Senescence Of Epithelial Cells Research Articles

Abstract C023: P21-positive senescent stromal cells promote prostate cancer immune suppression and progression that can be reversed by senolytic therapy

Abstract Cellular senescence is historically viewed as a tumor-suppressive cell cycle arrest program to limit outgrowth of pre-malignant cells. However, emerging evidence suggests senescent cell persistence contributes to immune suppression and cancer progression. Using prostate cancer patient samples and murine models, we find that p16+ senescent cells accumulate throughout malignant progression and are associated with immune suppression and poor survival. p16 ablation in mice eliminated senescent epithelial cells and reversed immune suppression mediated by immature myeloid cells but not prostate tumorigenesis. Single cell sequencing revealed an additional population of senescent p21+ fibroblasts and myeloid cells. Targeting BCL-xL or PD-L1 induced during senescence could remove both p16+ epithelial and p21+ stromal senescent cells, reactivate T cell immunity, and block tumor initiation as well as progression in advanced prostate cancer models. Our findings demonstrate that targeting heterogenous senescent populations can not only prevent malignant transformation but also yield therapeutic benefits in advanced prostate cancers through activating anti-tumor immunity. Citation Format: Lin Zhou, Kelly D. DeMarco, Katherine C. Murphy, Zhenpeng Wu, Jinping Li, Junhui Li, Calvin Johnson, Zhong Jiang, Shi Bai, Tianyi Ye, Karl Simin, Lihua Julie Zhu, Jason R Pitarresi, Hong Wu, Marcus Ruscetti. P21-positive senescent stromal cells promote prostate cancer immune suppression and progression that can be reversed by senolytic therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C023.

Effects of MMP2 and its inhibitor TIMP2 on DNA damage, apoptosis and senescence of human lens epithelial cells induced by oxidative stress.

Oxidative stress-induced lens epithelial cells (LECs) death plays a pivotal role in pathogenesis of age-related cataract (ARC), causing significant visual impairment. Apoptosis of porcine granulosa cells mediated by MMP2 is linked to DNA damage. The current study aimed to investigate the potential mechanism of MMP2 in DNA damage, apoptosis and senescence of lens epithelial cells caused by oxidative stress. HLE-B3 cells were treated with different doses of H2O2 for 24h, and CCK-8 was used to detect cell viability. Furthermore, western blotting was used to detect the expressions of MMP2, Bcl2, Bax, cleaved caspase3, γ-H2AX, p16, p21, and TIMP2. DCFH-DA staining was used to assess ROS levels. Moreover, EdU staining was used to detect cell proliferation, and flow cytometry was used to detect cell apoptosis. Then, 15A3 immunofluorescence staining and γ-H2AX staining were used to detect DNA damage. In addition, SA-β-gal staining was used to observe cell senescence. The present findings suggest that oxidative stress triggers damage to LECs viability and elevates the expression of MMP2. Furthermore, MMP2 interference attenuates H2O2-induced active damage, apoptosis, DNA damage, and cellular senescence in LECs. Additionally, TIMP2 expression is down-regulated in H2O2-induced LECs, which suppresses the expression of MMP2 induced by H2O2. These findings highlight the crucial role of MMP2 and TIMP2 in the modulation of oxidative stress-induced cellular responses in LECs. Collectively, TIMP2 alleviates H2O2-induced lens epithelial cell viability damage, apoptosis, DNA damage and cell senescence in LECs by inhibiting MMP2.

Tetrandrine Alleviates Pulmonary Fibrosis by Modulating Lung Microbiota-Derived Metabolism and Ameliorating Alveolar Epithelial Cell Senescence.

