Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders

Aging is the leading risk factor of chronic diseases including cardiovascular diseases and metabolic diseases. Emerging evidence suggests cellular senescence, one of the pillars of aging, plays a causal role in development of these diseases. While we previously reported that senescent cell elimination delays or prevents progression of several aging phenotypes in aged mice, the responsible types of senescent cells contributing to diseases in vivo are not yet fully elucidated. We made high fat diet-induced obesity model (60% fat diet, HFD) and examine the cell type of senescent cells. Single cell mass cytometry analysis using adipose tissue revealed that senescent endothelial cells expressed the DNA damage marker γH2AX and inflammatory factors such as TNFα and IL-6 (Figure 1). To test the hypothesis that senescent endothelial cells are a major contributor to chronic diseases, we developed Tie2-Cre; p16 Ink4a - LOX-ATTAC mice, which allows specific elimination of p16 Ink4a positive senescent endothelial cells upon administration of AP20187 (which only affects Tie2 -expressing p16 Ink4a positive cells carrying the conditionally-expressed ATTAC construct) (Figure 2). We found that targeted removal of these cells alleviated HFD-induced metabolic dysfunction with decrease of inflammatory cytokines (Figure 3). In contrast, transplanting senescent endothelial cells into lean mice caused adipose tissue inflammation and metabolic dysfunction. We found the inflammatory factors from senescent endothelial cells induced inflammation in preadipocytes and expand inflammation and targeting senescent endothelial cells may cut the expanding inflammation. Consistent with these findings, natural flavonoid fisetin, which we previously identified that can induce apoptosis selectively to senescent endothelial cells among other senescent cell types, reduced senescent cell amount in adipose tissue and improved glucose metabolism. Moreover, we confirmed the effect of fisetin against senescent endothelial cells in freshly isolated human omental adipose tissue from obese individuals. The fisetin-treated explants exhibited a significant reduction in both SA-β-gal-positive adipocytes and endothelial cells, as well as fewer p16-positive cells, indicating that fisetin induced apoptosis in senescent endothelial cells. Collectively, these data suggest that elimination of p16 Ink4a positive senescent endothelial cells may be a viable therapeutic strategy for alleviation of metabolic diseases.

Telomere shortening induces cellular senescence in proliferative cells. Yet, it is presently unclear how it is triggered in post‐mitotic cells such as cardiac myocytes. A new study by Anderson et al (2019) reports that during ageing of the heart, cellular senescence develops independently of telomere length, but is evoked by DNA damage, which preferentially accumulates at the telomere. Removal of senescent cells using senolytic drugs ameliorated cardiac hypertrophy and fibrosis and may inform novel approaches to improve the conditions for the ageing heart.

What Is Aging?

In the Limelight: December 2021

Changes of senescent cell accumulation and removal in skin tissue with ageing.

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.

Dysregulated cholesterol metabolism is critical in the pathogenesis of AMD. Cellular senescence contributes to the development of numerous age-associated diseases. In this study, we investigated the link between cholesterol burden and the cellular senescence of photoreceptors. Retinas from rod-specific ATP binding cassette subfamily A member 1 (Abca1) and G member 1 (Abcg1) (Abca1/g1-rod/-rod) knockout mice fed with a high-fat diet were analyzed for the signs of cellular senescence. Real-time quantitative PCR and immunofluorescence were used to characterize the senescence profile of the retina and cholesterol-treated photoreceptor cell line (661W). Inducible elimination of p16(Ink4a)-positive senescent cells (INK-ATTAC) mice or the administration of senolytic drugs (dasatinib and quercetin: D&Q) were used to examine the impact of senolytics on AMD-like phenotypes in Abca1/g1-rod/-rod retina. Increased accumulation of senescent cells as measured by markers of cellular senescence was found in Abca1/g1-rod/-rod retina. Exogenous cholesterol also induced cellular senescence in 661W cells. Selective elimination of senescent cells in Abca1/g1-rod/-rod;INK-ATTAC mice or by administration of D&Q improved visual function, lipid accumulation in retinal pigment epithelium, and Bruch's membrane thickening. Cholesterol accumulation promotes cellular senescence in photoreceptors. Eliminating senescent photoreceptors improves visual function in a model of retinal neurodegeneration, and senotherapy offers a novel therapeutic avenue for further investigation.

