Senescent cells suppress innate smooth muscle cell repair functions in atherosclerosis.

Senescent cells (SNCs) degenerate the fibrous cap that normally prevents atherogenic plaque rupture, a leading cause of myocardial infarction and stroke. Here we explored the underlying mechanism using pharmacological or transgenic approaches to clear SNCs in the Ldlr -/- mouse model of atherosclerosis. SNC clearance reinforced fully deteriorated fibrous caps in highly advanced lesions, as evidenced by restored vascular smooth muscle cell (VSMC) numbers, elastin content, and overall cap thickness. We found that SNCs inhibit VSMC promigratory phenotype switching in the first interfiber space of the arterial wall directly beneath atherosclerotic plaque, thereby limiting lesion entry of medial VSMCs for fibrous cap assembly or reinforcement. SNCs do so by antagonizing IGF-1 through the secretion of insulin-like growth factor-binding protein 3 (Igfbp3). These data indicate that the intermittent use of senolytic agents or IGFBP-3 inhibition in combination with lipid lowering drugs may provide therapeutic benefit in atherosclerosis.

Glomerular endothelial cell senescence drives age-related kidney disease through PAI-1.

The mechanisms underlying the development of glomerular lesions during aging are largely unknown. It has been suggested that senescence might play a role, but the pathophysiological link between senescence and lesion development remains unexplained. Here, we uncovered an unexpected role for glomerular endothelial cells during aging. In fact, we discovered a detrimental cross-talk between senescent endothelial cells and podocytes, through PAI-1. In vivo, selective inactivation of PAI-1 in endothelial cells protected glomeruli from lesion development and podocyte loss in aged mice. In vitro, blocking PAI-1 in supernatants from senescent endothelial cells prevented podocyte apoptosis. Consistently, depletion of senescent cells prevented podocyte loss in old p16 INK-ATTAC transgenic mice. Importantly, these experimental findings are relevant to humans. We showed that glomerular PAI-1 expression was predictive of poor outcomes in transplanted kidneys from elderly donors. In addition, we observed that in elderly patients, urinary PAI-1 was associated with age-related chronic kidney disease. Altogether, these results uncover a novel mechanism of kidney disease and identify PAI-1 as a promising biomarker of kidney dysfunction in allografts from elderly donors.

Progressive Cellular Senescence Mediates Renal Dysfunction in Ischemic Nephropathy.

BACKGROUND: Peripheral vascular diseases may induce chronic ischemia and cellular injury distal to the arterial obstruction. Cellular senescence involves proliferation arrest in response to stress, which can damage neighboring cells. Renal artery stenosis (RAS) induces stenotic-kidney dysfunction and injury, but whether these arise from cellular senescenceand their temporal pattern remain unknown. METHODS: Chronic renal ischemia was induced in transgenic INK-ATTAC and wild type C57BL/6 mice by unilateral RAS, and kidney function (in vivo micro-MRI) and tissue damage were assessed. Mouse healthy and stenotic kidneys were analyzed using unbiased single-cell RNA-sequencing. To demonstrate translational relevance, cellular senescence was studied in human stenotic kidneys. RESULTS: Using intraperitoneal AP20187 injections starting 1, 2, or 4 weeks after RAS, selective clearance of cells highly expressing p16Ink4a attenuated cellular senescence and improved stenotic-kidney function; however, starting treatment immediately after RAS induction was unsuccessful. Broader clearance of senescent cells, using the oral senolytic combination dasatinib and quercetin, in C57BL/6 RAS mice was more effective in clearing cells positive for p21 (Cdkn1a) and alleviating renal dysfunction and damage. Unbiased, single-cell RNA sequencing in freshly dissociated cells from healthy and stenotic mouse kidneys identified stenotic-kidney epithelial cells undergoing both mesenchymal transition and senescence. As in mice, injured human stenotic kidneys exhibited cellular senescence, suggesting this process is conserved. CONCLUSIONS: Maladaptive tubular cell senescence, involving upregulated p16 (Cdkn2a), p19 (Cdkn2d), and p21 (Cdkn1a) expression, is associated with renal dysfunction and injury in chronic ischemia. These findings support development of senolytic strategies to delay chronic ischemic renal injury.

CD38 ecto-enzyme in immune cells is induced during aging and regulates NAD+ and NMN levels.

Decreased NAD+ levels have been shown to contribute to metabolic dysfunction during aging. NAD+ decline can be partially prevented by knockout of the enzyme CD38. However, it is not known how CD38 is regulated during aging, and how its ecto-enzymatic activity impacts NAD+ homeostasis. Here we show that an increase in CD38 in white adipose tissue (WAT) and the liver during aging is mediated by accumulation of CD38+ immune cells. Inflammation increases CD38 and decreases NAD+. In addition, senescent cells and their secreted signals promote accumulation of CD38+ cells in WAT, and ablation of senescent cells or their secretory phenotype decreases CD38, partially reversing NAD+ decline. Finally, blocking the ecto-enzymatic activity of CD38 can increase NAD+ through a nicotinamide mononucleotide (NMN)-dependent process. Our findings demonstrate that senescence-induced inflammation promotes accumulation of CD38 in immune cells that, through its ecto-enzymatic activity, decreases levels of NMN and NAD+.