Tetrandrine (TET) is a minimally toxic drug extracted from the root of Stephania tetrandra. We previously demonstrated that TET could ameliorate pulmonary fibrosis (PF) by modulating autophagy. However, the mechanism behind TET's protective effects on PF remains unclear. In this study, we utilized 16S rRNA gene sequencing, nontargeted metabolomic analysis, and network pharmacology to identify changes in lung microbiota and metabolites that mediate alveolar epithelial cell senescence in bleomycin (BLM)-induced PF in mice. Additionally, we employed Western blot analysis, RT-PCR, and immunofluorescence staining to investigate the invitro and invivo effects of TET and its influential bacterial metabolites on PF. The TET intervention alleviated PF by regulating the compositions of lung microbial communities (Streptococcus, Micrococcus, Acinetobacter, Altererythrobacter, Atopostipes, Candidatus Cloacimonas, Clostridium sensu stricto 1, Sphingomonas, Listeria, Blautia, and Pseudomonas) and metabolites (3,4-dihydroxyphenylpropionic acid (3,4-DHPPA), 6-Aminonicotinamide, N-acetyl-5-methoxykynuramine, and resiniferatoxin). Through network pharmacological analysis, it was determined that 3,4-DHPPA played a crucial role in alleviating PF by further inhibiting the senescence of alveolar epithelial cells, a finding further validated in exvivo experiments. TET mitigated BLM-induced PF in murine models through the modulation of lung microbiota composition and metabolism. Specifically, TET augmented the level of the microbiota-derived metabolite, 3,4-DHPPA, which in turn attenuated alveolar epithelial cell senescence.

RXRα/MR signaling promotes diabetic kidney disease by facilitating renal tubular epithelial cells senescence and metabolic reprogramming

Cell senescence and metabolic reprogramming are significant features of diabetic kidney disease (DKD). However, the underlying mechanisms between cell senescence and metabolic reprogramming are poorly defined. Here, we report that retinoid X receptor α (RXRα), a key nuclear receptor transcription factor, regulates cell senescence and metabolic reprogramming in DKD. Through high-throughput sequencing, bioinformatic analysis and experimental validation, we confirmed the critical role of RXRα in promoting senescence and metabolic disturbances in renal tubular epithelial cells (RTECs) induced by lipid overload. In vivo, in situ injection of AAV9-shRxra into the kidney reduced proteinuria, RTECs senescence and insulin resistance in DKD mice. In vitro, knockdown of RXRα markedly improved G2/M phase arrest and suppressed the expression of senescence-associated secretory phenotypes (SASPs). Protein-protein interaction (PPI) analysis and unbiased bioinformatics were employed to identify the direct interactions between RXRα and the mineralocorticoid receptor (MR), which were subsequently validated through coimmunoprecipitation. Gene network analysis revealed the collaborative regulatory role of RXRα and MR in RTECs senescence. In an accelerated aging mouse model, an MR antagonist inhibited RXRα/MR signaling, improved RTECs senescence, and reduce interstitial fibrosis and lipid deposition in the kidneys. These findings indicate that inhibition of RXRα/MR signaling could alleviate cell senescence during metabolic disorders. Thus, our study revealed that RXRα/MR signaling serves as a critical regulatory factor mediating the crosstalk between cell senescence and metabolic reprogramming, shedding light on a novel mechanism for targeting cell senescence and metabolic dysregulation in DKD.

FYCO1 regulates autophagy and senescence via PAK1/p21 in cataract

ARC (Age-related cataract) is one of the leading causes of vision impairment and blindness; however, its pathogenesis remains unclear. FYCO1 (FYVE and coiled-coil domain containing 1) serves as an autophagy adaptor. The present study investigated the role of FYCO1 in cataract. Ultraviolet-B (UVB) irradiation was used to establish a cataract mice model. Hematoxylin and eosin (H&E) assay were used to observe lens morphology. Cell models were constructed by cultivating SRA 01/04 cells with H2O2 and UVB. Cell counting kit-8 (CCK8) and Senescence-associated β-galactosidase (SA-β-Gal) assay were performed to explore proliferation and senescence. The gene and protein expression were assessed by quantitative real-time PCR (qRT-PCR), western blot and immunofluorescence staining. We demonstrated lens structural damage and downregulation of FYCO1 in mice with UVB-induced cataracts. In vitro results revealed a deletion in autophagy levels along with the decrease of FYCO1 expression in human lens epithelial cells (HLECs) after H2O2 treatment, which was confirmed in vivo. The knockout of FYCO1 in the HLECs did not change basal autophagy and senescence but suppressed HLECs response in the induction of both. Further investigation indicated that FYCO1 knockout inhibited senescence and p21 levels by suppressing the expression of p21 activated kinase 1 (PAK1) in cataract cell models. This study has newly characterized the role of FYCO1 in UVB-induced cataracts and in oxidative stress, both of which are associated with ARCs. A novel association between FYCO1 and PAK1/p21 in lens epithelial cell autophagy, senescence, and cataractogenesis also appears to have been established.

