Senescence-associated Β-galactosidase Activity Research Articles

Novel Role of Mammalian Cell Senescence-Sustenance of Muscle Larvae of Trichinella spp.

Muscle larva of the parasitic nematode Trichinella spp. lives in a portion of muscle fibre transformed to a nurse cell (NC). Based on our previous transcriptomic studies, NC growth arrest was inferred to be accompanied by cellular senescence. In the current study, NC was proven to display the following markers of senescence: high senescence-associated β-galactosidase activity, lipid deposition, DNA damage, and cell cycle inhibition. Moreover, the nuclear localization of Activator Protein 1 (c-Fos, c-Jun, and FosB), as well as the upregulation of numerous AP-1 target genes in the NC, remained in accord with AP-1 recently identified as a master transcription factor in senescence. An increase in reactive oxygen species generation and the upregulation of antioxidant defence enzymes, including glutathione peroxidases 1 and 3, catalase, superoxide dismutases 1 and 3, and heme oxygenase 1, indicated an ongoing oxidative stress to proceed in the NC. Interestingly, antioxidant defence enzymes localized not only to the NC but also to the larva. These results allowed us to hypothesize that oxidative stress accompanying muscle regeneration and larval antigenic properties lead to the transformation of a regenerating myofibre into a senescent cell. Cellular senescence apparently represents a state of metabolism that sustains the long-term existence of muscle larva and ultimately provides it with the antioxidant capacity needed during the next host colonization. Senotherapy, a therapeutic approach aimed at selective elimination of senescent cells, can thus be viewed as potentially effective in the treatment of trichinosis.

Cell Cycle and Senescence Regulation by Podocyte Histone Deacetylase 1 and 2.

The loss of integrity of the glomerular filtration barrier results in proteinuria that is often attributed to podocyte loss. Yet how damaged podocytes are lost remains unknown. Germline loss of murine podocyte-associated Hdac1 and Hdac2 ( Hdac1/2 ) results in proteinuria and collapsing glomerulopathy due to sustained double-stranded DNA damage. Hdac1/2 deletion induces loss of podocyte quiescence, cell cycle entry, arrest in G1, and podocyte senescence, observed both in vivo and in vitro . Through the senescence secretory associated phenotype, podocytes secrete proteins that contribute to their detachment. These results solidify the role of HDACs in cell cycle regulation and senescence, providing important clues in our understanding of how podocytes are lost following injury. Intact expression of podocyte histone deacetylases (HDAC) during development is essential for maintaining a normal glomerular filtration barrier because of its role in modulating DNA damage and preventing premature senescence. Germline podocyte-specific Hdac1 and 2 ( Hdac1 / 2 ) double-knockout mice were generated to examine the importance of these enzymes during development. Podocyte-specific loss of Hdac1 / 2 in mice resulted in severe proteinuria, kidney failure, and collapsing glomerulopathy. Hdac1 / 2 -deprived podocytes exhibited classic characteristics of senescence, such as senescence-associated β-galactosidase activity and lipofuscin aggregates. In addition, DNA damage, likely caused by epigenetic alterations such as open chromatin conformation, not only resulted in podocyte cell-cycle entry as shown in vivo by Ki67 expression and by FUCCI-2aR mice, but also in p21-mediated cell-cycle arrest. Through the senescence secretory associated phenotype, the damaged podocytes secreted proinflammatory cytokines, growth factors, and matrix metalloproteinases, resulting in subsequent podocyte detachment and loss, evidenced by senescent podocytes in urine. Hdac1 / 2 plays an essential role during development. Loss of these genes in double knockout mice leads to sustained DNA damage and podocyte senescence and loss.

Dual BCL-XL/2 Protac 753B Not Only Potently Induces Apoptosis in Venetoclax-Resistant Primary AML Cells but Also Effectively Eliminates Chemotherapy-Induced Senescent AML Cells

