Senescence-associated Secretory Phenotype Factors Research Articles

MtDNA release promotes cGAS-STING activation and accelerated aging of postmitotic muscle cells

The mechanism regulating cellular senescence of postmitotic muscle cells is still unknown. cGAS-STING innate immune signaling was found to mediate cellular senescence in various types of cells, including postmitotic neuron cells, which however has not been explored in postmitotic muscle cells. Here by studying the myofibers from Zmpste24−/− progeria aged mice [an established mice model for Hutchinson-Gilford progeria syndrome (HGPS)], we observed senescence-associated phenotypes in Zmpste24−/− myofibers, which is coupled with increased oxidative damage to mitochondrial DNA (mtDNA) and secretion of senescence-associated secretory phenotype (SASP) factors. Also, Zmpste24−/− myofibers feature increased release of mtDNA from damaged mitochondria, mitophagy dysfunction, and activation of cGAS-STING. Meanwhile, increased mtDNA release in Zmpste24−/− myofibers appeared to be related with increased VDAC1 oligomerization. Further, the inhibition of VDAC1 oligomerization in Zmpste24−/− myofibers with VBIT4 reduced mtDNA release, cGAS-STING activation, and the expression of SASP factors. Our results reveal a novel mechanism of innate immune activation-associated cellular senescence in postmitotic muscle cells in aged muscle, which may help identify novel sets of diagnostic markers and therapeutic targets for progeria aging and aging-associated muscle diseases.

The SASP factor IL-6 sustains cell-autonomous senescent cells via a cGAS-STING-NFκB intracrine senescent noncanonical pathway.

Senescent cells produce a Senescence-Associated Secretory Phenotype (SASP) that involves factors with diverse and sometimes contradictory activities. One key SASP factor, interleukin-6 (IL-6), has the potential to amplify cellular senescence in the SASP-producing cells in an autocrine action, while simultaneously inducing proliferation in the neighboring cells. The underlying mechanisms for the contrasting actions remain unclear. We found that the senescence action does not involve IL-6 secretion nor the interaction with the receptor expressed in the membrane but is amplified through an intracrine mechanism. IL-6 sustains intracrine senescence interacting with the intracellular IL-6 receptor located in anterograde traffic specialized structures, with cytosolic DNA, cGAS-STING, and NFκB activation. This pathway triggered by intracellular IL-6 significantly contributes to cell-autonomous induction of senescence and impacts in tumor growth control. Inactivation of IL-6 in somatotrophic senescent cells transforms them into strongly tumorigenic in NOD/SCID mice, while re-expression of IL-6 restores senescence control of tumor growth. The intracrine senescent IL-6 pathway is further evidenced in three human cellular models of therapy-induced senescence. The compartmentalization of the intracellular signaling, in contrast to the paracrine tumorigenic action, provides a pathway for IL-6 to sustain cell-autonomous senescent cells, driving the SASP, and opens new avenues for clinical consideration to senescence-based therapies.

Relationship between gene expression associated with cellular senescence in cells from discarded wound dressings and wound healing: A retrospective cohort study

AimSenescent cells, inducing a senescence-associated secretory phenotype (SASP), lead to chronic inflammation in hard-to-heal wound tissue. However, eliminating senescent cells may impede normal wound healing due to their important role in the wound healing mechanism. Accordingly, we focused on wound exudates in chronic wounds, which contain many inflammation biomarkers consistent with SASP. Therefore, we hypothesized that senescent cells might be present in the exudates and induce chronic inflammation. This study investigated the relationship between gene expression associated with cellular senescence in exudates from pressure injuries and wound healing status. MethodsThis retrospective cohort study involved patients treated by a pressure injury team. We collected viable cells from wound dressings and analyzed gene expression. Pearson’s correlation coefficient was calculated between cellular senescence and SASP expression. The relationship between the gene expression of cellular senescence and the wound area reduction rate by the following week was examined using a mixed-effects model. ResultsCDKN1A—related to cellular senescence—was expressed in 96.3% of 54 samples, and CDKN1A expression and SASPs positively correlated (PLAU: r=0.68 and TNF: r=0.34). Low CDKN1A expression was statistically associated with a large wound area reduction rate (β=0.83, p < 0.01). ConclusionsGene expression of both cellular senescence and SASP factor in wound dressings suggests the presence of cellular senescence. Senescent cells in wound dressings could be associated with delayed wound healing in the following week.

