Senescence-associated Beta-galactosidase Activity Research Articles

Sub-lethal doses of chemotherapeutic agents induce senescence in T cells and upregulation of PD-1 expression.

Cellular senescence is a stable cell cycle arrest, usually in response to internal and/or external stress, including telomere dysfunction, abnormal cellular growth, and DNA damage. Several chemotherapeutic drugs, such as melphalan (MEL) and doxorubicin (DXR), induce cellular senescence in cancer cells. However, it is not clear whether these drugs induce senescence in immune cells. We evaluated the induction of cellular senescence in T cells were derived from human peripheral blood mononuclear cells (PBMNCs) in healthy donors using sub-lethal doses of chemotherapeutic agents. The PBMNCs were kept overnight in RPMI 1640 medium with 2% phytohemagglutinin and 10% fetal bovine serum and then cultured in RPMI 1640 with 20ng/mL IL-2 and sub-lethal doses of chemotherapeutic drugs (2μM MEL and 50nM DXR) for 48h. Sub-lethal doses of chemotherapeutic agents induced phenotypes associated with senescence, such as the formation of γH2AX nuclear foci, cell proliferation arrest, and induction of senescence-associated beta-galactosidase (SA-β-Gal) activity, (control vs. MEL, DXR; median mean fluorescence intensity (MFI) 1883 (1130-2163) vs. 2233 (1385-2254), 2406.5 (1377-3119), respectively) in T cells. IL6 and SPP1 mRNA, which are senescence-associated secretory phenotype (SASP) factors, were significantly upregulated by sublethal doses of MEL and DXR compared to the control (P = 0.043 and 0.018, respectively). Moreover, sub-lethal doses of chemotherapeutic agents significantly enhanced the expression of programmed death 1 (PD-1) on CD3 + CD4 + and CD3 + CD8 + T cells compared to the control (CD4 + T cells; P = 0.043, 0.043, and 0.043, respectively, CD8 + T cells; P = 0.043, 0.043, and 0.043, respectively). Our results suggest that sub-lethal doses of chemotherapeutic agents induce senescence in T cells and tumor immunosuppression by upregulating PD-1 expression on T cells.

Advances in the Phytochemical Characterisation and Bioactivities of Salvia aurea L. Essential Oil.

The Salvia L. genus (Lamiaceae) is largely used in the pharmaceutical and food industry. Several species of biological relevance are extensively employed in traditional medicine, including Salvia aurea L. (syn. S. africana-lutea L.), which is used as a traditional skin disinfectant and in wounds as a healing remedy; nevertheless, these properties have not been validated yet. The aim of the present study is to characterise S. aurea essential oil (EO), unveiling its chemical composition and validating its biological properties. The EO was obtained by hydrodistillation and subsequently analysed by GC-FID and GC-MS. Different biological activities were assessed: the antifungal effect on dermatophytes and yeasts and the anti-inflammatory potential by evaluating nitric oxide (NO) production and COX-2 and iNOS protein levels. Wound-healing properties were assessed using the scratch-healing test, and the anti-aging capacity was estimated through the senescence-associated beta-galactosidase activity. S. aurea EO is mainly characterised by 1,8-cineole (16.7%), β-pinene (11.9%), cis-thujone (10.5%), camphor (9.5%), and (E)-caryophyllene (9.3%). The results showed an effective inhibition of the growth of dermatophytes. Furthermore, it significantly reduced protein levels of iNOS/COX-2 and simultaneously NO release. Additionally, the EO exhibited anti-senescence potential and enhanced wound healing. Overall, this study highlights the remarkable pharmacological properties of Salvia aurea EO, which should be further explored in order to develop innovative, sustainable, and environmentally friendly skin products.

Senescent Secretome of Blind Mole Rat Spalax Inhibits Malignant Behavior of Human Breast Cancer Cells Triggering Bystander Senescence and Targeting Inflammatory Response

Subterranean blind mole rat, Spalax, has developed strategies to withstand cancer by maintaining genome stability and suppressing the inflammatory response. Spalax cells undergo senescence without the acquisition of senescence-associated secretory phenotype (SASP) in its canonical form, namely, it lacks the main inflammatory mediators. Since senescence can propagate through paracrine factors, we hypothesize that conditioned medium (CM) from senescent Spalax fibroblasts can transmit the senescent phenotype to cancer cells without inducing an inflammatory response, thereby suppressing malignant behavior. To address this issue, we investigated the effect of CMs of Spalax senescent fibroblasts on the proliferation, migration, and secretory profile in MDA-MB-231 and MCF-7 human breast cancer cells. The results suggest that Spalax CM induced senescence in cancer cells, as evidenced by increased senescence-associated beta-galactosidase (SA-β-Gal) activity, growth suppression and overexpression of senescence-related p53/p21 genes. Contemporaneously, Spalax CM suppressed the secretion of the main inflammatory factors in cancer cells and decreased their migration. In contrast, human CM, while causing a slight increase in SA-β-Gal activity in MDA-MB-231 cells, did not decrease proliferation, inflammatory response, and cancer cell migration. Dysregulation of IL-1α under the influence of Spalax CM, especially the decrease in the level of membrane-bound IL1-α, plays an important role in suppressing inflammatory secretion in cancer cells, which in turn leads to inhibition of cancer cell migration. Overcoming of SASP in tumor cells in response to paracrine factors of senescent microenvironment or anti-cancer drugs represents a promising senotherapeutic strategy in cancer treatment.

