Senescence Genes Research Articles

Comprehensive assessment of cellular senescence in intestinal immunity and biologic therapy response in ulcerative colitis

Ulcerative Colitis (UC) is an inflammatory disorder characterized by chronic intestinal inflammation and immune dysregulation. Despite a clear association between cellular senescence and chronic inflammation and immune dysregulation, the mechanisms underlying cellular senescence in UC remain unclear. We screened differentially expressed genes (DEGs) associated with cellular senescence in multiple UC datasets, performed immune infiltration analysis, and constructed clinical diagnostic models. Additionally, we investigated the relationship between key genes related to cellular senescence and disease remission in UC patients undergoing biologic therapy, validating their expression in a single-cell dataset. We identified six DEGs associated with cellular senescence (TWIST1, IGFBP5, MME, IFNG, ME1, FOS). Immune infiltration results indicated strong correlations of four of these genes with immune cells and pathways. Through WGCNA, GO, and KEGG analyses, we found that gene modules strongly associated with the expression of hub genes in cellular senescence were enriched in inflammation-related pathways. In the single-cell dataset, the expression of these six key genes exhibited similarities with Immune infiltration results. Additionally, we constructed a nomogram using these six genes for diagnosing UC, demonstrating good diagnostic capability and clinical efficacy. Kaplan–Meier survival analysis revealed a significant association between changes in the expression levels of these cell genes and disease remission in UC patients undergoing biologic therapy. This study utilizes bioinformatic analysis and machine learning to identify and analyze features associated with cellular senescence in multiple UC datasets. It provides insights into the role of cellular senescence in the premature onset of intestinal aging in UC and offers new perspectives for exploring novel therapeutic targets.

Senescence in Intervertebral Disc Degeneration: A Comprehensive Analysis Based on Bioinformatic Strategies.

Intervertebral disc degeneration (IDD) is a major cause for low back pain. Studies showed the association between senescence and degenerative diseases. Cell senescence can promote the occurrence and development of degenerative diseases through multiple mechanisms including inflammatory stress, oxidative stress and nutritional deprivation. The roles of senescence and senescence-associated genes (SAGs) remains unknown in IDD. Four differently expressed SAGs were identified as hub SAGs using "limma" package in R. We then calculated the immune infiltration of IDD patients, and investigated the relation between hub SAGs and immune infiltration. Enrichment analysis was performed to explore the functions of hub SAGs in IDD. Nomogram and LASSO model based on hub SAGs was constructed to predict the risk of severe degeneration (SD) for IDD patients. Subsequently, single cell analysis was conducted to describe the expression pattern of hub SAGs in intervertebral disc tissue. We identified ASPH, CCND1, IGFBP3 and SGK1 as hub SAGs. Further analysis demonstrated that the hub SAGs might mediate the development of IDD by regulating immune infiltration and multiple pathways. The LASSO model based on the four hub SAGs showed good performance in predicting the risk of SD. Single cell analysis revealed that ASPH, CCND1 and SGK1 mainly expressed in nucleus pulposus cells, while IGFBP3 mainly expressed in epithelial cells. Eleven candidate drugs targeting hub SAGS were predicted for IDD patients through Comparative Toxicogenomics Database (CDT). PCR and immunohistochemical analysis showed that the levels of four hub SAGs were higher in SD than MD (mild degeneration) patients. We performed a comprehensive analysis of SAGs in IDD, which revealed their functions and expression pattern in intervertebral disc tissue. Based on hub SAGs, we established a predictive model and explored the potential drugs. These findings provide new understandings of SAG mechanism and promising therapeutic strategies for IDD.

Role of Cellular Senescence Genes and Immune Infiltration in Sepsis and Sepsis-Induced ARDS Based on Bioinformatics Analysis.

Sepsis is the leading cause of death in critically ill patients; it results in multiorgan dysfunction, including acute respiratory distress syndrome (ARDS). Our study was conducted to determine the role of cellular senescence genes and immune infiltration in sepsis and sepsis-induced ARDS via bioinformatic analyses. Datasets GSE66890 and GSE145227 were obtained from the Gene Expression Omnibus (GEO) database and utilized for bioinformatics analyses. Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the differentially expressed genes (DEGs) were performed to identify the key functional modules. Two machine learning algorithms, least absolute shrinkage and selection operator (LASSO) and support vector machine recursive feature elimination (SVM-RFE), were used to screen for characteristic genes in sepsis and sepsis-induced ARDS. ROC curves were generated to evaluate the predictive ability of gene hubs. Differences in immune infiltration levels between the disease and control groups were compared via ssGSEA. The diagnostic value of the hub genes was verified via quantitative PCR (qPCR) in hospitalized patients. Four characteristic genes (ATM, CCNB1, CCNA1, and E2F2) were identified as biomarkers for the progression of sepsis-induced ARDS. E2F2 showed the highest predictive ability for the occurrence of ARDS in patients with sepsis. CD56bright and plasmacytoid dendritic cells exhibited high infiltration in the sepsis-induced ARDS group, whereas eosinophils, MDSCs, macrophages, and neutrophils exhibited low infiltration. In addition, ATM expression was lower in patients with sepsis than in those without sepsis (n = 6). Sepsis-induced ARDS is correlated with circulating immune responses, and the expression of ATM, CCNB1, CCNA1, and E2F2 may serve as potential diagnostic biomarkers and therapeutic targets in sepsis-induced ARDS.

