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• Research Paper Volume 13, Issue 14 pp 18852-18869

  Regulation of the IGF1 signaling pathway is involved in idiopathic pulmonary fibrosis induced by alveolar epithelial cell senescence and core fucosylation

  Relevance score: 6.6864614

  Wei Sun, Xiaoyan Jing, Xiaoyu Yang, Hui Huang, Qun Luo, Shu Xia, Ping Wang, Na Wang, Qian Zhang, Jian Guo, Zuojun Xu

  Keywords: IPF, aging, alveolar epithelial cell, core fucosylation, IGF-1

  Published in Aging on July 30, 2021

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  Idiopathic pulmonary fibrosis (IPF) mainly occurs in elderly people over the age of sixty. IPF pathogenesis is associated with alveolar epithelial cells (AECs) senescence. Activation of PI3K/AKT signaling induced by insulin-like growth factor 1 (IGF1) participates in AEC senescence and IPF by releasing CTGF, TGF-β1, and MMP9. Our previous study demonstrated that core fucosylation (CF) modification, catalyzed by a specific core fucosyltransferase (FUT8) can regulate the activation of multiple signaling pathways, and inhibiting CF can alleviate pulmonary fibrosis in mice induced by bleomycin. However, whether CF is involved in IGF1-mediated AEC senescence in IPF remains unclear. In this study, we found that the IGF1/PI3K/AKT signaling pathway was activated in IPF lung tissue. Meanwhile, CF was present in senescent AECs. We also showed that IGF1 could induce AECs senescence with enhanced CF in vivo and in vitro. Inhibiting CF alleviated AECs senescence and pulmonary fibrosis induced by IGF1. In addition, activation of IGF1/PI3K/AKT signaling depends on CF. In conclusion, this study confirmed that CF is an important target regulating the IGF1 signaling pathway in AEC senescence and IPF, which might be a candidate target to treat IPF in the future.

• Research Paper Volume 13, Issue 10 pp 13822-13845

  Development and validation of epithelial mesenchymal transition-related prognostic model for hepatocellular carcinoma

  Relevance score: 7.4527164

  Xuequan Wang, Ziming Xing, Huihui Xu, Haihua Yang, Tongjing Xing

  Keywords: hepatocellular carcinoma, epithelial cell transformation, prognosis

  Published in Aging on April 30, 2021

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  Epithelial cell transformation (EMT) plays an important role in the pathogenesis and metastasis of hepatocellular carcinoma (HCC). We aimed to establish a genetic risk model to evaluate HCC prognosis based on the expression levels of EMT-related genes. The data of HCC patients were collected from TCGA and ICGC databases. Gene expression differential analysis, univariate analysis, and lasso combined with stepwise Cox regression were used to construct the prognostic model. Kaplan–Meier curve, receiver operating characteristic (ROC) curve, calibration analysis, Harrell’s concordance index (C-index), and decision curve analysis (DCA) were used to evaluate the predictive ability of the risk model or nomogram. GO and KEGG were used to analyze differently expressed EMT genes, or genes that directly or indirectly interact with the risk-associated genes. A 10-gene signature, including TSC2, ACTA2, SLC2A1, PGF, MYCN, PIK3R1, EOMES, BDNF, ZNF746, and TFDP3, was identified. Kaplan–Meier survival analysis showed a significant prognostic difference between high- and low-risk groups of patients. ROC curve analysis showed that the risk score model could effectively predict the 1-, 3-, and 5-year overall survival rates of patients with HCC. The nomogram showed a stronger predictive effect than clinical indicators. C-index, DCA, and calibration analysis demonstrated that the risk score and nomogram had high accuracy. The single sample gene set enrichment analysis results confirmed significant differences in the types of infiltrating immune cells between patients in the high- and low-risk groups. This study established a new prediction model of risk gene signature for predicting prognosis in patients with HCC, and provides a new molecular tool for the clinical evaluation of HCC prognosis.

• Research Paper Volume 12, Issue 18 pp 18008-18018

  Pre-incubation with human umbilical cord derived mesenchymal stem cells-exosomes prevents cisplatin-induced renal tubular epithelial cell injury

  Relevance score: 6.5578084

  Zongying Li, Shuyi Cao

  Keywords: apoptosis, cisplatin, exosomes, renal tubular epithelial cell, viability nephrotoxicity

  Published in Aging on September 23, 2020

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  Purpose: The administration of cisplatin is limited due to its nephrotoxicity, and prevention of this nephrotoxicity of cisplatin is difficult. Mesenchymal stem cell (MSC)-derived exosomes have been implicated as a novel therapeutic approach for tissue injury.

  Results: In vitro, the NRK cells pre-incubated with HUMSC-exosomes increased the Cp-inhibited cell viability, proliferation activity, and the cell proportion in G1-phase and inhibited Cp-induced cell apoptosis. Furthermore, the expression levels of apoptotic marker proteins Bim, Bad, Bax, cleaved caspase-3, and cleaved caspase-9 induced by Cp in the NRK cells were decreased by pre-incubating with HUMSC-exosomes.

  Conclusion: Our findings indicated that the exosomes from HUMSCs can effectively increase the survival rate and inhibit cell apoptosis of NRK cells. Therefore, pre-treatment of HUMSC-exosomes may be a new method to improve the therapeutic effect of cisplatin.

  Patients and methods: Exosomes were isolated from human umbilical cord derived mesenchymal stem cells (HUMSCs). Co-culture of normal rat renal tubular epithelial cells (NRK) and the absorption of exogenous exosomes by NRK cells were examined in vitro. Then the NRK cells were incubated with exosomes from HUMSCs and cisplatin (Cp). Cells were harvested for MTT assay, cloning formation, flow cytometry, and Western blot.

• Research Paper Volume 12, Issue 6 pp 5516-5538

  Microarray analysis of verbenalin-treated human amniotic epithelial cells reveals therapeutic potential for Alzheimer’s Disease

  Relevance score: 5.992769

  Farhana Ferdousi, Shinji Kondo, Kazunori Sasaki, Yoshiaki Uchida, Nobuhiro Ohkohchi, Yun-Wen Zheng, Hiroko Isoda

  Keywords: Alzheimer’s disease, verbenalin, human amnion epithelial cell, microarray analysis, natural compound

  Published in Aging on March 29, 2020

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  Alzheimer’s disease (AD) has become a major world health problem as the population ages. There is still no available treatment that can stop or reverse the progression of AD. Human amnion epithelial cells (hAECs), an alternative source for stem cells, have shown neuroprotective and neurorestorative potentials when transplanted in vivo. Besides, studies have suggested that stem cell priming with plant-derived bioactive compounds can enhance stem cell proliferation and differentiation and improve the disease-treating capability of stem cells. Verbenalin is an iridoid glucoside found in medicinal herbs of Verbenaceae family. In the present study, we have conducted microarray gene expression profiling of verbenalin-treated hAECs to explore its therapeutic potential for AD. Gene set enrichment analysis revealed verbenalin treatment significantly enriched AD-associated gene sets. Genes associated with lysosomal dysfunction, pathologic angiogenesis, pathologic protein aggregation, circadian rhythm, age-related neurometabolism, and neurogenesis were differentially expressed in the verbenalin-treated hAECs compared to control cells. Additionally, the neuroprotective effect of verbenalin was confirmed against amyloid beta-induced neurotoxicity in human neuroblastoma SH-SY5Y cells. Our present study is the first to report the therapeutic potential of verbenalin for AD; however, further in-depth research in the in vitro and in vivo models are required to confirm our preliminary findings.

  

  (A) Volcano plot displaying DEGs between verbenalin-treated and untreated-control hAECs on day 7 (performed in Transcriptome Analysis Console version 4 software). The vertical axis (y-axis) corresponds to -log10 p-value of the ANOVA p-values, and the horizontal axis (x-axis) displays linear fold change. The red dots represent the up-regulated genes; the green dots represent the downregulated genes. (B) Distribution of fold changes in mRNA expression levels in verbenalin-treated hAECs (C) Pie chart showing the enriched (p < 0.05) tissue expressions by the DEGs between verbenalin-treated and untreated-control hAECs on day 7 (analyzed by DAVID online tool).

  
  
  

  

  (A) Significantly enriched cellular components for DEGs. (B) Top biological processes as per p-value (modified Fisher’s exact) by DEGs. (C) Significantly enriched KEGG pathways by DEGs (p < 0.05; modified Fisher’s exact test). All the gene ontology enrichment analyses were performed using DAVID online tool.

  
  
  

  

  Heat maps showing relative expression intensity of genes reported to be (A) upregulated in AD human brain, (B) strongly associated with late-onset of AD, (C) associated with neurodegenerative diseases in untreated control hAECs on day 0 and day 7, and in verbenalin-treated hAECs on day 7. (D) Boxplots for the relative ratios of gene intensity (genes presented in the heat maps) in day 7 control (Control_D7) and verbenalin-treated hAECs compared with day 0 control. Box ranges from 25^th to 75^th percentile, the line in the middle represents the median value, the whiskers represent the min, max, and mean values, and the error bar represents the SD. Significance was computed by One-way ANOVA for linear distribution and Mann-Whitney U test for nonlinear distribution. Heat maps were generated using Morpheus online tool.

  
  
  

  

  Effect of verbenalin treatment on the expressions of EGF, VEGF, and NRG1. The hAECs were treated with 20 μM of verbenalin (Ver) for 7 days, while the control cells were maintained in the placental basal medium. (A) Gene expressions were evaluated by real-time PCR. Each value represents the mean ± SD (n = 3). Asterisks refer to statistical significance (*p < 0.05, **p < 0.01) by One-way ANOVA as compared with control (Ctrl). (B) Boxplots of protein concentration (ng/ml) obtained by ELISA (n = 4). Box ranges from 25^th to 75^th percentile, the line in the middle represents the median value, the whiskers represent the min, max, and mean values, and the error bar represents the SD. The difference in protein concentration between treatment and control group was measured using One-way ANOVA for linear distribution.