Ginsenoside Rg2 Promotes the Proliferation and Stemness Maintenance of Porcine Mesenchymal Stem Cells through Autophagy Induction

Mesenchymal stem cells (MSCs) can be used as a cell source for cultivated meat production due to their adipose differentiation potential, but MSCs lose their stemness and undergo replicative senescence during expansion in vitro. Autophagy is an important mechanism for senescent cells to remove toxic substances. However, the role of autophagy in the replicative senescence of MSCs is controversial. Here, we evaluated the changes in autophagy in porcine MSCs (pMSCs) during long-term culture in vitro and identified a natural phytochemical, ginsenoside Rg2, that could stimulate pMSC proliferation. First, some typical senescence characteristics were observed in aged pMSCs, including decreased EdU-positive cells, increased senescence-associated beta-galactosidase activity, declined stemness-associated marker OCT4 expression, and enhanced P53 expression. Importantly, autophagic flux was impaired in aged pMSCs, suggesting deficient substrate clearance in aged pMSCs. Rg2 was found to promote the proliferation of pMSCs using MTT assay and EdU staining. In addition, Rg2 inhibited D-galactose-induced senescence and oxidative stress in pMSCs. Rg2 increased autophagic activity via the AMPK signaling pathway. Furthermore, long-term culture with Rg2 promoted the proliferation, inhibited the replicative senescence, and maintained the stemness of pMSCs. These results provide a potential strategy for porcine MSC expansion in vitro.

PS-BPB10-5: POSSIBLE INVOLVEMENT OF RENAL TUBULAR NFAT5 IN AGING-ASSOCIATED RENAL DYSFUNCTION AND SALT-SENSITIVE HYPERTENSION

Objective: Japan is currently facing a super-aging society, where an incidence of lifestyle-related diseases has become a greater social burden. The number of patients with chronic kidney disease (CKD) and hypertension is being increased with age; however, it is not clearly defined why and how aging causes renal dysfunction and hypertension. Nuclear factor of activated T-cells 5 (NFAT5) is a transcription factor that is activated upon hypertonic conditions as observed in the renal medulla. We have shown that the renal tubular cell-specific NFAT5 conditional knockout (KO) mice exhibit salt-sensitive hypertension, while the mice exhibit impaired urine concentrating ability and are susceptible to renal fibrosis. These phenotypes resemble aging-associated renal dysfunction, i.e., urine concentrating disorder, salt-sensitive hypertension, and renal fibrosis. We therefore investigated the possible involvement of NFAT5 in aging-related changes of the kidney. Methods: Male wild-type (WT) and KO mice (12 weeks old) were subjected to unilateral ureteral obstruction (UUO) or sham operation. After 7 days, mice were sacrificed and kidneys were removed for analysis. Renal fibrosis was evaluated by AZAN staining. Senescence-associated beta-galactosidase (SA-β;-Gal) activity was examined by fluorescence study in the whole kidney slices. mRNA expression for senescence-associated secretory phenotype (SASP)-related factors (TGF-β;1, COL1A1, ICAM1, PAI-1, p21, p16, and p53) in the whole kidney tissue was examined by real-time PCR. Results: AZAN staining showed that UUO induced renal fibrosis both in WT and KO mice. The UUO-induced fibrotic area was significantly larger in KO mice than in WT mice in the medulla. In sham-operated mice, overall SA-β;-Gal activity was larger in KO mice, in which the activity was dominant in the corticomedullary region. In UUO groups, overall SA-β;-Gal activity was low compared to sham-operated groups. The mRNA expression of SASP-related factors was not different between WT and KO mice in sham-operated groups. UUO upregulated the mRNA expression of all SASP-related factors both in WT and KO mice. In UUO groups, the expression of TGF-β;1, COL1A1, ICAM1, and PAI-1 was significantly larger in KO mice than in WT mice. The expression of p21, p16, and p53 tended to be larger in KO mice than in WT mice. Conclusions: Renal tubular NFAT5 deficiency showed senescence-related phenotypes at baseline. Deficit of renal tubular NFAT5 may further induce cell senescence upon insult, which might accelerate aging-associated renal inflammation and dysfunction in CKD.