Non-small cell lung cancer cells and concomitant cancer therapy induce a resistance-promoting phenotype of tumor-associated mesenchymal stem cells.

The tumor microenvironment gained attraction over the last decades as stromal cells significantly impact on tumor development, progression and metastasis, and immune evasion as well as on cancer therapy resistance. We previously reported that lung-resident mesenchymal stem cells (MSCs) were mobilized and activated in non-small cell lung cancer (NSCLC) progression and could even mediate radiation resistance in co-cultured NSCLC cells. We investigated how MSCs were affected by NSCLC cells in combination with cancer (radiation) therapy in indirect co-cultures using tumor-conditioned medium and Transwells or direct three-dimensional NSCLC-MSC spheroid co-cultures in order to unravel the resistance-mediating action of tumor-associated MSCs. Although no obvious phenotypic and functional alterations in MSCs following NSCLC co-culture could be observed, MSC senescence was induced following co-applied radiotherapy (RT). Global gene expression profiling, in combination with gene set enrichment analysis upon treatment, was used to confirm the senescent phenotype of irradiated MSC and to reveal relevant senescence-associated secretory phenotype (SASP) factors that could meditate NSCLC RT resistance. We identified senescent tumor-associated MSC-derived serine proteinase inhibitor (serpin) E1/PAI1 as potential SASP factor mediating NSCLC progression and RT resistance. Specified intra-tumor-stroma interactions and cell type-specific pro-tumorigenic functions could not only improve lung cancer classification but could even be used for a more precise profiling of individual patients, finally paving an additional way for the discovery of potential drug targets for NSCLC patients.

Exosomes Released from Senescent Cells and Circulatory Exosomes Isolated from Human Plasma Reveal Aging-associated Proteomic and Lipid Signatures.

Senescence emerged as a significant mechanism of aging and age-related diseases, offering an attractive target for clinical interventions. Senescent cells release a senescence-associated secretory phenotype (SASP), including exosomes that may act as signal transducers between distal tissues, propagating secondary or bystander senescence and signaling throughout the body. However, the composition of exosome SASP remains underexplored, presenting an opportunity for novel unbiased discovery. Here, we present a detailed proteomic and lipidomic analysis of exosome SASP using mass spectrometry from human plasma from young and older individuals and from tissue culture of senescent primary human lung fibroblasts. We identified ~1,300 exosome proteins released by senescent fibroblasts induced by three different senescence inducers causing most exosome proteins to be differentially regulated with senescence. In parallel, a human plasma cohort from young and old individuals revealed over 1,350 exosome proteins and 171 plasma exosome proteins were regulated when comparing old vs young individuals. Of the age-regulated plasma exosome proteins, we observed 52 exosome SASP factors that were also regulated in exosomes from the senescent fibroblasts, including serine protease inhibitors (SERPINs), Prothrombin, Coagulation factor V, Plasminogen, and Reelin. In addition, 247 lipids were identified with high confidence in all exosome samples. Following the senescence inducers, a majority of the identified phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin species increased significantly indicating cellular membrane changes. The most notable categories of significantly changed proteins were related to extracellular matrix remodeling and inflammation, both potentially detrimental pathways that can damage surrounding tissues and even induce secondary or bystander senescence. Our findings reveal mechanistic insights and potential senescence biomarkers, enabling a better approach to surveilling the senescence burden in the aging population and offering promising therapeutic targets for interventions.

NLRP1 inflammasome promotes senescence and senescence-associated secretory phenotype

BackgroundSenescence is a cellular aging-related process triggered by different stresses and characterized by the secretion of various inflammatory factors referred to as senescence-associated secretory phenotype (SASP), some of which are produced by the NLRP3 inflammasome. Here, we present evidence that the NLRP1 inflammasome is a DNA damage sensor and a key mediator of senescence.MethodsSenescence was induced in fibroblasts in vitro and in mice. Cellular senescence was assessed by Western blot analysis of several proteins, including p16, p21, p53, and SASP factors, released in the culture media or serum. Inflammasome components, including NLRP1, NLRP3 and GSDMD were knocked out or silenced using siRNAs.ResultsIn vitro and in vivo results suggest that the NLRP1 inflammasome promotes senescence by regulating the expression of p16, p21, p53, and SASP factors in a Gasdermin D (GSDMD)-dependent manner. Mechanistically, the NLRP1 inflammasome is activated in response to genomic damage detected by the cytosolic DNA sensor cGMP-AMP (cGAMP) synthase (cGAS).ConclusionOur findings show that NLRP1 is a cGAS-dependent DNA damage sensor during senescence and a mediator of SASP release through GSDMD. This study advances the knowledge on the biology of the NLRP1 inflammasome and highlights this pathway as a potential pharmcological target to modulate senescence.