METTL3-regulated m6A modification of lncRNA E230001N04Rik is involved in myofibroblast differentiation in arsenic-induced pulmonary fibrosis through promoting senescence of lung epithelial cells

Arsenic is a toxic agent that causes respiratory damage. Long non-coding RNAs (lncRNAs) are non-coding transcripts that adsorb specific miRNAs and regulate biological processes of human diseases. N6-Methyladenosine (m6A) is an internal modification of RNAs. However, there are few reports about lncRNAs and m6A modifications as co-regulators of pulmonary fibrosis. For 6 months, C57BL/6 mice were given water containing 0, 10, or 20 ppm arsenite. meRIP-seq and lncRNA-seq analyses showed that the m6A levels of the lncRNA E230001N04Rik were higher, and the levels of E230001N04Rik itself were lower in the high-dose arsenite group than in the controls. Murine lung epithelial 12 (MLE12) cells, exposed to 8 μM arsenite for 8 passages, had elevated METTL3 and miR-20b-3p and low E230001N04Rik. Arsenite induced cellular senescence, as demonstrated by secretion of factors related to the senescence-associated secretory phenotype (SASP). Arsenite-treated MLE12 cells co-cultured with primary lung fibroblasts (PLFs) caused myofibroblast differentiation. These data show that METTL3 reduces E230001N04Rik expression via controlling its m6A levels, which regulate miR-20b-3p and mediate the senescence of alveolar epithelial cells (AECs). Thereby, E230001N04Rik is involved in the arsenite-induced myofibroblast differentiation and in pulmonary fibrosis. These observations provide a prospective mechanism for chronic pulmonary disease caused by arsenite.

Single-cell analysis reveals alternations between the aged and young mice prostates

BackgroundAging of the male prostate is an inevitable process in which the prostate undergoes hyperplasia, and this growth may lead to compression of the urethra, resulting in voiding dysfunction and associated symptoms, and an increased risk of prostate cancer. Despite the significance of prostate aging, the molecular mechanisms involved are still not fully understood.MethodsProstate split by lobes from young (2 months) and aged (24 months) mice were collected for single-cell RNA sequencing (scRNA-seq) analysis. Tissues from both anterior prostate (AP) and ventral/dorsal/lateral prostate (VDLP) were included in the study. Data analysis included unsupervised clustering using the uniform manifold approximation and projection (UMAP) algorithm to identify distinct cell types based on marker gene expression. Differential gene expression analysis was performed to identify age-related changes in gene expression across different cell types. Functional enrichment analysis was conducted to elucidate biological pathways associated with differentially expressed genes. Additionally, cellular interactions and developmental trajectories were analyzed to characterize cellular dynamics during prostate aging.ResultsThe single-cell transcriptome analysis of the mouse prostate during aging revealed heterogeneity across various cell types and their changes during the aging process. We found a significant increase in the proportion of mesenchymal and immune cells in aged mice. Our study unveiled alterations in genes and pathways associated with cellular senescence, oxidative stress, and regeneration in epithelial cells. Furthermore, we observed that basal cells may undergo epithelial-mesenchymal transition (EMT) to become mesenchymal cells, particularly prominent in aged mice. Additionally, immune cells, notably macrophages and T cells, exhibited a heightened inflammatory response in aged mice.ConclusionIn summary, our study provides a comparative analysis of the single-cell transcriptome of the aged and young mice prostates, elucidating cellular and molecular changes between the aged and young mice prostates.

The Punicalagin Compound Mitigates Bronchial Epithelial Cell Senescence Induced by Cigarette Smoke Extract through the PAR2/mTOR Pathway.