Aging is the main risk factor for most chronic diseases, disabilities, and declining health. It has been proposed that senescent cells--damaged cells that have lost the ability to divide--drive the deterioration that underlies aging and age-related diseases. However, definitive evidence for this relationship has been lacking. The use of a progeroid mouse model (which expresses low amounts of the mitotic checkpoint protein BubR1) has been instrumental in demonstrating that p16(Ink4a)-positive senescent cells drive age-related pathologies and that selective elimination of these cells can prevent or delay age-related deterioration. These studies identify senescent cells as potential therapeutic targets in the treatment of aging and age-related diseases. Here, we describe how senescent cells develop, the experimental evidence that causally implicates senescent cells in age-related dysfunction, the chronic diseases and disorders that are characterized by the accumulation of senescent cells at sites of pathology, and the therapeutic approaches that could specifically target senescent cells.

Cellular senescence entails an irreversible growth arrest and a pro-inflammatory secretory phenotype, which contributes to aging-associated disorders such as atherosclerosis and diabetes, however, underlying mechanisms are largely unknown. In this study, we identified a novel protein, senescence-associated glycoprotein (SAGP), as a biomarker of cellular senescence and we also found that elimination of senescent cells targeting SAGP attenuated aging-associated disorders such as atherosclerosis and diabetes. First, we identified that SAGP as a senescent marker by microarray analysis of senescent human endothelial cells compared with young endothelial cells. The expression of SAGP was significantly increased in the aorta of chronological aging mice or ApoE-knockout mice. Then we measured SAGP expression in the patients registered in our hospital and found that mean SAGP expression was significantly higher in patients with atherosclerotic diseases compared to patients without atherosclerotic diseases.Recently, it is reported that elimination of senescent cells (senolysis) reversibly improved pathological aging phenotypes and also extended the lifespan. We established senolytic therapy targeting SAGP. We generated SAGP-DTR (diphtheria toxin receptor) transgenic mice, in which we could eliminate the SAGP- positive senescent cells using DT (diphtheria toxin). We found elimination of SAGP positive senescent cells significantly reduced the atherosclerotic plaque burden in the aorta of ApoE-KO mice and improved the glucose metabolism of dietary obese mice. For clinical implication, we then developed a cytotoxic vaccine targeting SAGP. Treatment with SAGP vaccine successfully eliminated SAGP positive senescent cells and attenuated atherosclerosis and metabolic dysfunction. These data indicate that targeting SAGP-positive cells could be a novel strategy for senolytic therapy.

Cigarette smoke (CS) leads to increased oxidative stress, inflammation, and exaggerated senescence, which are involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). While the role of cellular senescence in COPD is known, it is not clear if the removal of senescent cells could alleviate the disease symptoms. To test this, we used the novel mouse model‐p16‐3MR, and studied the effect of ganciclovir (GCV)‐mediated removal of senescent cells after chronic CS (3 months) and environmental tobacco smoke (ETS) (6 months) exposure to CS. Our results showed the reversal of CS‐induced cellular senescence on the clearance of p16+ senesced cells by GCV treatment. Interestingly, the clearance of p16+ senescent cells via GCV led to a decrease in the neutrophil counts in the BALF of GCV‐treated CS‐exposed p16‐3MR mice, as well as reversal of CS‐mediated airspace enlargement in p16‐3MR mice. Mice exposed to low dose ETS caused insignificant changes in the SA‐β‐Gal+ senescent cells and airspace enlargement. Overall, our data provide evidence for the role of lung cellular senescence on smoke exposure and clearance of senescent cells in p16‐3MR mice in the reversal of COPD/emphysema pathology with a possibility of senolytics as therapeutic interventions in COPD.