  
  
  

  

  Neuroprotective effects of verbenalin (Ver) on amyloid beta (Aβ)-induced toxicity in human neuroblastoma SH-SY5Y cells. (A) Cells were exposed to verbenalin at concentrations of 1, 5, 10, 20, and 40 μM for 72 h. The control cells were not treated. Cell viability was measured by the MTT assay and was calculated as a percentage of that in the control group (100%). The results are expressed as the means ± standard error of the mean (SEM) of independent experiments (n = 6, 96-well plate). ***p < 0.001 as compared to control. Cells were pre-treated with 20 μM verbenalin for 24 h and then exposed to 5 μM Aβ for 72 h. The results are expressed as the means ± standard error of the mean (SEM) of independent experiments (n = 6, 96-well plate). ^†p < 0.1, *p < 0.05, **p < 0.01 compared with the group exposed to Aβ only (ANOVA followed by Dunnett’s multiple comparisons test). (B) Cell viability was measured by the MTT assay and was calculated as a percentage of that in the control group (100%). (C) A bioluminescence assay was used to measure cellular ATP levels, and the results are shown as relative intracellular ATP levels. (D) Levels of intracellular reactive oxygen species (ROS) were measured using a fluorescence cell-based assay, and results are shown as relative intracellular ROS (n=4).

  
  
  

  

  Effect of verbenalin treatment on the expressions of EGF, VEGF, and NRG1 in Aβ-induced human neuroblastoma SH-SY5Y cells. (A) Gene expressions were evaluated by real-time PCR. Each value represents the mean ± SD (n = 4). (B) Boxplots of protein concentration (ng/ml) obtained by ELISA (n = 4). Box ranges from 25^th to 75^th percentile; the line in the middle represents the median value; the error bar represents the SD. Asterisks refer to statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001) by One-way ANOVA followed by Dunnett’s multiple comparisons test (for linear distribution) as compared with only Aβ-treated group.

  
  
  

• Research Paper Volume 12, Issue 1 pp 242-259

  Epithelial cell senescence induces pulmonary fibrosis through Nanog-mediated fibroblast activation

  Relevance score: 4.9274025

  Xiang Chen, Hongyang Xu, Jiwei Hou, Hui Wang, Yi Zheng, Hui Li, Hourong Cai, Xiaodong Han, Jinghong Dai

  Keywords: idiopathic pulmonary fibrosis (IPF), epithelial cell senescence, pulmonary fibroblast activation, wnt/β-catenin signalling, nanog

  Published in Aging on December 31, 2019

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  Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease tightly correlated with aging. The pathological features of IPF include epithelial cell senescence and abundant foci of highly activated pulmonary fibroblasts. However, the underlying mechanism between epithelial cell senescence and pulmonary fibroblast activation remain to be elucidated. In our study, we demonstrated that Nanog, as a pluripotency gene, played an essential role in the activation of pulmonary fibroblasts. In the progression of IPF, senescent epithelial cells could contribute to the activation of pulmonary fibroblasts via increasing the expression of senescence-associated secretory phenotype (SASP). In addition, we found activated pulmonary fibroblasts exhibited aberrant activation of Wnt/β-catenin signalling and elevated expression of Nanog. Further study revealed that the activation of Wnt/β-catenin signalling was responsible for senescent epithelial cell-induced Nanog phenotype in pulmonary fibroblasts. β-catenin was observed to bind to the promoter of Nanog during the activation of pulmonary fibroblasts. Targeted inhibition of epithelial cell senescence or Nanog could effectively suppress the activation of pulmonary fibroblasts and impair the development of pulmonary fibrosis, indicating a potential for the exploration of novel anti-fibrotic strategies.

  

  Increased epithelial cell senescence occurred in idiopathic pulmonary fibrosis (IPF). (A) The mRNA levels of p16 and p21 were measured by Q-PCR, **P < 0.01. (B) The protein levels of collagen I, p16 and p21 were measured by Western blot. (C) SA-β-gal activity was revealed by X-gal staining. The expression of p16 was determined by immunohistochemical analysis. (D, E) The normal lung (Con) and lung of a patient with idiopathic pulmonary fibrosis (IPF) were double stained with E-cadherin and p21 (D) or p16 (E) by immunofluorescence.

  
  
  

  

  Rapamycin could protect mice from bleomycin (BLM)-induced pulmonary fibrosis. Mice (n = 10 in each group) were intraperitoneally injected with vehicle (DMSO/PBS, 10%) or 5 mg/kg rapamycin every other day starting 7 days after administration of BLM (5 mg/kg). (A) Pulmonary fibrosis was determined by haematoxylin and eosin (H&E) staining. Collagen was revealed by Masson’s trichrome staining. The expression of p21 was measured by immunohistochemical analysis. (B) The protein levels of p16, p21, α-SMA and collagen I were detected by Western blot. The expression levels were quantified with ImageJ (n = 3). GAPDH was used as a loading control, *P < 0.05 and **P < 0.01. (C, D) The lung tissues were double stained with E-cadherin and p21 (C), α-SMA and collagen I (D) by immunofluorescence. The positive areas of p21 and collagen I were quantified by densitometry (n = 3), **P < 0.01.

  
  
  

  

  Epithelial cell senescence could induce pulmonary fibroblast activation via activating Wnt/β-catenin signalling. (A–C) MLE-12 cells were treated with bleomycin (BLM, 25 μg/ml) for the indicated times. (A, B) SA-β-gal staining and β-galactosidase activity measurement were performed to detect cellular senescence, * P < 0.05 and ** P < 0.01 vs. 0 h. (C) The protein levels of p21 and p16 were measured by Western blot. The expression levels were quantified with ImageJ (n = 3). GAPDH was used as a loading control, *P < 0.05 and **P < 0.01. (D–I) MLE-12 cells were pre-treated with or without BLM for 3 days. The medium was replaced by fresh medium without BLM and co-cultured with pulmonary fibroblasts for another 3 days. (D) The migration capacity of pulmonary fibroblasts was detected by using a wound-healing assay. Wound areas were calculated by ImageJ, **P < 0.01. (E) The proliferation ability of pulmonary fibroblasts was measured by EdU assay. The percentage of proliferating cells were calculated by ImageJ, **P < 0.01. (F) The protein levels of collagen I, vimentin and α-SMA were determined by Western blot. The expression levels were quantified with ImageJ (n = 3). GAPDH was used as a loading control, **P < 0.01. (G) Pulmonary fibroblasts were double stained with vimentin and collagen I by immunofluorescence. (H) Pulmonary fibroblasts were double stained with α-SMA and periostin by immunofluorescence. (I) The expression of β-catenin was measured by immunofluorescence. (J) MLE-12 cells were pre-treated with or without BLM for 3 days. MLE-12 cells were cultured with fresh medium without BLM for another 3 days. The supernatants were collected to culture pulmonary fibroblasts in the presence or absence of ICG-001. The expression of β-catenin, α-SMA and collagen I were examined by Western blot. The expression levels were quantified with ImageJ (n = 3). GAPDH was used as a loading control, *P < 0.05 and **P < 0.01.

  
  
  

  

  Rapamycin could suppress epithelial cell senescence and fibroblast activation via impairing the production of SASP. (A–E) MLE-12 cells were treated with bleomycin (BLM), followed by treatment with or without rapamycin for 3 days. (A) The expression of p16 and p21 were measured by Western blot. The expression levels were quantified with ImageJ (n = 3). GAPDH was used as a loading control, **P < 0.01. (B, C) The protein levels of p16 and p21 were detected by immunofluorescence. (D) The mRNA levels of IL-1β, IL-6, IL-8 and TNF-α were determined by Q-PCR, ** P < 0.01 vs. Con and ## P < 0.01 vs. BLM. (E, F) MLE-12 cells were treated as in Figure 4A and co-cultured with pulmonary fibroblasts in fresh medium for another 3 days. (E) The proliferation ability of pulmonary fibroblasts were measured by EdU assay. The percentage of proliferating cells was calculated by ImageJ, **P < 0.01. (F) The expression of α-SMA and collagen I were detected by Western blot. The expression levels were quantified with ImageJ (n = 3). GAPDH was used as a loading control, **P < 0.01.

  
  
  

  

  Aberrantly expressed Nanog in activated pulmonary fibroblasts and fibrotic lung tissues were mediated by Wnt/β-catenin. (A–C) The expression of Nanog in lung tissues derived from patients with idiopathic pulmonary fibrosis (IPF) was determined by Q-PCR (A), Western blot (B) and immunohistochemical analysis (C), ** P < 0.01 vs. Con. (D) The lung tissues of patients with IPF were double stained with α-SMA and Nanog by immunofluorescence. (E) The lung tissues derived from pulmonary fibrosis mouse models were double stained with α-SMA and Nanog via immunofluorescence. (F) The expression of Nanog in pulmonary fibroblasts isolated from fibrotic mouse lung tissues were measured by immunofluorescence. (G) Cells were treated as in Figure 3D. Pulmonary fibroblasts were double stained with α-SMA and Nanog by immunofluorescence. (H, I) Pulmonary fibroblasts were treated with Wnt3a for various durations. (H) The mRNA level of Nanog was detected by Q-PCR, * P < 0.05 and ** P < 0.01 vs. D0. (I) ChIP assays were performed by using chromatin isolated from Wnt3a treated pulmonary fibroblasts. The final DNA extracts were analysed by Q-PCR, ** P < 0.01.

