Percentage Of Senescent Cells Research Articles

Inhibition of the RXRA-PPARα-FABP4 signaling pathway alleviates vascular cellular aging by an SGLT2 inhibitor in an atherosclerotic mice model.

Atherosclerosis is the pathological cause of atherosclerotic cardiovascular disease (ASCVD), which rapidly progresses during the cellular senescence. Sodium-glucose cotransporter 2 inhibitors (SGLT2is) reduce major cardiovascular events in patients with ASCVD and have potential antisenescence effects. Here, we investigate the effects of the SGLT2 inhibitor dapagliflozin on cellular senescence in atherosclerotic mice. Compared with ApoE-/- control mice treated with normal saline, those in the ApoE-/- dapagliflozin group, receiving intragastric dapagliflozin (0.1 mg kg-1 d-1) for 14 weeks, exhibited the reduction in the total aortic plaque area (48.8%±6.6% vs. 74.6%±8.0%, P<0.05), the decrease in the lipid core area ((0.019±0.0037) mm2vs. (0.032±0.0062) mm2, P<0.05) and in the percentage of senescent cells within the plaques (16.4%±3.7% vs. 30.7%±2.0%, P<0.01), while the increase in the thickness of the fibrous cap ((21.6±2.1) µm vs. (14.6±1.5) µm, P<0.01). Transcriptome sequencing of the aortic arch in the mice revealed the involvement of the PPARα and the fatty acid metabolic signaling pathways in dapagliflozin's mechanism of ameliorating cellular aging and plaque progression. In vitro, dapagliflozin inhibited the expression of PPARα and its downstream signal FABP4, by which the accumulation of senescent cells in human aortic smooth muscle cells (HASMCs) was reduced under high-fat conditions. This effect was accompanied by a reduction in the intracellular lipid content and alleviation of oxidative stress. However, these beneficial effects of dapagliflozin could be reversed by the PPARα overexpression. Bioinformatics analysis and molecular docking simulations revealed that dapagliflozin might exert its effects by directly interacting with the RXRA protein, thereby influencing the expression of the PPARα signaling pathway. In conclusion, the cellular senescence of aortic smooth muscle cells is potentially altered by dapagliflozin through the suppression of the RXRA-PPARα-FABP4 signaling pathway, resulting in a deceleration of atherosclerotic progression.

A Comparative Analysis of the Age-dependent Features of Mesenchymal Stem Cells from Buffalo Adipose Tissue

Background: Mesenchymal stem cells are a potential candidate for regenerative medicine. Many factors may influence the physiological characteristics of stem cells that may lead to the alteration in their potential. Methods: The impact of age on mesenchymal stem cell characteristics was investigated in older buffaloes (aged over 10 years) and compared to younger counterparts (aged less than 2 years). Following isolation through enzymatic digestion, the mesenchymal stem cells were cultured in DMEM containing 12.5% FBS and antibiotics. Different variables indicating stem cell characteristics were evaluated at subsequent cultures. Result: The results revealed both groups displayed positive staining for alkaline phosphatase (AP) and expressed stem cell markers, including CD73, CD105 and OCT4, while remain negative for CD34. Furthermore, both sets of stem cells demonstrated the ability to differentiate into adipogenic, osteogenicand chondrogenic lineages. There were no notable variations observed between the two groups in terms of the cell yield, percentages of senescent cells, necrotic cells, dead cells and the apoptotic index. Expression of the P53 gene in RT-PCR amplicon in both groups confirms that stem cells are dividing normally as healthy cells and not tumor cells. Consequently, it was concluded from the present investigation that the age of the animal does not influence the characteristics of mesenchymal stem cells derived from adipose tissue. Thus, the stem cells derived from older and younger buffaloes are suited equally as a cell source for regenerative therapeutics.

Mitochondrial Role on Cellular Apoptosis, Autophagy, and Senescence during Osteoarthritis Pathogenesis.

Authors have demonstrated that apoptosis activation is a pathway related to cartilage degradation characteristics of the OA process. Autophagy is an adaptive response to protect cells from various environmental changes, and defects in autophagy are linked to cell death. In this sense, decreased autophagy of chondrocytes has been observed in OA articular cartilage. The aim of this work was to study the role of OA mitochondria in apoptosis, autophagy, and senescence, using OA and Normal (N) transmitochondrial cybrids. Results: OA cybrids incubated with menadione showed a higher percentage of late apoptosis and necrosis than N cybrids. Stimulation of cybrids with staurosporine and IL-1β showed that OA cybrids were more susceptible to undergoing apoptosis than N cybrids. An analysis of the antioxidant response using menadione on gene expression revealed a lower expression of nuclear factor erythroid 2-like 2 and superoxide dismutase 2 in OA than N cybrids. Activation of microtubule-associated protein 1A/1B-light chain 3 was reduced in OA compared to N cybrids. However, the percentage of senescent cells was higher in OA than N cybrids. Conclusion: This work suggests that mitochondria from OA patients could be involved in the apoptosis, autophagy, and senescence of chondrocytes described in OA cartilage.

The role and mechanism of engineered nanovesicles derived from hair follicle mesenchymal stem cells in the treatment of UVB-induced skin photoaging.

Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are effective in the treatment of skin photoaging; however, their low yield and functional decline with passage progression limit their clinical application. Cell-derived nanovesicles (CNVs) are potential alternatives that can address the limitations of EVs derived from MSCs and are conducive to clinical transformations. Hair follicle mesenchymal stem cells (HFMSCs), a type of MSCs, have demonstrated the function of repairing skin tissues; nevertheless, the efficacy of CNVs from HFMSCs (HFMSC-CNVs) in the treatment of skin photoaging remains unclear. Therefore, ultraviolet radiation B (UVB)-induced photoaging nude mice and human dermal fibroblasts (HDFs) were used as experimental models to investigate the therapeutic effects of HFMSC-CNVs in photoaging models. HFMSC-CNVs were successfully prepared using the mechanical extrusion method. UVB-induced nude mice and HDFs were used as experimental models of photoaging. Multiple approaches, including hematoxylin-eosin and Masson staining, immunohistochemistry, immunofluorescence, detection of reactive oxygen species (ROS), flow cytometry, western blotting, and other experimental methods, were combined to investigate the possible effects and mechanisms of HFMSC-CNVs in the treatment of skin photoaging. In the nude mouse model of skin photoaging, treatment with HFMSC-CNVs reduced UVB-induced skin wrinkles (p < 0.05) and subcutaneous capillary dilation, alleviated epidermis thickening (p < 0.001), and dermal thinning (p < 0.001). Furthermore, HFMSC-CNVs upregulated proliferating cell nuclear antigen (PCNA) expression (p < 0.05) and decreased the levels of ROS, β-galactosidase (β-Gal), and CD86 (p < 0.01). Invitro experiments, treatment with HFMSC-CNVs enhanced the cellular activity of UVB-exposed HDFs (p < 0.05), and reduced ROS levels and the percentage of senescent cells (p < 0.001), and alleviated cell cycle arrest (p < 0.001). HFMSC-CNVs upregulated the expression of Collagen I (Col I), SMAD2/3, transforming growth factor beta (TGF-β), catalase (CAT), glutathione peroxidase-1 (GPX-1), and superoxide dismutase-1 (SOD-1) (p < 0.05) and downregulated the expression of cycle suppressor protein (p53), cell cycle suppressor protein (p21), and matrix metalloproteinase 3 (MMP3) (p < 0.05). Conclusively, the anti-photoaging properties of HFMSC-CNVs were confirmed both invivo and invitro. HFMSC-CNVs exert anti-photoaging effects by alleviating cell cycle arrest, decreasing cellular senescence and macrophage infiltration, promoting cell proliferation and extracellular matrix (ECM) production, and reducing oxidative stress by increasing the activity of antioxidant enzymes.

Abstract 18638: Myocardial Infarction Induces Dysfunction of Endothelial Colony Forming Cells With Independence of Common Cardiovascular Comorbidities

Introduction: Endothelial Colony Forming Cells (ECFCs) present a dysfunctional and senescent phenotype in ischemic heart disease. However, the effect of myocardial infarction (MI), independently of common cardiovascular comorbidities (CCVC), on ECFCs functionality has not been evaluated up to date. Hypothesis: MI induces dysfunction of ECFCs with independence of CCVC Methods: To determine the potential effect of MI on ECFCs, human ECFCs (hECFCs) were isolated from peripheral blood from healthy volunteers (n=6), patients immediately after MI (n=6) (AMI-hECFCs) and patients 6 months after MI (n=6) (CMI-hECFCs). To determine the effect of MI on ECFCs impairment, isolated from the effect of common cardiovascular comorbidities that are found in patients, 6 healthy pigs were subjected to 90 minutes of myocardial ischemia by coronary balloon occlusion followed by reperfusion. Porcine ECFCs (pECFCs) (n=6) were isolated from blood samples before the MI and 1 month after the procedure (CMI-pECFCs) (n=6). For all groups, the following parameters were determined: days until colonies appearance, morphometry, percentage of senescent cells, and migratory, proliferatory and angiogenic capacities. Results: Both CMI-hECFCs and CMI-pECFCs showed a delayed time until colonies appearance (p-ANOVA=0,0117 and p-ANOVA=0,0159 respectively) but no differences were found in AMI-hECFCs. AMI- and CMI-hECFCs showed changes in their area and shape, but only shape changes were observed in CMI-pECFCs. In terms of senescence, both AMI- and CMI-hECFCs, but not CMI-pECFCs, presented an increment of senescence (p-ANOVA= 0,0011). In terms of functionality, AMI- and CMI-hECFCs and CMI-pECFCs showed a significant reduction of angiogenesis and proliferation, and only CMI-hECFCs showed an impaired migratory capacity. Conclusions: MI Induces dysfunction of ECFCs with independence of CCVC

P-317 Associations between endometrial microbiome composition and cellular senescence