PS-BPB01-9: OMEGA-3 FATTY ACIDS ATTENUATE OXIDATIVE STRESS-INDUCED DNA DAMAGE THROUGH NRF-2 IN VASCULAR ENDOTHELIAL CELLS

Objective: Impaired vasodilation is one of the characterizations of hypertension. The effects of the omega-3 fatty acids on blood pressure and vascular function have been examined in many randomized controlled trials. In this study, we investigated the effects of omega-3 fatty acids, particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on DNA damage, which has crucial effects on vascular endothelial cell function. Design and method: We examined the effects of EPA and DHA on H2O2-induced DNA damage response in human aortic endothelial cells (HAECs). DNA double-strand breaks (DSBs) were detected by immunofluorescence staining of the phosphorylated form of the histone H2AX (gamma-H2AX). Results: EPA and DHA significantly reduced accumulation of DSBs and phosphorylation of ATM, a serine/threonine kinase recruited and activated upon DSBs, in HAECs. Since EPA and DHA attenuated DNA damage without enhancement of DNA repair response, the effects of EPA and DHA on oxidative stress, a DNA damage inducer, were examined. CM-H2DCFDA measurement showed that EPA and DHA significantly reduced intracellular reactive oxygen species. In addition, mRNA levels of antioxidant molecules, such as heme oxygenase-1 and thioredoxin reductase 1, were significantly elevated by EPA and DHA. Silencing the gene encoding nuclear factor erythroid 2-related factor 2 (Nrf2) abrogated these increases. Furthermore, EPA and DHA reduced H2O2-induced senescence-associated beta-galactosidase activity, which was revoked by Nrf2 silencing. Conclusions: Our results suggest that EPA and DHA attenuate oxidative stress-induced DNA damage and cell senescence in vascular endothelial cells partly via antioxidant response mediated by Nrf-2. Therefore EPA and DHA may play a role in the prevention of impairment in vasodilation by their genome-protective properties.

Lin28b delays vasculature aging by reducing platelet-derived growth factor-beta resistance in senescent vascular smooth muscle cells

Background and aimsPlatelet-derived growth factor-β (PDGFB) is an important mediator of vascular smooth muscle cell (VSMC) proliferation, and PDGFB resistance is observed in senescent VSMCs. Lin28b is a stemness regulator in the embryo; however, its role in vasculature aging and VSMC senescence is unknown. We aimed to investigate whether Lin28b could restore the VSMC response to PDGFB and delay vasculature aging. MethodsApoE−/− mice were fed a high-fat diet for different weeks to establish an aging model. PDGFB resistance was observed using EdU staining in vessel culture in vitro. Quantitative polymerase chain reaction and in situ hybridization were used to detect let-7 expression. Senescence was identified by Western blotting, senescence-associated beta-galactosidase activity or Sudan Black B staining, and VSMC function was determined using CCK-8, migration, and enzyme-linked immunosorbent assays. ResultsVessels from aged mice showed poor responses to PDGFB stimulation compared with those from young mice; similar results were found in senescent VSMCs. The expression levels of Lin28b and PDGF receptor-β were downregulated in aging vasculature and senescent VSMCs, whereas let-7 family levels increased with aging and VSMC passage growth. Transfection of VSMCs with let-7c induced PDGFB resistance and accelerated VSMC senescence, whereas blocking let-7c restored PDGFB reactions in VSMCs. Overexpression of Lin28b protein by lentivirus resulted in the restoration of PDGFB reactions and delayed VSMC senescence, which was blocked by a let-7c mimic. ConclusionsThis study reveals the role of Lin28b in delaying vasculature aging by decreasing senescent VSMC PDGFB resistance mediated by let-7.

H2S contributed from CSE during cellular senescence suppresses inflammation and nitrosative stress

Aging involves the time-dependent deterioration of physiological functions attributed to various intracellular and extracellular factors. Cellular senescence is akin to aging and involves alteration in redox homeostasis. This is primarily marked by increased reactive oxygen/nitrogen species (ROS/RNS), inflammatory gene expression, and senescence-associated beta-galactosidase activity, all hallmarks of aging. It is proposed that gasotransmitters which include hydrogen sulfide (H2S), carbon monoxide (CO), and nitric oxide (NO), may affect redox homeostasis during senescence. H2S has been independently shown to induce DNA damage and suppress oxidative stress. While an increase in NO levels during aging is well established, the role of H2S has remained controversial. To understand the role of H2S during aging, we evaluated H2S homeostasis in non-senescent and senescent cells, using a combination of direct measurements with a fluorescent reporter dye (WSP-5) and protein sulfhydration analysis. The free intracellular H2S and total protein sulfhydration levels are high during senescence, concomitant to cystathionine gamma-lyase (CSE) expression induction. Using lentiviral shRNA-mediated expression knockdown, we identified that H2S contributed by CSE alters global gene expression, which regulates key inflammatory processes during cellular senescence. We propose that H2S decreases inflammation during cellular senescence by reducing phosphorylation of IκBα and the p65 subunit of nuclear factor kappa B (NF-κB). H2S was also found to reduce NO levels, a significant source of nitrosative stress during cellular senescence. Overall, we establish H2S as a key gasotransmitter molecule that regulates inflammatory phenotype and nitrosative stress during cellular senescence.