  
  
  

  

  Nanog silencing could suppress pulmonary fibroblast activation and impair the development of pulmonary fibrosis. (A–C) Pulmonary fibroblasts were transfected with LV-Nanog-siRNA and co-cultured with MLE-12 cells as in Figure 3D. (A) The mRNA levels of Nanog, Oct4 and Rex1 were measured by Q-PCR, ** P < 0.01 vs. Con and ## P < 0.01 vs. bleomycin (BLM). (B) The protein levels of collagen I and α-SMA were determined by Western blot. The expression levels were quantified with ImageJ (n = 3). GAPDH was used as a loading control, *P < 0.05 and **P < 0.01. (C) The expression of collagen I and α-SMA were further examined by immunofluorescence staining. (D–G) Mice were intratracheally injected with 5 × 10⁸ TU/ml LV-Nanog-siRNA or negative control (NC) 7 days after administration of BLM. Mice were sacrificed on day 21 after BLM instillation. (D) Pulmonary fibrosis was determined by haematoxylin and eosin (H&E) staining and collagen I was revealed by Sirius Red/Fast Green staining. (E) The mRNA levels of Nanog, α-SMA and collagen I were determined by Q-PCR, ** P < 0.01 vs. NC and ## P < 0.01 vs. NC + BLM. (F) The protein levels of Nanog, collagen I and α-SMA were measured by Western blot. The expression levels were quantified with ImageJ (n = 3). GAPDH was used as a loading control, *P < 0.05 and **P < 0.01. (G) The expression of α-SMA and collagen I were further confirmed by immunofluorescence staining.

  
  
  

• Research Paper Volume 11, Issue 24 pp 11844-11864

  Hydrogen sulfide attenuates mitochondrial dysfunction-induced cellular senescence and apoptosis in alveolar epithelial cells by upregulating sirtuin 1

  Relevance score: 6.012976

  Ruijuan Guan, Zhou Cai, Jian Wang, Mingjing Ding, Ziying Li, Jingyi Xu, Yuanyuan Li, Jingpei Li, Hongwei Yao, Wei Liu, Jing Qian, Bingxian Deng, Chun Tang, Dejun Sun, Wenju Lu

  Keywords: hydrogen sulfide, cigarette smoke extract, alveolar epithelial cell, mitochondria injury, senescence

  Published in Aging on December 23, 2019

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  Hydrogen sulfide (H₂S), an endogenous gaseous signal molecule, regulates many pathologies related to aging. Sirtuin 1 (SIRT1) has been shown to protect against mitochondrial dysfunction and other pathological processes, including premature senescence. This study was aimed to investigate whether and how H₂S attenuates senescence and apoptosis of alveolar epithelial cells via a SIRT1-dependent mechanism. Our results showed that treatment with sodium hydrosulfide (NaHS), a donor of H₂S, attenuated cigarette smoke extract (CSE)-induced oxidative stress, mitochondrial dysfunction, cellular senescence and apoptosis in A549 cells. This was associated with SIRT1 upregulation. SIRT1 activation by a pharmacological activator, SRT1720, attenuated CSE-induced oxidative stress and mitochondrial dysfunction in A549 cells. While SIRT1 inhibition by EX 527 or silencing by siRNA transfection significantly attenuated or abolished the ability of NaHS to reverse the CSE-induced oxidative stress, mitochondrial dysfunction and the imbalance of mitochondrial fusion and fission. Also, SIRT1 inhibition or silencing abolished the protection of NaHS against CSE-induced cellular senescence and apoptosis. In conclusion, H₂S attenuates CSE-induced cellular senescence and apoptosis by improving mitochondrial function and reducing oxidative stress in alveolar epithelial cells in a SIRT1-dependent manner. These findings provide novel mechanisms underlying the protection of H₂S against cigarette smoke-induced COPD.

  

  Effects of NaHS on cell viability and apoptosis in CSE-stimulated A549 cells. (A, B) A549 cells were treated with different doses of CSE or NaHS for 48h. The cells stimulated with vehicle only served as controls. Cell viability was detected by CCK-8 assay. ^*P<0.05, ^**P<0.01, significantly different from control cells. A549 cells were cultured with and without 3% CSE and/or 100, 200, or 400μM NaHS for 48 h. (C) Cell viability of A549 cells with different treatments was measured by CCK-8 assay. (D) A549 cells were stained with Hoechst 33258 after treating with and without 3% CSE and/or 400μM NaHS for 48 h, and were examined under the fluorescence microscopy. (E) The cells were double-stained with Annexin V-FITC and PI, and then the cellular apoptosis was determined by flow cytometry. (F) The ratio of apoptotic cells was statistically analyzed. (G, H) The protein levels of Bcl-2, Bax and Cleaved caspase 3 were detected using Western blot. ^**P<0.01, significantly different from control cells [3% CSE (-) and NaHS (-)]; ^#P<0.05, ^##P<0.01, significantly different from cells treated with 3% CSE only.

  
  
  

  

  Effects of NaHS on cell senescence in CSE-stimulated A549 cells. A549 cells were cultured with and without 3% CSE and/or 100, 200, or 400μM NaHS for 48 h. Cell senescence was performed by examining the (A) the SA–β-gal activity. (B) the mRNA level of p21 by Real-time PCR. (C, D) the protein levels of p53 and p21 by Western blot. (E–G) The mRNA levels of IL-6, IL-8 and MMP-2 were detected using Real-time PCR. ^**P<0.01, significantly different from control cells [3%CSE (-) and NaHS (-)]; ^#P<0.05, ^##P<0.01, significantly different from cells treated with 3%CSE only.

  
  
  

  

  Effects of NaHS on CSE-induced oxidative stress in A549 cells. A549 cells treated with and without 3% CSE and/or 400μM NaHS for 48 h. Representative microphotographs showing intracellular ROS (A) and mtROS (B) generation respectively. (C) A549 cells were cultured with and without 3% CSE and/or 100, 200, or 400μM NaHS for 48 h. Western blot was used to analyze the protein expression of FOXO3. ^**P<0.01, significantly different from control cells [3%CSE (-) and NaHS (-)];^##P<0.01, significantly different from cells treated with 3%CSE only.

  
  
  

  

  Effects of NaHS on mitochondrial function in CSE-stimulated A549 cells. A549 cells were cultured with and without 3% CSE and/or 100, 200, or 400μM NaHS for 48 h. (A) The bioenergetic profiles of A549 cells were measured by a Seahorse Extracellular Flux Analyzer, OCR in cells treated with oligomycin, FCCP, and rotenone and Antimycin A. (B) Quantitative analysis of basal respiration, ATP production, maximal respiratory and spare capacity is shown. (C) mtDNA copy number was measured by Real-time PCR. (D) The mRNA levels of COXI, COXII, COXIII, ATPase 6 and Cyto b were detected using Real-time PCR. ^*P<0.05, ^**P<0.01, significantly different from control cells [3%CSE (-) and NaHS (-)]; ^#P<0.05, ^##P<0.01, significantly different from cells treated with 3%CSE only.

  
  
  

  

  Effects of NaHS on SIRT1 mRNA and protein expressions in CSE-stimulated epithelial A549 cells. A549 cells were cultured with and without 3% CSE and/or 100, 200, or 400μM NaHS for 48 h. (A) The mRNA level of SIRT1 was detected using Real-time PCR. (B) The protein level of SIRT1 was detected using Western blot. (C) Immunofluorescence staining for SIRT1 was performed on A549 cells treated with and without 3% CSE and 400μM NaHS for 48 h. ^*P<0.05, ^**P<0.01, significantly different from control cells [3%CSE (-) and NaHS (-)]; ^#P<0.05, ^##P<0.01, significantly different from cells treated with 3%CSE only.

  
  
  

  

  Effects of SIRT1 on the NaHS-mediated reduction in the oxidative stress in CSE-stimulated A549 cells. A549 cells were cultured with SIRT1 inhibitor (EX 527) in the absence and presence of 3% CSE and NaHS for 48 h. (A) Generation of intracellular ROS was determined by the ROS Assay Kit. (B) Generation of mtROS was determined by the MitoSOX^TM Red Assay Kit.

  
  
  

  

  Effects of SIRT1 on the NaHS-mediated mitochondrial damage in CSE-stimulated A549 cells. A549 cells were cultured with SIRT1 inhibitor (EX527) in the absence and presence of 3% CSE and NaHS for 48 h. (A) The bioenergetic profiles of A549 cells were measured by a Seahorse Extracellular Flux Analyzer, OCR in cells treated with oligomycin, FCCP, and rotenone and Antimycin A. (B) Quantitative analysis of basal respiration, ATP production, maximal respiratory and spare capacity is shown. (C) Mitochondrial permeability potential was determined by JC-1 staining. Red fluorescence represented normal membrane potential, and green fluorescence represented mitochondrial membrane potential depolarization. ^*P<0.05, ^**P<0.01, significantly different from control cells [CSE (-), NaHS (-) and EX 527 (-)]; ^#P<0.05, ^##P<0.01, significantly different from cells treated with 3% CSE only; ^&&P<0.01, significantly different from cells treated with EX 527 only.

  
  
  

  

  Effects of SIRT1 on the NaHS-mediated the alteration of mitochondrial morphology and mitochondrial dynamics-related protein expression in CSE-stimulated A549 cells. A549 cells were cultured with SIRT1 inhibitor (EX 527) in the absence and presence of 3% CSE and NaHS for 48 h. (A) Representative images for visualization of the mitochondrial morphology in A549 cells. (B, C) Western blot was used to analyze the protein level of MFN1. (D) Real-time PCR was performed to examine the mRNA level of OPA1. (E) Immunofluorescence staining of FIS1 was performed. ^*P<0.05, ^**P<0.01, significantly different from control cells [3% CSE (-), NaHS (-) and EX 527 (-)]; ^##P<0.01, significantly different from cells treated with 3% CSE only; ^&&P<0.01, significantly different from cells treated with EX 527 only.