Tobacco smoke is an important inducer of airway epithelial cell aging. Punicalagin(PCG) is a natural anti-aging compound. The effect of PCG on tobacco smoke-induced airway epithelial cell senescence is unknown. Our study investigated whether PCG can treat the human bronchial epithelial cell line (BEAS-2B) aging by inhibiting the protease-activated receptor 2 (PAR2)/m- TOR pathway. Bioinformatics techniques were used to analyze the potential biological functions of PAR2. Molecular dynamics evaluated the binding ability of PCG and PAR2. The CCK8 assay was used to detect the cytotoxicity of CSE and PCG. The activity of the PAR2/mTOR pathway and the expression of the characteristic aging markers p16, p21, and SIRT1 are detected by qRT-PCR and Western blotting. Cell senescence was observed by Senescence-associated β-galactosidase (SA-β-gal) staining. The senescence-associated secretory phenotype (SASP): concentrations of interleukin IL-6, IL-8, and TNF- α were detected by ELISA. The GSE57148 bioinformatics analysis dataset showed that PAR2 regulates lung senescence through the mTOR signaling pathway. Molecular dynamics results found that PCG and PAR2 had a strong and stable binding force. CSE induces BEAS-2B cell senescence and activates the PAR2/mTOR pathway. Inhibition of PAR2 mitigated the senescence changes. In addition, PCG's pretreatment can significantly alleviate CSE-induced BEAS-2B cell senescence while inhibiting the PAR2/mTOR pathway. PCG has a therapeutic effect on the senescence of airway epithelial cells.

Lens capsule advanced glycation end products induce senescence in epithelial cells: Implications for secondary cataracts.

Posterior capsule opacification (PCO) is a common complication after cataract surgery. Residual lens epithelial cells (LECs) on the anterior lens capsule, after cataract surgery, migrate to the posterior lens capsule and undergo transdifferentiation into myofibroblast-like cells. Those cells synthesize excessive amounts of extracellular matrix and contribute to fibrosis during PCO. Cellular senescence, a phenomenon that increases with aging, has been implicated in several fibrotic diseases. Here, we have investigated the prevalence of senescent LECs within the lens posterior capsule and the ability of advanced glycation end products (AGEs) in lens capsules to induce senescence, contributing to PCO. Aged lens capsules from pseudophakic human cadaver eyes showed the presence of senescent LECs. In human capsular bags, LECs showed an age-dependent increase in senescence after 28 days of culture. Human LECs cultured on aged lens capsules for 3 days underwent senescence; this effect was not seen in LECs cultured on young lens capsules. Human LECs cultured on an AGE-modified extracellular matrix (ECM-AGEs) showed an AGE-concentration-dependent increase in the expression of senescence markers and reactive oxygen species (ROS) levels. Treatment with a RAGE antagonist and ROS inhibitor reduced the expression of senescence and fibrotic markers. Additionally, conditioned media from ECM-AGEs-treated cells induced the expression of fibrotic markers in naïve LECs. Together, these suggest that AGEs in the capsule induce senescence of LECs, which triggers the mesenchymal transition of neighboring non-senescent LECs and contributes to PCO.

Therapeutic potential of 18-β-glycyrrhetinic acid-loaded poly (lactic-co-glycolic acid) nanoparticles on cigarette smoke-induced in-vitro model of COPD

Chronic obstructive pulmonary disease (COPD) is strongly linked to cigarette smoke, which contains toxins that induce oxidative stress and airway inflammation, ultimately leading to premature airway epithelial cell senescence and exacerbating COPD progression. Current treatments for COPD are symptomatic and hampered by limited efficacy and severe side effects. This highlights the need to search for an optimal therapeutic candidate to address the root causes of these conditions. This study investigates the possible potential of poly (lactic-co-glycolic acid) (PLGA)-based nanoparticles encapsulating the plant-based bioactive compound 18-β-glycyrrhetinic acid (18βGA) as a strategy to intervene in cigarette smoke extract (CSE)-induced oxidative stress, inflammation, and senescence, in vitro. We prepared 18βGA-PLGA nanoparticles, and assessed their effects on cell viability, reactive oxygen species (ROS) production, anti-senescence properties (expression of senescence-associated β galactosidase and p21 mRNA), and expression of pro-inflammatory genes (CXCL-1, IL-6, TNF-α) and inflammation-related proteins (IL-8, IL-15, RANTES, MIF). The highest non-toxic concentration of 18βGA-PLGA nanoparticles to healthy human broncho epithelial cell line BCiNS1.1 was identified as 5 µM. These nanoparticles effectively mitigated cigarette smoke-induced inflammation, reduced ROS production, protected against cellular aging, and counteracted the effects of CSE on the expression of the inflammation-related genes and proteins. This study underscores the potential of 18βGA encapsulated in PLGA nanoparticles as a promising therapeutic approach to alleviate cigarette smoke-induced oxidative stress, inflammation, and senescence. Further research is needed to explore the translational potential of these findings in clinical and in vivo settings.

