Giorgadze, Nino

ABSTRACT The senescent cell (SC) fate is linked to aging, multiple disorders and diseases, and physical dysfunction. Senolytics, agents that selectively eliminate 30%–70% of SCs, act by transiently disabling the senescent cell antiapoptotic pathways (SCAPs), which defend those SCs that are proapoptotic and pro‐inflammatory from their own senescence‐associated secretory phenotype (SASP). Consistent with this, a JAK/STAT inhibitor, Ruxolitinib, which attenuates the pro‐inflammatory SASP of senescent human preadipocytes, caused them to become “senolytic‐resistant”. Administering senolytics to obese mice selectively decreased the abundance of the subset of SCs that is pro‐inflammatory. In cell cultures, the 30%–70% of human senescent preadipocytes or human umbilical vein endothelial cells (HUVECs) that are senolytic‐resistant (to Dasatinib or Quercetin, respectively) had increased p16 INK4a , p21 CIP1 , senescence‐associated β‐galactosidase (SAβgal), γH2AX, and proliferative arrest similarly to the total SC population (comprising senolytic‐sensitive plus‐resistant SCs). However, the SASP of senolytic‐resistant SCs entailed less pro‐inflammatory/apoptotic factor production, induced less inflammation in non‐senescent cells, and was equivalent or richer in growth/fibrotic factors. Senolytic‐resistant SCs released less mitochondrial DNA (mtDNA) and more highly expressed the anti‐inflammatory immune evasion signal, glycoprotein non‐melanoma‐B (GPNMB). Transplanting senolytic‐resistant SCs intraperitoneally into younger mice caused less physical dysfunction than transplanting the total SC population. Because Ruxolitinib attenuates SC release of proapoptotic SASP factors, while pathogen‐associated molecular pattern factors (PAMPs) can amplify the release of these factors rapidly (acting as “senosensitizers”), senolytic‐resistant and senolytic‐sensitive SCs appear to be interconvertible.

AbstractObjectiveThe aim of this study was to determine the effect of age of obesity onset on senescence‐related markers in abdominal (AB) and femoral (FEM) subcutaneous adipose tissue (SAT) before and after moderate (~10%) weight loss.MethodsAB and FEM SAT were collected from human females with childhood‐onset obesity (CO) or adult‐onset obesity (AO) before and after diet‐ and exercise‐induced weight loss. Immunofluorescence analysis of γH2AX/RAD51 (DNA damage/repair markers) and p53/p21 (senescence markers) was conducted in cultured preadipocytes, and senescence‐associated β‐galactosidase (SA‐β‐gal) activity was measured in SAT.ResultsCO had proportionately more AB and FEM preadipocytes with DNA damage (γH2AX+) and senescence markers (p53+ and/or p21+) than AO at baseline. The proportion of γH2AX+ FEM preadipocytes declined with weight loss in CO and was similar between groups after weight loss. The number of γH2AX foci in γH2AX+ preadipocytes decreased similarly between groups and regions with weight loss in parallel with an increase in RAD51. The proportion of p53+ and p21+ preadipocytes and SA‐β‐gal+ cells in SAT did not change with weight loss, but the total p21 intensity in p53+/p21+ FEM preadipocytes declined in AO.ConclusionsThese results provide preliminary evidence that females with CO have an accelerated preadipocyte aging state that improves with weight loss in terms of DNA damage but not senescence.

Abstract Decreased α-Klotho, a geroprotective factor, and increased senescent cell burden are both associated with early onset of physical disability, cognitive impairment, and premature all-cause mortality. It has been demonstrated that eliminating senescent cells can enhance physical function, cognition, and survival in mice, as does overexpressing α-Klotho. Mice with low α-Klotho exhibit accelerated senescent cell accumulation, recombinant α-Klotho decreases senescent cell burden and restores lifespan in these mice, and senescent epidermal cells are reduced in mice overexpressing α-Klotho. Here, we tested the hypothesis that senescent cells cause decreased α-Klotho and hence that reducing senescent cells can increase α-Klotho. Senescent cell conditioned medium (CM) reduced α-Klotho in cultured non-senescent human umbilical vein endothelial cells (HUVECs), renal tubular endothelial cells, and astrocytes. These effects of senescent CM were partially attenuated by neutralizing antibodies against the senescence-associated secretory phenotype (SASP) factors, activin A and IL-1α. Transplanting senescent cells into younger mice caused decreased urine and brain α-Klotho. Genetically reducing highly p16Ink4a-expressing cells in old INK-ATTAC mice or administering the senolytics, Dasatinib plus Quercetin (D+Q) or Fisetin (F), to young mice transplanted with senescent cells, young diet-induced obese (DIO) mice, or naturally-aged mice increased urine, kidney, and/or brain α-Klotho. Treating patients with idiopathic pulmonary fibrosis (IPF), a cellular senescence-related disease, with D+Q led to increased urinary α-Klotho. Thus, targeting senescent cells causes increases in the geroprotective factor α-Klotho, potentially amplifying the beneficial effects of senolytic drugs.