Abstract Study question Is there an association between the percentage of senescent cells and microbiome composition in human endometrium? Summary answer We found that cellular senescence is negatively associated with the relative abundance of certain bacterial taxa. What is known already Recent studies indicated that endometrial microbiome characteristics and certain levels of cellular senescence are essential for the establishment of normal endometrial receptivity and successful embryo implantation. The secretion of senescence-associated secretory phenotype (SASP) components is responsible for immune cell activation and might cause endometrial dysbiosis. On the other hand, some studies suggest that the presence of specific metabolic groups of bacteria and their metabolites could influence cellular stress response that might suppress the development of senescent cells. However, the direct relationship between the quantity of senescent cells and microbiome content in the endometrium has never been studied. Study design, size, duration This was a prospective observational cohort study performed between April 2021 and January 2023. Endometrial biopsies were collected from 30 women aged between 34 and 45 during the mid-luteal phase (LH + 7) in a natural cycle. Each biopsy was divided for genetic and immunohistochemical analyses. The exclusion criteria were history of recent inflammatory disease, chronic endometritis, recent antibiotic treatment, endocrinological disorders, autoimmune diseases, oncological diseases, moderate or severe endometriosis, adenomyosis, uterine hyperplasia, and endometrial polyps. Participants/materials, setting, methods We used immunohistochemical biomarker p16ink4a (MAD-000690QD–7, Master Diagnostica) to identify senescent cells. The percentages of positively stained cells in the endometrial stroma and in the luminal epithelium were calculated by HALO image analysis software (version 2.3, IndicaLabs). Endometrial microbiota composition was analyzed after DNA isolation from the endometrial samples using 16S rRNA (v4-v5 region) gene sequencing. Statistical analysis was performed by Spearman’s correlation test using SPSS v.21 (IBM Corp., Armonk, NY, USA). Main results and the role of chance A total of 271 distinct bacterial species and 668 genera were identified across the studied 30 endometrial samples. The percentage of p16+ senescent endometrial stromal cells ranged from 0 to 1.7%, with a mean value of 0.37%, while the percentage of p16+ luminal epithelial cells ranged between 2.5% and 74.0%, with a mean value of 21.5% Spearman analysis revealed significant negative correlation between the percentage of p16+ stromal cells and the relative abundance of Porphyromonas sp. (R = −0.41, p = 0.03) and Sphingobacterium sp. (R = −0.37, p = 0.05). The percentage of p16+ luminal senescent cells correlated negatively with the abundance of Micrococcus luteus (R = −0.44, p = 0.02), Moraxella osloensis (R = −0.37, p = 0.05), Rubellimicrobium sp. (R = −0.46, p = 0.02), Stenotrophomonas sp. (R = − 0.45, p = 0.03), Chryseobacterium sp. (R = −0.41, p = 0.04), Carnobacterium sp. (R = −0.40, p = 0.04), Deinococcus sp. (R = −0.38, p = 0.05), and Enhydrobacter sp. (R = − 0.37, p = 0.05). Limitations, reasons for caution The study was limited in sample size. The assessment includes only endometrial samples from the mid-luteal phase of the cycle. Wider implications of the findings We found that the level of senescence is negatively associated with the relative abundance of certain bacteria taxa in human endometrium, including some related to impaired reproductive function and early embryonic development arrest. These findings suggest a possible specific interaction between senescent cells and microbiome that might influence endometrial receptivity. Trial registration number The current research was funded by National Science Fund, Ministry of Education, Bulgaria, Contract № KP-06-N53/14/16.11.2021

Anti-aging effects of the pistachio extract on mesenchymal stem cells proliferation and telomerase activity

PurposeUsing mesenchymal stem cells (MSCs) is a promising method in regenerative medicine. Limited proliferation and aging process of MSC are the most common problems in MSCs application. In the present study, we intend to investigate the anti-aging properties of pistachio pericarp in bone marrow-derived MSCs of old male rats. Materials and methodsFirst, 1000, 2000, and 3000 µg/mL AEPP were used to treat MSCs derived from bone marrow for 24 h at 37 °C. Then, cell viability, population doubling time, the percentage of senescent cells, telomere length, telomerase activity, and the expression of TRF1 and RAP1 when bone marrow-derived MSCs treated with AEPP were investigated. ResultsThe results showed that cell viability increased when MSCs derived from bone marrow were treated with 2000 and 3000 µg/mL AEPP, indicating this extract may stimulate proliferation. The population doubling time was also enhanced with an increase in AEPP concentration. Importantly, an increase in AEPP concentration significantly reduced senescent cell percentage. Telomere length, telomerase activity, and the expression of anti-aging genes were significantly increased with the increase of AEPP dose. ConclusionTaken together, AEPP has been used as a natural compound with excellent proliferation and anti-aging ability in MSCs. As new therapeutic candidates with promising effects, it can be used with high safety by multiplying cells and delaying the aging process. However, more studies are needed and the anti-aging effects of this extract should be well confirmed in animal models and clinical trials.

Abstract 11965: Cellular Senescence Produces Diastolic Dysfunction and Cardiac Hypertrophy With Aging in Mice