SummaryCellular senescence plays an important role in organismal aging and age‐related diseases. However, it is challenging to isolate low numbers of senescent cells from small volumes of biofluids for downstream analysis. Furthermore, there is no technology that could selectively remove senescent cells in a high‐throughput manner. In this work, we developed a novel microfluidic chip platform, termed senescence chip, for ultrahigh‐throughput isolation and removal of senescent cells. The core component of our senescence chip is a slanted and tunable 3D micropillar array with a variety of shutters in the vertical direction for rapid cell sieving, taking advantage of the characteristic cell size increase during cellular senescence. The 3D configuration achieves high throughput, high recovery rate, and device robustness with minimum clogging. We demonstrated proof‐of‐principle applications in isolation and enumeration of senescent mesenchymal stem cells (MSCs) from undiluted human whole blood, and senescent cells from mouse bone marrow after total body irradiation, with the single‐cell resolution. After scale‐up to a multilayer and multichannel structure, our senescence chip achieved ultrahigh‐throughput removal of senescent cells from human whole blood with an efficiency of over 70% at a flow rate of 300 ml/hr. Sensitivity and specificity of our senescence chips could be augmented with implementation of multiscale size separation, and identification of background white blood cells using their cell surface markers such as CD45. With the advantages of high throughput, robustness, and simplicity, our senescence chips may find wide applications and contribute to diagnosis and therapeutic targeting of cellular senescence.

Cellular senescence entails an irreversible growth arrest and a pro-inflammatory secretory phenotype, which contributes to aging-associated disorders such as atherosclerosis and diabetes, however, underlying mechanisms are largely unknown. In this study, we identified a novel protein, senescence-associated glycoprotein (SAGP), as a biomarker of cellular senescence and we also found that elimination of senescent cells targeting SAGP attenuated aging-associated disorders such as atherosclerosis, diabetes and frailty. First, we identified that SAGP as a senescent marker by microarray analysis of senescent human endothelial cells compared with young endothelial cells. The expression of SAGP was significantly increased in the aorta of chronological aging mice and ApoE-knockout mice. Then we measured SAGP expression in the patients registered in our hospital and found that mean SAGP expression was significantly higher in patients with atherosclerotic diseases compared to patients without atherosclerotic diseases. These data suggest that SAGP would become the novel marker of cellular senescence and/or aging-associated disorders. We found SAGP co-localized with lysosome and bound to V-ATPase, proton pump in the acid organelles such as lysosome. The electron microscopy analysis revealed that the dysfunctional lysosomes were accumulated in SAGP knockdown endothelial cell. The genetic deletion of SAGP resulted in the increase of lysosomal pH and the suppression of mitochondrial autophagy, mitophagy. And this associated with the high level of mitochondrial reactive oxygen species (ROS) and promoted premature senescence in human endothelial cells. These data suggest that SAGP was induced by the lysosomal stress in the senescent cells to protects senescent cells by maintaining the lysosomal homeostasis. Recently, it is reported that elimination of senescent cells (senolysis) reversibly improved pathological aging phenotypes and also extended the lifespan. We established senolytic therapy targeting SAGP. We generated SAGP-DTR (diphtheria toxin receptor) transgenic mice, in which we could eliminate the SAGP- positive senescent cells using DT (diphtheria toxin). We found elimination of SAGP positive senescent cells significantly reduced the atherosclerotic plaque burden in the aorta of ApoE-KO mice and improved the glucose metabolism of dietary obese mice, indicating that SAGP could be a useful target for senolytic therapy. For clinical implication, we then developed a cytotoxic vaccine targeting SAGP. Treatment with SAGP vaccine successfully eliminated SAGP positive senescent cells and attenuated atherosclerosis and metabolic dysfunction. Surprisingly, administration of SAGP vaccine to Zmpste24-KO mice, premature aging mice, extended the lifespan. These data indicate that targeting SAGP-positive cells could be a novel strategy for senolytic therapy. Effect of SAGP vaccine Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Grant-in-Aid for Scientific Research by Japan Society for the Promotion of Science (JSPS)