LGG-14. INTEGRATED MULTI-OMICS EXPLORATION OF MAPK PATHWAY INHIBITION IN PEDIATRIC PILOCYTIC ASTROCYTOMA

Abstract Pilocytic astrocytomas (PA), the most common pediatric brain tumors, exhibit MAPK pathway alterations leading to its constitutive activation. They are also associated with low proliferation index due to the oncogene-induced senescence (OIS), sustained by senescence-associated secretory phenotype (SASP) factors. Little is known about the downstream consequences of MAPK activation leading to OIS-SASP, and of the molecular implications of MAPK pathway inhibition in senescent PA cells. Senescent DKFZ-BT66 cells derived from a primary KIAA::BRAF-fusion positive PA were used to generate RNA-sequencing and phospho-/proteomic datasets, before and after treatment with the MEK inhibitor (MEKi) trametinib for different time-spans. A multi-omics factor analysis tool (MEFISTO) and single sample gene set enrichment analysis (ssGSEA) were used to identify key OIS effectors and differentially regulated pathways upon MAPK inhibition. Trametinib treatment inhibited OIS and SASP signatures across all omics levels in the senescent DKFZ-BT66 cells, associated with reduced sensitivity towards senolytics drugs, indicating inhibition of senescence features upon MEKi. MEFISTO analysis identified key effectors involved in MAPK-induced OIS in the senescent PA cells. We built a pathway network using a prior knowledge network approach allowing to map the direct downstream effectors of MAPK leading to OIS-SASP. We identified several parallel pathways (JAK/STAT, p38, GSK3B) potentially involved in OIS and under the control of the MAPK pathway. In addition, ssGSEA showed that pathways related to upstream MAPK activators (SOS1, CK2, several RTKs) were predicted to be upregulated upon treatment, highlighting putative targets to be further investigated. Our data suggest that MAPKi reverses OIS signaling in senescent PA cells, while inducing the activation of MAPK upstream regulators, identifying putative co-targets for the treatment of PA. Further validation of the targetability of these pathways is pending. Furthermore, the identification of the MAPK-related OIS-SASP genes provides insight into the regulation of OIS-SASP by the MAPK pathway.

Luteoloside mitigates premature age-related macular degeneration by suppressing p53-p21-Rb1 axis: Insights from transcriptomic analysis, serum metabolomics and gut microbiota analysis

Transcriptomics of dry age-related macular degeneration (AMD) patients with premature aging revealed the upregulated pathways involved in glycerolipid metabolism, tyrosine metabolism, and pentose and glucuronate interconversion. To investigate natural strategies for modulating these implicated pathways, we examined the impact and underlying mechanism of luteoloside on premature AMD using a stress-induced premature senescence (SIPS)-associated AMD animal model in middle-aged mice that mimicked the dysregulated pathways observed in dry AMD patients with premature aging. Luteoloside supplementation resulted in a significant reduction in serum levels of the pro-inflammatory cytokine IL-1β and lipofuscin, along with increased serum activity of the antioxidant enzyme superoxide dismutase (SOD) and elevated levels of pigment epithelium-derived factor (PEDF), and preserved retinal thickness and structure in AMD mice. Furthermore, luteoloside supplementation effectively reversed the abnormal serum levels of metabolites, particularly by reducing harmful lysophosphatidylcholine (LysoPC) and increasing beneficial 4-guanidinobutanoic acid. In addition to its impact on metabolites, luteoloside modulated the composition of gut microbiota, promoting the enrichment of beneficial bacterial populations, including Lactobacillus, while reducing the abundance of harmful bacterial populations, including Bacteroides. Overall, our findings highlight the potential of luteoloside supplementation in regulating the dysregulated intestinal microbiota and metabolites in premature AMD, thereby reducing ocular levels of senescence-associated secretory phenotype (SASP) factors through the suppression of the p53-p21-retinoblastoma protein 1 (Rb1) axis.