  
  
  

  

  Effects of SIRT1 on the NaHS-mediated cellular senescence and apoptosis in CSE-stimulated A549 cells. A549 cells were cultured with SIRT1 inhibitor (EX527) in the absence and presence of 3% CSE and NaHS for 48 h. (A) Cell senescence was performed by examining the the SA–β-gal activity. (B) The cells were double-stained with Annexin V-FITC and PI, and then the cellular apoptosis was determined by flow cytometry. (C) The ratio of apoptotic cells was statistically analyzed. (D–G) Western blot was used to analyze the protein levels of p53, p21, Bcl-2 and Bax. ^**P<0.01, significantly different from control cells [3%CSE (-), NaHS (-) and EX 527 (-)]; ^#P<0.05, ^##P<0.01, significantly different from cells treated with 3%CSE only; ^&P<0.05, ^&&P<0.01, significantly different from cells treated with EX 527 only.

  
  
  

• Research Paper Volume 11, Issue 24 pp 12497-12531

  Age-related changes in eye lens biomechanics, morphology, refractive index and transparency

  Relevance score: 6.8934293

  Catherine Cheng, Justin Parreno, Roberta B. Nowak, Sondip K. Biswas, Kehao Wang, Masato Hoshino, Kentaro Uesugi, Naoto Yagi, Juliet A. Moncaster, Woo-Kuen Lo, Barbara Pierscionek, Velia M. Fowler

  Keywords: fiber cell, strain, epithelial cell, cataract, stiffness

  Published in Aging on December 16, 2019

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  Life-long eye lens function requires an appropriate gradient refractive index, biomechanical integrity and transparency. We conducted an extensive study of wild-type mouse lenses 1-30 months of age to define common age-related changes. Biomechanical testing and morphometrics revealed an increase in lens volume and stiffness with age. Lens capsule thickness and peripheral fiber cell widths increased between 2 to 4 months of age but not further, and thus, cannot account for significant age-dependent increases in lens stiffness after 4 months. In lenses from mice older than 12 months, we routinely observed cataracts due to changes in cell structure, with anterior cataracts due to incomplete suture closure and a cortical ring cataract corresponding to a zone of compaction in cortical lens fiber cells. Refractive index measurements showed a rapid growth in peak refractive index between 1 to 6 months of age, and the area of highest refractive index is correlated with increases in lens nucleus size with age. These data provide a comprehensive overview of age-related changes in murine lenses, including lens size, stiffness, nuclear fraction, refractive index, transparency, capsule thickness and cell structure. Our results suggest similarities between murine and primate lenses and provide a baseline for future lens aging studies.

  

  Side view pictures of mouse lenses between 2-30 months of age pre-compression, during coverslip compression (1, 5 and 10 coverslips) and post-compression, and the isolated lens nucleus. With age, the application of the same load compressed the older lenses less than young lenses. There is an overall increase in lens size and nucleus size with age. The axial diameter (red double-headed arrows) and the equatorial diameter (green double-headed arrows) for each lens were measured to calculate lens volume, lens aspect ratio, axial compressive strain, equatorial expansion strain, resilience and nuclear volume. In very old lenses (24-30 months), there is an area of optical discontinuity in the lens cortex (yellow arrowheads). Scale bar, 1mm.

  
  
  

  

  Lens volume and aspect ratio for mouse lenses between 2–30 months of age. Lines on the plots reflect mean ± SD of n = at least 8 lenses per age. The graph next to the data plots shows the 95% confidence interval. Any comparisons not crossing the dotted line are statistically significant (p < 0.05). (A) Lens volume (mm³) from mice between 2–30 months of age. The volume increases steadily between 2-8 months of age and more slowly after 8 months. (B) The lens aspect ratio (axial/equatorial diameter) drops slightly between 2 to 4 months of age and then remains unchanged with age. Mouse lenses become slightly more spherical between 2 and 4 months.

  
  
  

  

  Nuclear volume and fraction for mouse lenses between 2–30 months of age. Lines on the plots reflect mean ± SD of n = at least 8 lenses per age. The graph next to the data plots shows the 95% confidence interval. Any comparisons not crossing the dotted line are statistically significant (p < 0.05). (A) The volume (mm³) of the lens nucleus steadily increases with age. (B) Since nuclear volume increases more than lens volume with age, the nuclear fraction (nuclear/lens volume) increases with age.

  
  
  

  

  Lens stiffness and resilience for mouse lenses between 2–30 months of age. Plots reflect mean ± SD of n = at least 8 lenses per age. The graph next to the data plots shows the 95% confidence interval. Any comparisons not crossing the dotted line are statistically significant (p < 0.05). (A, B) Compression testing using sequential application of coverslips showed a steady decrease in axial and equatorial strain with age, indicating that lenses from older mice are stiffer. (C) Very old lenses from 30-month-old mice had increased resilience, calculated as the ratio of the pre-compression over post-compression axial diameter. Resilience for 30-month-old lenses was 98.8% ± 1.2% while resilience for younger lenses was ~94-96%.

  
  
  

  

  Live lens measurements of capsule thickness and anterior epithelial cell area and fixed lens measurements of cortical fiber cell width. Lines on the plots reflect mean ± SD of n = at least 6 lenses per age. Data from 2-month-old samples are reprinted from our previous publication [53]. *, p<0.05; **, p<0.01; ****, p<0.0001. (A) Lens capsule thickness increases between 2 months and older ages, but the thickness is unchanged after 4 months of age. WGA (lens capsule) is shown in green, and tdTomato signal (basal surface of anterior epithelial cells) is shown in red. (B) Anterior epithelial cell area is increased between 2 and 4 months and 12 months of age. Cell nuclei (Hoechst) is shown in blue, and tdTomato signal (lateral membrane of anterior epithelial cells) is shown in grayscale. (C) Cortical fiber cell width increases between 2-month-old and older lenses, but there is no increase in fiber cell width after 4 months of age. The fiber cells are numbered showing 11 full-width cells in the 2-month-old lens and 10 full-width cells in lenses that were 4 months and older. These measurements show that although mouse lenses continue to increase in size with age, capsule thickness and fiber cell size only increase until about 4 months of age. There is a mild increase in epithelial cell size up to 12 months of age.

  
  
  

  

  Lens images (top down view) from mice between 2-30 months of age in various B6 wild-type backgrounds. (A) B6-albino wild-type mice have clear lenses up to 8 months of age and develop small anterior opacities (arrowheads) by 12 months of age. Lenses from mice between 12-18 months develop cortical haziness (asterisks). Old lenses from mice between 24-30 months display ring cataracts (arrows) with a clear periphery and translucent, but not transparent, central regions. (B, C) Similar to B6-albino wild-type lenses, C57BL6 and B6SJL wild-type lenses also develop anterior opacities (arrowheads), cortical haziness (asterisks) and ring cataracts (arrows) at the same age as B6-albino wild-type mice. These images reveal that aged mouse lenses in the B6 genetic background develop cataracts around 12 months of age at the anterior pole and the lens cortex (haziness and ring opacity). Scale bars, 1mm.

  
  
  

  

  Whole lens staining for F-actin (phalloidin, green) and nuclei (DAPI, red) in 4-month-old and 18-month-old lenses. (A) The maximum intensity projection of the anterior lens epithelium and underlying fibers in the 4-month-old lens shows evenly distributed epithelial cell nuclei (DAPI) with a normal branched Y-suture (F-actin) under the epithelial cells. In contrast, there is an obvious defect at the apex of the 18-month-old lens with abnormal distribution of epithelial cell nuclei (DAPI, arrows) and a gap in the anterior suture (F-actin, arrowheads). (B) A 2D YZ projection of the 3D reconstruction of a Z-stack through the anterior epithelium and underlying fiber cells in the 4-month-old lens reveals tight adhesion of the anterior epithelial and fibers. In the 18-month-old lens near the fiber cell defect, the anterior epithelial cell layer is wrinkled and is depressed into the gap of the Y-suture. Although there was a defect in the epithelial cell sheet organization, there was no evidence of multilayered epithelial cells or abnormal epithelial cell proliferation in the 18-month-old lens. These results reveal that anterior cataracts in 18-month-old lenses are correlated with detachment and wrinkling of the anterior epithelial cells from the underlying Y-suture formed by fiber cells. Scale bars, 0.5mm in A and 20μm in B.

  
  
  

  

  Side view lens images and scanning electron microscopy (SEM) at various depths in 8-month-old and 24-month-old lenses. Boxed regions in green on the low magnification SEM image indicate the approximate location where high magnification images were obtained. Cortical, newly formed fiber cells are disorganized in the 24-month-old lens compared to orderly cortical fibers in the 8-month-old lens. Differentiating fiber cells in deeper cortex layers (~100–200μm from the surface) of the 24-month-old lens lack normal small protrusions and formed a distinct zone of compaction. The location of the zone of compaction is correlated with the ring opacity (red arrows, red box). Mature inner fiber cells (~200-400μm from the surface) are comparable between the 8- and 24-month-old lenses with large paddles and small protrusions. Scale bars, 1mm (lens picture and low magnification SEM) and 4μm (high magnification SEM).

  
  
  

  

  Transmission electron microscopy (TEM) of lens cross sections at various depths in 3–29-month-old lenses. Two neighboring cells in each panel are pseudo-colored green and yellow to show cell shape and size. In the 3- and 8-month-old lenses, fiber cells are hexagonal in shape and uniform in size from the periphery to the inner mature fiber cells. In the 12-month-old lens, the most peripheral fibers have lost their distinct hexagonal shape, but cells are still similarly sized between neighboring layers. In the 29-month-old lens, the cells have lost their characteristic hexagon cell shape and are highly variable in shape and size. There is also variability in electron density between neighboring cells in the 29-month-old lens with dark and light gray cells. Scale bar, 4μm.