Cellular Identification and Quantification of Senescence-Associated ß-Galactosidase Activity In Vivo.

Senescence-associated ß-galactosidase (hereafter SA-ß-gal) staining has now been employed for more than 20 years to identify the presence of senescent cells (Dimri et al., Proc Natl Acad Sci U S A 92:9363-9367, 1995). These cells, characterized by a permanent cell-cycle arrest (Hayflick and Moorhead, Exp Cell Res 25:585-621, 1961) and the production of a distinct secretory phenotype of cytokines, chemokines, and proteases (Coppe et al., PLoS Biol 6:2853-2868, 2008), have received much attention in recent years for their impacts on diverse biological processes. Here we describe a method to identify and quantify the specific cells that become senescent in vivo using transmission electron microscopy after SA-ß-gal staining that can be used in countless scenarios.

Senescent cells: a therapeutic target for cardiovascular disease.

Cellular senescence, a major tumor-suppressive cell fate, has emerged from humble beginnings as an in vitro phenomenon into recognition as a fundamental mechanism of aging. In the process, senescent cells have attracted attention as a therapeutic target for age-related diseases, including cardiovascular disease (CVD), the leading cause of morbidity and mortality in the elderly. Given the aging global population and the inadequacy of current medical management, attenuating the health care burden of CVD would be transformative to clinical practice. Here, we review the evidence that cellular senescence drives CVD in a bimodal fashion by both priming the aged cardiovascular system for disease and driving established disease forward. Hence, the growing field of senotherapy (neutralizing senescent cells for therapeutic benefit) is poised to contribute to both prevention and treatment of CVD.

Senescent cells: an emerging target for diseases of ageing.

Chronological age represents the single greatest risk factor for human disease. One plausible explanation for this correlation is that mechanisms that drive ageing might also promote age-related diseases. Cellular senescence, which is a permanent state of cell cycle arrest induced by cellular stress, has recently emerged as a fundamental ageing mechanism that also contributes to diseases of late life, including cancer, atherosclerosis and osteoarthritis. Therapeutic strategies that safely interfere with the detrimental effects of cellular senescence, such as the selective elimination of senescent cells (SNCs) or the disruption of the SNC secretome, are gaining significant attention, with several programmes now nearing human clinical studies.

Senescent intimal foam cells are deleterious at all stages of atherosclerosis.

Advanced atherosclerotic lesions contain senescent cells, but the role of these cells in atherogenesis remains unclear. Using transgenic and pharmacological approaches to eliminate senescent cells in atherosclerosis-prone low-density lipoprotein receptor-deficient (Ldlr-/-) mice, we show that these cells are detrimental throughout disease pathogenesis. We find that foamy macrophages with senescence markers accumulate in the subendothelial space at the onset of atherosclerosis, where they drive pathology by increasing expression of key atherogenic and inflammatory cytokines and chemokines. In advanced lesions, senescent cells promote features of plaque instability, including elastic fiber degradation and fibrous cap thinning, by heightening metalloprotease production. Together, these results demonstrate that senescent cells are key drivers of atheroma formation and maturation and suggest that selective clearance of these cells by senolytic agents holds promise for the treatment of atherosclerosis.

Naturally occurring p16(Ink4a)-positive cells shorten healthy lifespan.

Cellular senescence, a stress-induced irreversible growth arrest often characterized by expression of p16(Ink4a) (encoded by the Ink4a/Arf locus, also known as Cdkn2a) and a distinctive secretory phenotype, prevents the proliferation of preneoplastic cells and has beneficial roles in tissue remodelling during embryogenesis and wound healing. Senescent cells accumulate in various tissues and organs over time, and have been speculated to have a role in ageing. To explore the physiological relevance and consequences of naturally occurring senescent cells, here we use a previously established transgene, INK-ATTAC, to induce apoptosis in p16(Ink4a)-expressing cells of wild-type mice by injection of AP20187 twice a week starting at one year of age. We show that compared to vehicle alone, AP20187 treatment extended median lifespan in both male and female mice of two distinct genetic backgrounds. The clearance of p16(Ink4a)-positive cells delayed tumorigenesis and attenuated age-related deterioration of several organs without apparent side effects, including kidney, heart and fat, where clearance preserved the functionality of glomeruli, cardio-protective KATP channels and adipocytes, respectively. Thus, p16(Ink4a)-positive cells that accumulate during adulthood negatively influence lifespan and promote age-dependent changes in several organs, and their therapeutic removal may be an attractive approach to extend healthy lifespan.

Cellular senescence in aging and age-related disease: from mechanisms to therapy.