Urolithin A alleviates cell senescence by inhibiting ferroptosis and enhances corneal epithelial wound healing.

To analyze the therapeutic effect and mechanism of Urolithin A (UA) on delayed corneal epithelial wound healing. The C57BL/6 mice were continuously exposed to hyperosmotic stress (HS) for 7 days followed by the removal of central corneal epithelium to establish a delayed corneal epithelial wound healing model in vivo. In vitro, the human corneal epithelial cell line (HCE-T) was also incubated under HS. UA was administered in vivo and in vitro to study its effects on corneal epithelial cells. Senescence-associated β-galactosidase (SA-β-gal) staining was performed to detect the level of cell senescence. Transcriptome sequencing (RNA-seq) was conducted to elucidate the molecular mechanism underlying the effect of UA on corneal epithelial repair. Additionally, the expression of senescence-related and ferroptosis-related genes and the levels of lipid peroxides (LPO) and malondialdehyde (MDA) were measured. Hyperosmotic stress (HS) significantly increased the proportion of SA-β-gal staining positive cells in corneal epithelial cells and upregulated the expression of p16 and p21 (p < 0.0001). Topical application of UA decreased the accumulation of senescent cells in corneal epithelial wounds and promoted epithelial wound healing. The results of RNA-seq of HS-induced corneal epithelial cells showed that the ferroptosis pathway was significantly dysregulated. Further investigation revealed that UA decreased the level of oxidative stress in HCE-T cells, including the levels of LPO and MDA (p < 0.05). Inhibition of ferroptosis significantly prevented cellular senescence in HS-induced HCE-T cells. In this study, UA promoted HS-induced delayed epithelial wound healing by reducing the senescence of corneal epithelial cells through the inhibition of ferroptosis.

Krüppel-like transcription factor 14 alleviates alveolar epithelial cell senescence by inhibiting endoplasmic reticulum stress in pulmonaryfibrosis

Pulmonary fibrosis (PF) is defined as a specific form of chronic, progressive fibrosing interstitial pneumonia, occurring primarily in older adults with poor prognosis. Alveolar epithelial cell (AEC) senescence is the critical pathological mechanism of PF. However, the molecular mechanisms regulating AEC senescence in PF are incompletely understood. Herein, we provided evidence to support the function of Krüppel-like factor 14 (KLF14), a novel Krüppel-like transcription factor, in the regulation of AEC senescence during PF. We confirmed that the expression of KLF14 was up-regulated in PF patients and mice treated with bleomycin (BLM). KLF14 knockdown resulted in more pronounced structural disruption of the lung tissue and swelling of the alveolar septum, which led to significantly increased mortality in BLM-induced PF mice. Mechanistically, RNA-seq analysis indicated that KLF14 decreased the senescence of AECs by inhibiting endoplasmic reticulum (ER) stress. Furthermore, the pharmacological activation of KLF14 conferred protection against PF in mice. In conclusion, our findings reveal a protective role for KLF14 in preventing AECs from senescence and shed light on the development of KLF14-targeted therapeutics for PF.

Hsa-miR-3928–3p targets the CCL3/CCR5 axis to induce amniotic epithelial cell senescence involved in labor initiation

IntroductionSenescence in human amniotic epithelial cells (hAECs) and increased sterile inflammation in the amniotic cavity can lead to the initiation of term labor (TL). We investigated the possible roles of hsa-miR-3928–3p and chemokine ligand 3 (CCL3) in labor initiation and the underlying molecular mechanisms. MethodsMicroarray chip screening was used to analyse the differential expression of miRNAs in amniotic fluid exosomes from women in TL and term not-in-labor. The GEO and miRWalk databases were used to identify differential genes, and a dual luciferase assay was used to verify the relationship. Reverse transcription quantitative PCR (RT-qPCR) and immunofluorescence were used to determine the expression and localization of CCL3/CCR5 in fetal membranes. RT-qPCR and western blotting were used to detect the expression of CCL3/CCR5 in hAECs with hsa-miR-3928–3p knockdown/overexpression. Cell counting kit 8, flow cytometry, EdU proliferation, senescence-associated β-galactosidase, and enzyme-linked immunosorbent assays were performed to detect the impact of hsa-miR-3928–3p on hAEC function. Resultshsa-miR-3928–3p expression was downregulated in TL. CCL3 (macrophage inflammatory protein-1α) was identified as a differentially expressed target gene. hsa-miR-3928–3p targeted the 3′ UTR of CCL3. Downregulation of hsa-miR-3928–3p expression increased CCL3 expression. CCL3, via its CCR5 receptor, decreased the proliferation, but increased the senescence, apoptosis rate, secretion of inflammatory factors (IL-8, TNF-α, and IL-6), and expression of senescence-associated protein p21 in hAECs. Discussionhsa-miR-3928–3p negatively regulates CCL3, promoting hAEC senescence through the CCL3-CCR5 axis and inducing signals for labor initiation. These findings provide novel insights for labor initiation in clinical settings.