BCL-XL and BCL-2 (BCL-XL/2 ) are key antiapoptotic proteins and validated cancer targets. 753B is a novel BCL-XL/2 proteolysis targeting chimera (PROTAC) that targets both BCL-XL and BCL-2 to the Von Hippel-Lindau (VHL) E3 ligase, leading to BCL-XL/2 ubiquitination and degradation (Lv et al. Nat. Comm. 2021). It exhibits an improved potency against BCL-XL/2 leukemia cells while reduced toxicity to platelets compared to the dual BCL-XL/2 inhibitor navitoclax or ABT263 in vitro. Here we examined the potential therapeutic efficacy of 753B against venetoclax-resistant primary AML cells and chemotherapy-induced senescent AML cells in vitro and in vivo. We first evaluated the sensitivity of 24 genetically diverse leukemia cell lines (17 AML, 5 T-ALL, and 2 AML secondary to myeloproliferative neoplasms (MPN-AML) cell lines) to ABT263, DT2216 (a BCL-XL selective PROTAC) (Khan et.al Nat. Med, 2019) and 753B. 753B induced time and dose-dependent BCL-XL degradation in all lines with DC50 (the concentrations with 50% protein degradation) ranging from 0.01 μM to 0.54 μM. BCL-2 was degraded in all except Loucy cells, with DC50 ranging from 0.02 μM to >1 μM. 753B caused dose-dependent reduction of viability in AML cell lines by CellTiter-Glo (CTG) assay at 24hr (IC50 range 0.01μM - 27.35 μM). 753B was more potent than ABT-263 in 12/17 AML and 3/5 T-ALL lines, with the mean IC50 values of 0.10 μM and 0.48 μM, respectively. The degradation of BCL-XL/2 by 753B was accompanied with MCL-1 upregulation in 10/17 cell lines tested, which was reversely correlated with the IC50. Recent findings indicate that chemotherapy (Ara-C)-induced senescence is associated with chemoresistance (Duy et al., Cancer Discovery 2021), and BCL-XL was previously reported to be the primary survival factor of senescent cells (Chang et al.,Nat. Med.2016). As previously reported, Ara-C indeed induced cellular senescence (SnCs) in MOLM-14 and Kasumi-1 AML cells, with increased cell size, higher senescence-associated β-galactosidase activity, upregulation of cell cycle regulator proteins (p16, p21, p53), and expression of senescence-associated secretory phenotype (IL-8 and CCR5). 753B cleared chemotherapy-induced SnCs by induction of apoptosis. To explore the senolytic mechanism of 753B, we FACS-sorted senescence-high or -low populations based on C12-FDG positivity (fluorogenic substrate di-β-D-galactopyranoside). C12-FDG high senescent AML population expressed higher levels of BCL-XL/2, priming the cells to 753B-induced apoptosis. We next isolated blasts from 16 primary AML samples and tested their sensitivity to 753B. 753B potently reduced cells viability with median IC50 value of 0.23 mM (range 0.02 - 2.29 mM). Similar to cell lines, 753B was more potent in degrading BCL-XL and inducing apoptosis than DT2216 in primary AML cells. 753B showed potency comparable to ABT-263 in all tested AML samples, including seven venetoclax-resistant samples (median IC50, 753B 0.22 mM; ABT-263 0.13 mM). 753B degraded both BCL-XL and BCL-2 in primary myeloblasts in association with the induction of apoptosis. BCL-2 degradation was observed in 4 of 6 samples tested. To investigate the antileukemia activity of 753B in vivo, we developed a patient-derived xenograft (PDX) model using NSG mice injected with AML PDX #8550 (harboring FLT3-ITD, DNMT3A, IDH1, KIT, and NPM1 mutations). Mice were randomized into 2 groups to receive either vehicle or 753B (5 mg/kg intraperitoneally every 4 days) for 3 weeks after leukemia engraftment was confirmed (day 33). Mice tolerated 753B therapy well with no significant changes in body weight and no significant platelet toxicity ascertained by blood counts. 753B as a single agent reduced the circulating leukemia cell burden, serially measured by flow cytometry, and extended median survival time from 61 days to 71 days (P = 0.0023)(Fig. 1A). In summary, the novel BCL-XL/2 PROTAC 753B potently reduced cell viability in leukemia cell lines with diverse genetic background, in primary AML cells including venetoclax-resistant AML blasts, and extended survival of mice harboring human AML PDX in vivo without hematologic toxicity. Our data indicate that co-targeting of BCL-2 and BCL-XL may eliminate senescent AML cells surviving Ara-C chemotherapy, supporting the utility of future combination studies in leukemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Tiron Has Negative Effects on Osteogenic Differentiation via Mitochondrial Dysfunction in Human Periosteum-Derived Cells.

