Senescence-associated Beta-galactosidase Activity Research Articles

Inhibition of mTOR prevents glucotoxicity-mediated increase of SA-beta-gal, p16INK4a, and insulin hypersecretion, without restoring electrical features of mouse pancreatic islets.

An over-activation of the mechanistic target of rapamycin (mTOR) pathway promotes senescence and age-related diseases like type 2 diabetes. Besides, the regenerative potential of pancreatic islets deteriorates with aging. Nevertheless, the role of mTOR on senescence promoted by metabolic stress in islet cells as well as its relevance for electrophysiological aspects is not yet known. Here, we investigated whether parameters suggested to be indicative for senescence are induced in vitro in mouse islet cells by glucotoxicity and if mTOR inhibition plays a protective role against this. Islet cells exhibit a significant increase (~ 76%) in senescence-associated beta-galactosidase (SA-beta-gal) activity after exposure to glucotoxicity for 72h. Glucotoxicity does not markedly influence p16INK4a protein within 72h, but p16INK4a levels increase significantly after a 7-days incubation period. mTOR inhibition with a low rapamycin concentration (1nM) entirely prevents the glucotoxicity-mediated increase of SA-beta-gal and p16INK4a. At the functional level, reactive oxygen species, calcium homeostasis, and electrical activity are disturbed by glucotoxicity, and rapamycin fails to prevent this. In contrast, rapamycin significantly attenuates the insulin hypersecretion promoted by glucotoxicity by modifying the mRNA levels of Vamp2 and Snap25 genes, related to insulin exocytosis. Our data indicate an influence of glucotoxicity on pancreatic islet-cell senescence and a reduction of the senescence markers by mTOR inhibition, which is relevant to preserve the regenerative potential of the islets. Decreasing the influence of mTOR on islet cells exposed to glucotoxicity attenuates insulin hypersecretion, but is not sufficient to prevent electrophysiological disturbances, indicating the involvement of mTOR-independent mechanisms.

The protein composition of exosomes released by prostate cancer cells is distinctly regulated by androgen receptor-antagonists and -agonist to stimulate growth of target cells.

Prostate cancer (PCa) is a prevalent malignancy in men worldwide, ranking as the second leading cause of cancer-related death in Western countries. Various PCa hormone therapies, such as androgen receptor (AR)-antagonists or supraphysiological androgen level (SAL) reduce cancer cell proliferation. However, treated cells may influence the growth of neighboring cells through secreted exosomes in the tumor microenvironment (TME). Here, the change of protein content of exosomes secreted from PCa cells through treatment with different AR-antagonists or SAL has been analyzed. Isolation of exosomes via ultracentrifugation of treated human PCa LNCaP cells with AR-agonist and various AR-antagonists; analysis of cellular senescence by detection of senescence associated beta galactosidase activity (SA β-Gal); Western blotting and immunofluorescence staining; Mass spectrometry (MS-spec) of exosomes and bioinformatic analyses to identify ligand-specific exosomal proteins. Growth assays to analyze influence of exosomes on non-treated cells. MS-spec analysis identified ligand-specific proteins in exosomes. One thousand seventy proteins were up- and 52 proteins downregulated by SAL whereas enzalutamide upregulated 151 proteins and downregulated 42 exosomal proteins. The bioinformatic prediction indicates an up-regulation of pro-proliferative pathways. AR ligands augment hub factors in exosomes that include AKT1, CALM1, PAK2 and CTNND1. Accordingly, functional assays confirmed that the isolated exosomes from AR-ligand treated cells promote growth of untreated PCa cells. The data suggest that the cargo of exosomes is controlled by AR-agonist and -antagonists and distinct among the AR-antagonists. Further, exosomes promote growth that might influence the TME. This finding sheds light into the complex interplay between AR signaling and exosome-mediated communication between PCa cells.

