Nicotinamide adenine dinucleotide (NAD) levels decrease during aging and are involved in age-related metabolic decline. To date, the mechanism responsible for the age-related reduction in NAD has not been elucidated. Here we demonstrate that expression and activity of the NADase CD38 increase with aging and that CD38 is required for the age-related NAD decline and mitochondrial dysfunction via a pathway mediated at least in part by regulation of SIRT3 activity. We also identified CD38 as the main enzyme involved in the degradation of the NAD precursor nicotinamide mononucleotide (NMN) in vivo, indicating that CD38 has a key role in the modulation of NAD-replacement therapy for aging and metabolic diseases.

Aging is associated with increased cellular senescence, which is hypothesized to drive the eventual development of multiple comorbidities. Here we investigate a role for senescent cells in age-related bone loss through multiple approaches. In particular, we used either genetic (i.e., the INK-ATTAC 'suicide' transgene encoding an inducible caspase 8 expressed specifically in senescent cells) or pharmacological (i.e., 'senolytic' compounds) means to eliminate senescent cells. We also inhibited the production of the proinflammatory secretome of senescent cells using a JAK inhibitor (JAKi). In aged (20- to 22-month-old) mice with established bone loss, activation of the INK-ATTAC caspase 8 in senescent cells or treatment with senolytics or the JAKi for 2-4 months resulted in higher bone mass and strength and better bone microarchitecture than in vehicle-treated mice. The beneficial effects of targeting senescent cells were due to lower bone resorption with either maintained (trabecular) or higher (cortical) bone formation as compared to vehicle-treated mice. In vitro studies demonstrated that senescent-cell conditioned medium impaired osteoblast mineralization and enhanced osteoclast-progenitor survival, leading to increased osteoclastogenesis. Collectively, these data establish a causal role for senescent cells in bone loss with aging, and demonstrate that targeting these cells has both anti-resorptive and anabolic effects on bone. Given that eliminating senescent cells and/or inhibiting their proinflammatory secretome also improves cardiovascular function, enhances insulin sensitivity, and reduces frailty, targeting this fundamental mechanism to prevent age-related bone loss suggests a novel treatment strategy not only for osteoporosis, but also for multiple age-related comorbidities.

Testosterone supplementation increases muscle mass in older men but has not been shown to consistently improve physical function and activity. It has been hypothesized that physical exercise is required to induce the adaptations necessary for translation of testosterone-induced muscle mass gain into functional improvements. However, the effects of testosterone plus low intensity physical exercise training (T/PT) on functional performance and bioenergetics are unknown. In this pilot study, we tested the hypothesis that combined administration of T/PT would improve functional performance and bioenergetics in male mice late in life more than low-intensity physical training alone. 28-month old male mice were randomized to receive T/PT or vehicle plus physical training (V/PT) for 2 months. Compare to V/PT control, administration of T/PT was associated with improvements in muscle mass, grip strength, spontaneous physical movements, and respiratory activity. These changes were correlated with increased mitochondrial DNA copy number and expression of markers for mitochondrial biogenesis. Mice receiving T/PT also displayed increased expression of key elements for mitochondrial quality control, including markers for mitochondrial fission-and-fusion and mitophagy. Concurrently, mice receiving T/PT also displayed increased expression of markers for reduced tissue oxidative damage and improved muscle quality.