Tiron is a potent antioxidant that counters the pathological effects of reactive oxygen species (ROS) production due to oxidative stress in various cell types. We examined the effects of tiron on mitochondrial function and osteoblastic differentiation in human periosteum-derived cells (hPDCs). Tiron increased mitochondrial activity and decreased senescence-associated β-galactosidase activity in hPDCs; however, it had a detrimental effect on osteoblastic differentiation by reducing alkaline phosphatase (ALP) activity and alizarin red-positive mineralization, regardless of H2O2 treatment. Osteoblast-differentiating hPDCs displayed increased ROS production compared with non-differentiating hPDCs, and treatment with tiron reduced ROS production in the differentiating cells. Antioxidants decreased the rates of oxygen consumption and ATP production, which are increased in hPDCs during osteoblastic differentiation. In addition, treatment with tiron reduced the levels of most mitochondrial proteins, which are increased in hPDCs during culture in osteogenic induction medium. These results suggest that tiron exerts negative effects on the osteoblastic differentiation of hPDCs by causing mitochondrial dysfunction.

Inhibition of cellular senescence hallmarks by mitochondrial transplantation in senescence-induced ARPE-19 cells

Retinal pigment epithelium (RPE) damage is a major factor in age-related macular degeneration (AMD). The RPE in AMD shows mitochondrial dysfunction suggesting an association of AMD with mitochondrial function. Therefore, exogenous mitochondrial transplantation for restoring and replacing dysfunctional mitochondria may be an effective therapeutic strategy for AMD. Here, we investigated the effects of extrinsic mitochondrial transplantation on senescence-induced ARPE-19 cells. We demonstrated mitochondrial dysfunction in replicative senescence-induced ARPE-19 cells after repeated passage. Imbalanced mitophagy and mitochondrial dynamics resulted in increased mitochondrial numbers and elevated levels of mitochondrial and intracellular reactive oxygen species. Exogenous mitochondrial transplantation improved mitochondrial dysfunction and alleviated cellular senescence hallmarks, such as increased cell size, increased senescence-associated β-galactosidase activity, augmented NF-κB activity, increased inflammatory cytokines, and upregulated the cyclin-dependent kinase inhibitors p21 and p16. Further, cellular senescence properties were improved by exogenous mitochondrial transplantation in oxidative stress-induced senescent ARPE-19 cells. These results indicate that exogenous mitochondrial transplantation modulates cellular senescence and may be considered a novel therapeutic strategy for AMD.

Repeated Administration of Cisplatin Transforms Kidney Fibroblasts through G2/M Arrest and Cellular Senescence.

Cisplatin is a potent chemotherapeutic used for the treatment of many types of cancer, but it has nephrotoxic side effects leading to acute kidney injury and subsequently chronic kidney disease (CKD). Previous work has focused on acute kidney tubular injury induced by cisplatin, whereas the chronic sequelae post-injury has not been well-explored. In the present study, we established a kidney fibroblast model of CKD induced by repeated administration of cisplatin (RAC) as a clinically relevant model. In NRK-49F rat kidney fibroblasts, RAC upregulated α-smooth muscle actin (α-SMA) and fibronectin proteins, suggesting that RAC induces kidney fibroblast-to-myofibroblast transformation. RAC also enhanced cell size, including the cell attachment surface area, nuclear area, and cell volume. Furthermore, RAC induced p21 expression and senescence-associated β-galactosidase activity, suggesting that kidney fibroblasts exposed to RAC develop a senescent phenotype. Inhibition of p21 reduced cellular senescence, hypertrophy, and myofibroblast transformation induced by RAC. Intriguingly, after RAC, kidney fibroblasts were arrested at the G2/M phase. Repeated treatment with paclitaxel as an inducer of G2/M arrest upregulated p21, α-SMA, and fibronectin in the kidney fibroblasts. Taken together, these data suggest that RAC transforms kidney fibroblasts into myofibroblasts through G2/M arrest and cellular senescence.

Low-dose tributyltin triggers human chondrocyte senescence and mouse articular cartilage aging.

Tributyltin (TBT) is known as an endocrine-disrupting chemical. This study investigated the effects and possible mechanisms of TBT exposure on inducing human articular chondrocyte senescence in vitro at the human-relevant concentrations of 0.01-0.5μM and mouse articular cartilage aging in vivo at the doses of 5 and 25μg/kg/day, which were 5 times lower than the established no observed adverse effect level (NOAEL) and equal to NOAEL, respectively. TBT significantly increased the senescence-associated β-galactosidase activity and the protein expression levels of senescence markers p16, p53, and p21 in chondrocytes. TBT induced the protein phosphorylation of both p38 and JNK mitogen-activated protein kinases in which the JNK signaling was a main pathway to be involved in TBT-induced chondrocyte senescence. The phosphorylation of both ataxia-telangiectasia mutated (ATM) and histone protein H2AX (termed γH2AX) was also significantly increased in TBT-treated chondrocytes. ATM inhibitor significantly inhibited the protein expression levels of γH2AX, phosphorylatedp38, phosphorylatedJNK, p16, p53, and p21. TBT significantly stimulated the mRNA expression of senescence-associated secretory phenotype (SASP)-related factors, including IL-1β, TGF-β, TNF-α, ICAM-1, CCL2, and MMP13, and the protein expression of GATA4 and phosphorylated NF-κB-p65 in chondrocytes. Furthermore, TBT by oral gavage for 4weeks in mice significantly enhanced the articular cartilage aging and abrasion. The protein expression of phosphorylated p38, phosphorylated JNK, GATA4, and phosphorylated NF-κB-p65, and the mRNA expression of SASP-related factors were enhanced in the mouse cartilages. These results suggest that TBT exposure can trigger human chondrocyte senescence in vitro and accelerating mouse articular cartilage aging in vivo.

