February 2009
Volume 50, Issue 2
Free
Retinal Cell Biology  |   February 2009
Subtoxic Oxidative Stress Induces Senescence in Retinal Pigment Epithelial Cells via TGF-β Release
Author Affiliations
  • Alice L. Yu
    From the Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany; the
  • Rudolf Fuchshofer
    Department of Anatomy, University of Regensburg, Regensburg, Germany; the
  • Daniel Kook
    From the Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany; the
  • Anselm Kampik
    From the Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany; the
  • Hans Bloemendal
    Department of Biomolecular Chemistry NCMLS, Radboud University Njimegen, Njimegen, The Netherlands; and the
  • Ulrich Welge-Lüssen
    From the Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany; the
    Department of Ophthalmology, Friedrich-Alexander-University, Erlangen, Germany.
Investigative Ophthalmology & Visual Science February 2009, Vol.50, 926-935. doi:10.1167/iovs.07-1003
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Alice L. Yu, Rudolf Fuchshofer, Daniel Kook, Anselm Kampik, Hans Bloemendal, Ulrich Welge-Lüssen; Subtoxic Oxidative Stress Induces Senescence in Retinal Pigment Epithelial Cells via TGF-β Release. Invest. Ophthalmol. Vis. Sci. 2009;50(2):926-935. doi: 10.1167/iovs.07-1003.

      Download citation file:


      © 2016 Association for Research in Vision and Ophthalmology.

      ×
  • Supplements
Abstract

purpose. The goal of the present study was to determine whether oxidative stress and transforming growth factor (TGF)-β induce cellular senescence in human retinal pigment epithelial (RPE) cells.

methods. Cultured human RPE cells were exposed to 50 to 150 μM hydrogen peroxide (H2O2) for 1 and 2 hours or treated with 1.0 ng/mL TGF-β1 or -β2 for 12, 24, and 48 hours. Senescence-associated β-galactosidase (SA-β-Gal) activity was detected by histochemical staining. Expression of senescence-associated genes (apolipoprotein J [Apo J], connective tissue growth factor [CTGF], fibronectin, and SM22) was examined by real-time PCR and induction of signal transduction proteins (p21, p16, and pRb) by Western blot analysis. The effects of TGF-β blocking on the oxidative stress-induced expression of senescence-associated biomarkers were investigated by simultaneous incubation with neutralizing antibodies against the TGF-β1, -β2, and -β3 isoforms and the TGF-βII receptor.

results. H2O2 markedly increased the number of SA-β-Gal-positive cells to up to 89% and the expression of Apo J, CTGF, fibronectin, and SM22 by approximately three to fourfold. Treatment with TGF-β1 and -β2 showed similar changes. H2O2and TGF-β1 and -β2 markedly enhanced the expression of p21 but downregulated pRb. In contrast, they had no effect on p16 expression. Simultaneous treatment with neutralizing antibodies against the TGF-β1, -β2, and -β3 isoforms and the TGF-βII receptor prevented the oxidative stress–mediated elevation of senescence-associated biomarkers.