Cellular senescence is a cell fate program that entails essentially irreversible proliferative arrest in response to damage signals. Tumor necrosis factor-alpha (TNFα), an important pro-inflammatory cytokine secreted by some types of senescent cells, can induce senescence in mouse and human cells. However, downstream signaling pathways linking TNFα-related inflammation to senescence are not fully characterized. Using human umbilical vein endothelial cells (HUVECs) as a model, we show that TNFα induces permanent growth arrest and increases p21CIP1, p16INK4A, and SA-β-gal, accompanied by persistent DNA damage and ROS production. By gene expression profiling, we identified the crucial involvement of inflammatory and JAK/STAT pathways in TNFα-mediated senescence. We found that TNFα activates a STAT-dependent autocrine loop that sustains cytokine secretion and an interferon signature to lock cells into senescence. Furthermore, we show STAT1/3 activation is necessary for cytokine and ROS production during TNFα-induced senescence. However, inhibition of STAT1/3 did not rescue cells from proliferative arrest, but rather suppressed cell cycle regulatory genes and altered TNFα-induced senescence. Our findings suggest a positive feedback mechanism via the STAT pathway that sustains cytokine production and reveal a reciprocal regulatory role of JAK/STAT in TNFα-mediated senescence.

Senescent cells accumulate with aging and at sites of pathology in multiple chronic diseases. Senolytics are drugs that selectively promote apoptosis of senescent cells by temporarily disabling the pro-survival pathways that enable senescent cells to resist the pro-apoptotic, pro-inflammatory factors that they themselves secrete. Reducing senescent cell burden by genetic approaches or by administering senolytics delays or alleviates multiple age- and disease-related adverse phenotypes in preclinical models. Reported senolytics include dasatinib, quercetin, navitoclax (ABT263), and piperlongumine. Here we report that fisetin, a naturally-occurring flavone with low toxicity, and A1331852 and A1155463, selective BCL-XL inhibitors that may have less hematological toxicity than the less specific BCL-2 family inhibitor navitoclax, are senolytic. Fisetin selectively induces apoptosis in senescent but not proliferating human umbilical vein endothelial cells (HUVECs). It is not senolytic in senescent IMR90 cells, a human lung fibroblast strain, or primary human preadipocytes. A1331852 and A1155463 are senolytic in HUVECs and IMR90 cells, but not preadipocytes. These agents may be better candidates for eventual translation into clinical interventions than some existing senolytics, such as navitoclax, which is associated with hematological toxicity.

The uterine fibrosis contributes to gestational outcomes. Collagen deposition in the uterus is related to uterine aging. Senolytic therapies are an option for reducing health complications related to aging. We investigated effects of aging and the senolytic drug combination of dasatinib plus quercetin (D+Q) on uterine fibrosis. Forty mice, 20 young females (03-months) and 20 old females (18-months), were analyzed. Young (Y) and old (O) animals were divided into groups of 10 mice, with one treatment (T) group (YT and OT) and another control © group (YC and OC). Comparative analysis of Pi3k/Akt1/mTor and p53 gene expression and related microRNAs (miR34a, miR34b, miR34c, miR146a, miR449a, miR21a, miR126a, and miR181b) among groups was performed to test effects of age and treatment on collagen deposition pathways. Aging promoted downregulation of the Pi3k/Akt1/mTor signaling pathway (P = 0.005, P = 0.031, and P = 0.028, respectively) as well as a reduction in expression of miR34c (P = 0.029), miR126a (P = 0.009), and miR181b (P = 0.007). D+Q treatment increased p53 gene expression (P = 0.041) and decreased miR34a (P = 0.016). Our results demonstrate a role for the Pi3k/Akt1/mTor signaling pathway in uterine aging and suggest for the first time a possible anti-fibrotic effect in the uterus of D+Q senolytic therapy.