Primary oocytes with cellular senescence features are involved in ovarian aging in mice

In mammalian females, quiescent primordial follicles serve as the ovarian reserve and sustain normal ovarian function and egg production via folliculogenesis. The loss of primordial follicles causes ovarian aging. Cellular senescence, characterized by cell cycle arrest and production of the senescence-associated secretory phenotype (SASP), is associated with tissue aging. In the present study, we report that some quiescent primary oocytes in primordial follicles become senescent in adult mouse ovaries. The senescent primary oocytes share senescence markers characterized in senescent somatic cells. The senescent primary oocytes were observed in young adult mouse ovaries, remained at approximately 15% of the total primary oocytes during ovarian aging from 6 to 12 months, and accumulated in aged ovaries. Administration of a senolytic drug ABT263 to 3-month-old mice reduced the percentage of senescent primary oocytes and the transcription of the SASP factors in the ovary, in addition, led to increased numbers of primordial and total follicles and a higher rate of oocyte maturation. Our study provides experimental evidence that primary oocytes, a germline cell type that is arrested in meiosis, become senescent in adult mouse ovaries and that senescent cell clearance reduced primordial follicle loss and mitigated ovarian aging phenotypes.

METTL3 Promotes Nucleus Pulposus Cell Senescence in Intervertebral Disc Degeneration by Regulating TLR2 m6A Methylation and Gut Microbiota.

Nucleus pulposus cell (NPC) senescence in intervertebral disc (IVD) tissue is the major pathological cause of intervertebral disc degeneration (IDD). N6-methyladenosine (m6A) methylation and gut microbiota play important roles in the progression of IDD. This study investigated whether methyltransferase-like 3 (METTL3) regulates TLR2 m6A modification and gut microbiota to influence NPC senescence. An IDD rat model was established by lumbar IVD puncture and NPCs were challenged with IL-1β to mimic IVD injury. IDD rats and IL-1β-exposed NPCs were treated with METTL3-interfering lentivirus and the TLR2 agonist Pam3CSK4. Compositional changes in the rat gut microbiota were analyzed and fecal microbiota transplantation procedures were used. NPC senescence, cell cycle, and the expression of senescence-associated secretory phenotype (SASP) factors were assessed. The m6A enrichment of TLR2 and the binding of IGF2BP1 to TLR2 mRNA were examined. METTL3 and TLR2 were highly expressed in IDD rats. METTL3 silencing attenuated senescent phenotypes and reduced secretion of SASP factors. Pam3CSK4 reversed the beneficial effects of METTL3 silencing on NPC senescence and IVD injury. METTL3 stabilized TLR2 mRNA in an IGF2BP1-dependent manner. METTL3 silencing restored specific gut microbiota levels in IDD rats, which was further reversed by administration of Pam3CSK4. Fecal microbiota from METTL3 silenced IDD rats altered the pathological phenotypes of IDD rats. These results demonstrate the beneficial effects of METTL3 silencing on NPC senescence and amelioration of IVD injury, involving modulation of TLR2 m6A modification and gut microbiota. These findings support METTL3 silencing as a potential therapeutic target for IDD.

YAP/TAZ activation mediates PQ-induced lung fibrosis by sustaining senescent pulmonary epithelial cells.

Paraquat (PQ) is a widely used herbicide and a common cause of poisoning that leads to pulmonary fibrosis with a high mortality rate. However, the underlying mechanisms of PQ-induced pulmonary fibrosis and whether pulmonary epithelial cell senescence is involved in the process remain elusive. In this study, PQ-induced pulmonary epithelial cell senescence and Hippo-YAP/TAZ activation were observed in both C57BL/6 mice and human epithelial cells. PQ-induced senescent pulmonary epithelial cells promoted lung fibroblast transformation through secreting senescence-associated secretory phenotype (SASP) factors. Yap/Taz knockdown in mice lungs significantly decreased the expression of downstream profibrotic protein Ctgf and senescent markers p16 and p21, and alleviated PQ-induced pulmonary fibrosis. Interfering YAP/TAZ in senescent human pulmonary epithelial cells resulted in decreased expression of the anti-apoptosis protein survivin and elevated level of apoptosis. In conclusion, our findings reveal a novel mechanism by which the involvement of Hippo-YAP/TAZ activation in pulmonary epithelial cell senescence mediates the pathogenesis of PQ-induced pulmonary fibrosis, thereby offering novel insights and potential targets for the clinical management of PQ poisoning as well as providing the mechanistic insight of the involvement of Yap/Taz activation in cell senescence in pulmonary fibrosis and its related pulmonary disorders. The YIN YANG balance between cell senescence and apoptosis is important to maintain the homeostasis of the lung, the disruption of which will lead to disease.