Cellular senescence, a process that imposes permanent proliferative arrest on cells in response to various stressors, has emerged as a potentially important contributor to aging and age-related disease, and it is an attractive target for therapeutic exploitation. A wealth of information about senescence in cultured cells has been acquired over the past half century; however, senescence in living organisms is poorly understood, largely because of technical limitations relating to the identification and characterization of senescent cells in tissues and organs. Furthermore, newly recognized beneficial signaling functions of senescence suggest that indiscriminately targeting senescent cells or modulating their secretome for anti-aging therapy may have negative consequences. Here we discuss current progress and challenges in understanding the stressors that induce senescence in vivo, the cell types that are prone to senesce, and the autocrine and paracrine properties of senescent cells in the contexts of aging and age-related diseases as well as disease therapy.

Systematic Review of the Use of Phytochemicals for Management of Pain in Cancer Therapy.

Pain in cancer therapy is a common condition and there is a need for new options in therapeutic management. While phytochemicals have been proposed as one pain management solution, knowledge of their utility is limited. The objective of this study was to perform a systematic review of the biomedical literature for the use of phytochemicals for management of cancer therapy pain in human subjects. Of an initial database search of 1,603 abstracts, 32 full-text articles were eligible for further assessment. Only 7 of these articles met all inclusion criteria for this systematic review. The average relative risk of phytochemical versus control was 1.03 [95% CI 0.59 to 2.06]. In other words (although not statistically significant), patients treated with phytochemicals were slightly more likely than patients treated with control to obtain successful management of pain in cancer therapy. We identified a lack of quality research literature on this subject and thus were unable to demonstrate a clear therapeutic benefit for either general or specific use of phytochemicals in the management of cancer pain. This lack of data is especially apparent for psychotropic phytochemicals, such as the Cannabis plant (marijuana). Additional implications of our findings are also explored.

Spartan deficiency causes genomic instability and progeroid phenotypes.

Spartan (also known as DVC1 and C1orf124) is a PCNA-interacting protein implicated in translesion synthesis, a DNA damage tolerance process that allows the DNA replication machinery to replicate past nucleotide lesions. However, the physiological relevance of Spartan has not been established. Here we report that Spartan insufficiency in mice causes chromosomal instability, cellular senescence and early onset of age-related phenotypes. Whereas complete loss of Spartan causes early embryonic lethality, hypomorphic mice with low amounts of Spartan are viable. These mice are growth retarded and develop cataracts, lordokyphosis and cachexia at a young age. Cre-mediated depletion of Spartan from conditional knockout mouse embryonic fibroblasts results in impaired lesion bypass, incomplete DNA replication, formation of micronuclei and chromatin bridges and eventually cell death. These data demonstrate that Spartan plays a key role in maintaining structural and numerical chromosome integrity and suggest a link between Spartan insufficiency and progeria.

Senescence and apoptosis: dueling or complementary cell fates?

In response to a variety of stresses, mammalian cells undergo a persistent proliferative arrest known as cellular senescence. Many senescence-inducing stressors are potentially oncogenic, strengthening the notion that senescence evolved alongside apoptosis to suppress tumorigenesis. In contrast to apoptosis, senescent cells are stably viable and have the potential to influence neighboring cells through secreted soluble factors, which are collectively known as the senescence-associated secretory phenotype (SASP). However, the SASP has been associated with structural and functional tissue and organ deterioration and may even have tumor-promoting effects, raising the interesting evolutionary question of why apoptosis failed to outcompete senescence as a superior cell fate option. Here, we discuss the advantages that the senescence program may have over apoptosis as a tumor protective mechanism, as well as non-neoplastic functions that may have contributed to its evolution. We also review emerging evidence for the idea that senescent cells are present transiently early in life and are largely beneficial for development, regeneration and homeostasis, and only in advanced age do senescent cells accumulate to an organism's detriment.

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

Advanced age is the main risk factor for most chronic diseases and functional deficits in humans, but the fundamental mechanisms that drive ageing remain largely unknown, impeding the development of interventions that might delay or prevent age-related disorders and maximize healthy lifespan. Cellular senescence, which halts the proliferation of damaged or dysfunctional cells, is an important mechanism to constrain the malignant progression of tumour cells. Senescent cells accumulate in various tissues and organs with ageing and have been hypothesized to disrupt tissue structure and function because of the components they secrete. However, whether senescent cells are causally implicated in age-related dysfunction and whether their removal is beneficial has remained unknown. To address these fundamental questions, we made use of a biomarker for senescence, p16(Ink4a), to design a novel transgene, INK-ATTAC, for inducible elimination of p16(Ink4a)-positive senescent cells upon administration of a drug. Here we show that in the BubR1 progeroid mouse background, INK-ATTAC removes p16(Ink4a)-positive senescent cells upon drug treatment. In tissues--such as adipose tissue, skeletal muscle and eye--in which p16(Ink4a) contributes to the acquisition of age-related pathologies, life-long removal of p16(Ink4a)-expressing cells delayed onset of these phenotypes. Furthermore, late-life clearance attenuated progression of already established age-related disorders. These data indicate that cellular senescence is causally implicated in generating age-related phenotypes and that removal of senescent cells can prevent or delay tissue dysfunction and extend healthspan.