Background: Age is a major risk factor for heart failure with preserved ejection fraction (HFpEF); however, the underlying mechanisms are poorly understood. Cellular senescence is an aging and stress related response; senescent cells (SCs) secrete bioactive substances that produce a variety of pathological changes. The role of senescence in HFpEF is unknown. Objectives: To study the effect of targeted deletion of SCs on cardiac function, structure and left ventricular (LV) pressure in an aging mouse model of HFpEF. Methods: We studied INK ATTAC mice, in which SCs undergo targeted clearance following treatment with a dimerizing agent, AP20187 (AP). Mice were treated with blinded AP or vehicle from 12 months to 18 months of age. Cardiac function and structure were studied with echocardiography. Contractility parameters were measured with a Millar catheter. The percentage of SCs in different cardiac-cell populations was analyzed with the use of Fluorescence-Activated Cell Sorting (FACS). Results: From 12 to 18 months, LV mass/LV diameter at end diastole (LVDd) and the LV anterior wall thickness at end diastole (LVAWd) increased in the vehicle group while they decreased in the AP group (LV mass/LVDd: %Δvehicle=+7.9± 3%, %ΔAP=-2±1.2%, P=0.005; LVAWd: %Δvehicle=+5.3±2.5%, %ΔAP=-3.5±1.6%, P=0.007) (Fig1A, B). E/e’ (echo) and isovolumic relaxation time indicated diastolic dysfunction in vehicle mice, and improved with AP (Fig1C, D). GFP bicistronic with p16 indicated SCs; FACS-purified fibroblasts (FBs) showed reduced GFP fluorescence (P=0.056) in the AP group, whereas immune and endothelial cells were not affected, suggesting that the anti-HFpEF effects of SC-clearance might be mediated through the clearance of senescent FBs (Fig1E). Conclusions: Our study points to a significant role of SCs, likely senescent FBs, in the pathophysiology of HFpEF. Modulating senescence might provide the basis for novel therapeutic approaches to HFpEF prevention and/or therapy.

Ovarian cancer: Age, prognosis, and biologic underpinnings.

e17555 Background: The prognosis of ovarian cancer worsens markedly with age. Yet few data are available to explain this phenomenon. Two hypotheses can be made. Changes in pharmacokinetics (PK) and treatment tolerance limit the dosing of chemotherapy, and/or changes in tumor and host-tumor biology limit treatment efficacy. Objective:To identify PK, body composition, and biologic changes with age that may impact prognosis. We reported PK findings in a previous abstract (Extermann et al., ASCO 2021) In this abstract we focus on the biologic findings. Methods: We conducted a prospective cohort study of women with stage III/IV high-grade serous ovarian cancers treated with neoadjuvant intravenous carboplatin (C) AUC 5 every 3 weeks and paclitaxel (P) dosed either weekly at 80 mg/m2 or every 3 weeks at 175 mg/m2. Body composition was assessed using baseline CT scans at the L3 level. Pre- and post-chemotherapy plasma samples were assayed by ELISA for CRP, IL-6, TNF alpha, TNF-receptor 1 and 2, d-dimers, vitamins B12 and D, methylmalonic acid, and cortisol levels. Tissue samples pre- and post-chemotherapy were analyzed for gene expression, gene methylation, and immunohistochemistry for inflammatory markers and senescence. Toxicity was assessed as first cycle nadir ANC and G4 hematologic (H) or grade 3-4 non-hematologic (NH) toxicity by CTCAE 4.0 criteria over a 3 cycles follow-up. The correlation between continuous measures was assessed by Spearman correlation coefficient, and the association with toxicity was evaluated by Wilcoxon rank sum test. The association with relapse-free survival (RFS) and overall survival (OS) was calculated by Log-rank and Cox regression analyzes. Results: Seventeen patients, ages 38 to 86 (median 61) were eligible, 14 underwent surgery. Treatment delivery was high with a median relative dose intensity of 100%. The mean percentage of senescent cells (CAV-1 +) was 49.5% in the stroma, vs 12.8% in the tumor (p &lt; 0.001) prechemotherapy, and 15.7% in the stroma vs 21.4% in the tumor post-chemotherapy (pre-post stroma p &lt; 0.01). The percentage of PD-L1 positive cells and CTLA-4 positive cells was very low: mean &lt; 1% both pre-and post-chemotherapy. The results from gene expression and methylation showed an increase in the inflammatory pathways and a decrease in the apoptosis pathways over time. Vitamin D levels decreased with age. Prechemotherapy CD8 percentage in the stroma and post chemotherapy d-dimers were associated with RFS. Conclusions: Neoadjuvant treatment delivery was high in an academic setting. Our results provide insights in the underexplored area of biologic response to chemotherapy in patients with ovarian cancer. Although our results did not provide clear candidates to explain the worsening of prognosis with age, they offer clues to develop future research on this issue.

Accelerated Aging of T-Cell Subsets Among ART-Naïve HIV-Infected Chinese Men Who have Sex with Men: A Case-Control Study.

Evidence of lymphopoiesis, exhaustion, and premature aging in Chinese patients with human immunodeficiency virus (HIV) is very limited. To assess biological aging and immune senescence in Chinese healthy controls (HC) and ART-naïve HIV-infected men who have sex with men (MSM). This case-control study was conducted in Beijing Ditan Hospital from March 2018 to June 2019. The percentages of naïve (TN), central memory (TCM), effector memory (TEM), and terminally differentiated memory (TemRA) subsets of CD4 and CD8 T cells were studied, along with markers of senescence (CD28-CD57+) and activation (HLA-DR+). Telomere length of naïve (CD45RA+) and memory (CD45RO+) CD8 T cells were quantified by real-time PCR. A total of 26 HIV-infected and 20 age-matched HC MSM were included. Compared to the HC group, the CD4/CD8 ratio of the HIV-infected group was significantly reduced (0.30 vs. 1.70, P<0.001); significant differences emerged among all CD8 but not CD4 T cell subsets (all P<0.05). In the HIV-infected group, the percentages of senescent cells (CD28-CD57+) in TN, TCM, TEM, and TemRA subsets of CD8 T cells were higher (all P<0.05); while a significant difference was only found in naïve CD4 T cells (P<0.05). HLA-DR expression was increased significantly in all CD4 and CD8 T cell subsets. Both naïve (CD45RA+) and memory (CD45RO+) CD8 T cells in this population had significantly shorter telomere lengths (P<0.01) compared to the HC group. HIV-infected MSM exhibit signs of accelerated immune senescence and biological aging, which particularly affects the CD8 T-cell subsets.