TERT distal promoter GC islands are critical for telomerase and together with DNMT3B silencing may serve as a senescence-inducing agent in gliomas

Telomerase is reactivated in the majority of cancers. For instance, in gliomas, it is common that the TERT promoter is mutated. Research on telomere promoter GC islands have been focused primarily on proximal TERT promoter but little is known about the distal promoter. Therefore, in this study, we investigated the proximal and distal TERT promoter, in terms of DNA methylation. We did bisulfite sequencing in zebrafish tissue samples for the distal tert promoter. In the zebrafish brain tissues, we identified a hypomethylation site in the tert promoter, and found that this hypomethylation was associated with aging and shortened telomeres. Through site directed mutagenesis in glioma cell lines, we changed 10 GC spots individually, cloned into a reporter vector, and measured promoter activity. Finally, we silenced DNMT3B and measured telomerase activity along with vidaza and adriamycin treatments. Site directed mutagenesis of glioma cell lines revealed that each of the 10 GC spots are critical for telomerase activity. Changing GC to AT abolished promoter activity in all spots when transfected into glioma cell lines. Then, through silencing of DNMT3B, we observed a reduction in hTERT expression levels, while hTR remained the same, and a major increase in senescence-associated beta-galactosidase activity. Finally, we propose a model regarding the efficacy of two chemotherapeutic drugs, adriamycin and azacytidine, on gliomas. Here, we show that distal TERT promoter is critical; changing even one GC to AT abolishes TERT promoter activity. DNMT3B, a de novo methyltransferase, together with GC islands in distal TERT promoter plays an important role in regulation of telomerase expression and senescence.

Preclinical Evaluation of the Tumorigenic and Immunomodulatory Properties of Human Bone Marrow Mesenchymal Stromal Cell Populations with Clonal Trisomy 5.

Cytogenetic aberrations may emerge in human mesenchymal stromal cells (MSC) during ex vivo expansion for cell therapy. We have detected clonal trisomy 5 in two distinct autologous MSC products expanded from bone marrow which, based on the current quality control criteria, could not be released for clinical use. Although a safety concern, it is still unclear to what extent recurrent aneuploidies detected in MSC products may affect the threshold for neoplastic transformation or the medicinal properties of these cells. We have carried out an exploratory preclinical study to evaluate these MSC products with clonal trisomy 5, regarding their oncogenic and immunomodulatory potential. Cell population growth in vitro was reduced in MSC cultures with clonal trisomy 5 compared with the population growth of their euploid MSC counterparts, based on a lower cumulative population doubling level, reduced cell proliferation index, and increased senescence-associated beta-galactosidase activity. Subcutaneous injection of clinically relevant amount of MSC population, either with or without clonal trisomy 5, did not generate tumors in immunodeficient mice within a follow-up period of six months. Most importantly, MSC population with clonal trisomy 5 kept immunomodulatory properties upon interferon gamma (IFNγ) licensing, displaying overexpression of IDO, CXCL9, CXCL10, and CXCL11, in a similar fashion than that of IFNγ-licensed euploid MSC. Our findings suggest that bone marrow MSC products with clonal trisomy 5 may retain their therapeutic potential, based on poor tumor initiating capability and preserved immunomodulatory potency. This preclinical evidence may further support the definition of release criteria of autologous MSC products for cell therapy under critical clinical scenarios. This trial is registered with Clinical Study registration number: RBR-29x2pr.

Clearance of senescent cells with ABT-263 improves biological functions of synovial mesenchymal stem cells from osteoarthritis patients