  
  
  

  

  Whole lens staining for F-actin (phalloidin, green) and nuclei (DAPI, red) in 4-month-old and 18-month-old lenses reveal that the actin cytoskeleton in epithelial cells and the formation of organized meridional rows of hexagonal equatorial epithelial cells is similar between 4-month-old and 18-month-old lenses. (A) Single XY planes through anterior epithelial cells show similar F-actin staining and organization of equatorial epithelial cells between 4-month-old and 18-month-old lenses. These cells have a network of basal F-actin, membrane-adjacent F-actin and sequestered actin bundles near the lateral membrane, and polygonal arrays on the apical surface. (B) Single XY planes through the meridional rows at the lens equator reveals organized hexagonally-shaped epithelial cells with normal membrane-adjacent F-actin networks and a basal meshwork of F-actin in the 4-month-old and 18-month-old lenses. These data reveal that fiber cell shape changes and disorganization in older lenses is not due to altered shape or misalignment of equatorial epithelial cells. Scale bar, 20μm.

  
  
  

  

  3D mesh and 2D contour plots of the gradient of refractive index (GRIN) in whole eyes from mice between 2 weeks to 24 months of age. Plots are through the mid-sagittal plane and the mid-coronal plane passing through each central lens nucleus. The anterior of the eye (A), the posterior of the eye (P) and the lens (L) are marked on the mid-sagittal views of the 2-week-old eye. The dotted line through the 2D sagittal view of the 2-week-old eye represents the location of the mid-coronal 3D and 2D heat maps. All images are oriented in the same direction. Colors reflect the magnitude of refractive index from low refractive index in dark blue (1.30) to high refractive index in dark red (1.55). The areas with highest refractive index are the lens. Mouse lens GRIN profiles are two-tiered with a ring of indentation (bright yellow) clearly seen in the 3D mesh plots. There is a cap region of high refractive index (red and orange) and a bottom region (yellow and green). There is an increase in the size of the cap region with age. These data show that GRIN in mouse lens develops by 2 weeks of age, and there is a rapid increase and plateau of maximum refractive index at the center of the lens with age.

  
  
  

  

  A comparison of the diameter of the cap region of high refractive index and the diameter of the extracted lens nucleus. The images show a representative mid-sagittal 2D contour plot and a representative lens nucleus with double-headed arrows indicating measured diameters. The graph compares the cap diameter in the sagittal 2D GRIN plot to the diameter of the lens nucleus. Lines on the plots reflect mean ± SD of n = at least 3 lenses from different mice per age. There was no statistically significant difference between the cap and nucleus diameters indicating that the area of high refractive index is directly correlated with the hard and compact lens nucleus. Scale bars, 1mm.

  
  
  

  

  Average GRIN profiles along the visual axis and maximum refractive index in lenses from mice between 2 weeks to 24 months of age. Lines on the plots reflect mean ± SD of n = at least 3 lenses from different mice per age. The graph next to the data plots shows the 95% confidence interval. Any comparisons not crossing the dotted line are statistically significant (p < 0.05). (A) Average GRIN profiles increased in magnitude until about 6 months of age and then remained relatively unchanged with age. There is a statistically significant difference between profiles of different ages, except between the 12 months and 24 months profiles. (B) Max refractive index is ~1.55 in mouse lenses. Maximum refractive index rapidly increases until 6 months of age and then remains steady after 6 months of age.

  
  
  

  

  Wild-type mouse lenses in the B6 genetic background showed increased volume, nucleus size and overall stiffness, changes in cell morphology and microstructure along with appearance of anterior, cortical and ring cataracts with age. Lens volume and nucleus volume increase steadily with age. The shape and size of lens fiber cells become more disorganized in aged lenses. With age, mouse lenses develop anterior and cortical cataracts. Anterior cataracts are correlated with detachment of the anterior epithelial cells from the underlying fiber cells. Cortical ring opacities in the aged lenses are due to a zone of compaction in the cortical fiber cells leading to an optical discontinuity. While there is a steady increase in lens stiffness with age, resilience, or lens elasticity, is only increased in very old lenses. The maximum refractive index at the center of the lens (nucleus) increases rapidly until 6 months of age and reaches a plateau at 6 months. Lens capsule thickness and fiber cell width remain steady after 4 months of age, while epithelial cell area increases slightly between 4 and 12 months of age. Cartoons not all drawn to scale.

  
  
  

• Research Paper Volume 11, Issue 9 pp 2699-2723

  Laminin α4 overexpression in the anterior lens capsule may contribute to the senescence of human lens epithelial cells in age-related cataract

  Relevance score: 5.0775423

  Yu Yan, Haiyang Yu, Liyao Sun, Hanruo Liu, Chao Wang, Xi Wei, Fanqian Song, Hulun Li, Hongyan Ge, Hua Qian, Xiaoguang Li, Xianling Tang, Ping Liu

  Keywords: age-related cataract, anterior lens capsule, laminin α4, human lens epithelial cell, senescence, basement membrane

  Published in Aging on May 10, 2019

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  Senescence is a leading cause of age-related cataract (ARC). The current study indicated that the senescence-associated protein, p53, total laminin (LM), LMα4, and transforming growth factor-beta1 (TGF-β1) in the cataractous anterior lens capsules (ALCs) increase with the grades of ARC. In cataractous ALCs, patient age, total LM, LMα4, TGF-β1, were all positively correlated with p53. In lens epithelial cell (HLE B-3) senescence models, matrix metalloproteinase-9 (MMP-9) alleviated senescence by decreasing the expression of total LM and LMα4; TGF-β1 induced senescence by increasing the expression of total LM and LMα4. Furthermore, MMP-9 silencing increased p-p38 and LMα4 expression; anti-LMα4 globular domain antibody alleviated senescence by decreasing the expression of p-p38 and LMα4; pharmacological inhibition of p38 MAPK signaling alleviated senescence by decreasing the expression of LMα4. Finally, in cataractous ALCs, positive correlations were found between LMα4 and total LM, as well as between LMα4 and TGF-β1. Taken together, our results implied that the elevated LMα4, which was possibly caused by the decreased MMP-9, increased TGF-β1 and activated p38 MAPK signaling during senescence, leading to the development of ARC. LMα4 and its regulatory factors show potential as targets for drug development for prevention and treatment of ARC.

  

  Senescence associated markers increase with the grades of age-related cataract (ARC) in human anterior lens capsules (ALCs). Human ALCs graded before surgery for cataract severity using the Emery-Little Classification System of nuclear opacity grade were obtained from the anterior surface of cataractous lenses during surgery. (A) Photographs for representing immunoblot analysis of p53 and corresponding Coomassie brilliant blue (CBB) staining in cataractous ALCs of different grades and ages. (B) Relative expression levels of p53 immunoblots in cataractous ALCs. Quantification of immunoblots was processed using Image J. The figure depicts Pearson correlation between age and p53 protein expression (n = 144). (C) The correlation between patient age and ARC grades (Data were analyzed via One-way ANOVA). (D) The correlation between relative p53 expression and ARC grades (Data are analyzed via Wilcoxon Rank Sum Test). Data were shown as mean ± SD. *, p<0.05; **, p<0.01; ***, p<0.001 versus grade II group. #, p<0.05 versus grade III group.

  
  
  

  

  Excess laminins (LMs) in the senescent anterior lens capsules (ALCs) of age-related cataract (ARC). Representative photographs of hematoxylin and eosin (HE) staining of cataractous ALCs (A) and immunohistochemistry (IHC) of LMs in cataractous ALCs (B) (Scale bars: 50 μm). (C) Total LM in cataractous ALCs with different grades as detected by ELISA (Data were shown as mean ± SD and were analyzed by one-way ANOVA). ***, p<0.001 versus grade II group. The figure depicts Pearson correlation between total LM expression and relative p53 expression (n = 128) (D). (E) Immunoblot analysis of LM subunits in mixed cataractous ALCs (n = 10). (F) LM trimers in mixed cataractous ALCs as detected by LMα4. Immunoprecipitation-immunoblotting (IP-IB) using antibodies against LMα4 to precipitate LM trimers and antibodies against LMγ1, LMβ2 and LMβ1 for IB (n = 10).

  
  
  

  

  Premature senescence model of human lens epithelial cells (HLE B-3) induced by H₂O₂. Cells of the control group were cultured in medium only, whereas cells of senescent group were cultured in medium with H₂O₂ for 96 h. (A) Viabilities (left) of HLE B-3 cells treated with different concentrations of H₂O₂ (0–600 μM) for 96 h, as measured via an MTT assay. Morphologic changes (right) of HLE B-3 cells following a 96 h exposure to 400 μM H₂O₂. (B) Percentage of SA-β-gal-positive cells in HLE B-3 cells treated with different concentrations of H₂O₂ (0–600 μM) (left). SA-β-gal activity as measured by cell staining (right). (C) Immunoblot analysis of GLB1, p21 and P53 in HLE B-3 cells. (D) Immunofluorescence analysis of p21 (green) in HLE B-3 cell nuclei. (Scale bars: 100 μm). Data were shown as mean ± SD and are representative of 3 independent experiments.

  
  
  

  

  Elevated laminins (LMs) in senescent HLE B-3 cells and cell basement membranes (BMs) induced by H₂O₂. Cells (or cell BMs) of the control group were cultured in medium only, while cells (or cell BMs) of senescent group were cultured in medium with H₂O₂ (400 μM) for 96 h. (A) Total LM in HLE B-3 cells as detected by ELISA (data were analyzed by paired t-test). (B) Immunoblot analysis of 11 LM subunits, collagen 1α1, ATP1A1, MMP-9, and TGF-β1 in HLE B-3 cells. (C) Total LM in HLE B-3 cell BMs as detected by ELISA (data were analyzed by paired t-test). (D) SDS-PAGE analysis followed by CBB staining of HLE B-3 cell BMs. (E) Immunoblot analysis of LM subunits and MMP-9 in HLE B-3 cell BMs. (F) Immunofluorescence analysis of LMα4, LMβ3, and MMP-9 in HLE B-3 cell BMs (Scale bars: 50 μm). (G) TGF-β1 in HLE B-3 cell BMs, as detected by ELISA (data were analyzed using One-way ANOVA). Data are shown as mean ± SD. *, p<0.05; **, p<0.01 versus control group.