Ganoderma lucidum extract attenuates corticotropin-releasing hormone-induced cellular senescence in human hair follicle cells

Corticotropin-releasing hormone (CRH) is a key mediator in stress-induced hair growth inhibition. Here, we investigated the impact of stress-induced senescence and evaluated the potential of Ganoderma lucidum (GL) extract in mitigating CRH-induced senescence in human hair follicle cells (hHFCs). We show that CRH treatment increased the senescence-associated beta-galactosidase (SA-β-GAL) activity and reactive oxygen species (ROS) formation in hHFCs and suppressed alkaline phosphatase (ALP) activity and anagen-inducing genes. However, GL extract restored ALP activity and decreased the expression levels of anagen-related genes in CRH-treated hHFCs. It decreased SA-β-GAL activity, reduced ROS production, and prevented the phosphorylation of MAPK signaling pathways in CRH-related stress response. Moreover, GL reversed the CRH-induced inhibition of two-cell assemblage (TCA) elongation and Ki67 expression. GL extract attenuates stress-induced hair follicular senescence by delaying catagen entry and scavenging ROS. Our findings suggest that GL extract could be used for treating stress-induced hair loss.

Inhibition of DAPK3 Suppresses Radiation-Induced Cellular Senescence by Activation of a PGC1α-Dependent Metabolism Pathway in Brain Endothelial Cells.

In the brain, environmental changes, such as neuroinflammation, can induce senescence, characterized by the decreased proliferation of neurons and dendrites and synaptic and vascular damage, resulting in cognitive decline. Senescence promotes neuroinflammatory disorders by senescence-associated secretory phenotypes and reactive oxygen species. In human brain microvascular endothelial cells (HBMVECs), we demonstrate that chronological aging and irradiation increase death-associated protein kinase 3 (DAPK3) expression. To confirm the role of DAPK3 in HBMVEC senescence, we disrupted DAPK3 activity using small interfering RNA (siRNA) or a dominant-negative mutant (DAPK3-P216S), which reduced cellular senescence phenotypes, as assessed by changes in tube formation, senescence-associated beta-galactosidase activity, and cell proliferation. In endothelial cells, DAPK3 promotes cellular senescence by regulating the phosphorylation and inactivation of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α) via the protein kinase B pathway, resulting in the decreased expression of mitochondrial metabolism-associated genes, such as ATP5G1, BDNF, and COX5A. Our studies show that DAPK3 is involved in cellular senescence and PGC1α regulation, suggesting that DAPK3 regulation may be important for treating aging-related brain diseases or the response to radiation therapy.

Abstract 2978: Extracellular ATP induced senescence in human lung cancer cells

Abstract In tumors, extracellular ATP (eATP) concentrations are 1,000 to 10,000 times higher than is found in normal tissues and eATP is up taken through macropinocytosis leading to a large increase in intracellular ATP levels. eATP is also linked to the TGF-β signaling pathway as exocytosis of ATP is induced that is then used in an autocrine fashion activating the P2 × 7 purinergic receptor1. Through these mechanisms, eATP has also been recently shown to induce EMT, CSC formation, and drug resistance1. One of the mechanisms through which these processes may be induced is a senescent cell precursor2. Senescence is a generally irreversible epigenetically controlled process characterized by a complete exit from the cell cycle in response to either DNA damage, stressful conditions, or aging. Recent mounting evidence has shown that senescence can be a cancer promoting mechanism by entering this state in response to stress such as during chemotherapy, but they can utilize it to enhance their malignant capabilities, including the ability to escape their senescent state with heightened stem cell characteristics, which can lead to a higher chance of relapse after treatment2. Senescent cells have recently been named a hallmark of cancer3. This study shows the time dependent induction of a senescent like state in human lung cancer A549 cells after only 2 hours of treatment with 0.5mM eATP, the concentration found in the tumor microenvironment, through analysis of transcriptional markers using total polyA RNAseq as well as chromogenic and flow cytometric assays for senescence associated beta-galactosidase activity. Additionally, inhibitors of macropinocytosis and purinergic receptors have been used to gain insight into the mechanisms of action that this pathway takes. This demonstrates a novel use of ATP within the tumor microenvironment for aiding in cancer progression and drug resistance. After EMT, CSC formation, and drug resistance, senescence is added to the long list of eATP mediated cancer promoting processes. 1. Song J, Qian Y, Evers M, Nielsen CM, Chen X. Cancer Stem Cell Formation Induced and Regulated by Extracellular ATP and Stanniocalcin-1 in Human Lung Cancer Cells and Tumors. IJMS. 2022;23(23):14770. doi:10.3390/ijms232314770 2. Zhang D, Monteiro MJ, Liu J, Gu W. Mechanisms of cancer stem cell senescence: Current understanding and future perspectives. Clin Exp Pharma Physio. 2021;48(9):1185-1202. doi:10.1111/1440-1681.13528 3. Hanahan D. Hallmarks of Cancer: New Dimensions. Cancer Discovery. 2022;12(1):31-46. doi:10.1158/2159-8290.CD-21-1059 Citation Format: Ryan A. Ward, Arabella Hunt, Nora Anderson, Xiaozhuo Chen. Extracellular ATP induced senescence in human lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2978.