The BET protein inhibitor apabetalone (RVX-208) prevents doxorubicin-induced endothelial senescence

Abstract Background Accumulation of senescent endothelial cells (ECs) plays a pivotal role in cardiometabolic disorders and lifespan reduction(1). Yet, only a few senolytics are known to date, partly due to the poor grasp of the molecular mechanisms that control the senescence survival programme. Bromo-and extraterminal domain (BET) proteins, known epigenetic reader proteins, recognize acetylated histone tails and regulate gene transcription. RVX-208, an FDA-approved BET inhibitor, has shown to modulate transcriptional programs involved in inflammation, cancer, and renal disease(2). Yet, its potential role in EC senescence and cardio-oncology remains elusive. Purpose Our study aims to investigate whether pharmacological modulation of BET proteins by RVX-208 can mitigate doxorubicin-induced endothelial senescence. Methods Human aortic endothelial cells (HAECs) were exposed to different concentrations of Doxo (50nM-500nM) for 48 hours. Cell morphology changes, cell cycle arrest, senescence associated secreted phenotype (SASP) release, upregulation of SA-b gal activity, DNA damage response (gH2AX) and expression of p21, p16 and p53 were used as molecular markers of cellular senescence. Conditioned media was collected from Doxo-treated ECs and processed for molecular analyses or used for treatment of healthy, non-senescent ECs. To test whether RVX-208 exerts any senolytic or senomorphic effect, HAECs were treated with Doxo (100 nM) in presence or absence of RVX-208 (5mM-20mM) for 48 hrs. RNA-seq and proteomic profiling were performed in vehicle and Doxo-treated ECs. Results Doxo-treated ECs showed a dose-dependent increase in senescence markers (p16, p21 and p53) and DNA-damage response (gH2AX). FACS analysis revealed an accumulation of cells in G2/M phase in Doxo-treated ECs (100 nM). Of interest, treatment with RVX-208 (15mM) prevented the upregulation of senescent markers (p16, p21 and p53) while rescuing alterations in cell morphology and reducing the number of SA-b gal positive cells. RVX-208 prevented Doxo-induced upregulation of genes involved in inflammation, oxidative stress and mitochondrial dysfunction as shown by RNA-seq and proteomic analysis. Conditioned media from Doxo-treated ECs showed an enrichment of SASP-associated markers (IL6, CCL2, IL8, TIMP2 and PDGF-b) as shown by dot-blot arrays. Exposure of healthy ECs to conditioned media from senescent ECs recapitulated aging features. Notably, RVX-208 blunted the release of SASP markers (IL6, CCL2 and IL8) from Doxo-treated ECs. Mechanistically, we found enhanced BET protein occupancy (BRD4) with concomitant enrichment of activating chromatin marks (H3K27Ac and H3K4me) on the promoter of senescence genes COL1A2, CDKN1C and SESN3. Conclusion Targeting BET proteins with RVX-208 may offer a promising therapeutic strategy to prevent endothelial aging in cancer patients undergoing cardiotoxic therapies.

Senescence-related genes are associated with the immunopathology signature of American Tegumentary Leishmaniasis lesions and may predict progression to mucosal leishmaniasis.

The American Tegumentary Leishmaniasis (ATL) is caused by protozoans of the genus Leishmania and varies from mild localized cutaneous leishmaniasis (LCL) form to more severe manifestations such as the diffuse cutaneous leishmaniasis (DCL) form and the mucosal leishmaniasis (ML) form. Previously, we demonstrated the accumulation of senescent cells in skin lesions of patients with LCL. Moreover, lesional transcriptomic analyses revealed a robust co-induction of senescence and pro-inflammatory gene signatures, highlighting the critical role of senescent T cells in orchestrating pathology. In this work we hypothesized that senescent cells might operate differently among the ATL spectrum, potentially influencing immunopathological mechanisms and clinical outcome. We analysed previously published RNA-Seq datasets of skin biopsies of healthy subjects and lesional skin from DCL patients, LCL patients and LCL patients that, after treatment, progressed to mucosal leishmaniasis (MLP). Our findings demonstrate a robust presence of a CD8 T cell signature associated with both LCL and MLP lesions. Moreover, both inflammatory and cytotoxic signatures were significantly upregulated, showing a strong increase in MLP and LCL groups, but not DCL. The senescence signature was elevated between LCL and MLP groups, representing the only distinguishable signature of immunopathology between them. Interestingly, our analyses further revealed the senescence signature's capacity to predict progression from LCL to mucosal forms, which was not observed with other signatures. Both the senescence-signature score and specific senescence-associated genes demonstrated an increased capacity to predict mucosal progression, with correct predictions exceeding 97% of cases. Collectively, our findings contribute to a comprehensive understanding of immunosenescence in ATL and suggest that senescence may represent the latest and most important signature of the immunopathogenisis. This highlights its potential value in predicting disease severity.