conclusions. Oxidative stress, TGF-β1, and TGF-β2 are capable of inducing cellular senescence in cultured human RPE cells. Therefore, reduction of oxidative stress and minimizing TGF-β may help to prevent senescence-associated changes in the RPE as seen in early age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of severe visual impairment in elderly individuals. 1 2 However, the complex pathogenesis of this disease is poorly understood, and no efficient therapy or prevention exists to date. It has long been suspected that oxidative damage is involved in the pathogenesis of AMD. 3 One of the most compelling lines of evidence that oxidative damage plays a role in AMD has been provided by epidemiologic studies showing that smoking significantly increases the risk of AMD. 4 5 6 Furthermore, it has recently been shown that in selected individuals, the progression of the disease was reduced by the use of antioxidants and zinc supplements. 7 In addition, endogenous sources of oxidative damage to retinal pigment epithelial (RPE) cells include photoreceptor outer segment phagocytosis, peroxidized lipid membranes, and photo-oxidative reactive oxygen intermediates. 8  
The progressive oxidative damage of macromolecules resulting from exposure of cellular components to oxidative stress has long been implicated in aging and age-related diseases. 9 Of interest, a substantial body of evidence, mostly from work in human fibroblasts, indicates that oxidative stress can also induce or accelerate the development of cellular senescence. 10 This phenomenon has been collectively termed stress-induced premature senescence (SIPS), which usually begins 2 to 3 days after stress exposure. 11 12 13 One commonly used noncytotoxic oxidative stress agent, which has been shown to induce SIPS in human cell types such as diploid fibroblasts, endothelial cells, and melanocytes, is hydrogen peroxide (H2O2). 11 12 13 14 H2O2 is able to cross the plasma membrane. 8 In human RPE cells, intracellular H2O2 is mainly produced by lipid peroxidation from phagocytosed rod outer segments. 8  
Cells in SIPS remain alive for months and display several features of cellular senescence. These features include senescence-associated β-galactosidase (SA-β-Gal) activity, as well as changes in the expression levels of several senescence-associated genes, such as apolipoprotein J (Apo J), connective tissue growth factor (CTGF), fibronectin, and SM22 (transgelin). 11 15 16 Mechanistically, these typical changes of SIPS are assumed to be mediated via two major signal transduction pathways: p21-pRb and p16-pRb pathways. 12 15 17 Upregulation of both tumor suppressor proteins p21 and p16 subsequently leads to hypophosphorylation of the pRb protein and hence induction of senescence. 12  
Several findings suggest that transforming growth factor (TGF)-β is also capable of inducing cellular senescence. For instance, stimulation of human diploid fibroblasts with TGF-β1 triggers the appearance of biomarkers of SIPS such as SA-β-Gal activity and increases mRNA steady state levels of senescence-associated genes including Apo J, fibronectin, and SM22. 14 18 In vitro studies of different cellular systems have shown that TGF-β1 is inducible by oxidative stress. 14 18 19 Thus, it has been hypothesized that oxidative stress-induced premature senescence are triggered via an increased expression of TGF-β1. 14 18 20 21 Previous studies have demonstrated that cellular senescence occurs in RPE cells during the aging process in primates. 22 Furthermore, it has been shown in vitro that cellular senescence in human RPE cells is inducible by exposure to mild hyperoxia. 23 Whether human RPE cells undergo senescent changes in AMD is unclear yet. Histochemical studies have detected an increased expression of TGF-β in the RPE of patients with AMD. 24  
The purpose of this work was to examine whether oxidative damage or TGF-β promotes stress-induced premature senescence in human RPE cells. In the present study, we demonstrated for the first time that subtoxic levels of hydrogen peroxide induced SIPS and the release of TGF-β1 and -β2 in cultured human RPE cells. Treatment with TGF-β1 and -β2 alone showed similar changes compared to H2O2. Simultaneous incubation with neutralizing antibodies against the TGF-β1, -β2, and -β3 isoforms and the TGF-βII receptor (TGF-βΙΙR) prevented these age-related changes. Furthermore, we were able to show that H2O2 and TGF-β1 and -β2 could activate the senescence-associated p21-pRb pathway. These data indicate a hitherto unknown role of oxidative stress and TGF-β in senescence-associated changes in human RPE cells, which may be helpful in revealing new pathomechanical processes in AMD. 
Methods
Isolation of Human RPE Cells
Eight human donor eyes were obtained from the Munich University Hospital Eye Bank and processed within 4 to 16 hours after death. The donors ranged in age between 25 and 40 years. None of the donors had a known history of eye disease. Human RPE cells were harvested after the procedure, as described previously. 25 In brief, whole eyes were thoroughly cleansed in 0.9% NaCl solution, immersed in 5% polyvinylpyrrolidone iodine (Jodobac; Bode-Chemie, Hamburg, Germany), and rinsed again in NaCl solution. The anterior segment from each donor eye was removed, and the posterior poles were examined with the aid of a binocular stereomicroscope to confirm the absence of gross retinal disease. Next, the neural retinas were carefully peeled away from the RPE-choroid-sclera with fine forceps. The eye cup was rinsed with Ca2+ and Mg2+ -free Hanks’ balanced salt solution, and treated with 0.25% trypsin (Invitrogen-Gibco, Karlsruhe, Germany) for 1 hour at 37°C. The trypsin was aspirated and replaced with Dulbecco’s modified Eagle’s medium (DMEM; Biochrom, Berlin, Germany) supplemented with 20% fetal calf serum (FCS; Biochrom). A pipette was used to gently agitate the media, releasing the RPE into the media by avoiding damage to Bruch’s membrane. 