The ability of virally suppressive antiretroviral therapy use to extend the life span of people with HIV (PWH) implies that the age of PWH will also increase. Among PWH, extended survival comes at a cost of earlier onset and increased rates of aging-associated comorbidities and geriatric syndromes, with persistent inflammation and immune dysregulation consequent to chronic HIV infection and to antiretroviral therapy use contributing to an overall decrease in health span. The geroscience hypothesis proposes that the root causes of most aging-related chronic diseases and conditions is the aging process itself. Hence, therapeutically targeting fundamental aging processes could have a greater impact on alleviating or delaying aging-associated comorbidities than addressing each disease individually. Extending the geroscience hypothesis to PWH, we speculate that targeting basic mechanisms of aging will improve overall health with age. Clinical features and pathophysiologic mechanisms of chronic diseases in PWH qualitatively resemble those seen in older adults without HIV. Therefore, drugs that target any of the pillars of aging, including metformin, rapamycin, and nicotinamide adenine dinucleotide precursors, may also slow the rate of onset of age-associated comorbidities and geriatric syndromes in PWH. Drugs that selectively induce apoptosis of senescent cells, termed senolytics, may also improve health span among PWH. Preliminary evidence suggests that senescent cell burden is increased in PWH, implying that senescent cells are an excellent therapeutic target for extending health span. Recently initiated clinical trials evaluating senolytics in age-related diseases offer insights into the design and potential implementation of similar trials for PWH.

Global consumption of high-fat diets (HFD) is associated with an increased incidence of cardiometabolic syndrome and cardiac injury, warranting identification of cardioprotective strategies. Cardioprotective effects of quercetin (Q) have mostly been evaluated in ischemic heart disease models and attributed to senolysis. We hypothesized that Q could alleviate murine cardiac damage caused by HFD by restoring the myocardial microcirculation. C57BL/6J mice were fed standard chow or HFD for 6 months and then treated with Q (50 mg/kg) or vehicle 5-day biweekly for 10 additional weeks. Left ventricular (LV) cardiac function was studied in vivo using magnetic resonance imaging, and intramyocardial fat deposition, microvascular density, oxidative stress, and senescence were analyzed ex vivo. Additionally, direct angiogenic effects of Q were studied in vitro in HUVECs. HFD increased body weight, heart weight, total cholesterol, and triglyceride levels, whereas Q normalized heart weight and triglycerides. LV ejection fraction was lower in HFD vs. control mice (56.20 ± 15.8% vs. 73.38 ± 5.04%, respectively, P < 0.05), but improved in HFD + Q mice (67.42 ± 7.50%, P < 0.05, vs. HFD). Q also prevented cardiac fat accumulation and reduced HFD-induced cardiac fibrosis, cardiomyocyte hypertrophy, oxidative stress, and vascular rarefaction. Cardiac senescence was not observed in any group. In vitro, ox-LDL reduced HUVEC tube formation activity, which Q effectively improved. Quercetin may directly induce angiogenesis and decrease myocardial oxidative stress, which might account for its cardioprotective effects in the murine HFD-fed murine heart independently from senolytic activity. Furthermore, its beneficial effects might be partly attributed to a decrease in plasma triglycerides and intramyocardial fat deposition.

The COVID-19 pandemic has revealed the pronounced vulnerability of the elderly and chronically ill to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced morbidity and mortality. Cellular senescence contributes to inflammation, multiple chronic diseases, and age-related dysfunction, but effects on responses to viral infection are unclear. Here, we demonstrate that senescent cells (SnCs) become hyper-inflammatory in response to pathogen-associated molecular patterns (PAMPs), including SARS-CoV-2 spike protein-1, increasing expression of viral entry proteins and reducing antiviral gene expression in non-SnCs through a paracrine mechanism. Old mice acutely infected with pathogens that included a SARS-CoV-2-related mouse β-coronavirus experienced increased senescence and inflammation, with nearly 100% mortality. Targeting SnCs by using senolytic drugs before or after pathogen exposure significantly reduced mortality, cellular senescence, and inflammatory markers and increased antiviral antibodies. Thus, reducing the SnC burden in diseased or aged individuals should enhance resilience and reduce mortality after viral infection, including that of SARS-CoV-2.

Activin A, an inflammatory mediator implicated in cellular senescence-induced adipose tissue dysfunction and profibrotic kidney injury, may become a new target for the treatment of diabetic kidney disease (DKD) and chronic kidney diseases. We tested the hypothesis that human DKD-related injury leads to upregulation of activin A in blood and urine and in a human kidney cell model. We further hypothesized that circulating activin A parallels kidney injury markers in DKD.

