of ß-galactosidase) in all adenocarcinoma cell lines. Axitinib-induced senescence was unrelated to the expression of VEGFRs on cancer cells. ROS were involved in axitinib-induced senescence. Axitinib-induced senescent lung adenocarcinoma A549 cells were drastically lysed by ABT-263. In A549-xenografted mice, combination therapy with axitinib and ABT-263 significantly suppressed tumor growth with the induction of apoptotic cancer cells.