Osteochondroprogenitor cells and neutrophils expressing p21 and senescence markers modulate fracture repair.

Cells expressing features of senescence, including upregulation of p21 and p16, appear transiently following tissue injury, yet the properties of these cells or how they contrast with age-induced senescent cells remains unclear. Here, we used skeletal injury as a model and identified the rapid appearance following fracture of p21+ cells expressing senescence markers, mainly as osteochondroprogenitors (OCHs) and neutrophils. Targeted genetic clearance of p21+ cells suppressed senescence-associated signatures within the fracture callus and accelerated fracture healing. By contrast, p21+ cell clearance did not alter bone loss due to aging; conversely, p16+ cell clearance, known to alleviate skeletal aging, did not affect fracture healing. Following fracture, p21+ neutrophils were enriched in signaling pathways known to induce paracrine stromal senescence, while p21+ OCHs were highly enriched in senescence-associated secretory phenotype factors known to impair bone formation. Further analysis revealed an injury-specific stem cell-like OCH subset that was p21+ and highly inflammatory, with a similar inflammatory mesenchymal population (fibro-adipogenic progenitors) evident following muscle injury. Thus, intercommunicating senescent-like neutrophils and mesenchymal progenitor cells were key regulators of tissue repair in bone and potentially across tissues. Moreover, our findings established contextual roles of p21+ vs p16+ senescent/senescent-like cells that may be leveraged for therapeutic opportunities.

NEMO/NF-κB signaling functions as a double-edged sword in PanIN formation versus progression to pancreatic cancer

BackgroundPancreatic ductal adenocarcinoma (PDAC) is marked by a dismal survival rate, lacking effective therapeutics due to its aggressive growth, late-stage diagnosis, and chemotherapy resistance. Despite debates on NF-κB targeting for PDAC treatment, no successful approach has emerged.MethodsTo elucidate the role of NF-κB, we ablated NF-κB essential modulator (NEMO), critical for conventional NF-κB signaling, in the pancreata of mice that develop precancerous lesions (KC mouse model). Secretagogue-induced pancreatitis by cerulein injections was utilized to promote inflammation and accelerate PDAC development.ResultsNEMO deletion reduced fibrosis and inflammation in young KC mice, resulting in fewer pancreatic intraepithelial neoplasias (PanINs) at later stages. Paradoxically, however, NEMO deletion accelerated the progression of these fewer PanINs to PDAC and reduced median lifespan. Further, analysis of tissue microarrays from human PDAC sections highlighted the correlation between reduced NEMO expression in neoplastic cells and poorer prognosis, supporting our observation in mice. Mechanistically, NEMO deletion impeded oncogene-induced senescence (OIS), which is normally active in low-grade PanINs. This blockage resulted in fewer senescence-associated secretory phenotype (SASP) factors, reducing inflammation. However, blocked OIS fostered replication stress and DNA damage accumulation which accelerated PanIN progression to PDAC. Finally, treatment with the DNA damage-inducing reagent etoposide resulted in elevated cell death in NEMO-ablated PDAC cells compared to their NEMO-competent counterparts, indicative of a synthetic lethality paradigm.ConclusionsNEMO exhibited both oncogenic and tumor-suppressive properties during PDAC development. Caution is suggested in therapeutic interventions targeting NF-κB, which may be detrimental during PanIN progression but beneficial post-PDAC development.Graphical

Mechanistic role of RND3-regulated IL33/ST2 signaling on cardiomyocyte senescence