Cellular senescence, which is a major cause of tissue dysfunction with aging and multiple other conditions, is known to be triggered by p16Ink4a or p21Cip1 , but the relative contributions of each pathway toward inducing senescence are unclear. Here, we directly addressed this issue by first developing and validating a p21-ATTAC mouse with the p21Cip1 promoter driving a "suicide" transgene encoding an inducible caspase-8 which, upon induction, selectively kills p21Cip1 -expressing senescent cells. Next, we used the p21-ATTAC mouse and the established p16-INK-ATTAC mouse to directly compare the contributions of p21Cip1 versus p16Ink4a in driving cellular senescence in a condition where a tissue phenotype (bone loss and increased marrow adiposity) is clearly driven by cellular senescence-specifically, radiation-induced osteoporosis. Using RNA in situ hybridization, we confirmed the reduction in radiation-induced p21Cip1 - or p16Ink4a -driven transcripts following senescent cell clearance in both models. However, only clearance of p21Cip1 +, but not p16Ink4a +, senescent cells prevented both radiation-induced osteoporosis and increased marrow adiposity. Reduction in senescent cells with dysfunctional telomeres following clearance of p21Cip1 +, but not p16Ink4a +, senescent cells also reduced several of the radiation-induced pro-inflammatory senescence-associated secretory phenotype factors. Thus, by directly comparing senescent cell clearance using two parallel genetic models, we demonstrate that radiation-induced osteoporosis is driven predominantly by p21Cip1 - rather than p16Ink4a -mediated cellular senescence. Further, this approach can be used to dissect the contributions of these pathways in other senescence-associated conditions, including aging across tissues.

The role of hepatic cell senescence in human non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) is not well understood. To examine this, we performed liver biopsies and extensive characterization of 58 individuals with or without NAFLD/NASH. Here, we show that hepatic cell senescence is strongly related to NAFLD/NASH severity, and machine learning analysis identified senescence markers, the BMP4 inhibitor Gremlin 1 in liver and visceral fat, and the amount of visceral adipose tissue as strong predictors. Studies in liver cell spheroids made from human stellate and hepatocyte cells show BMP4 to be anti-senescent, anti-steatotic, anti-inflammatory and anti-fibrotic, whereas Gremlin 1, which is particularly highly expressed in visceral fat in humans, is pro-senescent and antagonistic to BMP4. Both senescence and anti-senescence factors target the YAP/TAZ pathway, making this a likely regulator of senescence and its effects. We conclude that senescence is an important driver of human NAFLD/NASH and that BMP4 and Gremlin 1 are novel therapeutic targets.

Although cellular senescence drives multiple age-related co-morbidities through the senescence-associated secretory phenotype, in vivo senescent cell identification remains challenging. Here, we generate a gene set (SenMayo) and validate its enrichment in bone biopsies from two aged human cohorts. We further demonstrate reductions in SenMayo in bone following genetic clearance of senescent cells in mice and in adipose tissue from humans following pharmacological senescent cell clearance. We next use SenMayo to identify senescent hematopoietic or mesenchymal cells at the single cell level from human and murine bone marrow/bone scRNA-seq data. Thus, SenMayo identifies senescent cells across tissues and species with high fidelity. Using this senescence panel, we are able to characterize senescent cells at the single cell level and identify key intercellular signaling pathways. SenMayo also represents a potentially clinically applicable panel for monitoring senescent cell burden with aging and other conditions as well as in studies of senolytic drugs.

Wound healing is an essential physiological process for restoring normal skin structure and function post-injury. The role of cellular senescence, an essentially irreversible cell cycle state in response to damaging stimuli, has emerged as a critical mechanism in wound remodeling. Transiently-induced senescence during tissue remodeling has been shown to be beneficial in the acute wound healing phase. In contrast, persistent senescence, as observed in chronic wounds, contributes to delayed closure. Herein we describe a chronic wound murine model and its cellular senescence profile, including the senescence-associated secretory phenotype.