Cellular senescence is defined as a state of irreversible growth arrest and is accompanied by changes of both cell morphology and gene expression. Although accumulation of senescent vascular endothelial cells impair the vessel homeostasis and promote atherosclerotic diseases, underlying mechanisms are largely unknown. In this study, we identified a novel protein, senescence-associated glycoprotein (SAGP), as a biomarker of cellular senescence and we found modulation of SAGP or elimination of senescent cells targeting SAGP would become a novel therapy for atherosclerotic diseases. We found that SAGP expression was significantly increased in human endothelial cells undergoing replicative senescence compared with young endothelial cells. We also found SAGP expression in aorta was significantly increased both in chronological aging mice or ApoE knockout mice. Furthermore, we measured SAGP expression in patients registered in our hospital and found that mean SAGP expression was significantly higher in patients with atherosclerotic diseases compared to patients without atherosclerotic diseases.These data suggest that SAGP would become a novel cellular senescence and/or atherosclerotic disease marker. Genetic deletion of SAGP resulted in high level of mitochondrial reactive oxygen species (ROS) and promoted premature senescence in human endothelial cells. And this associated with suppression of mitochondrial autophagy, mitophagy. We found SAGP co-localized with lysosome by immunocytochemistry. In addition, the electron microscopy analysis revealed that the dysfunctional lysosomes were accumulated in SAGP knockdown endothelial cell, suggesting that SAGP maintain lysosomal homeostasis. Next, wegenerated ApoE-KO/ SAGP overexpression mice and found that atherosclerotic plaque burden was attenuated in these double-transgenic mice. In contrast, SAGP/ApoE double knockout mice showed progression in atherosclerosis. These data suggest that modulation of SAGPwould become a new therapeutic target for atherosclerotic diseases. SAGP vaccine Recently, it is reported that elimination of senescent cells (senolysis) reversibly improved pathological aging phenotypes and also extended the lifespan. We have taken another approach for atherosclerotic diseases, senolytic therapy targeting SAGP. We generated SAGP-DTR (diphtheria toxin receptor) transgenic mice, in which we could eliminate the SAGP- positive senescent cells using DT (diphtheria toxin). We found elimination of SAGP positive senescent cells significantly reduced the atherosclerotic plaque burden, indicating that SAGP would become a useful target for senolytic therapy. We then developed a cytotoxic vaccine targeting SAGP. Treatment with SAGP vaccine successfully eliminated SAGP positive senescent cells. Administration of SAGP vaccine to ApoE-KO mice significantly reduced atherogenesis. These data indicate that targeting SAGP-positive cells could become a strategy for senolytic therapy.

Cellular senescence and its association with aldose reductase promote cyst growth in autosomal dominant polycystic kidney disease.

Parkinson's disease (PD) is an age-related neurodegenerative disease, and the removal of senescent cells has been proved to be beneficial for improving age-associated pathologies in neurodegeneration disease. In this study, chiral gold nanoparticles (NPs) with different helical directions were synthesized to selectively induce the apoptosis of senescent cells under light illumination. By modifying anti-B2MG and anti-DCR2 antibodies, senescent microglia cells could be cleared by chiral NPs without damaging the activities of normal cells under illumination. Notably, l-P+ NPs exhibited about a 2-fold higher elimination efficiency than d-P− NPs for senescent microglia cells. Mechanistic studies revealed that the clearance of senescent cells was mediated by the activation of the Fas signaling pathway. The in vivo injection of chiral NPs successfully confirmed that the elimination of senescent microglia cells in the brain could further alleviate the symptoms of PD mice in which the alpha-synuclein (α-syn) in cerebrospinal fluid (CFS) decreased from 83.83 ± 4.76 ng mL−1 to 8.66 ± 1.79 ng mL−1 after two months of treatment. Our findings suggest a potential strategy to selectively eliminate senescent cells using chiral nanomaterials and offer a promising strategy for alleviating PD.