WNK1-OSR1 Signaling Regulates Angiogenesis-Mediated Metastasis towards Developing a Combinatorial Anti-Cancer Strategy.

Lysine-deficient protein kinase-1 (WNK1) is critical for both embryonic angiogenesis and tumor-induced angiogenesis. However, the downstream effectors of WNK1 during these processes remain ambiguous. In this study, we identified that oxidative stress responsive 1b (osr1b) is upregulated in endothelial cells in both embryonic and tumor-induced angiogenesis in zebrafish, accompanied by downregulation of protein phosphatase 2A (pp2a) subunit ppp2r1bb. In addition, wnk1a and osr1b are upregulated in two liver cancer transgenic fish models: [tert x p53−/−] and [HBx,src,p53−/−,RPIA], while ppp2r1bb is downregulated in [tert x p53−/−]. Furthermore, using HUVEC endothelial cells co-cultured with HepG2 hepatoma cells, we confirmed that WNK1 plays a critical role in the induction of hepatoma cell migration in both endothelial cells and hepatoma cells. Moreover, overexpression of OSR1 can rescue the reduced cell migration caused by shWNK1 knockdown in HUVEC cells, indicating OSR1 is downstream of WNK1 in endothelial cells promoting hepatoma cell migration. Overexpression of PPP2R1A can rescue the increased cell migration caused by WNK1 overexpression in HepG2, indicating that PPP2R1A is a downstream effector in hepatoma. The combinatorial treatment with WNK1 inhibitor (WNK463) and OSR1 inhibitor (Rafoxanide) plus oligo-fucoidan via oral gavage to feed [HBx,src,p53−/−,RPIA] transgenic fish exhibits much more significant anticancer efficacy than Regorafenib for advanced HCC. Importantly, oligo-fucoidan can reduce the cell senescence marker-IL-1β expression. Furthermore, oligo-fucoidan reduces the increased cell senescence-associated β-galactosidase activity in tert transgenic fish treated with WNK1-OSR1 inhibitors. Our results reveal the WNK1–OSR1–PPP2R1A axis plays a critical role in both endothelial and hepatoma cells during tumor-induced angiogenesis promoting cancer cell migration. By in vitro and in vivo experiments, we further uncover the molecular mechanisms of WNK1 and its downstream effectors during tumor-induced angiogenesis. Targeting WNK1–OSR1-mediated anti-angiogenesis and anti-cancer activity, the undesired inflammation response caused by inhibiting WNK1–OSR1 can be attenuated by the combination therapy with oligo-fucoidan and may improve the efficacy.

Aberrant methylation of Serpine1 mediates lung injury in neonatal mice prenatally exposed to intrauterine inflammation

BackgroundIntrauterine inflammation (IUI) alters epigenetic modifications in offspring, leading to lung injury. However, the epigenetic mechanism underlying IUI-induced lung injury remains uncertain. In the present study, we aim to investigate the effect of IUI on lung development, and to identify the key molecule involved in this process and its epigenetic regulatory mechanism.ResultsSerpine1 was upregulated in the lung tissue of neonatal mice with IUI. Intranasal delivery of Serpine1 siRNA markedly reversed IUI-induced lung injury. Serpine1 overexpression substantially promoted cell senescence of both human and murine lung epithelial cells, reflected by decreased cell proliferation and increased senescence-associated β-galactosidase activity, G0/G1 cell fraction, senescence marker, and oxidative and DNA damage marker expression. IUI decreased the methylation level of the Serpine1 promoter, and methylation of the promoter led to transcriptional repression of Serpine1. Furthermore, IUI promoted the expression of Tet1 potentially through TNF-α, while Tet1 facilitated the demethylation of Serpine1 promoter. DNA pull-down and ChIP assays revealed that the Serpine1 promoter was regulated by Rela and Hdac2. DNA demethylation increased the recruitment of Rela to the Serpine1 promoter and induced the release of Hdac2.ConclusionIncreased Serpine1 expression mediated by DNA demethylation causes lung injury in neonatal mice with IUI. Therefore, therapeutic interventions targeting Serpine1 may effectively prevent IUI-induced lung injury.