P02.15.A INVESTIGATING THE ROLE OF CELLULAR SENESCENCE IN BRIDGING GLIOBLASTOMA AND ALZHEIMER’S DISEASE

Abstract BACKGROUND With changing demographics, age-related diseases like neurodegenerative disorders and cancer are becoming increasingly prevalent. We previously demonstrated that over 50% of glioblastoma (GB) patients show Alzheimer’s Disease (AD)-like changes in the tumor-adjacent cortex. However, whether these diseases are biologically linked remains unclear. Here, we investigate cellular senescence, a hallmark of aging, as a shared mechanism. MATERIAL AND METHODS We utilized single cell and single nucleus RNA sequencing datasets from 110 GB patients (GBmap core atlas) and 89 individuals with varying stages of AD (SEA-AD MTG atlas). Following data integration, we assessed senescent cells using three established gene lists, performed differential gene expression and conducted gene set enrichment analysis. Senescence scores were compared across different cell types and patients. Co-expression modules with senescence-associated genes were identified using weighted gene co-expression network analysis (WGCNA). Quantitative neuropathological analysis of published measurements (Iba1, AT8, 6E10) complemented the molecular findings. RESULTS Senescence was found in both GB and AD patient cells including cell types of the microenvironment. Notably, the variability in scores per patient was significantly higher in GB as compared to AD cell types, indicating greater inter-patient heterogeneity. Microglia were specifically enriched for senescence gene sets among glial cells. Subset analysis of microglia through data harmonization identified a proinflammatory GB microglia subtype that shared high similarity with AD microglia and had highest senescence scores. Notably, the increase in senescent microglia was associated with progression to advanced disease stages in both diseases. WGCNA identified 3 consensus modules of senescence-associated genes that were coexpressed with disease-specific genes, like PTEN implicated phosphorylation in the AD module and SPP1 for extracellular matrix remodeling in the GB module. All senescence associated modules showed increased expression in more advanced disease stages. Quantitative analysis of immunohistochemistry measurements in the AD dataset confirmed that samples with a high senescence score showed a significantly higher density of tau pathology and an altered microglia morphology. CONCLUSION Our study identifies cellular senescence as one connection between GB and AD pathology by utilizing atlas-level transcriptomic datasets. Particularly microglia upregulate senescence signatures with disease progression. The co-expression of senescence-associated genes within disease-specific modules underscores the complexity of senescence in this context.

Transcriptomic markers of biological aging in breast cancer survivors: a longitudinal study.

The purpose of this study was to examine the impact of breast cancer therapy on biological aging as measured by expression of genes for cellular senescence (p16INK4a, SenMayo), DNA damage response, and proinflammatory senescence-associated secretory phenotype. This longitudinal, observational study evaluated women diagnosed with breast cancer (stage 0-III) prior to radiation therapy (RT) and/or chemotherapy (CT) and at repeated visits out to 2 years. Peripheral blood mononuclear cell gene expression was assessed using RNA sequencing on quality-verified RNA. Longitudinal data were analyzed using mixed linear models and a zero-inflated 2-part model. Women (mean age = 55.5 years) receiving CT with or without RT (n = 73) had higher odds (odds ratio = 2.97, 95% confidence interval = 1.52 to 5.8) of having detectable p16INK4a following treatment compared with RT (n = 76) or surgery alone (n = 37). The proportion of women expressing 16INK4a over the follow-up period increased in all treatment groups (P < .001), with no interaction by treatment. All groups also increased over time in DNA damage response (P < .001), SenMayo (P < .001), and senescence-associated secretory phenotype (P < .001). Groups differed in the pattern of increase over time with statistically significant quadratic time by group differences for CT with or without RT compared with RT alone for DNA damage response (P = .05), SenMayo (P = .006), and the senescence-associated secretory phenotype (P = .02). Results revealed activation of genes associated with biological aging in women with breast cancer from diagnosis through early survivorship, including DNA damage response, cell senescence, and the inflammatory secretome. Increases were evident across cancer treatments, although women receiving CT showed sustained increases, whereas RT exhibited slowing at later time points. Overall, findings suggest that women treated for breast cancer are aging within their immune cells.

Mapping epidermal and dermal cellular senescence in human skin aging.

Single-cell RNA sequencing and spatial transcriptomics enable unprecedented insight into cellular and molecular pathways implicated in human skin aging and regeneration. Senescent cells are individual cells that are irreversibly cell cycle arrested and can accumulate across the human lifespan due to cell-intrinsic and -extrinsic stressors. With an atlas of single-cell RNA-sequencing and spatial transcriptomics, epidermal and dermal senescence and its effects were investigated, with a focus on melanocytes and fibroblasts. Photoaging due to ultraviolet light exposure was associated with higher burdens of senescent cells, a sign of biological aging, compared to chronological aging. A skin-specific cellular senescence gene set, termed SenSkin™, was curated and confirmed to be elevated in the context of photoaging, chronological aging, and non-replicating CDKN1A+ (p21) cells. In the epidermis, senescent melanocytes were associated with elevated melanin synthesis, suggesting haphazard pigmentation, while in the dermis, senescent reticular dermal fibroblasts were associated with decreased collagen and elastic fiber synthesis. Spatial analysis revealed the tendency for senescent cells to cluster, particularly in photoaged skin. This work proposes a strategy for characterizing age-related skin dysfunction through the lens of cellular senescence and suggests a role for senescent epidermal cells (i.e., melanocytes) and senescent dermal cells (i.e., reticular dermal fibroblasts) in age-related skin sequelae.