Hyperinflammatory responses are pivotal in the cardiomyocyte senescence pathophysiology, with IL33 serving as a crucial pro-inflammatory mediator. Our previous findings highlighted RND3's suppressive effect on IL33 expression. This study aims to explore the role of RND3 in IL33/ST2 signaling activation and in cardiomyocyte senescence. Intramyocardial injection of exogenous IL33 reduces the ejection fraction and fractional shortening of rats, inducing the appearance of senescence-associated secretory phenotype (SASP) in myocardial tissues. Recombinant IL33 treatment of AC16 cardiomyocytes significantly upregulated expression of SASP factors like IL1α, IL6, and MCP1, and increased the p-p65/p65 ratio and proportions of SA-β-gal and γH2AX-positive cells. NF-κB inhibitor pyrrolidinedithiocarbamate ammonium (PDTC) and ST2 antibody astegolimab treatments mitigated above effects. RND3 gene knockout H9C2 cardiomyocytes using CRISPR/Cas9 technology upregulated IL33, ST2L, IL1α, IL6, and MCP1 levels, decreased sST2 levels, and increased SA-β-gal and γH2AX-positive cells. A highly possibility of binding between RND3 and IL33 proteins was showed by molecular docking and co-immunoprecipitation, and loss of RND3 attenuated ubiquitination mediated degradation of IL33; what's more, a panel of ubiquitination regulatory genes closely related to RND3 were screened using qPCR array. In contrast, RND3 overexpression in rats by injection of AAV9-CMV-RND3 particles inhibited IL33, ST2L, IL1α, IL6, and MCP1 expression in cardiac tissues, decreased serum IL33 levels, and increased sST2 levels. These results suggest that RND3 expression in cardiomyocytes modulates cell senescence by inhibiting the IL33/ST2/NF-κB signaling pathway, underscoring its potential as a therapeutic target in cardiovascular senescence.

Analysis of the relationship between age-related erythrocyte dysfunction and fatigue.

With the declining birth rates and aging societies in developed countries, the average age of the working population is increasing. Older people tend to get tired more easily, so prevention of fatigue is important to improve the quality of life for older workers. This study aimed to assess the mechanism of fatigue in older people, especially focused on relation between dysfunction of erythrocyte and fatigue. Total power (TP), which is the value of autonomic nerve activity, was measured as a value of fatigue and significantly decreased in workers with aging. As properties of senescent erythrocytes, the erythrocyte sedimentation rate and damaged erythrocytes population increased with aging and correlated with TP. These results suggested that the accumulation of damaged erythrocytes contributes to fatigue. Recent studies revealed that senescence-associated secretory phenotype (SASP), a phenomenon in which senescent cells secrete a variety of cytokines, affected hematopoiesis in bone marrow. We analyzed the effects of SASP factors on erythropoiesis and found that Interleukin -1α (IL-1α) suppressed erythrocyte differentiation of hematopoietic stem cells in vitro. We also showed that IL-1α levels in human blood and saliva increase with aging, suggesting the possibility that IL-1α level in saliva can be used to predict the decline in hematopoietic function.

GPX4 Over-expression Is Associated with Increased Senescence Burden and Metabolic Dysfunction

Chronic inflammation is a hallmark of obesity, diabetes, and cardiovascular disease (CVD), contributing to morbidity and mortality worldwide. Adipose tissue inflammation is commonly observed in obesity-related diseases and is considered a critical driver of metabolic dysfunction. Cellular senescence, a state of cell cycle arrest, accumulates in adipose tissues in obesity and contributes to tissue inflammation through the production of pro-inflammatory factors known as the SASP (senescence associated secretory phenotype). Novel therapeutics targeting senescent cells have been shown to improve metabolic function in obesity, although the underlying mechanisms of senescent cell accumulation and clearance are not well known. Here, we compared a genetically modified mouse model that overexpresses glutathione peroxidase 4 (GPX4), with wildtype (WT) littermate mice. GPX4 is an antioxidant, selenoenzyme, and a key regulator of ferroptosis, an iron-dependent form of regulated cell death. Previous in vitro studies have shown that activation of ferroptosis through inhibition of GPX4 is associated with a decrease in senescence burden, suggesting that ferroptosis may serve as a novel senolytic pathway. We hypothesized that overexpression of GPX4 in the transgenic (TG) mice will reduce ferroptosis, increase senescence burden, and impair metabolic function compared to WT mice. To determine the effects of ferroptosis on metabolic function, GPX4 WT and TG mice were fed either a 60% high fat diet (HFD) or normal chow (NC) ad libitum for 8-10 weeks. We found higher body, liver, and white adipose tissue (WAT) mass in GPX4 TG compared to WT mice fed NC and HFD (Interaction all p&lt;0.0003, 0.0001, 0.0044, n=8-10/group). After HFD, T cell infiltration in the WAT was higher in GPX4 TG mice compared to WT (Interaction all p&lt;0.0253), and both CD8 and CD4 were significantly different from NC controls, contributing to metabolic dysfunction in these animals (p=0.027,0.207, n=4/group). Mice fed HFD demonstrated increased glucose intolerance compared to NC controls (p=0.029, p=0.0216, n=8-12/group), however, the TG HFD mice were trending towards higher glucose intolerance compared to WT HFD (p=0.17). Compared to NC and HFD fed WT mice, there was increased gene expression for the senescence markers, p16 (all p&lt;0.0041) and p21(all p&lt;0.002), as well as increased expression of SASP factors, CXCL2 (all p&lt;0.0055), TNFa (all p&lt;0.0159), and PAI1 (all p&lt;0.05, n=5-7/group) in the HFD fed TG mice, indicating increased senescence burden in the TG HFD mice. Malondialdehyde (MDA), a byproduct and marker of ferroptosis, was elevated in the HFD fed WT mice (p=0.0002) compared to all other groups, indicating increased ferroptosis. HFD fed TG mice also had increased expression of ferritin (all p&lt;0.0079, n=5/group) compared to all other groups, suggesting resistance to ferroptosis that may contribute to the increased senescence burden in the TG HFD group. Together these data demonstrate that increases in cellular senescence and impairments in metabolic function after HFD are exacerbated by increased GPX4 expression, suggesting that GPX4 may be a novel target to decrease senescence and improve metabolic function through induction of the ferroptosis pathway. Funded in part by awards from National Institute of Health Awards R01 AG 060395, R01 AG048366, T32 HL139451-05, T32 HL007576, R01 AG077751, Nora Eccles Treadwell Foundation (NEFT), and Veteran's Affairs Merit Review Award I01 BX004492. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Delineating the heterogeneity of senescence-induced-functional alterations in hepatocytes