RNAa-mediated epigenetic attenuation of the cell senescence via locus specific induction of endogenous SIRT1

SIRT1, a known regulator of cellular senescence, is a therapeutic target for age related disorders and its upregulation is a strategy to improve the cell therapeutic potentials of human mesenchymal stem cell (MSCs). Knockdown of natural antisense transcripts via small activating RNAs (RNAa) is an emerging approach for safe and locus specific gene regulation. We have recently identified a natural antisense transcript at human SIRT1 locus (SIRT1-NAT), the expression of which shows a negative correlation with that of SIRT1. To test the hypothetic upregulation of SIRT1 via knockdown of SIRT1-NAT, in this study we designed a single stranded oligonucleotide (SIRT1-antagoNAT) against the antisense transcript, transfection of which efficiently knocked down the SIRT1-NAT and induced SIRT1 transcription in human MSCs. In addition, activation of SIRT1 transfection via knockdown of SIRT1-NAT in human MSCs enhanced their proliferation and differentiation potentials, reduced senescence associated β-galactosidase activity and reversed the senescence associated molecular alterations. Our findings introduce an RNAa mediated approach for epigenetic induction of endogenous SIRT1 and the consequent attenuation of senescence. Further studies should evaluate the therapeutic potentials of this approach against various age related disorders.

Establishment of In Vitro Models by Stress-Induced Premature Senescence for Characterizing the Stromal Vascular Niche in Human Adipose Tissue.

Acting as the largest energy reservoir in the body, adipose tissue is involved in longevity and progression of age-related metabolic dysfunction. Here, cellular senescence plays a central role in the generation of a pro-inflammatory environment and in the evolution of chronic diseases. Within the complexity of a tissue, identification and targeting of senescent cells is hampered by their heterogeneity. In this study, we generated stress-induced premature senescence 2D and 3D in vitro models for the stromal vascular niche of human adipose tissue. We established treatment conditions for senescence induction using Doxorubicin (Dox), starting from adipose-derived stromal/stem cells (ASCs), which we adapted to freshly isolated microtissue-stromal vascular fraction (MT-SVF), where cells are embedded within their native extracellular matrix. Senescence hallmarks for the established in vitro models were verified on different cellular levels, including morphology, cell cycle arrest, senescence-associated β-galactosidase activity (SA-βgal) and gene expression. Two subsequent exposures with 200 nM Dox for six days were suitable to induce senescence in our in vitro models. We demonstrated induction of senescence in the 2D in vitro models through SA-βgal activity, at the mRNA level (LMNB1, CDK1, p21) and additionally by G2/M phase cell cycle arrest in ASCs. Significant differences in Lamin B1 and p21 protein expression confirmed senescence in our MT-SVF 3D model. MT-SVF 3D cultures were composed of multiple cell types, including CD31, CD34 and CD68 positive cells, while cell death remained unaltered upon senescence induction. As heterogeneity and complexity of adipose tissue senescence is given by multiple cell types, our established senescence models that represent the perivascular niche embedded within its native extracellular matrix are highly relevant for future clinical studies.

Endothelial senescence mediates hypoxia-induced vascular remodeling by modulating PDGFB expression.

Uncontrolled accumulation of pulmonary artery smooth muscle cells (PASMCs) to the distal pulmonary arterioles (PAs) is one of the major characteristics of pulmonary hypertension (PH). Cellular senescence contributes to aging and lung diseases associated with PH and links to PH progression. However, the mechanism by which cellular senescence controls vascular remodeling in PH is not fully understood. The levels of senescence marker, p16INK4A and senescence-associated β-galactosidase (SA-β-gal) activity are higher in PA endothelial cells (ECs) isolated from idiopathic pulmonary arterial hypertension (IPAH) patients compared to those from healthy individuals. Hypoxia-induced accumulation of α-smooth muscle actin (αSMA)-positive cells to the PAs is attenuated in p16fl/fl-Cdh5(PAC)-CreERT2 (p16iΔEC) mice after tamoxifen induction. We have reported that endothelial TWIST1 mediates hypoxia-induced vascular remodeling by increasing platelet-derived growth factor (PDGFB) expression. Transcriptomic analyses of IPAH patient lungs or hypoxia-induced mouse lung ECs reveal the alteration of senescence-related gene expression and their interaction with TWIST1. Knockdown of p16INK4A attenuates the expression of PDGFB and TWIST1 in IPAH patient PAECs or hypoxia-treated mouse lungs and suppresses accumulation of αSMA–positive cells to the supplemented ECs in the gel implanted on the mouse lungs. Hypoxia-treated mouse lung EC-derived exosomes stimulate DNA synthesis and migration of PASMCs in vitro and in the gel implanted on the mouse lungs, while p16iΔEC mouse lung EC-derived exosomes inhibit the effects. These results suggest that endothelial senescence modulates TWIST1-PDGFB signaling and controls vascular remodeling in PH.