Maize stigmas react differently to self- and cross-pollination and fungal invasion.

During sexual reproduction in flowering plants, tip-growing pollen tubes travel from the stigma inside the maternal tissues of the pistil towards ovules. In maize (Zea mays L.), the stigma is highly elongated, forming thread-like strands known as silks. Only compatible pollen tubes successfully penetrate and grow through the transmitting tract of the silk to reach the ovules. Like pollen, fungal spores germinate at the surface of silks and generate tube-like structures (hyphae) penetrating silk tissue. To elucidate commonalities and differences between silk responses to these distinctive invading cells, we compared growth behavior of the various invaders as well as the silk transcriptome after self-pollination, cross-pollination and infection using two different fungi. We report that self-pollination triggers mainly senescence genes, whereas incompatible pollen from Tripsacum dactyloides leads to downregulation of rehydration, microtubule, and cell wall-related genes, explaining the slower pollen tube growth and arrest. Invasion by the ascomycete Fusarium graminearum triggers numerous defense responses including the activation of monolignol biosynthesis and NAC as well as WRKY transcription factor genes, whereas responses to the basidiomycete Ustilago maydis are generally much weaker. We present evidence that incompatible pollination and fungal infection trigger transcriptional reprograming of maize silks cell wall. Pathogen invasion also activates the phytoalexin biosynthesis pathway.

The senolytic agent ABT263 ameliorates osteoporosis caused by active vitamin D insufficiency through selective clearance of senescent skeletal cells

Background/ObjectiveActive vitamin D insufficiency accelerates the development of osteoporosis, with senescent bone cells and the senescence-associated secretory phenotype (SASP) playing crucial roles. This study aimed to investigate whether the senolytic agent ABT263 could correct osteoporosis caused by active vitamin D insufficiency by selectively clearing senescent cells. MethodsBone marrow mesenchymal stem cells (BM-MSCs) from young and aged mice were treated with ABT263 in vitro, and 1,25(OH)2D-insufficient (Cyp27b1+/−) mice were administered ABT263 in vivo. Cellular, molecular, imaging, and histopathological analyses were performed to compare treated cells and mice with control groups. ResultsABT263 induced apoptosis in senescent BM-MSCs by downregulating Bcl2 and upregulating Bax expression. It also induced apoptosis in senescent BM-MSCs from 1,25(OH)2D-insufficient mice. ABT263 administration corrected bone loss caused by 1,25(OH)2D insufficiency by increasing bone density, bone volume, trabecular number, trabecular thickness, and collagen synthesis. It also enhanced osteoblastic bone formation and reduced osteoclastic bone resorption in vivo. ABT263 treatment corrected the impaired osteogenic action of BM-MSCs by promoting their proliferation and osteogenic differentiation. Furthermore, it corrected oxidative stress and DNA damage caused by 1,25(OH)2D insufficiency by increasing SOD-2 and decreasing γ-H2A.X expression. Finally, ABT263 corrected bone cell senescence and SASP caused by 1,25(OH)2D insufficiency by reducing the expression of senescence and SASP-related genes and proteins. ConclusionABT263 can correct osteoporosis caused by active vitamin D insufficiency by selectively clearing senescent skeletal cells, reducing oxidative stress, DNA damage, and SASP, and promoting bone formation while inhibiting bone resorption. These findings provide new insights into the potential therapeutic application of senolytic agents in the treatment of osteoporosis associated with active vitamin D insufficiency. The translational potential of this articleThis study highlights the therapeutic potential of ABT263, a senolytic compound, in treating osteoporosis caused by active vitamin D insufficiency. By selectively eliminating senescent bone cells and their associated SASP, ABT263 intervention demonstrates the ability to restore bone homeostasis, prevent further bone loss, and promote bone formation. These findings contribute to the growing body of research supporting the use of senolytic therapies for the prevention and treatment of age-related bone disorders. The translational potential of this study lies in the development of novel therapeutic strategies targeting cellular senescence to combat osteoporosis, particularly in cases where vitamin D insufficiency is a contributing factor. Further clinical studies are warranted to validate the efficacy and safety of ABT263 and other senolytic agents in the treatment of osteoporosis in humans.