BackgroundOsteoarthritis (OA) is an age-related joint disease characterized by progressive cartilage loss. Synovial mesenchymal stem cells (MSCs) are anticipated as a cell source for OA treatment; however, synovial MSC preparations isolated from OA patients contain many senescent cells that inhibit cartilage regeneration through their senescence-associated secretory phenotype (SASP) and poor chondrogenic capacity. The aim of this study was to improve the biological function of OA synovial MSCs by removing senescent cells using the senolytic drug ABT-263.MethodsWe pretreated synovial MSCs derived from 5 OA patients with ABT-263 for 24 h and then evaluated senescence-associated beta-galactosidase (SA-β-gal) activity, B cell lymphoma 2 (BCL-2) activity, apoptosis, surface antigen expression, colony formation ability, and multipotency.ResultsThe ABT-263 pretreatment significantly decreased the percentage of SA-β-gal-positive cells and BCL-2 expression and induced early- and late-stage apoptosis. Cleaved caspase-3 was expressed in SA-β-gal-positive cells. The pretreated MSCs formed greater numbers of colonies with larger diameters. The expression rate of CD34 was decreased in the pretreated cells. Differentiation assays revealed that ABT-263 pretreatment enhanced the adipogenic and chondrogenic capabilities of OA synovial MSCs. In chondrogenesis, the pretreated cells produced greater amounts of glycosaminoglycan and type II collagen and showed lower expression of senescence markers (p16 and p21) and SASP factors (MMP-13 and IL-6) and smaller amounts of type I collagen.ConclusionPretreatment of synovial MSCs from OA patients with ABT-263 can improve the function of the cells by selectively eliminating senescent cells. These findings indicate that ABT-263 could hold promise for the development of effective cell-based OA therapy.

Pcsk6 Deficiency Promotes Cardiomyocyte Senescence by Modulating Ddit3-Mediated ER Stress.

Cardiac aging is a critical determinant of cardiac dysfunction, which contributes to cardiovascular disease in the elderly. Proprotein convertase subtilisin/kexin 6 (PCSK6) is a proteolytic enzyme important for the maintenance of cardiac function and vascular homeostasis. To date, the involvement of PCSK6 in cardiac aging remains unknown. Here we report that PCSK6 expression decreased in the hearts of aged mice, where high levels cyclin dependent kinase inhibitor 2A (P16) and cyclin dependent kinase inhibitor 1A (P21) (senescence markers) were observed. Moreover, PCSK6 protein expression was significantly reduced in senescent rat embryonic cardiomyocytes (H9c2) induced by D-galactose. Pcsk6 knockdown in H9c2 cells increased P16 and P21 expression levels and senescence-associated beta-galactosidase activity. Pcsk6 knockdown also impaired cardiomyocyte function, as indicated by increased advanced glycation end products, reactive oxygen species level, and apoptosis. Overexpression of PCSK6 blunted the senescence phenotype and cellular dysfunction. Furthermore, RNA sequencing analysis in Pcsk6-knockdown H9c2 cells identified the up-regulated DNA-damage inducible transcript 3 (Ddit3) gene involved in endoplasmic reticulum (ER) protein processing. Additionally, DDIT3 protein levels were remarkably increased in aged mouse hearts. In the presence of tunicamycin, an ER stress inducer, DDIT3 expression increased in Pcsk6-deficient H9c2 cells but reduced in PCSK6-overexpressing cells. In conclusion, our findings indicate that PCSK6 modulates cardiomyocyte senescence possibly via DDIT3-mediated ER stress.

Abstract TP11: G-quadruplexes Regulate The Senescence Factor Cysteine-rich Angiogenic Inducer-61 In Primary Cerebral Endothelial Cells

Senescence in the cerebral endothelium has been proposed as a mechanism that can drive dysfunction of the cerebral vasculature, which precedes vascular dementia. Cysteine-rich angiogenic inducer 61 (Cyr61/CCN1) is a matricellular protein secreted by cerebral endothelial cells (CEC). CCN1 induces senescence in fibroblasts. Whether CCN1 contributes to senescence of CEC and how it is regulated require further study. Aging has been associated with the formation of four-stranded Guanine-quadruplexes (G4s) in G-rich motifs of DNA and RNA. G4 stabilization regulates transcription and translation either by upregulation or downregulation depending on the gene target. Previously, we showed that aged mice treated with a G4-stabilizing compound had enhanced senescence-associated (SA) phenotypes in their brains, and exhibited cognitive deficits. The expression of human CCN1 can be upregulated via the stabilization of G4s. A sequence in the 3’- untranslated region (UTR) of the CCN1 mRNA has the ability to fold in to G4 in vitro . Thus, we hypothesize that CCN1 is upregulated with aging and induces senescence, and that G4 stabilization upregulates CCN1 in cultured primary CEC. We used whole brain lysates, cerebral vessel fractions, and cultured primary CEC from young (4-months old, mo) and aged (18-mo) female mice to determine the CCN1 levels. SA phenotypes were determined by high-resolution fluorescence microscopy in cultured primary CEC, and we used Thioflavin T to recognize RNA G4s for fluorescence spectra. We found that CCN1 levels were increased in the brains of aging mice (p=0.0314, n=5), in cerebral vessel fractions (p=0.001, n=5), and in cultured primary CEC derived from aged mice (p=0.012, n=3), compared with those derived from young mice. In cultured CEC, a recombinant CCN1 protein induced SA phenotypes, such as SA beta-galactosidase activity (p=0.003, n=3), and double-strand DNA damage (p=0.016, n=3). Furthermore, CCN1 levels were upregulated by a G4 ligand (p=0.0103, n=3), and a G-rich motif in the 3’-UTR of the Ccn1 mRNA folded into a G4 (p=0.008, n=4). In conclusion , we demonstrate that CCN1 induces senescence in cultured primary CEC, and we propose G4 stabilization as a novel mechanism that regulates the SASP component CCN1.