Human RPE Cell Culture
The human RPE cell suspension was added to a 50-mL flask (Falcon, Wiesbaden, Germany) containing 20 mL of DMEM supplemented with 20% FCS and maintained at 37°C and 5% CO2. Epithelial origin was confirmed by immunohistochemical staining for cytokeratin with a pancytokeratin antibody (Sigma-Aldrich, Deisenhofen, Germany). 26 The cells were tested and found free of contaminating macrophages (anti-CD11; Sigma-Aldrich) and endothelial cells (anti-von Willebrand factor; Sigma-Aldrich). After reaching confluence, primary RPE cells were subcultured and maintained in DMEM supplemented with 10% FCS at 37°C and in 5% CO2. Confluent primary RPE cells of passage 3 to 5 were used for the experiments. For induction of oxidative stress, the cells were washed, incubated overnight in serum-free medium, and subsequently incubated with 50 to 150 μM H2O2 in fresh serum-free DMEM for 1 to 2 hours. Thereafter, the medium was changed and replaced by fresh serum-free DMEM for 12, 24, and 48 hours. Control cells were incubated under identical conditions without H2O2 treatment in the medium. To investigate the effects of TGF-β1 and -β2, the cells were washed, and the medium was changed to serum-free DMEM. After 24 hours incubation, the latter medium was replaced by fresh serum-free DMEM supplemented with 1.0 ng/mL active TGF-β1 and -β2 (R&D Systems, Wiesbaden, Germany). Under these conditions, the cells were incubated 12, 24, and 48 hours, respectively. In control cultures, the medium was changed at the same time points but neither TGF-β1 nor -β2 was added. To prove our hypothesis that the oxidative stress-induced release of TGF-β1 and -β2 into the medium is responsible for the increase of senescence biomarkers, RPE cells were simultaneously incubated with neutralizing antibodies against the TGF-β1, -β2, and -β3 isoforms (mAb-240; R&D Systems), diluted in serum-free DMEM at 3 μg/mL or with neutralizing antibodies against the TGF-βII receptor (TGF-βIIR; AF-241NA; R&D Systems) diluted in serum-free DMEM at 10 μg/mL. For control experiments, cells were incubated with IgG at a concentration of 10 μg/mL. The tetrazolium dye-reduction assay (MTT; 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; Sigma-Aldrich) was used to test cell viability before and at the end of the treatment. The results did not reveal any signs of increased cell death in oxidative stress or TGF-β-treated cells (data not shown). All experiments were performed at least in triplicate in RPE cultures from three donors. 
SA-β-Gal Activity
The proportion of RPE cells positive for the SA-β-Gal activity was determined as described by Dimri et al. 27 Briefly, treated RPE cells were washed twice with phosphate-buffered saline (PBS) and fixed with 2% formaldehyde and 0.2% glutaraldehyde in PBS at pH 6.0 at room temperature (RT) for 4 minutes. Cells were then washed twice with PBS and incubated under light protection for 8 hours at 37°C with fresh SA-β-Gal staining solution (1 mg/mL 5-bromo-4-chloro-3-indoyl-β-d-galactopyranoside [X-gal], 40 mM citric acid/sodium phosphate [pH 6.0], 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgCl2 diluted in PBS). The cells were then examined for the development of blue color and photographed at low magnification (200×) using a light microscope. All experiments were performed at least in triplicate in RPE cultures from three donors. 
RNA Isolation and Real-Time PCR
Total RNA was isolated from 10-cm Petri dishes by the guanidium thiocyanate-phenol-chloroform extraction method (Stratagene, Heidelberg, Germany). The structural integrity of the RNA samples was confirmed by electrophoresis in 1% Tris-acetate-EDTA (TAE)-agarose gels. Yield and purity were determined photometrically. After RNA isolation, mRNA was transcribed to cDNA via reverse transcription. This cDNA was then used for specific real-time PCR. Quantification of human Apo J, CTGF, fibronectin, SM22, and TGF-β1, -β2 and -β3 mRNA was performed with specific primers (Table 1)during 40 cycles with a thermocycler (LightCycler System; Roche Diagnostics, Mannheim, Germany). Primers and probes were found with a computer program (ProbeFinder, ver. 2.04; Roche Diagnostics). The standard curve was obtained from probes of three different untreated human RPE cell cultures. To normalize differences of the amount of total RNA added to each reaction, 18S rRNA (Table 1)was simultaneously processed in the same sample as an internal control. The levels of Apo J, CTGF, fibronectin, SM22, and TGF-β1, -β2, and -β3 mRNA were determined as the relative ratio (RR), which was calculated by dividing the level of mRNA by the level of the 18S rRNA housekeeping gene in the same samples. TGF-β3 could not be amplified from cultured human RPE cells (data not shown). All experiments were performed at least in triplicate in RPE cultures from three donors. 