Repurposing Drugs for Senotherapeutic Effect: Potential Senomorphic Effects of Female Synthetic Hormones.

Repurposing previously approved drugs may fast track the route to the clinic for potential senotherapeutics and improves the inefficiency of the clinical drug development pipeline. We performed a repurposing screen of 240 clinically approved molecules in human primary dermal fibroblasts for their effects on CDKN2A expression. Molecules demonstrating effects on CDKN2A expression underwent secondary screening for senescence-associated beta galactosidase (SAB) activity, based on effect size, direction, and/or molecule identity. Selected molecules then underwent a more detailed assessment of senescence phenotypes including proliferation, apoptosis, DNA damage, senescence-associated secretory phenotype (SASP) expression, and regulators of alternative splicing. A selection of the molecules demonstrating effects on senescence were then used in a new bioinformatic structure-function screen to identify common structural motifs. In total, 90 molecules displayed altered CDKN2A expression at one or other dose, of which 15 also displayed effects on SAB positivity in primary human dermal fibroblasts. Of these, 3 were associated with increased SAB activity, and 11 with reduced activity. The female synthetic sex hormones-diethylstilboestrol, ethynyl estradiol and levonorgestrel-were all associated with a reduction in aspects of the senescence phenotype in male cells, with no effects visible in female cells. Finally, we identified that the 30 compounds that decreased CDKN2A activity the most had a common substructure linked to this function. Our results suggest that several drugs licensed for other indications may warrant exploration as future senotherapies, but that different donors and potentially different sexes may respond differently to senotherapeutic compounds. This underlines the importance of considering donor-related characteristics when designing drug screening platforms.

Predisposal of Interferon Regulatory Factor 1 Deficiency to Accumulate DNA Damage and Promote Osteoarthritis Development in Cartilage.

Interferon regulatory factor 1 (IRF1) is a transcriptional regulator conventionally associated with immunomodulation. Recent molecular analyses mapping DNA binding sites of IRF1 have suggested its potential function in DNA repair. However, the physiologic significance of this noncanonical function remains unexplored. Here, we investigated the role of IRF1 in osteoarthritis (OA), a condition marked by senescence and chronic joint inflammation. OA progression was examined in wild-type and Irf1-/- mice using histologic assessments and microcomputed tomography analysis of whole-joint OA manifestations and behavioral assessments of joint pain. An integrated analysis of assay for transposase-accessible chromatin with sequencing and whole transcriptome data was conducted for the functional assessment of IRF1 in chondrocytes. The role of IRF1 in DNA repair and senescence was investigated by assaying γ-H2AX foci and senescence-associated beta-galactosidase activity. Our genome-wide investigation of IRF1 footprinting in chondrocytes revealed its primary occupancies in the promoters of DNA repair genes without noticeable footprint patterns in those of interferon-responsive genes. Chondrocytes lacking IRF1 accumulated irreversible DNA damage under oxidative stress, facilitating their entry into cellular senescence. IRF1 was down-regulated in the cartilage of human and mouse OA. Although IRF1 overexpression did not elicit an inflammatory response in joints or affect OA development, genetic deletion of Irf1 caused enhanced chondrocyte senescence and exacerbated post-traumatic OA in mice. IRF1 offers DNA damage surveillance in chondrocytes, protecting them from oxidative stress associated with OA risk factors. Our study provides a crucial and cautionary perspective that compromising IRF1 activity renders chondrocytes vulnerable to cellular senescence and promotes OA development.