Sirtuin-1 attenuates cadmium-induced renal cell senescence through p53 deacetylation

Cadmium (Cd), the common environmental pollutant, primarily targets at renal proximal tubules and induces nephrotoxicity. Cellular senescence, a phenomenon of cell growth arrest and a characteristics of maladaptive cell self-repair, is associated with renal disease progression. However, whether and how Cd induces renal tubular cells premature senescence is unknown. In our study, we found that Cd induced kidney damage and dysfunctions, which correlated with exacerbated tubular cell senescence, evidenced by increased senescence-associated β-galactosidase activity, the upregulated protein expression of p53 and p21Waf1/Cip1 proteins, and elevated expression and secretion of cytokines in human proximal tubular epithelial HK-2 cells in vitro and in Cd-treated mice in vivo. Moreover, a S-phase arrest and decrease in Edu positive rate were found in Cd-treated HK-2 cells. Mechanistically, Cd suppressed the expression and activity of Sirtuin-1 (SIRT1), an anti-senescence deacetylase, resulting in the accumulation of acetylated p53 and upregulation of p21Waf1/Cip1. Activation of SIRT1 significantly abolished Cd-induced premature senescence and S-phase arrest. Finally, silencing p21Waf1/Cip1 efficiently delayed premature senescence and recovered cell cycle progression. These findings indicate that Cd promotes tubular cells senescence and impairs tubular cells regeneration, resulting in kidney dysfunctions, which could be ameliorated by SIRT1 activation.

Inorganic arsenic exposure-induced premature senescence and senescence-associated secretory phenotype (SASP) in human hepatic stellate cells

Exposure to inorganic arsenic has been known to induce cancers in various organs, however, the underlying mechanisms remain unclear. Premature senescence refers to the irreversible growth arrest induced by stress stimuli. The senescence-associated secretory phenotype (SASP), particularly in fibroblasts, has been shown to promote cancer development. In this study, we examined whether arsenite exposure causes premature senescence and induction of SASP in liver fibroblasts using the human hepatic stellate cell line, LX-2. Exposure of LX-2 cells to 5 or 7.5 μM of sodium arsenite for 144 h induced the features of senescence in the cells, including morphological changes, growth inhibition, increased senescence-associated β-galactosidase activity, increased P21 gene expression, and decreased LAMINB1 gene expression. The mRNA expressions of SASP factors, such as MMP1, MMP3, IL-8, IL-1β, and CXCL1, were also highly upregulated. The wound healing assay revealed that the conditioned medium from LX-2 cells with arsenite-induced senescence increased the migration activity of cells of the human hepatoma cell line, Huh-7. Gene expression data of liver cancer samples from the Human Protein Atlas showed that high expression levels of the SASP factors that were upregulated in the cells with arsenite-induced senescence were strongly associated with a poor prognosis. In addition, the cellular levels of γ-H2AX, a DNA double-strand break marker, were increased by arsenite exposure, suggesting that DNA damage could contribute to premature senescence induction. These results show that arsenite exposure induces premature senescence in hepatic stellate cells and suggest that the SASP factors from the senescent cells promote hepatic carcinogenesis.