  
  
  

  

  MMP-9 reduces cell senescence and laminin (LM) deposition induced by H₂O₂. Senescent HLE B-3 cells [or cell basement membranes (BMs)] were cultured in medium with H₂O₂ (400 μM) for 96 h. (A-I) HLE B-3 cells were treated with H₂O₂ only, or in combination with indicated plasmid. (A) Immunoblot analysis of MMP-9 in HLE B-3 cells. (B-C) Percentage of SA-β-gal-positive cells (B) and protein expression of GLB1 (C) in HLE B-3 cells. (D) Total LM in HLE B-3 cells, detected by ELISA. (E) Protein expressions of TGF-β1 and LM subunits in HLE B-3 cells, detected by IB. (F) Total LM in HLE B-3 cell BMs, detected by ELISA. (G) SDS-PAGE analysis followed by CBB staining of HLE B-3 cell BMs. (H) Immunoblot analysis of LMα4 and LMα1 in HLE B-3 cell BMs. (I) Immunofluorescence analysis of LMα4 (green) and LMβ3 (green) in HLE B-3 cell BMs (Scale bars: 50 μm). (J-M) HLE B-3 cells were treated with H₂O₂ only, or in combination with indicated siRNA. (J) The percentage of SA-β-gal-positive cells in HLE B-3 cells. (K) Total LM in HLE B-3 cells, as detected by ELISA. (L) Immunoblot analysis of LM subunits and TGF-β1 in HLE B-3 cells. (M) Total LM in HLE B-3 cell BMs, as detected by ELISA. Data are shown as mean ± SD and were analyzed using paired t-test. *, p<0.05; ***, p<0.001.

  
  
  

  

  TGF-β1 enhances cell senescence and laminin (LM) deposition induced by H₂O₂. Senescent HLE B-3 cells [or cell basement membranes (BMs)] were cultured in medium with H₂O₂ (400 μM) for 96 h. (A-G) HLE B-3 cells were treated with H₂O₂ only, or in combination with LY2109761 (5 µM) or SB431542 (10 µM) for 72 h. (A-C) Percentage of SA-β-gal-positive cells (A), protein expression of GLB1 (B) and immunofluorescence analysis of p21 (C) in HLE B-3 cells. (D) Total LM in HLE B-3 cells, as detected by ELISA. (E) Immunoblot analysis of LM subunits and MMP-9 in HLE B-3 cells. (F) Total LM in HLE B-3 cell BM, as detected by ELISA. (G) Immunofluorescence analysis of LMα4 (green) and LMβ3 (green) in HLE B-3 cell BMs (Scale bars: 100 μm). (H) HLE B-3 cells treated with TGF-β1 (15 ng/ml) for 96 h. Protein expression levels of TGF-β1, p21, MMP-9 and LM subunits in HLE B-3 cells analyzed via IB. Data were shown as mean ± SD and were analyzed using paired t-test. *, p<0.05; **, p<0.01.

  
  
  

  

  Interactions between laminin α4 (LMα4) and the activated p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway in cell senescence. (A-E) Cells were treated with sheep anti-LMα4 globular domain antibodies (2 μg/ml) for 96 h, while cells treated with sheep IgG (2 μg/ml) were selected as the control group. (A) Percentage of SA-β-gal-positive cells. (B) Migratory abilities of HLE B-3 cells. (C) Cell viabilities of HLE B-3 cells measured by CCK-8 assay. (D) Total TGF-β1 in HLE B-3 cells detected by ELISA. (E) Immunoblot analysis of p-p38, collagen 1α1, MMP-9, and LMs in HLE B-3 cells. (F) HLE B-3 cells treated with 400 μM H₂O₂ for 96 h only, or in combination with indicated siRNA. Immunoblot analysis of LMα4, p21, p53, TGF-β1, MMP-9 and ATP1A1 in HLE B-3 cells. (G-I) HLE B-3 cells treated with H₂O₂ (400 μM) only for 96 h, or in combination with SB203580 (30 μM). (G) Immunoblot analysis of p-p38 and T-p38 (total p38) in HLE B-3 cells. (H) Percentage of SA-β-gal-positive cells. (I) Immunoblot analysis of TGF-β1, collagen 1α1, MMP-9 and LMs in HLE B-3 cells. (J) HLE B-3 cells were treated with H₂O₂ (400 μM) only for 96 h, or in combination with indicated siRNA. Immunoblot analysis of MMP-9, p-p38 and LMα4 in HLE B-3 cells. (K-L) HLE B-3 cells were treated with H₂O₂ (400 μM) only for 96 h, or in combination with R)-(+)-Limonene (1000 μM). (K) Percentage of SA-β-gal-positive cells. (L) Immunoblot analysis of p-p38, T-p38, MMP-9 and LMα4 in HLE B-3 cells. Data were shown as mean ± SD and were analyzed using the paired t-test. *, p<0.05.

  
  
  

  

  Elevated TGF-β1 and laminin α4 (LMα4) in the cataractous anterior lens capsules (ALCs) with senescence. Human ALCs, graded before surgery for cataract severity by the Emery-Little Classification System of nuclear opacity grade, were obtained from the anterior surface of cataractous lenses during surgery. (A) Total TGF-β1 in human ALCs with ARC of grade II and grade V groups, as detected by ELISA. The figure depicts a Pearson correlation between TGF-β1 expression and senescence (n = 22) (B). (C) LMα4 subunit in human ALC groups groups with ARC grades II and V, as detected by ELISA. The figure depicts a Pearson correlation between LMα4 expression and senescence (n = 64) (D). (E) Association between LMα4 and total LM in ALCs with ARC (n = 60). (F) Association of LMα4 and TGF-β1 in ALCs with ARC (n = 9). Data were shown as mean ± SD and were analyzed using the Wilcoxon Rank Sum Test. *, p<0.05.

  
  
  

  

  Summary of anterior lens capsular proteins and possible regulatory mechanism in age-related cataract (ARC). Schematic diagram of reactive oxygen species related senescent lens epithelial cells and senescent anterior lens capsules along with up-regulation of total LM, LMα4 and TGF-β1 and down-regulation of MMP-9.

  
  
  

• Research Paper Volume 9, Issue 2 pp 524-546

  Compound effects of aging and experimental FSGS on glomerular epithelial cells

  Relevance score: 5.305703

  Remington R.S. Schneider, Diana G. Eng, J. Nathan Kutz, Mariya T. Sweetwyne, Jeffrey W. Pippin, Stuart J. Shankland

  Keywords: kidney disease, glomerulus, parietal epithelial cell, podocyte, epithelial to mesenchymal transition, Collagen IV

  Published in Aging on February 17, 2017

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  Advanced age portends a poorer prognosis in FSGS. To understand the impact of age on glomerular podocytes and parietal epithelial cells (PECs), experimental FSGS was induced in 3m-old mice (20-year old human age) and 27m-old mice (78-year old human age) by abruptly depleting podocytes with a cytopathic anti-podocyte antibody. Despite similar binding of the disease-inducing antibody, podocyte density was lower in aged FSGS mice compared to young FSGS mice. Activated PEC density was higher in aged versus young FSGS mice, as was the percentage of total activated PECs. Additionally, the percentage of glomeruli containing PECs with evidence of phosphorylated ERK and EMT was higher in aged FSGS mice. Extracellular matrix, measured by collagen IV and silver staining, was higher in aged FSGS mice along Bowman’s capsule. However, collagen IV accumulation in the glomerular tufts alone and in glomeruli with both tuft and Bowman’s capsule accumulation were similar in young FSGS and aged FSGS mice. Thus, the major difference in collagen IV staining in FSGS was along Bowman’s capsule in aged mice. The significant differences in podocytes, PECs and extracellular matrix accumulation between young mice and old mice with FSGS might explain the differences in outcomes in FSGS based on age.

  

  Albuminuria was higher in aged mice at baseline and in FSGS. (A-D) Sheep IgG staining. Sheep IgG staining confirmed the equal distribution of anti-glomerular antibody in the glomerular tuft in both young (Y) and aged (A) mice. Images were taken at 100x. (A'-D') 400x close-up images of glomeruli from (A-D). Sheep IgG deposition (purple color) was not seen in baseline mice as expected (A,B,A',B'). Animals given FSGS with the anti-glomerular antibody showed sheep IgG deposition in the glomerular tufts (C, D, C’, D'). (E) Albumin to creatinine ratios (ACR). ACRs (µg/mg) for young mice (black circles), and aged mice (white circles), at baseline prior to FSGS and at days 7, 14, 21 and 28 post-FSGS. ACR increased acutely for both young and aged animals to peak at D7, followed by gradual recovery for 21 days. Aged mice started with higher ACR at baseline and finished with significantly higher ACR at D28.

  
  
  

  

  Podocyte density was lower at baseline in aged mice, and in aged mice with FSGS. (A-C) Quantification of podocyte density. Graphs A and B show the average podocyte density in podocytes per glomerular volume (µm³) for individual animals in OC and JM glomeruli respectively. Graph C shows podocyte density for individual animals when OC and JM glomeruli are combined, which serves as a representation of the entire section. Podocyte density was lower in aged baseline mice than young baseline mice in glomeruli of the OC (A), JM (B), and when combined (C). Aged FSGS mice also had lower podocyte density in OC (A), JM (B), and combined (C) glomeruli than young FSGS mice, despite young mice experiencing a larger magnitude of podocyte depletion with FSGS. (D-G) PAS/p57 double staining. Representative images of glomeruli at 20x magnification, with higher magnifications shown in D’-G’ of the glomerulus marked by solid black square. Podocytes were identified by p57⁺ staining (brown color, nuclear) against the pink PAS counterstain.