Phenotypes and ontogeny of senescent hepatic stellate cells in metabolic dysfunction-associated steatohepatitis

Hepatic stellate cells (HSCs) are the key drivers of fibrosis in metabolic dysfunction-associated steatohepatitis (MASH), the fastest growing cause of hepatocellular carcinoma (HCC) worldwide. HSCs are heterogenous, and a senescent subset of HSCs is implicated in hepatic fibrosis and HCC. Administration of anti-uPAR (urokinase-type plasminogen activator receptor) CAR T cells has been shown to deplete senescent HSCs and attenuate fibrosis in murine models. However, the comprehensive features of senescent HSCs in MASH, as well as their cellular ontogeny have not been characterized; hence, we aimed to comprehensively characterize and define the origin of HSCs in human and murine MASH. To comprehensively characterize the phenotype and ontogeny of senescent HSCs in human and murine MASH, we integrated senescence-associated beta galactosidase activity with immunostaining, flow cytometry and single-nucleus RNA sequencing (snRNAseq). We integrated the immunohistochemical profile with a senescence score applied to snRNAseq data to characterize senescent HSCs and mapped the evolution of uPAR expression in MASH. Using pseudotime trajectory analysis, we establish that senescent HSCs arise from activated HSCs. While uPAR is expressed in MASH, the magnitude and cell-specificity of its expression evolve with disease stage. In early disease, uPAR is more specific to activated and senescent HSCs, while it is also expressed by myeloid-lineage cells, including Trem2+ macrophages and myeloid-derived suppressor cells, in late disease. Furthermore, we identify novel surface proteins expressed on senescent HSCs in human and murine MASH that could be exploited as therapeutic targets. These data define features of HSC senescence in human and murine MASH, establishing an important blueprint to target these cells as part of future antifibrotic therapies. Hepatic stellate cells (HSCs) are the primary drivers of scarring in chronic liver diseases. As injury develops, a subset of HSCs become senescent; these cells are non-proliferative and pro-inflammatory, thereby contributing to worsening liver injury. Here we show that senescent HSCs are expanded in MASH (metabolic dysfunction-associated steatohepatitis) in humans and mice, and we trace their cellular origin from the activated HSC subset. We further characterize expression of uPAR (urokinase plasminogen activated receptor), a protein that marks senescent HSCs, and report that uPAR is also expressed by activated HSCs in early injury, and in immune cells as liver injury advances. We have integrated high-resolution single-nucleus RNA sequencing with immunostaining and flow cytometry to identify five other novel proteins expressed by senescent HSCs, including mannose receptor CD206, which will facilitate future therapeutic development.

Titan mice as a model to test interventions that attenuate frailty and increase longevity.

Wild-type murine models for aging research have lifespans of several years, which results in long experimental duration and late output. Here we explore the short-lived non-inbred Titan mouse (DU6) as a mouse model to test longevity interventions. We show that Titan mice exhibit increased frailty and senescence-associated beta-galactosidase activity at an early age. Dietary intervention attenuates the frailty progression of Titan mice. Additionally, cyclic administration of the senolytic drug Navitoclax at an early age increases the lifespan and reduces senescence-associated beta-galactosidase activity. Our data suggests that Titan mice can serve as a cost-effective and timely model for longevity interventions in mammals.

High-throughput assessment of cellular senescence.

Cellular senescence is a molecular process that is activated in response to a large variety of distinct stress signals. Mechanistically, cellular senescence is characterized by an arrest in cell cycle accompanied by phenotypic adaptations and physiological alterations including changes in the secretory profile of senescent cells termed the senescence-associated secretory phenotype (SASP). Here we describe a detailed, automation- compatible method for the detection of senescence-associated beta galactosidase (SA-β-gal) activity as a hallmark of cellular senescence using a conventional fluorescent microscope equipped with a transmitted light module. Moreover, we outline a protocol for the automated analysis of cellular senescence using convolutional neural networks (CNNs) and mathematical morphology. In sum, we provide a toolset for the high throughput assessment of cellular senescence based on light microscopy and automated image analysis.