Cathepsin F is a potential marker for senescent human skin fibroblasts and keratinocytes associated with skin aging

Cellular senescence is characterized by cell cycle arrest and the senescence-associated secretory phenotype (SASP) and can be triggered by a variety of stimuli, including deoxyribonucleic acid (DNA) damage, oxidative stress, and telomere exhaustion. Cellular senescence is associated with skin aging, and identification of specific markers of senescent cells is essential for development of targeted therapies. Cathepsin F (CTSF) has been implicated in dermatitis and various cancers and participates in cell immortalization through its association with Bcl family proteins. It is a candidate therapeutic target to specifically label and eliminate human skin fibroblasts and keratinocytes immortalized by aging and achieve skin rejuvenation. In this study, we investigated whether CTSF is associated with senescence in human fibroblasts and keratinocytes. In senescence models, created using replicative aging, ionizing radiation exposure, and the anticancer drug doxorubicin, various senescence markers were observed, such as senescence-associated β-galactosidase (SA-β-gal) activity, increased SASP gene expression, and decreased uptake of the proliferation marker BrdU. Furthermore, CTSF expression was elevated at the gene and protein levels. In addition, CTSF-positive cells were abundant in aged human epidermis and in some parts of the dermis. In the population of senescent cells with arrested division, the number of CTSF-positive cells was significantly higher than that in the proliferating cell population. These results suggest that CTSF is a candidate for therapeutic modalities targeting aging fibroblasts and keratinocytes.

Alteration of E2F2 Expression in Governing Endothelial Cell Senescence.

Endothelial cell senescence has a vital implication for vascular dysfunction, leading to age-related cardiovascular disease, especially hypertension and atherosclerosis. E2F transcription factor 2 (E2F2) plays a critical role in cell proliferation, differentiation, and DNA damage response. Up to date, no study has ever connected E2F2 to vascular endothelial cell senescence. Here, we demonstrate that E2F2 is involved in endothelial cellular senescence. We found that E2F2 expression is decreased during the replicative senescence of human umbilical vein endothelial cells (HUVECs) and the aortas of aged mice. The knockdown of E2F2 in young HUVECs induces premature senescence characterized by an increase in senescence-associated β-galactosidase (SA-β-gal) activity, a reduction in phosphorylated endothelial nitric oxide synthase (p-eNOS) and sirtuin 1 (SIRT1), and the upregulation of senescence-associated secretory phenotype (SASP) IL-6 and IL-8. The lack of E2F2 promoted cell cycle arrest, DNA damage, and cell proliferation inhibition. Conversely, E2F2 overexpression reversed the senescence phenotype and enhanced the cellular function in the senescent cells. Furthermore, E2F2 deficiency downregulated downstream target genes including CNNA2, CDK1, and FOXM1, and overexpression restored the expression of these genes. Our findings demonstrate that E2F2 plays an indispensable role in endothelial cell senescence.

Resolvin E1 attenuates doxorubicin-induced cardiac fibroblast senescence: A key role for IL-1β

Cardiac fibroblasts (CFs) undergo senescence in reaction to different stressors, leading to a poor prognosis of cardiac disease. Doxorubicin (Doxo) is an antineoplastic drug with strong cardiotoxic effects, which induces IL-1β secretion and thus, triggers a potent pro-inflammatory response. Doxo induces CFs senescence; however, the mechanisms are not fully understood. Different pharmacological strategies have been used to eliminate senescent cells by inducing their apoptosis or modifying their secretome. However, Resolvin E1 (RvE1), a lipid derivative resolutive mediator with potent anti-inflammatory effects has not been used before to prevent CFs senescence.CFs were isolated from adult male C57BL/6J mice and subsequently stimulated with Doxo, in the presence or absence of RvE1. Senescence-associated β-galactosidase activity (SA-β-gal), γ-H2A.X, p53, p21, and senescence-associated secretory phenotype (SASP) were evaluated. The involvement of the NLRP3 inflammasome/interleukin-1 receptor (IL-1R) signaling pathway on CFs senescence was studied using an NLRP3 inhibitor (MCC950) and an endogenous IL-1R antagonist (IR1A).Doxo is able to trigger CFs senescence, as evidenced by an increase of γ-H2A.X, p53, p21, and SA-β-gal, and changes in the SASP profile. These Doxo effects were prevented by RvE1. Doxo triggers IL-1β secretion, which was dependent on NLRP3 activation. Doxo-induced CFs senescence was partially blocked by MCC950 and IR1A. In addition, IL-1β also triggered CFs senescence, as evidenced by the increase of γ-H2A.X, p53, p21, SA-β-gal activity, and SASP. All these effects were also prevented by RvE1 treatment. ConclusionThese data show the anti-senescent role of RvE1 in Doxo-induced CFs senescence, which could be mediated by reducing IL-1β secretion.

PGRMC1 Regulates Cellular Senescence via Modulating FOXO1 Expression in Decidualizing Endometrial Stromal Cells.