Comparison of the Expression Levels of Senescence Genes between Human Normal Kidney Cells and Kidney Tumor Cells According to Age

Comparison of the Expression Levels of Senescence Genes between Human Normal Kidney Cells and Kidney Tumor Cells According to Age

Abstract C147: Impact of genetic ancestry in triple-negative breast cancer subtypes

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive BC subtype characterized by significant molecular and clinical heterogeneity, influenced by genetic, phenotypic, and environmental factors, as well as interactions of the tumor with its surrounding milieu. A significant racial disparity has been observed between African American (AA) and European American (EA) women regarding the incidence and clinical progression of TNBC. TNBC frequently occurs in young parous women who self-identify as AA and in women who harbor a germline BRCA1 mutation. Notably, AA women are diagnosed with TNBC at a younger age and exhibit a higher risk of developing stage IV disease. Studies examining gene expression signatures that distinguish AA and EA TNBC have reported distinct tumor-associated immunologic profiles in AA patients. We have developed a novel cell type-driven subtyping system based on a racially diverse TNBC sample set that identified three TNBC subtypes, each with distinct biology and prognosis: CC1- immune signaling/T-cell response; CC2- pro- migratory/inflammatory; CC3- fatty acid and hormone and nuclear receptor signaling. In this study, we propose to examine ancestry-inferred differences within each TNBC subtype between patients with African and European ancestry. Methods: RNA-Sequencing data for the Louisiana Tumor Registry, a TNBC cohort of 250 diverse women [EA: 123 and AA: 127], was obtained from our collaborators at Louisiana State University Health Sciences Center. Genetic admixture was performed on 227/250 patients using the method described by Pakstis, A. J. et al. Differentially expressed genes (DEGs) were identified in pairwise comparisons at p-adj ≤ 0.05. Survival was assessed using log-rank tests. Results: We investigated DEGs within each subtype between patients with &amp;gt;60% African (Aa, n=102) or &amp;gt;60% European ancestry (Ea, n=99) (identified via admixture analysis). In all three subtypes, upregulated genes, and their associated enrichment in Aa were related to immune pathways, while DEGs in Ea patients were related to epithelial cell regulation. In CC1, Aa was associated with the upregulation of immunoglobulin genes involved in immunoglobulin production and antigen recognition, while Ea was associated with the upregulation of embryonic development genes. In CC2, Aa showed enriched complement activation and immunoglobulin genes, while Ea exhibited upregulation of basal cytokeratin and growth factor EGFR. In CC3, Aa was associated with the upregulation of inflammatory genes, whereas Ea showed upregulation of cellular senescence genes. Survival analysis demonstrated favorable outcomes for Aa in CC1 and less favorable outcomes for Aa in CC2. Conclusion: Our study highlights significant ancestry-inferred differences in gene expression within TNBC subtypes, with Aa patients showing immune pathways enrichment and Ea patients exhibiting differential epithelial cell regulation. These findings underscore the importance of considering genetic ancestry in TNBC prognosis and treatment strategies. Citation Format: Zahra Mesrizadeh, Kavitha Mukund, Fokrul Hossain, Jovanny Zabaleta, Denise Danos, Luis De Valle, Xiao-Cheng Wu, Chindo Hicks, Augusto Ochoa, Lucio Miele, Victoria L. Seewaldt, Shankar Subramaniam. Impact of genetic ancestry in triple-negative breast cancer subtypes [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C147.

IL-10 deficiency aggravates cell senescence and accelerates BLM-induced pulmonary fibrosis in aged mice via PTEN/AKT/ERK pathway

BackgroundPulmonary fibrosis (PF) is an aging-related progressive lung disorder. The aged lung undergoes functional and structural changes termed immunosenescence and inflammaging, which facilitate the occurrence of fibrosis. Interleukin-10 (IL-10) is a potent anti-inflammatory and immunoregulatory cytokine, yet it remains unclear how IL-10 deficiency-induced immunosenescence participates in the development of PF.MethodsFirstly we evaluated the susceptibility to fibrosis and IL-10 expression in aged mice. Then 13-month-old wild-type (WT) and IL-10 knockout (KO) mice were subjected to bleomycin(BLM) and analyzed senescence-related markers by PCR, western blot and immunohistochemistry staining of p16, p21, p53, as well as DHE and SA-β-gal staining. We further compared 18-month-old WT mice with 13-month-old IL-10KO mice to assess aging-associated cell senescence and inflamation infiltration in both lung and BALF. Moreover, proliferation and apoptosis of alveolar type 2 cells(AT2) were evaluated by FCM, immunofluorescence, TUNEL staining, and TEM analysis. Recombinant IL-10 (rIL-10) was also administered intratracheally to evaluate its therapeutic potential and related mechanism. For the in vitro experiments, 10-week-old naïve pramily lung fibroblasts(PLFs) were treated with the culture medium of 13-month PLFs derived from WT, IL-10KO, or IL-10KO + rIL-10 respectively, and examined the secretion of senescence-associated secretory phenotype (SASP) factors and related pathways.ResultsThe aged mice displayed increased susceptibility to fibrosis and decreased IL-10 expression. The 13-month-old IL-10KO mice exhibited significant exacerbation of cell senescence compared to their contemporary WT mice, and even more severe epithelial-mesenchymal transition (EMT) than that of 18 month WT mice. These IL-10 deficient mice showed heightened inflammatory responses and accelerated PF progression. Intratracheal administration of rIL-10 reduced lung CD45 + cell infiltration by 15%, including a 6% reduction in granulocytes and a 10% reduction in macrophages, and increased the proportion of AT2 cells by approximately 8%. Additionally, rIL-10 significantly decreased α-SMA and collagen deposition, and reduced the expression of senescence proteins p16 and p21 by 50% in these mice. In vitro analysis revealed that conditioned media from IL-10 deficient mice promoted SASP secretion and upregulated senescence genes in naïve lung fibroblasts, which was mitigated by rIL-10 treatment. Mechanistically, rIL-10 inhibited TGF-β-Smad2/3 and PTEN/PI3K/AKT/ERK pathways, thereby suppressing senescence and fibrosis-related proteins.ConclusionsIL-10 deficiency in aged mice leads to accelerated cell senescence and exacerbated fibrosis, with IL-10KO-PLFs displaying increased SASP secretion. Recombinant IL-10 treatment effectively mitigates these effects, suggesting its potential as a therapeutic target for PF.Graphical

Aging-Associated Molecular Changes in Human Alveolar Type I Cells.