Clearing senescent cells extends healthspan in mice. Using a hypothesis-driven bioinformatics-based approach, we recently identified pro-survival pathways in human senescent cells that contribute to their resistance to apoptosis. This led to identification of dasatinib (D) and quercetin (Q) as senolytics, agents that target some of these pathways and induce apoptosis preferentially in senescent cells. Among other pro-survival regulators identified was Bcl-xl. Here, we tested whether the Bcl-2 family inhibitors, navitoclax (N) and TW-37 (T), are senolytic. Like D and Q, N is senolytic in some, but not all types of senescent cells: N reduced viability of senescent human umbilical vein epithelial cells (HUVECs), IMR90 human lung fibroblasts, and murine embryonic fibroblasts (MEFs), but not human primary preadipocytes, consistent with our previous finding that Bcl-xl siRNA is senolytic in HUVECs, but not preadipocytes. In contrast, T had little senolytic activity. N targets Bcl-2, Bcl-xl, and Bcl-w, while T targets Bcl-2, Bcl-xl, and Mcl-1. The combination of Bcl-2, Bcl-xl, and Bcl-w siRNAs was senolytic in HUVECs and IMR90 cells, while combination of Bcl-2, Bcl-xl, and Mcl-1 siRNAs was not. Susceptibility to N correlated with patterns of Bcl-2 family member proteins in different types of human senescent cells, as has been found in predicting response of cancers to N. Thus, N is senolytic and acts in a potentially predictable cell type-restricted manner. The hypothesis-driven, bioinformatics-based approach we used to discover that dasatinib (D) and quercetin (Q) are senolytic can be extended to increase the repertoire of senolytic drugs, including additional cell type-specific senolytic agents.

Preadipocytes are present in adipose tissues throughout adult life that can proliferate and differentiate into mature adipocytes in response to environmental cues. Abnormal increase in adipocyte number or size leads to fat tissue expansion. However, it is now recognized that adipocyte hypertrophy is a greater risk factor for metabolic syndrome whereas fat tissue that continues to produce newer and smaller fat cells through preadipocyte differentiation is "metabolically healthy". Because adipocyte hypertrophy is often associated with increased oxidant stress and low grade inflammation, both are linked to disturbed cellular redox, we tested how preadipocyte differentiation may be regulated by beta-mercaptoethanol (BME), a pharmacological redox regulator and radical scavenger, using murine 3T3-F442A preadipocytes as the cell model. Effects of BME on adipogenesis were measured by microphotography, real-time PCR, and Western analysis. Our data demonstrated that preadipocyte differentiation could be regulated by extracellular BME. At an optimal concentration, BME enhanced expression of adipogenic gene markers and lipid accumulation. This effect was associated with BME-mediated down-regulation of inflammatory cytokine expression during early differentiation. BME also attenuated TNFalpha-induced activation of NFkappaB in differentiating preadipocytes and partially restored TNFalpha-mediated suppression on adipogenesis. Using a non-adipogenic HEK293 cell line transfected with luciferase reporter genes, we demonstrated that BME reduced basal and TNFalpha-induced NFkappaB activity and increased basal and ciglitazone-induced PPARgamma activity; both may contribute to the pro-adipogenic effect of BME in differentiating F442A preadipocytes.