MTOR potentiates senescent phenotypes and primary cilia formation after cisplatin-induced G2 arrest in retinal pigment epithelial cells

Cisplatin, a platinum-based anticancer drug, is used to treat several types of cancer. Despite its effectiveness, cisplatin-induced side effects have often been reported. Although cisplatin-induced toxicities, such as apoptosis and/or necrosis, have been well studied, the fate of cells after exposure to sublethal doses of cisplatin needs further elucidation. Treatment with a sublethal dose of cisplatin induced cell cycle arrest at the G2 phase in retinal pigment epithelial cells. Following cisplatin withdrawal, the cells irreversibly exited the cell cycle and became senescent. Notably, the progression from the G2 to the G1 phase occurred without mitotic entry, a phenomenon referred to as mitotic bypass, resulting in the accumulation of cells containing 4N DNA content. Cisplatin-exposed cells exhibited morphological changes associated with senescence, including an enlarged size of cell and nucleus and increased granularity. In addition, the senescent cells possessed primary cilia and persistent DNA lesions. Senescence induced by transient exposure to cisplatin involves mTOR activation. Although transient co-exposure with an mTORC1 inhibitor rapamycin did not prevent mitotic bypass and entry into senescence, it delayed the progression of senescence and attenuated senescent phenotypes, resulting in shorter primary cilia formation. Conclusively, cisplatin induces senescence in retinal pigment epithelial cells by promoting mTOR activation.

Network pharmacology and experimental verification revealing valnemulin alleviates DSS-induced ulcerative colitis by inhibiting intestinal senescence

Ulcerative colitis (UC) is a chronic inflammatory disease that is increasing in prevalence globally. Senescence is characterized by a specific chronic, low-grade, “sterile” inflammatory state known as inflammaging, suggesting that senescence may exacerbate the severity of UC. However, the link between UC and senescence remains unclear. Valnemulin (VAL) is a semi-synthetic derivative of a naturally occurring diterpenoid antibiotic (pleuromutilin), which can inhibit peptidyl transferase. Studies investigating the potential of valnemulin to inhibit senescence and alleviate colitis are currently limited. In this study, we revealed that dextran sulfate sodium (DSS), an inducer of UC, induces senescence in both colon epithelial NCM460 cells and colon tissues. Additionally, VAL, identified from a compound library, exhibited robust anti-senescence activity in DSS-treated NCM460 cells. Identified in our study as an anti-senescence agent, VAL effectively mitigated DSS-induced UC and colonic senescence in mice. Through network pharmacology analysis and experimental validation, the potential signaling pathway (AMPK/NF-κB) for VAL in treating UC was identified. We discovered that DSS significantly inhibited the AMPK signaling pathway and activated the NF-κB signaling pathway. However, supplementation with VAL remarkably restored AMPK activity and inhibited the NF-κB signaling pathway, which led to the inhibition of senescence. In summary, our study demonstrated that DSS-induced UC stimulates the senescence of colonic tissues, and VAL can effectively alleviate DSS-induced colonic damage and reduce colonic senescence. Our research findings provide a new perspective for targeting anti-senescence in the treatment of UC.

Mechanical Stretch Induces Senescence of Lung Epithelial Cells and Drives Fibroblast Activation by Paracrine Mechanisms.