Multi-combination therapy for temozolomide-resistant GBM: identification of temozolomide/small molecule inhibitor combinations that target the MDM2/p53 and PI3K-AKT/mTOR networks

Introduction: Glioblastoma (GBM) is the most aggressive malignant brain cancer in adults. Induction of the DNA damage response pathway by Temozolomide (TMZ), a DNA alkylating agent, activates p53 resulting in apoptosis. GBM can adapt by upregulatingthe pro-survival pathway regulator Protein Kinase B (AKT), which phosphorylates murine double minute 2 (MDM2) resulting in increased MDM2-mediated p53 ubiquitination. We hypothesize that a combination treatment of blood-brain-barrier penetrant small molecule inhibitors (SMIs) to AKT (GDC-0068) and MDM2 protein-protein interaction inhibitor (RG7388), will stabilize p53 expression and potentiate TMZ-mediated effects in a recurrent p53wt GBM xenoline.&#x0D; Methods: Dose response assays followed by Calcusyn statistical analysis determined optimal combination dose ratios. Incucyte imaging analyzedconfluence throughout a treatment cycle. Cellular response was characterized by: 1) Western blotting 2) Flow cytometry with SPiDER β-Gal and FITC Annexin V/ Propidium Iodide to quantify senescent and apoptotic cells, respectively 3) p53 siRNA knockdown to examine p53 dependency in treated cells.&#x0D; Results: Combination index identified synergismof TMZ in the presence of AKT and MDM2 inhibitors at clinically achievable concentrations. Incucyte confirmed a low-dose triple combination significantly inhibited tumor growth. Western blots detected low expression of cleaved PARP and elevated expression of p53 and p21 in RG7388-treated cells compared to vehicle, suggesting senescence-related growth inhibition. SPiDER β-Gal and FITC Annexin V/PI assays confirmed a high percentage of senescent cells and minimal apoptosis following combination treatment compared to vehicle or single SMI-treated cells. P53 siRNA knockdown confirmed that cell growth inhibition is p53 dependent in treated cells.&#x0D; Conclusion: This study provides rationale for targeting p53 in recurrent p53wt GBM and reveals that senescence could function as a potential therapeutic resistance mechanism. In future studies, targeting of the MDM2-p53 network in the presence of a SM senescence inhibitor will be evaluated to determine if this increases GBM cell death.

CIGARETTE SMOKE EXTRACT INDUCES DNA DAMAGE AND ACCELERATES CELLULAR SENESCENCE IN HUMAN ENDOTHELIAL CELLS

Abstract Objective: Elevated blood pressure and tobacco smoking are causes of preventable mortality. The effects of tobacco smoking on blood pressure are complex and chronic effects of tobacco smoking are inconsistent. We reported that DNA damage was increased in smokers’ blood cells compared to non-smokers. Recently, it has been reported that DNA damage is involved in cellular senescence. In this study, we investigated whether cigarette smoke extract (CSE) exerts DNA damage and cellular senescence in endothelial cells and the physiological influence of DNA damage. Design and method: The effect of CSE extracted from tobacco smoke was examined in human umbilical vein endothelial cells (HUVEC). CSE was added to HUVEC and DNA damage formation was quantified by fluorescence immunostaining; DNA single strand breaks (SSBs) with RPA 2 and double-strand breaks (DSBs) with phosphorylated histone H2AX as indices. Cellular senescence was quantified by senescence-associated -b galactosidase activity. The mRNA expression of inflammatory cytokines was quantified by real-time RT PCR. Results: SSBs increased at 24 hours after CSE stimulation, and DSBs increased significantly at 72 hours after stimulation. Continuous stimulation with CSE for 7 days resulted in an accumulation of cytosolic DNA (percentage of cells with cytosolic DNA; control: 3.4 ± 1.1, 7 days stimulation: 11.8 ± 1.8, p &lt; 0.01)and accelerated cellular senescence (percentage of senescent cells; control: 32.5 ± 3.4, 7 days stimulation: 61.5 ± 5.2, p &lt; 0.01). The mRNA expression of inflammatory cytokines, such as IL-6 and IL-1a was increased and prolonged by continuous CSE stimulation. Conclusions: CSE was shown to induce DNA damage, accumulation of cytosolic DNA, cellular senescence and inflammation of vascular endothelial cells.