An Ex Vivo Aorta Culture Model to Study Vascular Cellular Senescence.

Animal studies on vascular aging pose a few limitations. One of the most important reasons for this is the absence of a fast and efficient model of vascular tissue aging. In this study, ex vivo aortic culture and Matrigel subcutaneous implantation are combined to develop a new model for studying vascular cellular senescence. Eight-week-old C57BL/6J mice are used to obtain aortas. Bleomycin is used to induce aortas senescence in vitro. Then, aortas are transplanted to the acceptor mice with Matrigel. Senescence is evaluated using western blotting, quantitative polymerase chain reaction, and senescence-associated beta-galactosidase activity. Inflammatory cytokines are detected using Luminex Liquid Suspension Chip. RNA levels are analyzed by transcriptome sequencing. The results revealed that vessels in the bleomycin group exhibited significant senescence than those in the control group that can be enhanced by stripping vessel adventitia. The levels of cytokines such as interleukin (IL-2, IL-1β, and IL-6 increased significantly in the ex vivo model. Furthermore, transcriptome sequencing revealed 56 significantly differentially expressed genes (DEGs) in ex vivo model vessels compared with those in naturally aging aortas. In conclusion, this study introduces a cost-effective and time-saving vessel senescence model for vascular cellular senescence.

Asiaticoside Prevents Oxidative Stress and Apoptosis in Endothelial Cells by Activating ROS-dependent p53/Bcl-2/Caspase-3 Signaling Pathway.

Asiaticoside (AC) is a triterpenoid saponin found in Centella asiatica (L.) urban extract that has a wide range of pharmacological properties. Our previous study demonstrated that AC could promote angiogenesis in diabetic wounds, but the specific mechanisms remain unknown. This study aimed to examine the effectiveness and mechanism of AC on human umbilical vein endothelial cells (HUVECs) exposed to tert-butyl hydroperoxide (t-BHP) toxicity. Senescence was confirmed using senescence-associated betagalactosidase (SA-β-gal) activity and expression of the cell cycle phase markers p16 and p21. The levels of SOD, NO, MDA, GSH-Px, and ROS were tested. Furthermore, several cell death-related genes and proteins (p53, Bax, Bcl-2 and Caspase-3) were assessed with RT-qPCR and Western blotting. AC significantly reduced SA-β-gal activity, with both the suppression of cellcycle inhibitors p16 and p21. We also found that the induced oxidative stress and apoptosis caused by t-BHP treatment resulted in the decrease of antioxidant enzymes activities, the surge of ROS and MDA, the up-regulation of p53, Bax and caspase-3, and the decrease of SOD, NO, GSH-Px and Bcl-2. These biochemical changes were all reversed by treatment with varying doses of AC. AC alleviates t-BHP-induced oxidative injury and apoptosis in HUVECs through the ROS-dependent p53/Bcl-2/Caspase-3 signaling pathway. It may be a potential antioxidant applied in metabolic disorders and pharmaceutical products.

Unripe Carica papaya Fresh Fruit Extract Protects against Methylglyoxal-Mediated Aging in Human Dermal Skin Fibroblasts.

The glycolytic metabolite methylglyoxal (MGO) initiates the formation of advanced glycation end products and oxidative stress, leading to cellular senescence and skin aging. This study focuses on the anti-aging properties of unripe Carica papaya L. (UCP) fresh fruit extract on MGO-induced human dermal fibroblast senescence. We pretreated human foreskin fibroblasts with UCP before incubating them with MGO (400 μM) for 72 h. We used the glycation inhibitor aminoguanidine hydrochloride (AG) as the positive control. Senescent fibroblasts were detected using senescence-associated beta-galactosidase activity and collagen type I expression (COL1A1). We investigated the changes in the Akt, JNK/p38 mitogen-activated protein kinase (MAPK), c-Jun, and nuclear factor kappa B (NF-κB) signaling pathways using Western blotting. UCP significantly suppressed MGO-induced senescent fibroblasts (from 20.90±2.00% to 11.78±2.04%) when compared with the baseline level at 7.10±0.90% (P<0.05). While COL1A1 was diminished by 43.35±1.56% (P<0.001) in the MGO-treated fibroblasts, UCP and AG could recover COL1A1 to 63.22±4.78% and 64.39±3.34%, respectively. MGO triggered overactivation of Akt, JNK/p38 MAPK, c-Jun, and NF-κB by 2.10±0.09, 8.10±0.37, 6.60±0.29, 2.18±0.23, and 3.74±0.37 folds, respectively. UCP and AG significantly abolished these changes. Consistently, MGO increased matrix metalloproteinase-1 (MMP-1) levels by 2.58±0.04 folds, which was significantly suppressed by UCP and AG pretreatment to 1.87±0.11 and 1.69±0.07 folds, respectively. In summary, UCP controlled MGO-induced fibroblast senescence by suppressing the JNK/c-Jun/MMP and p38/NF-κB/COL1A1 pathways, similar to the action of the glycation inhibitor AG. Therefore, UCP can be considered a functional fruit for preventing and delaying skin aging.