Human alveolar type I (AT1) cells are specialized epithelial cells that line the alveoli in the lungs where gas exchange occurs. The primary function of AT1 cells is not only to facilitate efficient gas exchange between the air and the blood in the lungs, but also to contribute to the structural integrity of the alveoli to maintain lung function and homeostasis. Aging has notable effects on the structure, function, and regenerative capacity of human AT1 cells. However, our understanding of the molecular mechanisms driving these age-related changes in AT1 cells remains limited. Leveraging a recent single-cell transcriptomics dataset we generated on healthy human lungs, we identified a series of significant molecular alterations in AT1 cells from aged lungs. Notably, the aged AT1 cells exhibited increased cellular senescence and chemokine gene expression, alongside diminished epithelial features such as decreases in cell junctions, endocytosis, and pulmonary matrisome gene expression. Gene set analyses also indicated that aged AT1 cells were resistant to apoptosis, a crucial mechanism for turnover and renewal of AT1 cells, thereby ensuring alveolar integrity and function. Further research on these alterations is imperative to fully elucidate the impact on AT1 cells and is indispensable for developing effective therapies to preserve lung function and promote healthy aging.

Abstract P162: Senolytic Treatment Attenuates Palmitic Acid-Induced Microglial Activation: Therapeutic Implications for Obesity-Induced Sympathoexcitation

Objective: Microglial dysfunction has been previously implicated in the pathogenesis of obesity-induced neuroinflammation and sympathoexcitation, both of which significantly increase the risk for cardiovascular diseases. However, the mechanisms underlying obesity-induced glial dysfunction are not clear. In the current study, we investigated the role of cellular senescence that culminates in irreversible proliferative arrest and inflammatory phenotype, in obesity-induced glial dysfunction. Methods: Human microglial cells (n=6 wells/group/expt) were treated with 35 μM palmitic acid (PA) for 24 hours to mimic an obese microenvironment. Subsequently, the cells were treated with senolytic drugs, Dasatinib (250 nM) and Quercetin (100 μM) (D+Q) for 48 hours after PA treatment. Real-time PCR, lipid profiling, and senescence-associated beta-galactosidase (SAβGAL) were performed to evaluate changes in microglia to PA and D+Q treatment. Immunofluorescence staining evaluated microglial activation and vesicular glutamate transporter (VGLUT1) expression. Results: Microglial activation was confirmed by upregulation of Iba1 and CD11b in PA-treated cells. Significant upregulation of senescence genes (p16 and p21), senescence-associated secretory phenotypes (SASPs such as TNFα, MMP3), and SAβGAL staining in the PA treatment group indicating cellular senescence. PA treatment, significantly upregulated glutamate transporters EAAT1 and EAAT2 suggesting a potential adaptive response to counteract excitotoxicity. Bodipy and Oil-Red-O (ORO) staining revealed increased lipid accumulation in PA group, indicating altered lipid metabolism. D+Q group showed a significant decrease in senescence markers (p16 and p21) and SAβGAL staining, along with a reduction in lipid markers such as perilipin 1 and 2, and decreases in activated microglial markers IBA1 and CD11b. A significant decrease in EAAT1 expression was also observed. Immunofluorescence showed a significant decrease in VGLUT1 expression in the D+Q compared to PA-treated group. Conclusions: The findings suggest that palmitic acid-induced changes in human microglial cells can contribute to oxidative stress, cellular senescence, and alterations in glutamate clearance pathway potentially leading to sympathetic overactivity in obesity. Senolytic treatment (D+Q) mitigated PA-induced lipid accumulation and inflammatory changes in the microglial cells indicating a promising therapeutic strategy for managing sympathoexcitation in obesity.

Generation of a conditional cellular senescence model using proximal tubule cells and fibroblasts from human kidneys

Emerging evidence highlights cellular senescence’s pivotal role in chronic kidney disease (CKD). Proximal tubule epithelial cells (PTECs) and fibroblasts are major players in CKD and serve as cellular sources of senescence. The generation of a conditionally immortalized human kidney cell model would allow to better understand the specific mechanisms and factors associated with cellular senescence in a controlled setting, devoid of potential confounding factors such as age and comorbidities. In addition, the availability of human kidney cell lines for preclinical research is sparse and most cell lines do not reflect their in vivo counterparts due to their altered behavior as immortalized cancer-like cells. In this study, PTECs and fibroblasts from human kidneys were isolated and transduced with doxycycline-inducible simian virus 40 large T antigen (SV40LT) vector. By comparing their gene expression with single-cell RNA sequencing data from human kidneys, the newly produced human kidney cell lines demonstrated significant resemblances to their in vivo counterparts. As predicted, PTECs showed functional activity and fibroblasts responded to injury with fibrosis. Withdrawal of the immortalizing factor doxycycline led to p21+ cell-cycle arrest and the key hallmarks of senescence. The obtained senescence gene set largely overlapped between both cell lines and with the previously published SenMayo set of senescence-associated genes. Furthermore, crosstalk experiments showed that senescent PTECs can cause a profibrotic response in fibroblasts by paracrine actions. In 76 human kidney sections, the number of p21+ cells correlated with the degree of fibrosis, age and reduced glomerular filtration, validating the role of senescence in CKD. In conclusion, we provide a novel cellular ex vivo model to study kidney senescence which can serve as a platform for large scale compounds testing.