Impact of senescence on the transdifferentiation process of human hepatic progenitor-like cells.

BACKGROUNDSenescence is characterized by a decline in hepatocyte function, with impairment of metabolism and regenerative capacity. Several models that duplicate liver functions in vitro are essential tools for studying drug metabolism, liver diseases, and organ regeneration. The human HepaRG cell line represents an effective model for the study of liver metabolism and hepatic progenitors. However, the impact of senescence on HepaRG cells is not yet known. AIMTo characterize the effects of senescence on the transdifferentiation capacity and mitochondrial metabolism of human HepaRG cells.METHODSWe compared the transdifferentiation capacity of cells over 10 (passage 10 [P10]) vs P20. Aging was evaluated by senescence-associated (SA) beta-galactosidase activity and the comet assay. HepaRG transdifferentiation was analyzed by confocal microscopy and flow cytometry (expression of cluster of differentiation 49a [CD49a], CD49f, CD184, epithelial cell adhesion molecule [EpCAM], and cytokeratin 19 [CK19]), quantitative PCR analysis (expression of albumin, cytochrome P450 3A4 [CYP3A4], γ-glutamyl transpeptidase [γ-GT], and carcinoembryonic antigen [CEA]), and functional analyses (albumin secretion, CYP3A4, and γ-GT). Mitochondrial respiration and the ATP and nicotinamide adenine dinucleotide (NAD+)/NAD with hydrogen (NADH) content were also measured. RESULTSSA β-galactosidase staining was higher in P20 than P10 HepaRG cells; in parallel, the comet assay showed consistent DNA damage in P20 HepaRG cells. With respect to P10, P20 HepaRG cells exhibited a reduction of CD49a, CD49f, CD184, EpCAM, and CK19 after the induction of transdifferentiation. Furthermore, lower gene expression of albumin, CYP3A4, and γ-GT, as well as reduced albumin secretion capacity, CYP3A4, and γ-GT activity were reported in transdifferentiated P20 compared to P10 cells. By contrast, the gene expression level of CEA was not reduced by transdifferentiation in P20 cells. Of note, both cellular and mitochondrial oxygen consumption was lower in P20 than in P10 transdifferentiated cells. Finally, both ATP and NAD+/NADH were depleted in P20 cells with respect to P10 cells.CONCLUSIONSA mitochondrial dysfunction may limit the transdifferentiation potential of HepaRG cells, with consequent impairment of metabolic and regenerative properties, which may alter applications in basic studies.

Metformin Protects Against Inflammation, Oxidative Stress to Delay Poly I:C-Induced Aging-Like Phenomena in the Gut of an Annual Fish.

Metformin, a clinical agent of type 2 diabetes, is reported as a potential geroprotector. Viral infection induces phenotypes of senescence in human T cells, and polyinosinic:polycytidylic acid (poly I:C), a viral mimic, induces upregulation of senescence-associated beta-galactosidase (SA-β-gal) activity in the ovary of the annual fish Nothobranchius guentheri. However, the effects and mechanisms of metformin on poly I:C-induced aging-like phenomena are poorly understood in vertebrates. In this study, the activity of SA-β-gal increased in the gut of 12-month-old fish and poly I:C-injected 6-month-old fish, compared to 6-month-old control fish, indicating that poly I:C induces aging-like phenomena in the gut of the fish. Metformin supplementation retarded accumulation of SA-β-gal in the gut of old fish and poly I:C-treated young fish. The results of qPCR analysis showed that metformin reduced NF-κB-mediated inflammatory response including the decreased level of proinflammatory cytokine IL-8 and increased expression of anti-inflammatory cytokine IL-10 in the gut of the fish with natural aging and poly I:C-injected 6-month-old fish. Metformin also exhibited antioxidant effects, as it reduced reactive oxygen species production that is associated with the upregulation of FoxO3a and PGC-1α in the gut of 6-month-old fish with poly I:C injection. Expression of AMPK and SIRT1 was reduced in the gut of 6-month-old fish with poly I:C treatment, and feeding metformin reversed these declines. Taken together, the present study suggested that poly I:C injection led to aging-like phenomena in the gut and metformin activated AMPK and SIRT1 to reduce NF-κB-mediated inflammation and resist oxidative stress via enhanced expression of FoxO3a and PGC-1α and finally delayed gut aging in vertebrates.