Dapagliflozin impedes endothelial cell senescence by activating the SIRT1 signaling pathway in type 2 diabetes

BackgroundSodium-glucose cotransporter 2 inhibitors (SGLT2i) clinically reduce atherosclerosis and lower blood pressure. However, their impact on endothelial dysfunction in type 2 diabetes (T2D) remains unclear. In this study, we investigated the protective effect and underlying mechanism of the SGLT2 inhibitor dapagliflozin in diabetes. MethodsVascular reactivity was measured to assess the vasoprotective effect of dapagliflozin in a mouse model of high glucose (HG)-induced T2D. Pulse wave velocity was measured to quantify arterial stiffness. Protein expression was assessed by western blotting and immunofluorescence, oxidative stress was evaluated using dihydroethidium, nitric oxide was evaluated using the Griess reaction, and cellular senescence was assessed based on senescence-associated beta-galactosidase (SA‐β‐gal) activity and the expression of senescence markers. Furthermore, the endothelial nitric oxide synthase (eNOS) acetylation status was determined and eNOS interactions with SIRT1 were evaluated by coimmunoprecipitation assays. ResultsDapagliflozin protected against impaired endothelium-dependent vasorelaxation and improved arterial stiffness in the mouse model of T2D; mouse aortas had significantly reduced levels of senescence activity and senescence-associated inflammatory factors. HG-induced increases in senescence activity, protein marker levels, and oxidative stress in vitro were all ameliorated by dapagliflozin. The decreases in eNOS phosphorylation and nitric oxide (NO) production in senescent endothelial cells were restored by dapagliflozin. SIRT1 expression was reduced in HG-induced senescent endothelial cells, and dapagliflozin restored SIRT1 expression. SIRT1 inhibition diminished the antisenescence effects of dapagliflozin. Coimmunoprecipitation showed that SIRT1 was physically associated with eNOS, suggesting that the effects of dapagliflozin are dependent on SIRT1 activation. ConclusionThese findings indicate that dapagliflozin protects against endothelial cell senescence by regulating SIRT1 signaling in diabetic mice.

Dapagliflozin prevents oxidative stress-induced endothelial dysfunction via sirtuin 1 activation

Recent studies have demonstrated that dapagliflozin, a sodium-glucose cotransporter type 2 (SGLT2) inhibitor, prevents endothelial dysfunction; however, direct effects of dapagliflozin on the endothelium under oxidative stress and the underlying mechanism of action are not completely understood. This study aimed to define the role and related mechanisms of dapagliflozin in hydrogen peroxide (H2O2)-induced endothelial dysfunction. The endothelium-dependent vasorelaxation effect of dapagliflozin was assessed in an organ bath study. Endothelial dysfunction was assessed using protein expression level and phosphorylation of endothelial nitric oxide synthase (eNOS), nitric oxide (NO), reactive oxygen species (ROS), senescence-associated beta-galactosidase (SA-β-gal) activity, and senescence marker proteins (p21, p53). Co-immunoprecipitation and protein acetylation were performed to detect protein interactions. Dapagliflozin exerted a direct vasorelaxant effect in the aortic rings of C57BL/6 J mice. Furthermore, there was a significant improvement in endothelium-dependent vasorelaxation in dapagliflozin-treated diabetic mice compared to vehicle controls. Moreover, intracellular ROS levels and ONOO- levels, increased by H2O2, were reduced by dapagliflozin. Importantly, dapagliflozin inhibited H2O2-induced senescence in the human umbilical vein endothelial cells (HUVECs), as indicated by reduced SA‐β‐gal, p21, and p53. Mechanistically, dapagliflozin reversed the H2O2-mediated inhibition of eNOS serine phosphorylation and sirtuin 1 (SIRT1) expression in endothelial cells. In particular, SIRT1-mediated eNOS deacetylation is reportedly involved in dapagliflozin-enhanced eNOS activity. These findings indicate that dapagliflozin ameliorates endothelial dysfunction by restoring eNOS activity, restoring NO bioavailability, and reducing ROS generation via SIRT1 activation in oxidative stress-stimulated endothelial cells.