  
  
  

  

  Parietal epithelial cell (PEC) activation was highest in aged FSGS mice. PECs were identified by PAX8 staining (green color, nuclear), and the subset of PECs undergoing activation were identified by CD44 staining (red color, cytoplasmic). (A-C) Quantitation of the percentage of PECs that are activated. (A) In outer cortical (OC) glomeruli, the percentage of PECs that were activated (PAX8⁺CD44⁺) along Bowman’s capsule (BC) was higher in aged mice at baseline and in FSGS. (B) In juxta-medullary (JM) glomeruli, the percentage of activated PECs were similar at baseline, but higher in aged FSGS mice compared to young FSGS mice. (C) When OC and JM glomeruli were combined, aged FSGS mice had the highest percentage of PECs that were activated. (D-G) Representative images of Pax8 and CD44 staining on BC. Images taken at 400x by confocal microscopy for PAX8 (green, nuclear), CD44 (red, cytoplasmic) and DAPI (blue, nuclear) staining. (D’-G’) Higher magnification of the white square shown above. Solid arrow shows examples of PAX8 staining; dashed arrow shows examples of CD44 below (D’-G’). As shown in the above graphs, the percentage of PECs along Bowman’s capsule that are activated was higher in aged baseline mice, and increased further at D28 of FSGS in aged mice.

  
  
  

  

  Activated PECs migrated from Bowman’s capsule to the glomerular tuft in FSGS. (A-C) Quantitation showing the percentage of glomeruli with activated PECs (PAX8⁺CD44⁺) on the glomerular tuft. Aged FSGS mice had the highest percentage of activated PECs on the tufts of outer cortical (OC) (A), juxta-medullary (B) and combined OC and JM (C) glomeruli. (D-G) Representative images of Pax8 and CD44 staining on tuft. Images of glomeruli (400x) taken by confocal microscopy, showing staining for PAX8 (PEC marker, green, solid arrows), CD44 (activation marker, red, dashed arrows) and DAPI (nuclei, blue). Glomeruli are marked by the dashed line. (D’-G’) Higher power images of the area demarcated by the solid square shown above. PAX8 staining was detected along Bowman’s capsule in young baseline (D, D’) and aged baseline (E, E’) mice, but not in the glomerular tuft. (F, F’) In young FSGS mice, activated PECs were not readily detected on glomerular tufts. (G, G’) Activated PECs were detected on the glomerular tuft of aged FSGS mice. These results show that activated PECs were detected on the tuft of a subset of aged FSGS glomeruli.

  
  
  

  

  Percentage of glomeruli with phosphorylated-ERK stained PECs was highest in aged baseline mice with FSGS. (A-C) Quantitation showing the percentage of glomeruli with pERK staining of PECs along Bowman’s capsule. Aged mice and aged mice with FSGS had a higher percentages of glomeruli with pERK staining along Bowman’s capsule when compared to their respective young baseline and young FSGS mice in outer cortical glomeruli (OC) (A), juxta-medullary glomeruli (B) and combined OC and JM glomeruli(C). Overall, aged FSGS mice had the highest percentage of glomerular with pERK staining (C). (D-G) Representative images of pERK staining along Bowman’s capsule. Representative images of glomeruli at 100x magnification, with 400x magnifications shown in D’-G’ of the glomeruli marked by solid black square. Dashed arrows indicate pERK negative and solid arrows indicated pERK positive glomeruli (100x) and PECs (400x).

  
  
  

  

  EMT marker staining along Bowman’s capsule was highest in aged FSGS mice. (A-C) Quantitation showing the percentage of glomeruli with α-SMA (EMT marker) staining along BC. There was no significant difference in young and aged mice at baseline in OC (A), JM (B), or combined (C) glomeruli. In OC (A), JM (B), and combined (C) glomeruli, α-SMA staining increased with disease in aged animals, while only in the JM (B) was α-SMA staining significantly increased in young mice, likely due to large variation within the sample groups. (D-G) Representative images of glomeruli with alpha-SMA staining along BC taken at 40x. Frequency and intensity α-SMA staining increased with disease (D vs. F, E vs. G), despite similar levels between young and aged animals at baseline (D vs. E). (D’-G’) Higher power images of the area demarcated by the solid square shown above, emphasizing the increase in α-SMA staining of cells along BC (F’, G’).

  
  
  

  

  Extracellular matrix accumulation was higher in Bowman’s capsule of aged FSGS mice. (A-L) Collagen IV (Col IV) staining. Representative images taken at 40x of Col IV staining (brown color) along Bowman’s capsule only (A-D, solid arrows), glomerular tuft only (E-H, dashed arrows), or along Bowman’s capsule and the glomerular tuft (I-L, solid and dashed arrows respectively). (M-X) Jones’(Silver) staining. Representative images taken at 40x of Jones’ basement membrane staining along BC only (M-P, yellow solid arrow), in the glomerular tuft only (Q-T, dashed yellow arrow), and both along BC and in the tuft (U-X, solid and dashed yellow arrows respectively) confirmed the staining patterns of Col IV.

  
  
  

• Research Paper Volume 2, Issue 1 pp 28-42

  Attainment of polarity promotes growth factor secretion by retinal pigment epithelial cells: Relevance to age-related macular degeneration

  Relevance score: 4.9274025

  Shozo Sonoda, Parameswaran G. Sreekumar, Satoru Kase, Christine Spee, Stephen J Ryan, Ram Kannan, David R Hinton

  Keywords: retinal pigment epithelial cell, cell polarity, VEGF-A, PEDF, BMP-4, age-related macular degeneration

  Published in Aging on December 27, 2009

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  The antiangiogenic and neurotrophic growth factor, pigment epithelial derived factor (PEDF), and the proangiogenic growth factor, vascular endothelial growth factor-A (VEGF), are released from retinal pigment epithelial (RPE) cells where they play a critical role in the pathogenesis of age-related macular degeneration (AMD). Since RPE polarity may be altered in advanced AMD, we studied the effect of polarization of differentiated, human RPE monolayer cultures on expression and secretion of PEDF and VEGF. Polarized RPE demonstrated apical microvilli, expression of tight junction proteins, apical localization of Na/K- ATPase, and high transepithelial resistance (490 ± 17 Ω•cm²). PEDF secretion was about 1000 fold greater than that for VEGF in both polarized and non-polarized cultures. Polarization of the RPE monolayer increased PEDF secretion, which was predominantly apical, by 34 fold (p<0.02) and VEGF secretion, which was predominantly basolateral, by 5.7 fold (p<0.02). Treatment of non-polarized RPE cultures with bone morphogenetic protein-4 (BMP-4) had no effect on PEDF or VEGF secretion, but resulted in a dose-dependent >2-fold increase in basolateral VEGF secretion (p<0.05) in polarized cultures. Our data show that polarity is an important determinant of the level of PEDF and VEGF secretion in RPE and support the contention that loss of polarity of RPE in AMD results in marked loss of neurotrophic and vascular support for the retina potentially leading to photoreceptor loss and blindness.

  

  Evidence for tight junction proteins and polarity in fetal RPE cells cultured on Transwell filters for 6 weeks. (A, B) Immunofluorescence staining of tight junction proteins ZO-1 and occludin. (C) Localization of Na/K- ATPase to the apical plasma membrane as shown in the confocal vertical (X-Z) section (white arrow). (D) Well differentiated apical microvilli observed by scanning electron microscopy (SEM). (E) Well developed microvilli (mv), localization of pigment on the apical side (asterisks), nuclei on basal side (N), and presence of tight-junctional complexes (arrows) by transmission electron microscopy (TEM).

  
  
  

  

  Secretion from the polarized RPE cells represent the sum of experimentally determined apical and basolateral secretion values, normalized for total cellular protein. The total secretion increased 34 fold for PEDF and 5.7 fold for VEGF-A (A). Analysis of cellular protein (B) and mRNA (C) showed that expression in polarized human RPE was higher compared to nonpolarized RPE cells.

  
  
  

  

  Human polarized RPE cells on transwells isolated from 3 different donors preferentially secreted PEDF (A) to the apical side of the tissue and VEGF-A (B) to the basolateral side. The bars represent average of 2 determinations for each donor with variation in each sample <5%.

  
  
  

  

  Staining for PEDF is more intense in polarized RPE as compared to nonpolarized RPE. The apical region shows much higher PEDF expression in polarized cells.

  
  
  

  

  (A, B, C) Expression of p27 (green) and its localization to nuclei (blue). (D, E, F). Polarized RPE cultures show lack of expression of Ki-67 (green) in the nuclei. Nuclei counterstained blue with DAPI.

  
  
  

  

  (A) Transepithelial resistance (TER) of polarized human RPE monolayers and effect of rhBMP-4 treatment. TER values in human RPE monolayers, maintained for 1 month in 1% FBS-containing medium, averaged 490 ±17 Ω. cm² (mean ± SEM, n=48). The TER measurements in polarized RPE cells exposed to rhBMP-4 treatment for 24 h showed no significant difference (P>0.05) versus untreated controls (n=9/group). (B) Expression levels of tight junction proteins, ZO-1 and occludin were not significantly different between the BMP-4 treated and the untreated control groups. (C) No significant cell death was observed by TUNEL staining in highly polarized RPE cells of both untreated control and 100ng/ml BMP-4 treatment groups.

  
  
  

  

  Secretion of VEGF-A (A) and PEDF (C) are presented along with the corresponding cellular VEGF-A (B) and cellular PEDF (D) from three different donors. Data are presented as fold difference as compared to untreated controls. The cellular concentrations of VEGF-A and PEDF did not differ from untreated controls for the entire BMP-4 concentration range.

  
  
  

  

  Fold change over control values calculated from ELISA analysis is presented to account for inter donor variations. (A) The increase in VEGF-A secretion after treatment with BMP-4 from the apical domain was not statistically significant (p>0.05). (B) An increase in VEGF-A secretion from the basolateral domain was found even with the lowest dose used (10ng/ml) which increased further in a dose-dependent fashion. Asterisk indicates that VEGF-A secretion with 75 and 100ng/ml BMP-4 treatment was significantly higher than that of control (p<0.05). (C) The cellular levels of VEGF-A were not significantly affected by BMP-4 treatment. (D, E, F) No significant change was observed for PEDF secretion either at the apical domain or the basolateral domain and in cellular PEDF levels. Data are mean±SEM from four different donors.