Abstract 3195: Hijacked senescence secretomes as 'immune primers' of antiangiogenic TKI efficacy

Abstract VEGF receptor tyrosine kinase inhibitors (VEGFR TKIs) and immune checkpoint inhibitors (ICIs) are approved together as a treatment regimen for multiple metastatic cancers, yet the mechanistic basis for this combinatory benefit remains unclear. Previously we have shown that resistance to VEGFR TKI treatment can transiently hijack the secretory machinery typically associated with senescence - the process of cellular aging. Here we report that these senescence-associated secretory programs can also drive immune cell activation, potentially ‘priming' the tumor for PD-1 pathway inhibition. Using a novel live-cell sorting method based on C12FDG - a substrate for senescence-associated beta-galactosidase activity - we isolated senescence-marker (SM) expressing VEGFR TKI-treated cells for transcriptomic analysis. SM+ cell populations were enriched for senescence, immune, and interferon secretory processes, with a unique gene signature validated using published preclinical and clinical datasets. Notably, SM+ cells were found to be more sensitive to CD8 T-cell mediated tumor inhibition in vivo and ex vivo and be sensitive to PD-L1 blockade and inhibitors of mTOR, a key secretory regulator. Together, these results suggest VEGFR TKI controlled secretory programs contributing to resistance can simultaneously prime the tumor microenvironment for immune cell activation, providing an explanation for improved effects of antiangiogenic and immunotherapy combinations in patients. Citation Format: Melissa Dolan, Yuhao Shi, James W. Hill, Michalis Mastri, Cristina Vaghi, Joseph Barbi, Sebastien Benzekry, John M. Ebos. Hijacked senescence secretomes as 'immune primers' of antiangiogenic TKI efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3195.

Adaption of Lung Fibroblasts to Fluoro-Edenite Fibers: Evaluation of Molecular and Physiological Dynamics.

The fluoro-edenite fibrous amphibole was identified as an environmental pollutant associate to risk of carcinogenicity. In Sicily (Italy), it represents a public health issue because fluoro-edenite fibers are present in the soil of Biancavilla, a town located on the south-west slopes of the volcano Etna. Since the relationship between exposure to fluoro-edenite and the onset of lung disorders have been documented, in vitro studies were performed to clarify the mechanisms of damage, but most aspects remain unknown. Here, we focus on the effects of mineral fibers in a primary culture of lung fibroblasts. We supposed that the cells react to fluoro-edenite exposure by establishing a process of adaption that could modify their metabolic activity, their proliferation, and their physiological functions, as the production of extracellular matrix (ECM) components. To verify our hypothesis, we used immunofluorescence, cell proliferation, senescence, apoptosis, scratch, Western blot, Reverse transcription-polymerase chain reaction (RT-PCR), and evaluation of extracellular matrix components assays. Results demonstrated that lung fibroblasts react to fluoro-edenite by a down-regulation of mitochondrial activity, a reduction of cell growth and migration, and a resistance to apoptosis. These elements suggested the induction of a premature senescent phenotype that was confirmed by senescence-associated beta-galactosidase (SA-β-Gal) activity, and by the analysis of ECM elements. We found an unbalance of collagens ratio, and changes in matrix metalloproteinase3 production and release. Our data suggest that fluoro-edenite-induced senescence of lung fibroblasts could be an early and underestimated step that may drive fibroblasts toward a fibrotic and carcinogenic phenotype.

Dendranthema zawadskii var. lucidum (Nakai) J.H. Park Extract Inhibits Cellular Senescence in Human Dermal Fibroblasts and Aging-Related Inflammation in Rats

Senescence is the phenomenon by which physiological functions of organisms degenerate with time. Cellular senescence is marked by an inhibition of cell cycle progression. Beta-galactosidase accumulates in the lysosomes of aged cells. In this study, human dermal fibroblast cells (HDFs) were treated with 0.5 μM doxorubicin for 4 h to induce cellular senescence. Senescence-associated beta-galactosidase (SA-β-gal) activity was then measured 72 h after treatment with aerial parts of Dendranthema zawadskii var. lucidum (Nakai) J.H. Park (DZ) extract. Treatment with DZ extract significantly decreased SA-β-gal activity in a dose-dependent manner in HDFs. Additionally, DZ extract treatment reduced age-related oxidative stress and inflammation in the aortas of aged rats. The reactive oxygen species (ROS) levels in aortas of aged control rats were higher than those in young rats. However, DZ extract-fed aged rats showed significantly lower ROS levels than the aged control rats. When the aged rats were treated with DZ extract at either 0.2 or 1.0 mg∙kg−1∙day−1, NF-κB levels in aorta tissue decreased significantly compared to those in aorta tissue of the aged control rats without DZ treatment. In addition, DZ extract-fed aged rat aortas showed significant reductions in expression of iNOS and COX-2 induced by NF-κB translocation. Therefore, these results suggest that DZ effectively inhibited senescence-related NF-κB activation and inflammation. DZ extract may have a role in the prevention of the vascular inflammatory responses that occur during vascular aging.