Background and aimCellular senescence of hepatocytes involves permanent cell cycle arrest, disrupted cellular bioenergetics, resistance to cell death, and the release of pro-inflammatory cytokines. This ‘zombie-like’ state perpetuates harmful effects on tissues and holds potential implications for liver disease progression. Remarkably, senescence exhibits heterogeneity, stemming from two crucial factors: the inducing stressor and the cell type. As such, our present study endeavors to characterize stressor-specific changes in senescence phenotype, its related molecular patterns, and cellular bioenergetics in primary mouse hepatocytes (PMH) and hepatocyte-derived liver organoids (HepOrgs).MethodsPMH, isolated by collagenase-perfused mouse liver (C57B6/J; 18–23 weeks), were cultured overnight in William’s E-medium supplemented with 2% FBS, l-glutamine, and hepatocyte growth supplements. HepOrgs were developed by culturing cells in a 3D matrix for two weeks. The senescence was induced by DNA damage (doxorubicin, cisplatin, and etoposide), oxidative stress (H2O2, and ethanol), and telomere inhibition (BIBR-1532), p53 activation (nutlin-3a), DNA methyl transferase inhibition (5-azacitidine), and metabolism inhibitors (galactosamine and hydroxyurea). SA-β galactosidase activity, immunofluorescence, immunoblotting, and senescence-associated secretory phenotype (SASP), and cellular bioenergetics were used to assess the senescence phenotype.ResultsEach senescence inducer triggers a unique combination of senescence markers in hepatocytes. All senescence inducers, except hydroxyurea and ethanol, increased SA-β galactosidase activity, the most commonly used marker for cellular senescence. Among the SASP factors, CCL2 and IL-10 were consistently upregulated, while Plasminogen activator inhibitor-1 exhibited global downregulation across all modes of senescence. Notably, DNA damage response was activated by DNA damage inducers. Cell cycle markers were most significantly reduced by doxorubicin, cisplatin, and galactosamine. Additionally, DNA damage-induced senescence shifted cellular bioenergetics capacity from glycolysis to oxidative phosphorylation. In HepOrgs exposed to senescence inducers, there was a notable increase in γH2A.X, p53, and p21 levels. Interestingly, while showing a similar trend, SASP gene expression in HepOrgs was significantly higher compared to PMH, demonstrating a several-fold increase.ConclusionIn our study, we demonstrated that each senescence inducer activates a unique combination of senescence markers in PMH. Doxorubicin demonstrated the highest efficacy in inducing senescence, followed by cisplatin and H2O2, with no impact on apoptosis. Each inducer prompted DNA damage response and mitochondrial dysfunction, independent of MAPK/AKT.Graphical

Cell type-dependent modulation of senescence features using Weo electrolyzed water.