Fat distribution changes with aging. Inherent changes in fat cell progenitors may contribute because fat cells turn over throughout life. To define mechanisms, gene expression was profiled in preadipocytes cultured from epididymal and perirenal depots of young and old rats. 8.4% of probe sets differed significantly between depots, particularly developmental genes. Only 0.02% differed with aging, despite using less stringent criteria than for comparing depots. Twenty-five genes selected based on fold change with aging were analyzed in preadipocytes from additional young, middle-aged, and old animals by polymerase chain reaction. Thirteen changed significantly with aging, 13 among depots, and 9 with both. Genes involved in inflammation, stress, and differentiation changed with aging, as occurs in fat tissue. Age-related changes were greater in perirenal than epididymal preadipocytes, consistent with larger declines in replication and adipogenesis in perirenal preadipocytes. Thus, age-related changes in preadipocyte gene expression differ among depots, potentially contributing to fat redistribution and dysfunction.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by interstitial remodelling, leading to compromised lung function. Cellular senescence markers are detectable within IPF lung tissue and senescent cell deletion rejuvenates pulmonary health in aged mice. Whether and how senescent cells regulate IPF or if their removal may be an efficacious intervention strategy is unknown. Here we demonstrate elevated abundance of senescence biomarkers in IPF lung, with p16 expression increasing with disease severity. We show that the secretome of senescent fibroblasts, which are selectively killed by a senolytic cocktail, dasatinib plus quercetin (DQ), is fibrogenic. Leveraging the bleomycin-injury IPF model, we demonstrate that early-intervention suicide-gene-mediated senescent cell ablation improves pulmonary function and physical health, although lung fibrosis is visibly unaltered. DQ treatment replicates benefits of transgenic clearance. Thus, our findings establish that fibrotic lung disease is mediated, in part, by senescent cells, which can be targeted to improve health and function.

The incidence of non-alcoholic fatty liver disease (NAFLD) increases with age. Cellular senescence refers to a state of irreversible cell-cycle arrest combined with the secretion of proinflammatory cytokines and mitochondrial dysfunction. Senescent cells contribute to age-related tissue degeneration. Here we show that the accumulation of senescent cells promotes hepatic fat accumulation and steatosis. We report a close correlation between hepatic fat accumulation and markers of hepatocyte senescence. The elimination of senescent cells by suicide gene-meditated ablation of p16Ink4a-expressing senescent cells in INK-ATTAC mice or by treatment with a combination of the senolytic drugs dasatinib and quercetin (D+Q) reduces overall hepatic steatosis. Conversely, inducing hepatocyte senescence promotes fat accumulation in vitro and in vivo. Mechanistically, we show that mitochondria in senescent cells lose the ability to metabolize fatty acids efficiently. Our study demonstrates that cellular senescence drives hepatic steatosis and elimination of senescent cells may be a novel therapeutic strategy to reduce steatosis.

Menopause is a natural physiological process in older women that is associated with reduced estrogen production and results in increased risk for obesity, diabetes, and osteoporosis. 17α-estradiol (17α-E2) treatment in males, but not females, reverses several metabolic conditions associated with advancing age, highlighting sexually dimorphic actions on age-related pathologies. In this study we sought to determine if 17α-E2 could prevent ovariectomy (OVX)-mediated detriments on adiposity and bone parameters in females. Eight-week-old female C57BL/6J mice were subjected to SHAM or OVX surgery and received dietary 17α-E2 during a six-week intervention period. We observed that 17α-E2 prevented OVX-induced increases in body weight and adiposity. Similarly, uterine weight and luminal cell thickness were decreased by OVX and prevented by 17α-E2 treatment. Interestingly, 17α-E2 prevented OVX-induced declines in tibial metaphysis cancellous bone. And similarly, 17α-E2 improved bone density parameters in both tibia and femur cancellous bone, primarily in OVX mice. In contrast, to the effects on cancellous bone, cortical bone parameters were largely unaffected by OVX or 17α-E2. In the non-weight bearing lumbar vertebrae, OVX reduced trabecular thickness but not spacing, while 17α-E2 increased trabecular thickness and reduced spacing. Despite this, 17α-E2 did improve bone volume/tissue volume in lumbar vertebrae. Overall, we found that 17α-E2 prevented OVX-induced increases in adiposity and changes in bone mass and architecture, with minimal effects in SHAM-operated mice. We also observed that 17α-E2 rescued uterine tissue mass and lining morphology to control levels without inducing hypertrophy, suggesting that 17α-E2 could be considered as an adjunct to traditional hormone replacement therapies.