Metformin Modulates Doxorubicin‐induced Senescence Phenotype in Endothelial Cells

Background Doxorubicin (DOX) is a cardiotoxic chemotherapeutic agent. In addition to cardiotoxicity, DOX treatment is associated with endothelial dysfunction and increased markers of vascular aging in cancer survivors, contributing to DOX-induced cardiovascular complications. Metformin, an anti-diabetic drug, has been shown to have anti-aging effects in different models of aging. Therefore, in our current work, we first established an in vitro model of DOX-induced senescence in EA.hy926 human endothelial-derived cells and Human Umbilical Vein Endothelial Cells (HUVECs). Then, we identified the effects of metformin on DOX-induced senescence phenotype. Methods Both HUVECs and EA.hy926 human endothelial-derived cells were treated with increasing DOX concentrations for 24 hours. Thereafter, DOX was removed and cells were incubated in DOX free media for another 72-120 hours. Protein expression of senescence markers were measured using Western blot analysis. Cell cycle analysis was assessed using flow cytometry. Senescence-associated beta-galactosidase (SA-beta GAL) activity was measured by SA-beta GAL assay. Next, cells were incubated with DOX +/- increasing metformin concentrations. Effects of metformin on DOX-induced senescence markers and cell cycle were assessed. To delineate the molecular mechanisms, the effects of metformin on major signaling pathways were determined. Results DOX significantly induced expression of senescence markers in endothelial cells in a concentration-dependent manner. Moreover, DOX caused cell cycle arrest in the G2/M phase and increased SA-beta GAL staining. Metformin corrected DOX-induced upregulation of senescence markers, which was associated with a significant inhibition of the JNK pathway. Intriguingly, metformin had no effect on DOX-induced cell cycle arrest and slightly corrected the increased expression of SA-beta GAL. Conclusions Our current work shows that metformin modulates DOX-induced senescence phenotype in endothelial cells, suggesting it may protect against DOX-induced vascular aging and endothelial dysfunction.

Angiotensin II-induced upregulation of SGLT1 and 2 contributes to human microparticle\u2010stimulated endothelial senescence and dysfunction: protective effect of gliflozins

BackgroundSodium-glucose cotransporter 2 (SGLT2) inhibitors reduced cardiovascular risk in type 2 diabetes patients independently of glycemic control. Although angiotensin II (Ang II) and blood-derived microparticles are major mediators of cardiovascular disease, their impact on SGLT1 and 2 expression and function in endothelial cells (ECs) and isolated arteries remains unclear.Methods ECs were isolated from porcine coronary arteries, and arterial segments from rats. The protein expression level was assessed by Western blot analysis and immunofluorescence staining, mRNA levels by RT-PCR, oxidative stress using dihydroethidium, nitric oxide using DAF-FM diacetate, senescence by senescence-associated beta-galactosidase activity, and platelet aggregation by aggregometer. Microparticles were collected from blood of patients with coronary artery disease (CAD-MPs).ResultsAng II up-regulated SGLT1 and 2 protein levels in ECs, and caused a sustained extracellular glucose- and Na+-dependent pro-oxidant response that was inhibited by the NADPH oxidase inhibitor VAS-2780, the AT1R antagonist losartan, sotagliflozin (Sota, SGLT1 and SGLT2 inhibitor), and empagliflozin (Empa, SGLT2 inhibitor). Ang II increased senescence-associated beta-galactosidase activity and markers, VCAM-1, MCP-1, tissue factor, ACE, and AT1R, and down-regulated eNOS and NO formation, which were inhibited by Sota and Empa. Increased SGLT1 and SGLT2 protein levels were observed in the rat aortic arch, and Ang II- and eNOS inhibitor-treated thoracic aorta segments, and were associated with enhanced levels of oxidative stress and prevented by VAS-2780, losartan, Sota and Empa. CAD-MPs promoted increased levels of SGLT1, SGLT2 and VCAM-1, and decreased eNOS and NO formation in ECs, which were inhibited by VAS-2780, losartan, Sota and Empa.ConclusionsAng II up-regulates SGLT1 and 2 protein expression in ECs and arterial segments to promote sustained oxidative stress, senescence and dysfunction. Such a sequence contributes to CAD-MPs-induced endothelial dysfunction. Since AT1R/NADPH oxidase/SGLT1 and 2 pathways promote endothelial dysfunction, inhibition of SGLT1 and/or 2 appears as an attractive strategy to enhance the protective endothelial function.