Aging promotes accumulation of senescent and multiciliated cells in human endometrial epithelium.

What changes occur in the endometrium during aging, and do they impact fertility? Both the transcriptome and cellular composition of endometrial samples from women of advanced maternal age (AMA) are significantly different from that of samples from young women, suggesting specific changes in epithelial cells that may affect endometrial receptivity. Aging is associated with the accumulation of senescent cells in aging tissues. Reproductive aging is mostly attributed to the decline in ovarian reserve and oocyte quality, whereas the endometrium is a unique complex tissue that is monthly renewed under hormonal regulation. Several clinical studies have reported lower implantation and pregnancy rates in oocyte recipients of AMA during IVF. Molecular studies have indicated the presence of specific mutations within the epithelial cells of AMA endometrium, along with altered gene expression of bulk endometrial tissue. Endometrial transcriptome profiling was performed for 44 women undergoing HRT during the assessment of endometrial receptivity before IVF. Patients younger than 28 years were considered as the young maternal age (YMA) group (age 23-27 years) and women older than 45 years were considered as the AMA group (age 47-50 years). Endometrial biopsies were obtained on Day 5 of progesterone treatment and RNA was extracted. All endometrial samples were evaluated as being receptive based on the expression of 68 common endometrial receptivity markers. Endometrial samples from another 24 women classified into four age groups (YMA, intermediate age group 1 (IMA1, age 29-35), intermediate age group 2 (IMA2, age 36-44), and AMA) were obtained in the mid-secretory stage of a natural cycle (NC) and used for validation studies across the reproductive lifespan. A total of 24 HRT samples (12 YMA and 12 AMA) were subject to RNA sequencing (RNA-seq) and differential gene expression analysis, 20 samples (10 YMA and 10 AMA) were used for qPCR validation, and 24 NC samples (6 YMA, 6 IMA1, 6 IMA2 and 6AMA) were used for RNA-seq validation of AMA genes across the woman's reproductive lifespan. Immunohistochemistry (IHC) was used to confirm some expression changes at the protein level. Computational deconvolution using six endometrial cell type-specific transcriptomic profiles was conducted to compare the cellular composition between the groups. Comparisons between YMA and AMA samples identified a lower proportion of receptive endometria in the AMA group (P = 0.007). Gene expression profiling identified 491 differentially expressed age-sensitive genes (P adj < 0.05) that revealed the effects of age on endometrial epithelial growth and receptivity, likely contributing to decreased reproductive performance. Our results indicate that changes in the expression of the cellular senescence marker p16INK4a and genes associated with metabolism, inflammation, and hormone response are involved in endometrial aging. Importantly, we demonstrate that the proportion of multi-ciliated cells, as discovered based on RNA-seq data deconvolution and tissue IHC results, is affected by endometrial aging, and propose a putative onset of age-related changes. Furthermore, we propose that aging has an impact on the transcriptomic profile of endometrial tissue in the context of endometrial receptivity. The raw sequencing data reported in this article are deposited at the Gene Expression Omnibus under accession code GSE236128. This retrospective study identified changes in the endometrium of patients undergoing hormonal replacement and validated these changes using samples obtained during a NC. However, future studies must clarify the importance of these findings on the clinical outcomes of assisted reproduction. The findings reported in this study have important implications for devising future strategies aimed at improving fertility management in women of advanced reproductive age. This research was funded by the Estonian Research Council (grant no. PRG1076), Horizon 2020 innovation grant (ERIN, grant no. EU952516), Enterprise Estonia (grant no. EU48695), MSCA-RISE-2020 project TRENDO (grant no. 101008193), EU 874867 project HUTER, the Horizon Europe NESTOR grant (grant no. 101120075) of the European Commission, the EVA specialty program (grant no. KP111513) of the Maastricht University Medical Center (MUMC+), MICIU/AEI/10.13039/501100011033 and FEDER, EU projects Endo-Map (grant no. PID2021-12728OB-100), ROSY (grant no. CNS2022-135999), and the National Science Fund of Bulgaria (grant no. KII-06 H31/2). The authors declare no competing interests.

The Matrix Protein Tropoelastin Prolongs Mesenchymal Stromal Cell Vitality and Delays Senescence During Replicative Aging.