Analysis of TGF-β1 and -β2 in Cell Supernatants
Levels of TGF-β1 and -β2 in cell supernatants were determined by using a TGF-β1 and -β2 specific sandwich enzyme-linked immunosorbent assay (ELISA; Quantikine; R&D System). As the ELISA only measured levels of the active protein, activation of the supernatants was undertaken to measure the total (latent + active) levels of both proteins. Briefly, cell supernatants were activated with 1.0 N HCl and subsequently neutralized with 1.2 N NaOH/0.5 M HEPES and analyzed using ELISA. All experiments were performed at least in triplicate in RPE cultures from three donors. The intra- and interassay coefficients of variation determined in our laboratory were 9.8% and 16%, respectively. 
Protein Extraction and Western Blot Analysis of p21, p16, and pRb
Cells grown on 35-mm tissue culture dishes were washed twice with ice-cold PBS, collected, and lysed in RIPA cell lysis buffer. After centrifugation (19,000g for 30 minutes at 4°C) in a microfuge, the supernatants were transferred to fresh tubes and stored at −70°C for future use. The protein content was measured by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL). Denatured proteins (2 μg) were separated under reducing conditions for p21 and p16 by electrophoresis using 12% SDS-polyacrylamide gels and for pRb by electrophoresis with 7.5% SDS-polyacrylamide gels. Thereafter, the proteins were transferred with semidry blotting onto a polyvinyl difluoride membrane (Roche) and probed with a mouse monoclonal antibody directed against p21 (DCS60; Cell Signaling Technology, Beverly, MA), rabbit polyclonal antibody against p16 ([C-20]:sc-468; Santa Cruz Biotechnology, Santa Cruz, CA), and goat polyclonal antibody against pRb ([Ser 795]:sc-21875, Santa Cruz Biotechnology), as described before. 28 These antibodies were used at a dilution of 1:200, respectively. Chemiluminescence was detected with the imager (LAS-1000; RayTest, Pforzheim, Germany). Exposure times ranged between 1 and 20 minutes. Quantification was performed on a computer (AIDA software; RayTest). All experiments were performed at least in triplicate in RPE cultures from three donors. 
Assessment of Intracellular Reactive Oxygen Species
Intracellular levels of reactive oxygen species (ROS) are an important biomarker for oxidative stress. Production of ROS was assessed with the fluorescent dye 2′7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Invitrogen). 29 30 31 Nonfluorescent H2DCF is oxidized to highly fluorescent 2′,7′-dichlorofluorescein (DCF) in the presence of intracellular ROS. Therefore, increased fluorescence indicates increased intracellular ROS levels. In our experiments, cells at selected time points after stimulation with H2O2 and TGF-β1 and -β2 were washed with DMEM and incubated with 20 μM H2DCFDA at 37°C for 30 minutes in the dark. The dye was then removed and the cells were washed three times with PBS. Afterward, the cells were scanned with a fluorescence reader (Tecan, Crailsheim, Germany) at 485-nm excitation and 535-nm emission. A blank (unlabeled cells) was measured and subtracted from all readings. 
Assessment of Lipid Peroxidation
Oxidative stress can be assessed by markers of lipid peroxidation. A sensitive and specific assay for lipid peroxidation is based on metabolic incorporation of the fluorescent oxidation-sensitive fatty acid, cis-parinaric acid (PNA), a natural 18-carbon fatty acid with four conjugated double bonds, into membrane phospholipids of cells). 29 32 Oxidation of PNA results in disruption of the conjugated double-bond system that cannot be resynthesized in mammalian cells. Therefore, lipid peroxidation was estimated by measuring loss of cis-parinaric acid (PNA) fluorescence. Briefly, treated cells were incubated with 10 μM PNA (Invitrogen-Molecular Probes, Eugene, OR) at 37°C for 30 minutes in the dark. The media was then removed and the cells washed three times with PBS. Afterward, the cells were scraped into 2 mL PBS with a rubber policeman. The suspension was then added to a fluorescence cuvette and measured at 312-nm excitation and 455-nm emission. A blank (unlabeled cells) was measured and subtracted from all readings. 