CBIO-08. ASTROCYTE SENESCENCE CONTRIBUTES TO SEX DIFFERENCES IN GLIOBLASTOMA INCIDENCE AND OUTCOME

Abstract Sex differences in incidence and outcome are consistently observed in cancer, including in the most common malignant brain tumor – glioblastoma (GBM). Women are less likely to develop GBM and have a survival advantage compared to men. Previous research from our lab identified the proteins Rb, p16, and p21 as key mediators of female protection against cellular transformation. Strikingly, these proteins are all primary components of the senescence response, a potent cell-intrinsic anti-cancer mechanism that results in permanent cell cycle arrest, preventing proliferation of damaged cells. We hypothesized that sex differences in GBM incidence and outcome are mediated by differences in senescence induction after DNA damage in male and female cells. Using both wildtype astrocytes and a GBM model, we found that females have a lower threshold for senescence induction in response to oxidative stress, telomere shortening, and treatment with chemotherapy or radiation. Following irradiation, female GBM model cells had higher levels of p21 expression, and p21 levels correlated with the percentage of senescent cells. Knocking out p21 eliminated sex differences in the senescence response following irradiation. Achieving a better understanding of mechanisms of senescence and how they differ in male and female cells could advance development of senescence-directed therapies and potentially improve treatment for brain tumors.

Photobiomodulation with 590 nm Wavelength Delays the Telomere Shortening and Replicative Senescence of Human Dermal Fibroblasts In Vitro.

Background: Cellular senescence is one of the major factors contributing to the aging process. Photobiomodulation (PBM) is known to trigger an array of cellular responses, but there are no data on how it affects the process of cellular senescence. In this study, we analyze the effect of PBM on the cellular senescence and telomere dynamics. Methods: Human dermal fibroblasts were irradiated by a panel of light-emitting diodes with 590 nm and dose 30 J/cm2 accumulated over 1200 sec repeated in 4-day cycle within 40 days. After the last cycle of PBM treatment, the difference in number of senescent cells between PBM treated groups end nontreated control groups was measured by senescent sensitive β-galactosidase assay, and the difference in average telomere length between the experimental end control groups was analyzed using relative human telomere length quantitative Polymerase Chain Reaction (qPCR) assay. Results: After 10 cycles of irradiation, the percentage of senescent cells in PBM-treated cultures was 19.7% ± 4.5%, p < 0.05 smaller than the percentage of senescent cells in the control group, and their relative telomere length was 1.19 ± 0.09-fold, p < 0.05 greater than nontreated controls. Conclusions: Our study demonstrates for the first time that PBM with appropriate parameters can delay the attrition of the telomeres and the entry of cells into senescence, suggesting a potential involvement of telomerase reactivation. A hypothetical mechanism for this light-induced antiaging effect is discussed.

γ- and δ-Tocotrienols interfere with senescence leading to decreased viability of cells.

Senescence is an irreversible permanent cell cycle arrest accompanied by changes in cell morphology and physiology. Bioactive compounds including tocotrienols (vitamin E) can affect important biological functions. The aim of this study was to investigate how γ- and δ-tocotrienols can affect stress-induced premature senescence. We established two different models of premature stress senescence by induction of senescence with either hydrogen peroxide or etoposide in human lung fibroblasts MRC-5 (ECACC, England). We observed increased percentage of cells with increased SA-β-galactosidase activity, decreased cell viability/proliferation and increased level of p21 in both models. In addition, γ-tocotrienol or δ-tocotrienol (both at concentrations of 150, 200 and 300μM) were added to the cells along with the inductor of senescence (cotreatment). We have found that this cotreatment led to the decrease of cell viability/proliferation in both models of premature stress senescence, but did not change the percentage of senescent cells. Moreover, we detected no expression of caspase-3 or apoptotic DNA fragmentation in any models of premature stress senescence after the cotreatment with γ- as well as δ-tocotrienols. However, an increased level of autophagic protein LC-3 II was detected in cells with hydrogen peroxide-induced senescence after the cotreatment with γ-tocotrienol as well as δ-tocotrienol. In case of etoposide-induced senescence only δ-tocotrienol cotreatment led to an increased level of LC-3 II protein in cells. According to our work δ-tocotrienol is more effective compound than γ-tocotrienol.

ASSOCIATION BETWEEN THE PERCENTAGE OF p16-POSITIVE SENESCENT CELLS AND THE EXPRESSION OF ESTROGEN AND PROGESTERONE RECEPTORS IN HUMAN ENDOMETRIUM DURING THE MID-LUTEAL PHASE

Progesterone and estrogen secretion and the expression of their receptors are strongly connected with the cyclic changes in endometrium and its receptivity. We previously reported that the frequency of occurrence of p16-positive senescent cells in human endometrium appear to be also associated with endometrial receptivity and successful embryo implantation. The objective of this study was to analyze the relationship between the percentage of senescent cells and estrogen and progesterone receptors expression in human endometrium during the mid-luteal phase of the menstrual cycle. Prospective observational study. This study includes 383 women who had an endometrial biopsy during the mid-lutheal phase (7 days after LH surge) of the natural cycles. Tissue sections were stained immunohistochemically for ER, PR and p16 using the following monoclonal antibodies: progesterone receptor (PR) (NCL-PGR-312, Leica Microsystems), 1:100 dilution; estrogen receptor (ER) (NCL-L-ER-6F11, Leica Microsystems), 1:100 dilution; p16 ink4a antibody (MAD-000690QD–7, Master Diagnostica), ready-to-use. The percentage of positively stained p16 cells and the levels of ER/PR expression were evaluated by Image-J software (NIH, Maryland, USA) in multiple endometrial sections. Statistical analysis was performed by Spearman’s correlation test using SPSS v.21 (IBM Corp., Armonk, NY, USA). No correlation was found between the frequencies of occurrence of senescent p16-positive stromal, luminal and glandular epithelial cells and PR (p>0.05). In contrast, the glandular expression of ER showed low, but a significant correlation with the percentage of p16+ glandular epithelial cells (R=0.34; p=0.03). In conclusion, we found that the increased cell senescence in endometrial glands during the mid-luteal phase is associated with increased expression of glandular ER in the mid-luteal phase of the cycle. These results suggest an estrogen related change of endometrial senescent cells during the period of embryo implantation.