Severe lung injury requiring mechanical ventilation may lead to secondary fibrosis. Senescence, a cell response characterized by cell cycle arrest and a shift towards a proinflammatory/profibrotic phenotype, is one of the involved mechanisms. Here, we explore the contribution of mechanical stretch as trigger of senescence of the respiratory epithelium and its link with fibrosis. Human lung epithelial cells and fibroblasts were exposed in vitro to mechanical stretch, and senescence assessed. In addition, fibroblasts were exposed to culture media preconditioned by senescent epithelial cells and their activation was studied. Transcriptomic profiles from stretched, senescent epithelial cells and activated fibroblasts were combined to identify potential activated pathways. Finally, the senolytic effects of digoxin were tested in these models. Mechanical stretch induced senescence in lung epithelial cells, but not in fibroblasts. This stretch-induced senescence has specific features compared to senescence induced by doxorubicin. Fibroblasts were activated after exposure to supernatants conditioned by epithelial senescent cells. Transcriptomic analyses revealed notch signaling as a potential responsible for the epithelial-mesenchymal crosstalk, as blockade of this pathway inhibits fibroblast activation. Treatment with digoxin reduced the percentage of senescent cells after stretch and ameliorated the fibroblast response to preconditioned media. These results suggest that lung fibrosis in response to mechanical stretch may be caused by the paracrine effects of senescent cells. This pathogenetic mechanism can be pharmacologically manipulated to improve lung repair.

Lysosomal-Associated Protein Transmembrane 5, Tubular Senescence, and Progression of CKD.

Tubular senescence is a major determinant of chronic kidney disease (CKD) and identification of potential therapeutic targets involved in senescent tubular epithelial cells has clinical importance. Lysosomal-associated protein transmembrane 5 (LAPTM5) is a key molecule related to T and B cell receptor expression and inflammation. However, the expression pattern of LAPTM5 in the kidney and the contribution of LAPTM5 to the development of CKD keep unknown. LAPTM5-/- mice and tubule specific-LAPTM5 knockout mice were used to examine the role of LAPTM5 in tubular senescence by establishing different experimental mouse CKD models. LAPTM5 expression was significantly induced in the kidney, especially in proximal tubules and distal convoluted tubules, from mice with aristolochic acid nephropathy, bilateral ischemia/reperfusion injury (IRI)-induced CKD or unilateral ureter obstruction (UUO). Tubule-specific deletion of LAPTM5 inhibited senescence of tubular epithelial cells and alleviated tubulointerstitial fibrosis in aged mice. Moreover, LAPTM5 deficiency ameliorated kidney injury and tubular senescence in mice with CKD. Mechanistically, LAPTM5 inhibited ubiquitination of NICD1 by mediating WWP2 lysosomal degradation, then leading to cellular senescence in tubular epithelial cells. Notably, we also observed a higher expression of LAPTM5 in tubules from individuals with CKD and the level of LAPTM5 was correlated with kidney fibrosis and tubular senescence in people with CKD. LAPTM5 contributed to tubular senescence by regulating WWP2/NICD1 signaling pathway and exacerbated kidney injury during the progression of CKD.

Curcumin analogue EF24 prevents alveolar epithelial cell senescence to ameliorate idiopathic pulmonary fibrosis via activation of PTEN

BackgroundTreating Idiopathic pulmonary fibrosis (IPF) remains challenging owing to its relentless progression, grim prognosis, and the scarcity of effective treatment options. Emerging evidence strongly supports the critical role of accelerated senescence in alveolar epithelial cells (AECs) in driving the progression of IPF. Consequently, targeting senescent AECs emerges as a promising therapeutic strategy for IPF. PurposeCurcumin analogue EF24 is a derivative of curcumin and shows heightened bioactivity encompassing anti-inflammatory, anti-tumor and anti-aging properties. The objective of this study was to elucidate the therapeutic potential and underlying molecular mechanisms of EF24 in the treatment of IPF. MethodsA549 and ATII cells were induced to become senescent using bleomycin. Senescence markers were examined using different methods including senescence-associated β-galactosidase (SA-β-gal) staining, western blotting, and q-PCR. Mice were intratracheally administrated with bleomycin to induce pulmonary fibrosis. This was validated by micro-computed tomography (CT), masson trichrome staining, and transmission electron microscope (TEM). The role and underlying mechanisms of EF24 in IPF were determined in vitro and in vivo by evaluating the expressions of PTEN, AKT/mTOR/NF-κB signaling pathway, and mitophagy using western blotting or flow cytometry. ResultsWe identified that the curcumin analogue EF24 was the most promising candidate among 12 compounds against IPF. EF24 treatment significantly reduced senescence biomarkers in bleomycin-induced senescent AECs, including SA-β-Gal, PAI-1, P21, and the senescence-associated secretory phenotype (SASP). EF24 also effectively inhibited fibroblast activation which was induced by senescent AECs or TGF-β. We revealed that PTEN activation was integral for EF24 to inhibit AECs senescence by suppressing the AKT/mTOR/NF-κB signaling pathway. Additionally, EF24 improved mitochondrial dysfunction through induction of mitophagy. Furthermore, EF24 administration significantly reduced the senescent phenotype induced by bleomycin in the lung tissues of mice. Notably, EF24 mitigates fibrosis and promotes overall health benefits in both the acute and chronic phases of IPF, suggesting its therapeutic potential in IPF treatment. ConclusionThese findings collectively highlight EF24 as a new and effective therapeutic agent against IPF by inhibiting senescence in AECs.