  
  
  

  

  Expression of VEGF-A (A) and PEDF (B) mRNA in polarized fetal RPE cells vs controls was analyzed by real-time PCR. BMP-4 treatment caused an increase in VEGF-A gene expression, especially at 50, 75, and 100ng/ml BMP-4 treatment which was significantly different from controls (p<0.05). PEDF mRNA did not change with BMP-4 dose for the BMP-4 dose range studied.

  
  
  

• Research Perspective Volume 1, Issue 8 pp 740-745

  What determines the switch between atrophic and neovascular forms ofage related macular degeneration? - the role of BMP4 induced senescence

  Relevance score: 5.8888335

  DanHong Zhu, Xuemei Deng, Jing Xu, David R Hinton

  Keywords: BMP4, age related macular degeneration, senescence, retinal pigment epithelial cell, oxidative stress

  Published in Aging on August 12, 2009

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  Age-related macular degeneration (AMD), the leadingcause of blindness in the elderly, targets the retinal pigment epithelium(RPE), a monolayer of cells at the back of the eye. As AMD progresses, itcan develop into two distinct forms of late AMD: "dry," atrophic AMD,characterized by RPE senescence and geographic RPE loss, and "wet,"neovascular AMD, characterized by RPE activation with abnormal growth ofchoroidal vessels. The genetic and molecular pathways that lead to thesediverse phenotypes are currently under investigation. We have found thatbone morphogenetic protein-4 (BMP4) is differentially expressed in atrophicand neovascular AMD. In atrophic AMD, BMP4 is highly expressed in RPE, andmediates oxidative stress induced RPE senescencein vitro via Smadand p38 pathways. In contrast, in neovascular AMD lesions, BMP4 expressionin RPE is low, possibly a result of local expression of pro-inflammatorymediators. Thus, BMP4 may be involved in the molecular switch determiningwhich phenotypic pathway is taken in the progression of AMD.

  

  Diagram illustrating the progression of early age related macular degeneration (AMD) into 2 divergent late stages and the potential role of BMP4 as a switch between these pathways. Chronic stressors such as oxidative stress can promote the expression of BMP4 in the retinal pigment epithelium (RPE) and induce RPE senescence as part of the phenotype of early AMD. If BMP4 expression is sustained, it could lead to RPE apoptosis and geographic atrophy. In other individuals, activation of the senescence activated secretory pathway and expression of pro-inflammatory mediators could result in increased expression of interleukin (IL)-8, decreased expression of BMP4 and increased expression of vascular endothelial growth factor (VEGF) resulting in neovascular AMD with choroidal angiogenesis.

  
  
  

  

  Expression of BMP4 in late stages of age related macular degeneration (AMD). Immunohistochemical stains for BMP4 (red chromogen) in retinal pigment epithelium (RPE)/choroid tissue sections from donor eyes with hematoxylin counterstain. In (A) a control individual without AMD shows no apparent BMP4 staining in RPE or choroid. In (B) an individual with late dry AMD, away from a region of geographic atrophy shows prominent BMP4 immunoreactivity in RPE and in the accumulated drusen material between the RPE and the choroid. In (C) an individual with neovascular form of late AMD shows no apparent BMP4 staining in the RPE or the neovascular lesion between the RPE and retina. In (D) an individual with neovascular form of late AMD that further progressed to scar with loss of neovascular channels shows re-expression of BMP4 staining in cells within and adjacent to the lesion. Note loss of most cells in RPE layer. The institutional review board (IRB) of the University of Southern California approved our use of human donor eyes. All procedures conformed to the Declaration of Helsinki forresearch involving human subjects.

  
  
  

  

  IL-8 protein concentration in culture medium measured by ELISA. ARPE-19 cells were treated with 150 uM H₂O₂ in culture medium with 10% fetal bovine serum for 2 hours and allowed to recover in stressor-free ARPE medium for 22 hours. The procedure was repeated to generate the next treatment cycle. The twice treated cells were allowed to stay in 1% serum ARPE medium for 72 hours after stress before proceeding to further analytic assays. The culture media from control and senescent RPE cells were collected and used directly for ELISA measurement. IL-8 secretion level was measured in pg/ml using human IL-8 ELISA Kit (BioLegend, Inc., San Diego, CA) according to manufacturer's instructions. The level of IL-8 secretion shown here was averaged from a triplicate of each sample and from 3 independent repeats of H₂O₂ treatments. Student's t test was used for statistical analysis (**; p < 0.0005).

  
  
  

• Research Paper pp undefined-undefined

  Core fucosylation regulates alveolar epithelial cells senescence through activating of transforming growth factor-β pathway in pulmonary fibrosis

  Relevance score: 6.2735853

  Yu Jiang, Zhongzhen Wang, Jinying Hu, Wei Wang, Na Zhang, Lili Gao

  Keywords: cell senescence, idiopathic pulmonary fibrosis, core fucosylation, alveolar epithelial cell

  Published in Aging on Invalid Date

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  Idiopathic pulmonary fibrosis (IPF), a fatal disorder associated with aging, has a terrible prognosis. However, the potential causes of IPF remain a riddle. In this study, we designed to explore whether the modification of the core fucosylation (CF) can ameliorate pulmonary fibrosis by targeting alveolar epithelial cells (AECs) senescence. First, we verified that cellular senescence occurs in the bleomycin-induced lung fibrosis mice models and CF modifications accompanying senescent AECs in pulmonary fibrosis. Next, both gain- and loss- of function research on CF were performed to elucidate its role in promoting AECs senescence and triggering pulmonary fibrosis in vitro. Notably, using alveolar epithelial cell-specific FUT8 conditional knockout mouse models, however, inhibition of cellular senescence by deleting the FUT8 gene could attenuate pulmonary fibrosis in vivo. Finally, blocking the CF modification of transforming growth factor -β type I receptor (TGF-βR I) could reduce the activation of downstream transforming growth factor -β (TGF-β) pathways in AECs senescence both in vivo and in vitro. This study reveals that CF is a crucial interventional target for the treatment of pulmonary fibrosis. Blocking CF modification contributes importantly to inhibiting AECs senescence resulting in pulmonary fibrosis lessen.

• Research Paper pp undefined-undefined

  Modulating in vitro lung fibroblast activation via senolysis of senescent human alveolar epithelial cells

  Relevance score: 6.5578084

  Joseph S. Spina, Tracy L. Carr, Lucy A. Phillips, Heather L. Knight, Nancy E. Crosbie, Sarah M. Lloyd, Manisha A. Jhala, Tony J. Lam, Jozsef Karman, Meghan E. Clements, Tovah A. Day, Justin D. Crane, William J. Housley

  Keywords: cellular senescence, fibrosis, senolytic, senomorphic, SASP, alveolar epithelial cell

  Published in Aging on Invalid Date

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  Idiopathic pulmonary fibrosis (IPF) is an age-related disease with poor prognosis and limited therapeutic options. Activation of lung fibroblasts and differentiation to myofibroblasts are the principal effectors of disease pathology, but damage and senescence of alveolar epithelial cells, specifically type II (ATII) cells, has recently been identified as a potential trigger event for the progressive disease cycle. Targeting ATII senescence and the senescence-associated secretory phenotype (SASP) is an attractive therapeutic strategy; however, translatable primary human cell models that enable mechanistic studies and drug development are lacking. Here, we describe a novel system of conditioned medium (CM) transfer from bleomycin-induced senescent primary alveolar epithelial cells (AEC) onto normal human lung fibroblasts (NHLF) that demonstrates an enhanced fibrotic transcriptional and secretory phenotype compared to non-senescent AEC CM treatment or direct bleomycin damage of the NHLFs. In this system, the bleomycin-treated AECs exhibit classical hallmarks of cellular senescence, including SASP and a gene expression profile that resembles aberrant epithelial cells of the IPF lung. Fibroblast activation by CM transfer is attenuated by pre-treatment of senescent AECs with the senolytic Navitoclax and AD80, but not with the standard of care agent Nintedanib or senomorphic JAK-targeting drugs (e.g., ABT-317, ruxolitinib). This model provides a relevant human system for profiling novel senescence-targeting therapeutics for IPF drug development.

• Research Paper pp undefined-undefined

  Natural variation in macrophage polarization and function impact pneumocyte senescence and susceptibility to fibrosis

  Relevance score: 6.7509475

  Eun Joo Chung, Seokjoo Kwon, Uma Shankavaram, Ayla O. White, Shaoli Das, Deborah E. Citrin

  Keywords: senescence, macrophage, alveolar epithelial cell Type II, strain

  Published in Aging on Invalid Date

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  Radiation-induced pulmonary fibrosis (RIPF), a late adverse event of radiation therapy, is characterized by infiltration of inflammatory cells, progressive loss of alveolar structure, secondary to the loss of pneumocytes and accumulation of collagenous extracellular matrix, and senescence of alveolar stem cells. Differential susceptibility to lung injury from radiation and other toxic insults across mouse strains is well described but poorly understood. The accumulation of alternatively activated macrophages (M2) has previously been implicated in the progression of lung fibrosis. Using fibrosis prone strain (C57L), a fibrosis-resistant strain (C3H/HeN), and a strain with intermediate susceptibility (C57BL6/J), we demonstrate that the accumulation of M2 macrophages correlates with the manifestation of fibrosis. A comparison of primary macrophages derived from each strain identified phenotypic and functional differences, including differential expression of NADPH Oxidase 2 and production of superoxide in response to M2 polarization and activation. Further, the sensitivity of primary AECII to senescence after coculture with M2 macrophages was strain dependent and correlated to observations of sensitivity to fibrosis and senescence in vivo. Taken together, these data support that the relative susceptibility of different strains to RIPF is closely related to distinct senescence responses induced through pulmonary M2 macrophages after thoracic irradiation.