#4067 HYPOXIC PRECONDITIONING FOR THE IMPROVEMENT OF THE REGENERATIVE CAPACITY OF MESENCHYMAL STEM CELLS AND ITS APPLICATION IN HYPERTENSIVE KIDNEY DISEASE

Abstract Background and Aims Autologous mesenchymal stem/stromal cells (MSCs) have emerged as a therapeutic option for many chronic diseases. Hypertensive kidney disease (HKD) might impair MSCs’ reparative ability by altering important biomolecular properties of the cell, but the characteristics of this impairment are unclear. In our previous pre-clinical studies, hypoxic preconditioning (HPC) enhanced MSC angiogenesis and reduced senescence through global gene and protein expression. Thus, we hypothesize that HPC would improve human MSCs by enhancing their functionality and angiogenesis, creating an anti-inflammatory and anti-senescence environment in MSCs from healthy control (HC), hypertensive patients (HTN), and hypertensive kidney disease patients (HKD). Method Human MSCs were collected from abdominal subcutaneous fat tissue biopsy of HC, HTN, and HKD patients (n=12 each). Samples were collected from healthy volunteers with no history of hypertension or kidney disease (HC), patients diagnosed with hypertension controlled with antihypertensive drugs (HTN), and hypertensive patients with eGFR&amp;lt;60 mL/min/1.73 m2 (HKD). MSCs were harvested and cultured in Normoxic (20% O2) or Hypoxic (1% O2) conditions for 24-48 hours. MSC functionality was measured by migration and proliferation. Angiogenic and inflammatory secretome in conditioned media was assessed along with MSC senescence by senescence-associated beta-galactosidase (SA-beta-gal) activity. In addition, transcriptome analysis using RNA-sequencing (n=3/group) and quantitative PCR (qPCR) were performed to evaluate gene expression. Results At baseline, Normoxic HTN-MSCs interestingly had higher proliferation compared to HC. HC-MSC subjected to HPC showed increased proliferation (Figure 1A). SA-β-gal activity tended to decrease after HPC, particularly in the HC group (P = .06). HPC did not affect the release of the pro-angiogenic protein VEGF from MSCs, but increased EGF in HC-MSC, and decreased HGF in HC and HKD MSCs. HPC upregulated the pro-angiogenetic genes, inflammatory gene, and a few senescence genes (Figure 1F). Conclusion HPC has a more favorable functional effect on HC- than on HKD-MSC, reflected in increased proliferation and EGF release, and tends to decrease senescence, whereas it has little effect on HTN or HKD MSCs. These observations may assist in developing novel therapeutic strategies to improve the regenerative capacity of MSCs in patients with hypertensive kidney disease.

Premature senescence of the liver in Alagille patients.