Polystyrene microplastics facilitate renal fibrosis through accelerating tubular epithelial cell senescence

Microplastics (MPs), emerging contaminants, are easily transported and enriched in the kidney, suggesting the kidney is susceptible to the toxicity of MPs. In this study, we explored the toxicity of MPs, including unmodified polystyrene (PS), negative-charged PS-SO3H, and positive-charged PS-NH2 MPs, in mice models for 28 days at a human equivalent concentration. The results showed MPs significantly increased levels of UREA, urea nitrogen (BUN), creatinine (CREA), and uric acid (UA) levels in serum and white blood cells, protein, and microalbumin in urine. In the kidney, MPs triggered persistent inflammation and renal fibrosis, which was caused by the increased senescence of tubular epithelial cells. Moreover, we identified the critical role of the Klotho/Wnt/β-catenin signaling pathway in the process of MPs induced senescence of tubular epithelial cells, promoting the epithelial-mesenchymal transformation of epithelial cells. MPs supported the secretion of TGF-β1 by senescent epithelial cells and induced the activation of renal fibroblasts. On the contrary, restoring the function of Klotho can alleviate the senescence of epithelial cells and reverse the activation of fibroblasts. Thus, our study revealed new evidence between MPs and renal fibrosis, and adds an important piece to the whole picture of the plastic pollution on people's health.

Mesenchymal stem cells-derived exosomes alleviate senescence of retinal pigment epithelial cells by activating PI3K/AKT-Nrf2 signaling pathway in early diabetic retinopathy

Diabetic retinopathy (DR) is a major cause of vision loss and blindness in adults. Cellular senescence was involved in the pathogenesis of early-stage DR and is positively correlated with progression. Thus, our study aimed at exploring the effect and potential mechanism of Mesenchymal stem cells-derived exosomes (MSCs-EXOs) on Retinal Pigment Epithelial (RPE) cells senescence at an early stage of DR in vivo and in vitro. ARPE-19 cells were incubated in high glucose (HG) medium mixed with MSCs-EXOs to observe the changes in cell viability. Senescence-associated β-galactosidase (SA-β-gal) staining, Western blot and qRT-PCR were used to assess the expression of senescence-related genes and antioxidant mediators. Quantitative Real-Time polymerase chain reaction (qRT-PCR), Optical coherence tomography (OCT) Hematoxylin and eosin (HE) staining and Electroretinogram (ERG) were respectively used to verify cellular senescence, the structure and function of the retina. Our findings demonstrated that MSCs-EXOs inhibited HG-induced senescence in ARPE-19 cells. Furthermore, MSCs-EXOs reduced HG-induced cell apoptosis and oxidative stress levels while promoting cell proliferation. Mechanistically, HG suppressed PI3K/AKT phosphorylation as well as nuclear factor erythroid 2-related factor 2 (Nrf2) expression along with its downstream target gene expression in ARPE-19 cells. However, MSCs-EXOs reversed these changes by alleviating cellular senescence while enhancing antioxidant activity. In line with our results in vitro, MSCs-EXOs significantly ameliorated hyperglycemia-induced senescence in DR mice by downregulating mRNA expression of P53, P21, P16, and SASP. Additionally, MSCs-EXOs improved the functional and structural integrity of the retina in DR mice. Our study revealed the protective effect of MSCs-EXOs on cellular senescence, offering new insights for the treatment of DR.