Hepatocyte growth factor (HGF) and stem cell factor (SCF) maintained the stemness of human bone marrow mesenchymal stem cells (hBMSCs) during long-term expansion by preserving mitochondrial function via the PI3K/AKT, ERK1/2, and STAT3 signaling pathways

BackgroundMesenchymal stem cells (MSCs) have a limited self-renewal ability, impaired multi-differentiation potential, and undetermined cell senescence during in vitro series expansion. To address this concern, we investigated the effects of the microenvironment provided by stem cells from human exfoliated deciduous teeth (SHED) in maintaining the stemness of human bone marrow mesenchymal stem cells (hBMSCs) and identified the key factors and possible mechanisms responsible for maintaining the stemness of MSCs during long-term expansion in vitro.MethodsThe passage 3 (P3) to passage 8 (P8) hBMSCs were cultured in the conditioned medium from SHED (SHED-CM). The percentage of senescent cells was evaluated by β-galactosidase staining. In addition, the osteogenic differentiation potential was analyzed by reverse transcription quantitative PCR (RT-qPCR), Western blot, alizarin red, and alkaline phosphatase (ALP) staining. Furthermore, RT-qPCR results identified hepatocyte growth factor (HGF) and stem cell factor (SCF) as key factors. Thus, the effects of HGF and SCF on mitochondrial function were assessed by measuring the ROS and mitochondrial membrane potential levels. Finally, selected mitochondrial-related proteins associated with the PI3K/AKT, ERK1/2, and STAT3 signaling pathways were investigated to determine the effects of HGF and SCF in preserving the mitochondrial function of hBMSCs during long-term expansion.ResultsSHED-CM had significantly enhanced the cell proliferation, reduced the senescent cells, and maintained the osteogenesis and pro-angiogenic capacity in P8 hBMSCs during long-term expansion. In addition, hBMSCs treated with 100 ng/ml HGF and 10 ng/ml SCF had reduced ROS levels and preserved mitochondrial membrane potential compared with P8 hBMSCs during long-term expansion. Furthermore, HGF and SCF upregulated the expression of mitochondrial-related proteins associated with the PI3K/AKT, ERK1/2, and STAT3 signaling pathways, possibly contributing to the maintenance of hBMSCs stemness by preserving mitochondrial function.ConclusionBoth HGF and SCF are key factors in maintaining the stemness of hBMSCs by preserving mitochondrial function through the expression of proteins associated with the PI3K/AKT, ERK1/2, and STAT3 signaling pathways. This study provides new insights into the anti-senescence capability of HGF and SCF, as well as new evidence for their potential application in optimizing the long-term culture of MSCs.

Safety, tolerability, pharmacokinetics, and clinical outcomes following treatment of painful knee osteoarthritis with senolytic molecule UBX0101