Alagille syndrome (ALGS) is an autosomal dominant disease characterized by a multisystem involvement including bile duct paucity and cholestasis, caused by JAG1 or NOTCH2 mutations in most of the cases. Jagged1-Notch2 interactions are known to be crucial for intrahepatic biliary tract development, but the Notch signaling pathway is also involved in the juxtacrine transmission of senescence and in the induction and modulation of the senescence-associated secretory phenotype (SASP). Our aim was to investigate premature senescence and SASP in ALGS livers. Liver tissue from ALGS patients was prospectively obtained at the time of liver transplantation (n = 5) and compared to control livers (n = 5). We evidenced advanced premature senescence in the livers of five JAG1 mutated ALGS pediatric patients through increased senescence-associated beta-galactosidase activity (p<0.05), increased p16 and p21 gene expression (p<0.01), and increased p16 and γH2AX protein expression (p<0.01). Senescence was located in hepatocytes of the whole liver parenchyma as well as in remaining bile ducts. The classical SASP markers TGF-β1, IL-6, and IL-8 were not overexpressed in the livers of our patients. We demonstrate for the first time that ALGS livers display important premature senescence despite Jagged1 mutation, underlying the complexity of senescence and SASP development pathways.

Modulation of Cellular Senescence in HEK293 and HepG2 Cells by Ultrafiltrates UPla and ULu Is Partly Mediated by Modulation of Mitochondrial Homeostasis under Oxidative Stress.

Protein probes, including ultrafiltrates from the placenta (UPla) and lung (ULu) of postnatal rabbits, were investigated in premature senescent HEK293 and HepG2 cells to explore whether they could modulate cellular senescence. Tris-Tricine-PAGE, gene ontology (GO), and LC-MS/MS analysis were applied to describe the characteristics of the ultrafiltrates. HEK293 and HepG2 cells (both under 25 passages) exposed to a sub-toxic concentration of hydrogen peroxide (H2O2, 300 μM) became senescent; UPla (10 μg/mL), ULu (10 μg/mL), as well as positive controls lipoic acid (10 μg/mL) and transferrin (10 μg/mL) were added along with H2O2 to the cells. Cell morphology; cellular proliferation; senescence-associated beta-galactosidase (SA-β-X-gal) activity; expression of senescence biomarkers including p16 INK4A (p16), p21 Waf1/Cip1 (p21), HMGB1, MMP-3, TNF-α, IL-6, lamin B1, and phospho-histone H2A.X (γ-H2AX); senescence-related gene expression; reactive oxygen species (ROS) levels; and mitochondrial fission were examined. Tris-Tricine-PAGE revealed prominent detectable bands between 10 and 100 kDa. LC-MS/MS identified 150-180 proteins and peptides in the protein probes, and GO analysis demonstrated a distinct enrichment of proteins associated with "extracellular space" and "proteasome core complex". UPla and ULu modulated senescent cell morphology, improved cell proliferation, and decreased beta-galactosidase activity, intracellular and mitochondrial ROS production, and mitochondrial fission caused by H2O2. The results from this study demonstrated that UPla and Ulu, as well as lipoic acid and transferrin, could protect HEK293 and HepG2 cells from H2O2-induced oxidative damage via protecting mitochondrial homeostasis and thus have the potential to be explored in anti-aging therapies.

A novel role for YPEL2 in mediating endothelial cellular senescence via the p53/p21 pathway

Yippee-like 2 (YPEL2) is expressed in tissues and organs enriched in vascular networks, such as heart, kidney, and lung. However, the roles of YPEL2 in endothelial cell senescence and the expression of YPEL2 in atherosclerotic plaques have not yet been investigated. Here, we report the essential role of YPEL2 in promoting senescence in human umbilical vein endothelial cells (HUVECs) and the upregulation of YPEL2 in human atherosclerotic plaques. YPEL2 was significantly upregulated in both H2O2-induced senescent HUVECs and the arteries of aged mice. Endothelial YPEL2 deficiency significantly decreased H2O2-increased senescence-associated beta-galactosidase (SA-β-gal) activity and reversed H2O2-inhibited cell viability. Additionally, endothelial YPEL2 knockdown reduced H2O2-promoted THP-1 cell adhesion to HUVECs and downregulated ICAM1 and VCAM1 expression. Mechanistic studies divulged that the p53/p21 pathway was involved in YPEL2-induced cellular senescence. We conclude that YPEL2 promotes cellular senescence via the p53/p21 pathway and that YPEL2 expression is elevated in atherosclerosis. These findings reveal YPEL2 as a potential therapeutic target in aging-associated diseases.

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.