Electrolyzed-reduced water has powerful antioxidant properties with constituents that scavenge reactive oxygen species (ROS), which are known to be produced by several intrinsic and extrinsic processes. When there is an imbalance between ROS production and antioxidant defenses, oxidative stress occurs. Persistent oxidative stress leads to cellular senescence, an important hallmark of aging, and is involved in several age-related conditions and illnesses. This study aims to investigate whether Weo electrolyzed water (WEW) could modulate the phenotype of senescent cells. We compared normal human lung fibroblasts (BJ) and breast cancer cells (T47D) treated with hydrogen peroxide (H2O2) to induce senescence. We assessed the molecular impact of WEW on markers of cellular senescence, senescence-associated secretory phenotype (SASP) factors, and stress response genes. Treatment with WEW modulated markers of cellular senescence, such as the senescence-associated β-galactosidase (SA-β-gal) activity, EdU incorporation and p21 expression, similarly in both cell types. However, WEW modulated the expression of SASP factors and stress response genes in a cell type-dependent and opposite fashion, significantly decreasing them in BJ cells, while stimulating their expression in T47D cells. Reduction in the expression of SASP factors and stress-related genes in BJ cells suggests that WEW acts as a protective factor, thereby reducing oxidative stress in normal cells, while making cancer cells more sensitive to the effects of cellular stress, thus increasing their elimination and consequently reducing their deleterious effects. These findings suggest that, due to its differential effects as a senomorphic factor, WEW could have a positive impact on longevity and age-related diseases.

Senolytic and senomorphic agent procyanidin C1 alleviates structural and functional decline in the aged retina

Increased cellular senescence burden contributes in part to age-related organ dysfunction and pathologies. In our study, using mouse models of natural aging, we observed structural and functional decline in the aged retina, which was accompanied by the accumulation of senescent cells and senescence-associated secretory phenotype factors. We further validated the senolytic and senomorphic properties of procyanidin C1 (PCC1) both in vitro and in vivo, the long-term treatment of which ameliorated age-related retinal impairment. Through high-throughput single-cell RNA sequencing (scRNA-seq), we comprehensively characterized the retinal landscape after PCC1 administration and deciphered the molecular basis underlying the senescence burden increment and elimination. By exploring the scRNA-seq database of age-related retinal disorders, we revealed the role of cellular senescence and the therapeutic potential of PCC1 in these pathologies. Overall, these results indicate the therapeutic effects of PCC1 on the aged retina and its potential use for treating age-related retinal disorders.

Celastrol mediates CAV1 to attenuate pro-tumorigenic effects of senescent cells

BackgroundCellular senescence is an emerging hallmark of cancers, primarily fuels cancer progression by expressing senescence-associated secretory phenotype (SASP). Caveolin-1 (CAV1) is a key mediator of cell senescence. Previous studies from our group have evidenced that the expression of CAV1 is downregulated by Celastrol (CeT). PurposeTo investigate the impact of CeT on cellular senescence and its subsequent influence on post-senescence-driven invasion, migration, and stemness of clear cell renal cell carcinoma (ccRCC). Study design and methodsThe expression levels of CAV1, canonical senescence markers, and markers associated with epithelial-mesenchymal transition (EMT) and stemness in clinical samples were assessed through Pearson correlation analysis. Senescent cell models were induced using DOX, and their impact on migration, invasion, and stemness was evaluated. The effects of CeT treatment on senescent cells and their pro-tumorigenic effects were examined. Subsequently, the underlying mechanism of CeT were explored using lentivirus transfection and CRISPR/Cas9 technology to silence CAV1. ResultsIn human ccRCC clinical samples, the expression of the canonical senescence markers p53, p21, and p16 are associated with ccRCC progression. Senescent cells facilitated migration, invasion, and enhanced stemness in both ccRCC cells and ccRCC tumor-bearing mice. As expected, CeT treatment reduced senescence markers (p16, p53, p21, SA-β-gal) and SASP factors (IL6, IL8, CXCL12), alleviating cell cycle arrest. However, it did not restore the proliferation of senescent cells. Additionally, CeT suppressed senescence-driven migration, invasion, and stemness. Further investigations into the underlying mechanism demonstrated that CAV1 is a critical mediator of cell senescence and represents a potential target for CeT to attenuate cellular senescence. ConclusionsThis study presents a pioneering investigation into the intricate interplay between cellular senescence and ccRCC progression. We unveil a novel mechanism of CeT to mitigate cellular senescence by downregulating CAV1, thereby inhibiting the migration, invasion and stemness of ccRCC driven by senescent cells. These findings provide valuable insights into the underlying mechanisms of CeT and its potential as a targeted therapeutic approach for alleviating the aggressive phenotypes associated with senescent cells in ccRCC.