Purpose: To report the Results of a Phase 1 study that assessed the safety, pharmacokinetics (PK), and clinical outcomes of single dose, intra-articular (IA) UBX0101 in patients (pts) with painful knee osteoarthritis (OA). In a previous observational biomarker study of patients with painful knee OA, the percentage of senescent cells (SnCs) in synovial biopsies positively correlated with disease severity, synovitis severity, and knee pain. UBX0101 is an MDM2/p53 interaction inhibitor that can induce apoptosis of senescent synovial fibroblasts. Reducing the number of SnCs from painful OA joints with UBX0101 may reduce pain and create a pro-regenerative environment. Methods: This was a Phase 1, double-blind, randomized, placebo-controlled, single, ascending dose (SAD) study in 48 pts randomized 3:1 to IA UBX0101 (dose range of 0.1 to 4.0 mg) or placebo IA. Key eligibility criteria included knee OA by ACR criteria, Kellgren-Lawrence (KL) grade 1-4, and mean daily pain between 4-9 on a Numeric Rating Scale (NRS, 0-10). Clinical outcomes through 12 weeks included NRS and total WOMAC with pain, function, and stiffness sub-scores derived from the Knee Injury and Osteoarthritis Outcome Score (KOOS) Survey. PK modeling was used to pre-define low (0.1 to 0.4 mg) and high (1.0 to 4.0 mg) dose groups, predicted to achieve knee concentrations below and above 50% effective concentration (EC50) pharmacological thresholds for senolysis, respectively. The SAD study design and sample size were chosen for evaluation of safety. Statistical significance for efficacy endpoints in this Phase 1 SAD study size was considered to be p<0.1. WOMAC sub-scores were item-averaged (i.e., each was normalized to a 0-4 Likert scale). A minimal clinically important difference (MCID) for the WOMAC pain sub-score was considered to be a within-subject reduction in the pain sub-score of ≥0.5. In addition, the rate and extent of pain reduction was determined using the area under the effect curve (AUC) for both NRS and WOMAC pain sub-score. Odds ratios for achieving response were determined using logistic regression models controlling for baseline NRS score and baseline WOMAC sub-score. Results: The SAD study population was balanced regarding patient characteristics and baseline outcome measure values; mean age was 62 years, 67% were female, 89.6% white, mean WOMAC pain sub-score at baseline was 1.96 ± 0.46. Single IA doses of UBX0101 up to 4 mg were well-tolerated. Most adverse events (AEs) were mild. No serious AEs occurred and no AEs led to discontinuation. The plasma PK of UBX0101 following single IA injection demonstrated minor inter-patient variability at all dose groups and systemic concentrations were low and further minimized by an estimated 4-hour elimination half-life. Improvements in pain and function were dose-dependent, clinically meaningful, and durable through 12 weeks for the high doses group of UBX0101 (table). Greater proportions of pts in the high doses group achieved decreases from baseline in the WOMAC pain sub-score equivalent to the MCID, and twice the MCID, at Week 12 compared to the placebo and low dose groups. The odds ratio of 3.68 versus placebo of having a 70% reduction from baseline in the WOMAC pain sub-score in the high doses group was nearly statistically significant (p=0.106, alpha=0.1). The magnitude and duration of pain reduction as measured by the mean (standard deviation) WOMAC pain AUC from baseline to Week 12 was 4.94 (3.66) in the high doses group (p=0.034 vs placebo), 3.32 (3.62) in the low doses group, and 2.28 (2.35) in the placebo group. Conclusions: Single IA doses of UBX0101 up to 4.0 mg were well-tolerated by patients with painful knee OA. Systemic exposure was well described by a one-compartment model with first order absorption. The activity of UBX0101 is supported by the dose-dependent, clinically meaningful, and durable improvements in pain and function observed. Senolysis with IA UBX0101 may be an important future therapeutic option for pts with knee OA.Tabled 1Clinical Outcomes at Week 12UBX0101EndpointPlaceboInjection(n=14)Low doses,0.1-0.4mg (n=16)High doses,1.0-4.0mg (n=18)WOMAC Pain (0-4)Baseline, mean1.872.141.86LSM Change from BL to Week 12 (95% CI)-0.74 (-1.14, -0.34)-0.49 (-0.89, -0.10)-1.09 (-1.38, -0.80)p-value0.430.07Mean Daily Pain NRS (0-10)Baseline, mean6.476.296.31LSM Change from BL to Week 12 (95% CI)-1.96 (-3.14, -0.77)-2.66 (-3.78, -1.55)-3.95 (-4.74, -3.16)p-value0.42<0.01WOMAC Function (0-4)Baseline, mean1.932.051.94LSM Change from BL to Week 12 (95% CI)-0.72 (-1.12, -0.31)-0.49 (-0.88, -0.10)-1.05 (-1.36, -0.74)p-value0.430.13WOMAC= Western Ontario and McMasters Universities Osteoarthritis Index (Likert) 0-4, LSM= Least-squares mean, BL=Baseline, CI=Confidence Interval, NRS=Numeric rating scale 0-10 Open table in a new tab WOMAC= Western Ontario and McMasters Universities Osteoarthritis Index (Likert) 0-4, LSM= Least-squares mean, BL=Baseline, CI=Confidence Interval, NRS=Numeric rating scale 0-10

Alpha-mangostin decreased cellular senescence in human umbilical vein endothelial cells.

The hyperglycemic condition in diabetes induces cellular senescence in vascular endothelial cells and causes cardiovascular complications. Alpha-mangostin is a xanthone found in Garcinia mangostana, and has shown protective effects in metabolic syndrome. In this study, the anti-senescence effects of alpha-mangostin in the hyperglycemic condition are investigated. HUVECs were incubated with high glucose for 6days and co-treated by metformin or alpha-mangostin. After 6days, cell viability, reactive oxygen species, the percentage of senescent cells, secretory interleukin-6, and the expression of SIRT1, AMPK, p53 and p21 were measured. High glucose (60mM) significantly decreased cellular viability and increased reactive oxygen species and cellular senescence through the reduction of senescence-associated β-galactosidase activity. Moreover, high glucose increased the protein levels of p53, acetyl-p53 and p21. The protein levels of SIRT1 and total AMPK were decreased by high glucose. High glucose increased the secretion of IL-6. Alpha-mangostin (1.25μM) and metformin (50μM) reversed the toxic effects of high glucose in HUVECs. These results show that alpha-mangostin, similar to metformin, has anti-senescence effects in high-glucose conditions, which is probably due to its antioxidant activity through the SIRT1 pathway. Alpha-mangostin has previously shown anti-inflammatory effects and metabolic status improvement in animal and clinical studies. Therefore, this natural agent can be considered as a supplement to prevent vascular complications caused by high glucose in patients with diabetes. Graphical abstract.

Amyloid β and Tumorigenesis: Amyloid β-Induced Telomere Dysfunction in Tumor Cells

Amyloid β and Tumorigenesis: Amyloid β-Induced Telomere Dysfunction in Tumor Cells