Results
Hydrogen Peroxide–Induced SA-β-Gal Activity
Human RPE cells were treated for 1 or 2 hours with 50, 100, or 150 μM H2O2 (Fig. 1) . The proportion of RPE cells positive for SA-β-Gal activity was determined in three independent experiments 12, 24, and 48 hours after stress exposure (Fig. 1C) . Untreated control cells showed 3% to 6% of SA-β-Gal-positive RPE cells (Figs. 1A 1C) . Exposure to H2O2 at all investigated concentrations and exposure and postincubation times markedly increased the number of SA-β-Gal-positive RPE cells (Fig. 1C) . The most pronounced effect was observed when the cells were kept for 48 hours after treatment with 150 μM H2O2 for either 1 or 2 hours (Figs. 1B 1C) . Under these conditions, the number of SA-β-Gal-positive RPE cells ranged from 78% ± 8% to 89% ± 6% (Figs. 1B 1C) . We have not observed any differences in SA-β-Gal staining in RPE cell cultures from different donors (data not shown). 
Hydrogen Peroxide–Induced mRNA Expression of Four Senescence-Associated Genes
Since H2O2 exposure of the cells for 1 or 2 hours produced comparable results, we have depicted only the effects of H2O2 treatment for 1 hour in our further experiments. As the most pronounced effect was observed after incubation of RPE cells with 150 μM H2O2, real-time PCR analyses of the four senescence-associated genes were performed with this treatment strategy (Fig. 2) . The signals generated in untreated control cells after 12 hours were set to 100% (Fig. 2) . Untreated control cells kept in serum-free medium for 24 and 48 hours showed similar results (data not shown). The mRNA expression of Apo J (Fig. 2A) , CTGF (Fig. 2B) , fibronectin (Fig. 2C) , and SM22 (Fig. 2D)were investigated in three independent experiments 12, 24, and 48 hours after H2O2 exposure. The most pronounced effects were seen in RPE cells 48 hours after 1 hours of incubation with 150 μM H2O2 (Fig. 2) . In these cells, the mRNA expression of Apo J increased by 3.2 ± 0.5-fold (Fig. 2A) , CTGF by 3.1 ± 0.5-fold (Fig. 2B) , fibronectin by 3.7 ± 0.4 fold (Fig. 2C) , and SM22 by 2.8 ± 0.3 fold (Fig. 2D)
Hydrogen Peroxide–Induced TGF-β1 and -β2 Expression
For determination of TGF-β1 and -β2 mRNA expression, real-time PCR analyses were conducted (Figs. 3A 3B) . The signals generated in untreated control cells were set to 100% (Figs. 3A 3B) . Twelve hours after a 1-hour exposure to 50, 100, or 150 μM H2O2, there was a dose-dependent increase of TGF-β1 mRNA expression to a maximum of 3.8 ± 0.1-fold (Fig. 3A) . Accordingly, there was a dose-dependent increase of TGF-β2 mRNA up to 5.6 ± 0.9-fold under the same conditions (Fig. 3B) . Extension of postincubation times to 24 (Figs. 3A 3B)and 48 hours (data not shown) showed comparable results. TGF-β3 could not be amplified from cultured human RPE cells (data not shown). 