Cellular senescence leads to the functional decline of regenerative cells such as mesenchymal stromal/stem cells (MSCs), which gives rise to chronic conditions and contributes to poor cell therapy outcomes. Aging tissues are associated with extracellular matrix (ECM) dysregulation, including loss of elastin. However, the role of the ECM in modulating senescence is underexplored. In this work, it is shown that tropoelastin, the soluble elastin precursor, is not only a marker of young MSCs but also actively preserves cell fitness and delays senescence during replicative aging. MSCs briefly exposed to tropoelastin exhibit upregulation of proliferative genes and concurrent downregulation of senescence genes. The seno-protective benefits of tropoelastin persist during continuous, long-term MSC culture, and significantly extend the MSC replicative lifespan. Tropoelastin-expanded MSCs further maintain youth-associated phenotype and function compared to age-matched controls, including preserved clonogenic potential, minimal senescence-associated beta-galactosidase activity, maintained cell sizes, reduced expression of senescence markers, suppressed secretion of senescence-associated factors, and increased production of youth-associated proteins. This work points to the utility of exogenously-supplemented tropoelastin for manufacturing MSCs that robustly maintain regenerative potential with age. It further reveals the active role of classical structural ECM proteins in driving cellular age-associated fitness, potentially leading to future interventions for aging-related pathologies.

Abstract Tu076: N4-acetylcytidine-regulated Cardiomyocyte Ferroptosis Mediates Cardiac Benefits of Exercise in Aging

Introduction: Advanced age is a major risk factor for heart diseases. Exercise training has emerged as an effective intervention to improve functional outcomes in age-related cardiac dysfunction. N-acetyltransferase (NAT10), the only known N4-acetylcytidine (ac4C) ‘writer’ enzyme, has recently been implicated in heart failure. However, the role of NAT10 in cardiac aging and the cardiac benefits of exercise on age-related cardiac dysfunction are unclear. Hypothesis: NAT10 is necessary and sufficient for cardiac benefits of exercise in aging. Methods: Ten-week-old (young) or twenty-month-old (aged) mice received sedentary (Sed) or voluntary wheel running (Run) for eight weeks. AAV9 encoding cardiac troponin-T-driven NAT10 was tail vein-injected to overexpress NAT10 in cardiomyocytes (CMs). Results: Compared with young mice, aged mice exhibited increased E/e’ (Young: -14.68±1.09 vs Aged: -20.16±1.61, n=8, p=0.03) and decreased global longitudinal strain (GLS, Young: -20.04±1.47 vs Aged: -15.88±0.84, n=8, p=0.04) with preserved ejection fraction. Aged hearts also displayed increased ferroptosis (reduced GPX4 and FSP1, p&lt;0.05 vs Sed) and senescence (increased gene expression of p16, p21, and PAI, all p&lt;0.05). In aged mice, running improved diastolic function (E/e’, Sed: -20.16±1.61 vs Run: -15.4±1.06, n=8, p=0.04; GLS, Sed: -15.88±0.84 vs Run: -20.66±1.02, n=8, p=0.02), reduced cardiac ferroptosis (increased GPX4 and FSP1, p&lt;0.05 vs Sed), and decreased senescence gene expression. Cardiac NAT10 was downregulated in aged mice and was upregulated by exercise (all p&lt;0.05). In aged mice, AAV9 injection for eight weeks led to ~6-fold increase in cardiac NAT10, resulting in reduced E/e’ (AAV9-NAT10: -22.67±1.03 vs Control: -26.33±1.12, n=7, p=0.03) and increased GLS (AAV9-NAT10: -19.23±0.93 vs Control: -15.26±1.01, n=7, p=0.04) as well as reduced senescence gene expression (reduced p16, p21, and PAI, p&lt;0.05 vs AAV9-control). Mechanistically, NAT10 functioned by increasing ferritin ac4C deposition and promoting its translation, thereby inhibiting CM ferroptosis. Conclusions: NAT10 regulates cardiac aging through ferritin, and it mediates cardiac benefits of exercise in aging. NAT10 is a promising therapeutic target for combating cardiac aging.

West Nile Virus-Induced Expression of Senescent Gene Lgals3bp Regulates Microglial Phenotype within Cerebral Cortex.

Microglia, the resident macrophages of the central nervous system, exhibit altered gene expression in response to various neurological conditions. This study investigates the relationship between West Nile Virus infection and microglial senescence, focusing on the role of LGALS3BP, a protein implicated in both antiviral responses and aging. Using spatial transcriptomics, RNA sequencing and flow cytometry, we characterized changes in microglial gene signatures in adult and aged mice following recovery from WNV encephalitis. Additionally, we analyzed Lgals3bp expression and generated Lgals3bp-deficient mice to assess the impact on neuroinflammation and microglial phenotypes. Our results show that WNV-activated microglia share transcriptional signatures with aged microglia, including upregulation of genes involved in interferon response and inflammation. Lgals3bp was broadly expressed in the CNS and robustly upregulated during WNV infection and aging. Lgals3bp-deficient mice exhibited reduced neuroinflammation, increased homeostatic microglial numbers, and altered T cell populations without differences in virologic control or survival. These data indicate that LGALS3BP has a role in regulating neuroinflammation and microglial activation and suggest that targeting LGALS3BP might provide a potential route for mitigating neuroinflammation-related cognitive decline in aging and post-viral infections.