Adipocytes accumulate within and around skeletal muscle with advancing age. Adipose progenitor cells, preadipocytes, are prone to cellular senescence, a state of stable growth arrest associated with the secretion of cytokines, chemokines and matrix remodeling proteins, collectively referred to as the senescence‐associated secretory phenotype (SASP). Considerable evidence implicates the SASP in age‐related tissue inflammation and degeneration; however, its impact on skeletal muscle is poorly understood. Herein, we examined the effects of conditioned media (CM) collected from healthy human (control CM) and senescent by irradiation (senescent CM) preadipocytes on human skeletal muscle cells (SkMCs). Compared to control CM, senescent CM had more than 10‐fold higher concentrations of IL‐6, IL‐1β, IL‐8, PAI‐1, MCP‐1 and TNFα as quantified by a multiplexed antibody‐based assay. Senescent CM increased expression of IL‐6 (5‐fold), IL‐1α (1.6‐fold), IL‐1β (3.5‐fold), MCP‐1 (2.5‐fold), PAI‐1 (1.7‐fold) and MMP‐3 (3‐fold) in differentiated SkMCs relative to control CM as measured by qPCR (all p < 0.05). Senescent CM increased proliferation of undifferentiated SKMCs by 20%; however, inhibited differentiation relative to control CM per the fusion index. Preliminary data further suggest senescent CM compromises insulin‐ and IGF‐1‐mediated activation of Akt. Collectively, our data suggest that the SASP of senescent preadipocytes may drive inflammation in SkMCs and compromise their differentiation and, potentially, response to metabolic an anabolic stimuli. These effects may contribute to age‐related changes in skeletal muscle health and regeneration. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Inflammation is a hallmark of aging and a likely contributor to skeletal muscle loss ( sarcopenia ). We investigated whether the inflammatory phenotype of differentiated primary skeletal muscle‐derived cells (SkMDCs) from a 17‐year‐old (young) differed from that of a 68‐year‐old (older) donor. Under basal conditions, expression of genes encoding pro‐inflammatory cytokines was in fact lower in older compared to younger SkMDCs. However, in response to lipopolysaccharide (LPS), these factors were more highly upregulated in older cells. Specifically, in young compared to older SkMDCs, interleukin‐6 (IL‐6) increased 3‐ vs. 9‐fold, tumor necrosis factor‐alpha (TNF‐alpha) increased 11‐ vs. 80‐fold, and IL‐1alpha increased 14‐ vs. 21‐fold (all differences were p < 0.01). Given adipose tissue is a primary source of pro‐inflammatory stimuli and infiltrates skeletal muscle with age, we hypothesized that secreted factors from senescent preadipocytes drive a similar molecular phenotype to LPS. Exposure of SkMDCs to conditioned medium from senescent‐preadipocytes increased expression IL‐6 and TNF‐alpha in young and even more so in older cells (both p < 0.05). Thus, there are inherent differences in the molecular inflammatory phenotype and its response between young and older SkMDCs. These data also suggest that the age‐related accumulation of senescent preadipocytes may contribute to sarcopenia.

Infiltration of fat cells into skeletal muscle is a hallmark of aging that has been associated with insulin resistance and the pathogenesis of type 2 diabetes mellitus (T2DM). The mechanisms by which fat cells affect the health and metabolic state of skeletal muscle are poorly understood. Given the evidence for cross‐talk between these tissues and the secretory phenotype of senescent preadipocytes, we first utilized real time‐PCR to examine the expression of inflammatory cytokines in primary human skeletal muscle‐derived cells (SkMDCs) in response to conditioned medium (CM) from either control human preadipocytes (Normal CM) or from human preadipocytes driven to senescence by irradiation (Senescent CM). Exposure of SkMDCs to either Normal CM or Senescent CM for 24 hours significantly increased their expression of IL‐6 (5.1±1.7‐and 14.1±4.7‐fold, respectively), TNFα (1.3±0.2‐and 2.1±0.67‐fold, respectively) and MCP‐1 (2.9±1.1‐and 6.8±2.3‐fold, respectively). Next, we investigated the effects of CM on insulin signaling in SkMDCs via Western blot analysis. Exposure of SKMDCs to preadipocyte CM significantly blunted insulin‐stimulated phosphorylation of its downstream effectors, including Akt (14–24%, p<0.05). Finally, we examined the effects of CM on the proliferative capacity of undifferentiated SkMDCs. Senescent CM inhibited SkMDC proliferation by more than 30% (p < 0.05). In conclusion, factors secreted by preadipocytes, and in particular, senescent preadipocytes, negatively impact the inflammatory and metabolic phenotype and the proliferative capacity of skeletal muscle cells. Elucidation of the biologic materials that are secreted from fat cells and mediate these effects may lead to novel therapeutic strategies for the treatment of age‐related conditions, including T2DM. The authors acknowledge the support of Mayo Clinic and a generous gift from Robert and Arlene Kogod.