We used ELISA analyses to detect active (Figs. 3C 3D)and total (Figs. 3E 3F)TGF-β1 and -β2 protein release into culture medium after H2O2 treatment. Levels of TGF-β1 and -β2 protein in untreated control cells showed a basal secretion of less than 50 pg/mL of active TGF-β1 (Fig. 3C)or active TGF-β2 (Fig. 3D)and less than 100 pg/mL of total TGF-β1 (Fig. 3E)or total TGF-β2 (Fig. 3F) . Active TGF-β1 (Fig. 3C)and TGF-β2 (Fig. 3D)protein secretion increased to approximately two to threefold 24 hours and 48 hours (data not shown) after treatment with 150 μM H2O2 for 1 hour. Under the same conditions, total TGF-β1 amount was even upregulated more than 10-fold (Fig. 3E) . Total TGF-β2 was increased more than 20-fold as early as 12 hours after treatment with 150 μM H2O2 for 1 hour (Fig. 3F)
TGF-β1 and -β2–Induced SA-β-Gal Activity and Senescence-Associated Biomarkers
To investigate whether TGF-β1 and -β2 are capable of inducing cellular senescence, cultured human RPE cells were treated with 1.0 ng/mL TGF-β1 and -β2 for 12, 24, and 48 hours in three independent experiments (Figs. 4 5) . In untreated control cells, the number of SA-β-Gal-positive cells was approximately 5% (Figs. 4A 4C) . The number of SA-β-Gal-positive cells increased to 57% ± 9% and 68% ± 6% after treatment with TGF-β1 for 24 and 48 hours (Fig. 4C)and to 88% ± 10% and 96% ± 3% after treatment with TGF-β2 for 24 and 48 hours (Figs. 4B 4C)
Real-time PCR was used to determine the mRNA levels of the four senescence-related genes Apo J, CTGF, fibronectin, and SM22 (Fig. 5) . The signals generated in untreated control cells after 12 hours were set to 100% (Fig. 5) . Untreated control cells kept in serum-free medium for 24 and 48 hours showed similar results (data not shown). Expression of all four senescence-associated genes was markedly upregulated after stimulation with 1.0 ng/mL TGF-β1 or -β2 for 12, 24, and 48 hours and reached its maximal level 24 hours after stress exposure (Fig. 5) . Apo J mRNA expression was maximally increased by 2.5 ± 0.5-fold after TGF-β1 and by 3.5 ± 0.2-fold after TGF-β2 treatment (Fig. 5A) . CTGF was maximally overexpressed by 3.6 ± 0.5-fold after TGF-β1 and by 4.8 ± 0.8-fold after TGF-β2 (Fig. 5B) , fibronectin was elevated by 2.7 ± 0.5-fold after TGF-β1 and by 4.7 ± 0.8-fold after TGF-β2 (Fig. 5C) , and SM22 was increased by 1.9 ± 1.0-fold after TGF-β1 and by 4.9 ± 0.8-fold after TGF-β2 treatment (Fig. 5D)
TGF-β1 and -β2 and Stress-Induced Premature Senescence
Based on these results, we speculate that TGF-β1 and -β2 may be responsible for the stress-induced premature senescence. We neutralized TGF-β1, -β2, and -βIIR and investigated the appearance of stress-induced premature senescence biomarkers (Figs. 6 7) . Incubation of RPE cells with neutralizing antibodies against the TGF-β1, -β2, and -β3 isoforms and TGF-βIIR showed no significant modification of the percentage of SA-β-Gal-positive cells under normal conditions when compared with control nonstressed cells supplemented with 10 μg/mL IgG (Figs. 6 7) . In cells treated with 150 μM H2O2 for 2 hours alone, there was a significant increase in SA-β-Gal activity detected 48 hours after stress exposure (Fig. 6) . Treatment with 1.0 ng/mL TGF-β1 or TGF-β2 for 48 hours also led to a marked elevation in the number of SA-β-Gal-positive cells (Fig. 6) . Simultaneous incubation of H2O2-treated RPE cells with neutralizing antibodies against the TGF -β1, -β2, and -β3 isoforms or against TGF-βIIR displayed a significantly decreased proportion of SA-β-Gal-positive cells compared with that in cells solely treated with H2O2 (Fig. 6)
Furthermore, we examined the role of TGF-β1 and -β2 for the stress-induced mRNA expression of Apo J (Fig. 7A) , CTGF (Fig. 7B) , fibronectin (Fig. 7C) , and SM22 (Fig. 7D)by real-time PCR analyses. The signals generated in untreated control cells were set to 100% (Figs. 7) . Forty-eight hours after exposure of the cells to 150 μM H2O2 for 2 hours, the steady state levels of mRNA of the four genes were found to be significantly increased compared with that in untreated control cells (Figs. 7) . For all the genes tested, a much lower expression was observed when the H2O2-treated cells were simultaneously treated with neutralizing antibodies against the TGF -β1, -β2, and -β3 isoforms or TGF-βΙΙR (Figs. 7)