The appropriate differentiation of endometrial stromal cells (ESCs) into decidual cells is required for embryo implantation and subsequent placentation into humans. Decidualization is accompanied by the appearance of senescent-like cells. We recently reported the secretory phase-specific downregulation of endometrial progesterone receptor membrane component 1 (PGRMC1) and enhanced decidualization upon PGRMC1 knockdown and inhibition in cultured ESCs. However, it remains unknown whether PGRMC1 is involved in cellular senescence during decidualization. Here, we showed that the small interfering RNA (siRNA)-mediated knockdown of PGRMC1 and the inhibition of PGRMC1 by AG-205 increased the expression of the transcription factor forkhead box protein O1 (FOXO1) and the senescence-associated β-galactosidase activity in cAMP analog- and progesterone-treated ESCs. Furthermore, the knockdown of FOXO1 repressed the decidual senescence induced by siRNA-based PGRMC1 knockdown or AG-205 treatment. Taken together, the decreased PGRMC1 expression in ESCs may accelerate decidualization and cellular senescence via the upregulation of FOXO1 expression for appropriate endometrial remodeling and embryo implantation during the secretory phase.

Quercetin Alleviates Pulmonary Fibrosis in Mice Exposed to Silica by Inhibiting Macrophage Senescence.

Quercetin exerts anti-inflammatory, anti-oxidant and other protective effects. Previous studies have shown that senescent cells, such as fibroblasts and type II airway epithelial cells, are strongly implicated in the development of pulmonary fibrosis pathology. However, the role of senescent macrophages during silicosis remains unclear. We investigated the effects of quercetin on macrophage senescence and pulmonary fibrosis, and explored underlying mechanisms. Mice were randomized to six model groups. Vitro model was also established by culturing RAW264.7 macrophages with silica (SiO2). We examined the effects of quercetin on fibrosis, senescence-associated β-galactosidase (SA-β-Gal) activity, and senescence-specific genes (p16, p21, and p53). We showed that quercetin reduced pulmonary fibrosis and inhibited extracellular matrix (ECM) formation. Quercetin also attenuated macrophage senescence induced by SiO2 both in vitro and in vivo. In addition, quercetin significantly decreased the expressions of the senescence-associated secretory phenotype (SASP), including proinflammatory factors (interleukin-1α (Il-1α), Il-6, tumor necrosis factor-α (TNF-α), and transforming growth factor-β1 (TGF-β1)) and matrix metalloproteinases (MMP2, MMP9, and MMP12). In conclusion, quercetin mediated its anti-fibrotic effects by inhibiting macrophage senescence, possibly via SASP.

Vanadium pentoxide induced oxidative stress and cellular senescence in human lung fibroblasts

Both environmental exposure to vanadium pentoxide (V2O5, V+5 for its ionic counterparts) and fibroblast senescence are associated with pulmonary fibrosis, but whether V+5 causes fibroblast senescence remains unknown. We found in a dose-response study that 2–40 μM V+5 caused human lung fibroblasts (HLF) senescence with increased senescence-associated β-galactosidase activity and p16 expression, while cell death occurred at higher concentration (LC50, 82 μM V+5). Notably, measures of reactive oxygen species (ROS) production with fluorescence probes showed no association of ROS with V+5-dependent senescence. Preloading catalase (polyethylene-conjugated), a H2O2 scavenger, did not alleviate the cellular senescence induced by V+5. Analyses of the cellular glutathione (GSH) system showed that V+5 oxidized GSH, increased GSH biosynthesis, stimulated cellular GSH efflux and increased protein S-glutathionylation, and addition of N-acetyl cysteine inhibited V+5-elevated p16 expression, suggesting that thiol oxidation mediates V+5-caused senescence. Moreover, strong correlations between GSSG/GSH redox potential (Eh), protein S-glutathionylation, and cellular senescence (R2 > 0.99, p < 0.05) were present in V+5-treated cells. Studies with cell-free and enzyme-free solutions showed that V+5 directly oxidized GSH with formation of V+4 and GSSG in the absence of O2. Analyses of V+5 and V+4 in HLF and culture media showed that V+5 was reduced to V+4 in cells and that a stable V+4/V+5 ratio was rapidly achieved in extracellular media, indicating ongoing release of V+4 and reoxidation to V+5. Together, the results show that V+5-dependent fibroblast senescence is associated with a cellular/extracellular redox cycling mechanism involving the GSH system and occurring under conditions that do not cause cell death. These results establish a mechanism by which environmental vanadium from food, dietary supplements or drinking water, can cause or contribute to lung fibrosis in the absence of high-level occupational exposures and cytotoxic cell death.