Introduction: Senescent cells with essentially irreversible loss of replicative potential accumulate in various tissues with aging. Cellular senescence is associated with a pro‐inflammatory senescence‐associated secretory phenotype which the Janus‐associated kinase (JAK) pathway plays a role in. We hypothesized that senescent preadipocytes contribute to age‐related dysfunction of healthy cells. Methods: Preadipocytes from lean subjects were X‐irradiated to induce senescence. We co‐cultured non‐senescent cells with senescent cells or cultured them in conditioned medium (CM) from senescent preadipocytes to test the inflammation, differentiation and insulin sensitivity. Macrophage migration in response to CM was assayed as well. Moreover, we treated old or obese mice with Ruxolitinib, a JAK1/2 inhibitor, to test the beneficial effect. Results: The adipogenesis of preadipocytes was impaired by exposure to senescent cells. Macrophage migration was greater in response to senescent preadipocytes. Inflammation was increased in both adipose tissue and preadipocytes exposed to CM from senescent cells. All these adverse effect could be partially rescued by JAK inhibitor. In addition, senescent preadipocytes inhibited insulin‐induced phosphor‐AKT in adipose tissue and HepG2 cells. Insulin‐induced glycogen synthesis was also inhibited in HepG2 cells. Two months of Ruxolitinib treatment reduced inflammation, reversed age‐related lipodystrophy and improved physical function in frail, 24 month old mice. It also improved insulin sensitivity in diet‐induced obese mice. Conclusion: Senescent preadipocytes contribute to age‐related impairment in adipogenesis, fat tissue inflammation, and insulin resistance. Grant Funding Source : supported by NIH AG13925 and Robert and Arlene Kogod Center on Aging in Mayo Clinic.

Senescent cells exacerbate COVID-19 Cellular senescence is a state elicited in response to stress signals and is associated with a damaging secretory phenotype. The number of senescent cells increases with advanced age and this in turn drives age-related diseases. Camell et al. show that senescent cells have an amplified inflammatory response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (see the Perspective by Cox and Lord). This response is communicated to nonsenescent cells, suppressing viral defense mechanisms and increasing the expression of viral entry proteins. In old mice infected with a SARS-CoV-2–related virus, treatment with senolytics to reduce the senolytic cell burden reduced mortality and increased antiviral antibodies. Science , abe4832, this issue p. eabe4832 ; see also abi4474, p. 281

Abstract Aging is the biggest risk factor for the most serious chronic diseases and disabilities. Cellular senescence, a state in which cells stop dividing but release factors that damage other cells, may contribute to both age-related and chronic diseases. Removal of senescent cells from aged mice has been shown to delay aging and age–related disabilities. Our goal was to determine the ability of potential senolytic agents to remove senescent cells in a primate model. Several agents and combinations were tested including Fisetin, Navitoclax, combined Dasatinib and Quercetin, and combined Dasatinib and Fisetin. Here we describe the Dasatinib and Fisetin trial. Dasatinib is an FDA approved oral anticancer drug that has been used to treat chronic myelogenous leukemia in humans. Fisetin is a flavonoid that can be found in many plants, particularly strawberries, and acts as a coloring agent. After baseline measurements, six older (mean age=21 years) female rhesus monkeys (Macaca mulatta) were given a combined oral dose of Dasatinib (5 mg/kg) and Fisetin (100 mg/kg) on two consecutive days. Animals were additionally assessed at 1- and 7-weeks following dosing. At 7 weeks post dosing, there were fewer (p&lt;0.05) p16+ cells in the epidermis compared to baseline. Similarly, there was a reduction (p&lt;0.05) in p21+ cells in the epidermis at 1- and 7-weeks post dosing compared to baseline. There were no negative outcomes associated with treatment. This study provides preliminary evidence for the senolytic potential of combined Dasatinib and Fisetin treatment and indicates that pharmacological mitigation of age-related changes is possible.