Effect of Hydrogen Peroxide, TGF-β1- and -β2 on Cellular Senescence
In untreated control cells, the expression of p21, p16, and pRb protein was set to 100% (Figs. 8) . Western blot analysis revealed a marked increase of p21 expression by approximately fourfold 48 hours after exposure of the cells to 150 μM H2O2 for 2 hours (Fig. 8A) . A similar elevation of p21 expression was observed after treatment of the cells with 1.0 ng/mL TGF-β1 or -β2 for 24 hours (Fig. 8A) . In contrast, there was no effect on the expression of p16 after oxidative stress or TGF-β1 or -β2 treatment (Fig. 8B) . As both signal pathways, p21 and p16, are known to induce senescence by hypophosphorylation of pRb, we investigated whether changes in p21 expression are parallel with changes in the pRb protein. Forty-eight hours after exposure of RPE cells to 150 μM H2O2 for 2 hours, there was a marked decrease in pRb expression to 21% ± 8% compared with that in untreated control cells (Fig. 8C) . Treatment with 1.0 ng/mL TGF-β1 or -β2 for 24 hours led to a 3- to 4-fold reduction of pRb compared with that in untreated control cells (Fig. 8C)
Hydrogen Peroxide, TGF-β1, and TGF-β2 Effects on Intracellular ROS after 12, 24, or 48 Hours
In our experiments, the production of intracellular ROS was assessed with the fluorescent dye 2′7′-dichlorodihydrofluorescein diacetate. We could not observe any increase in the dye 12, 24 (data not shown), or 48 (Fig. 9A)hours after exposure to 50, 100 (data not shown) or 150 μM H2O2 (Fig. 9A)or after TGF-β1 and -β2 treatment for 12, 24 (data not shown), or 48 (Fig. 9A)hours. 
Hydrogen Peroxide, TGF-β1, and TGF-β2 Effects on Lipid Peroxidation after 12, 24, or 48 hours
In our experiments, lipid peroxidation of the cytoplasm membrane of cultured RPE cells was assessed by increased loss of cis-parinaric acid (PNA) fluorescence. We observes a decrease of PNA fluorescence 12, 24 (data not shown), and 48 (Fig. 9B)hours after exposure to 50, 100 (data not shown) or 150 μM H2O2 (Fig. 9B)or after TGF-β1 and -β2 treatment for 12, 24 (data not shown), and 48 (Fig. 9B)hours. 
Discussion
Distinctive features including a significant increase in SA-β-Gal activity and elevated expression of senescence-related genes (such as apolipoprotein J [Apo J], connective tissue growth factor [CTGF], fibronectin, and SM22) characterize SIPS. In the present study, we showed for the first time that acute sublethal treatment with hydrogen peroxide (H2O2) induced these senescence-associated changes and the release of TGF-β1 and -β2 in cultured human RPE cells. Treatment with TGF-β1 and -β2 alone showed similar changes compared with subtoxic levels of hydrogen peroxide. Using neutralizing antibodies against the TGF -β1, -β2, -β3 isoforms and TGF-βII receptor (TGF-βIIR) minimized these age-related changes. 
In various cellular systems, it has been shown that oxidative stress leads to an increase in SA-β-Gal staining. 11 33 Frippiat et al. 14 have demonstrated an elevation of SA-β-Gal expression in human fibroblasts triggered by H2O2-induced release of TGF-β1. Cellular senescence identified by positive staining of SA-β-Gal was also detected in vitro in late-passage RPE cultures 34 35 and in vivo in the RPE cells of old primate eyes. 22 In our experiments, treatment of human RPE cultures with H2O2, TGF-β1, and TGF-β2 led to a significant increase of the proportion of SA-β-Gal-positive cells. An increased expression of SA-β-Gal staining in cultured RPE cells was previously described after hyperoxia. 23 These observations are in line with our results, since it is believed that hyperoxia mediates its effects via production of reactive oxygen intermediates and thus oxidative stress. 36  
Besides positive SA-β-Gal staining, we detected an overexpression of senescence-related genes such as Apo J, CTGF, fibronectin and SM22 after H2O2, TGF-β1, and TGF-β2 treatment of cultured human RPE cells. Apo J belongs to the group of cellular chaperones. 37 38 39 This protein has been found in increased amounts in the RPE and drusen of patients with age-related macular degeneration (AMD). 40 41 The reason for this increased expression of Apo J in the RPE is still a matter of debate. We observed a marked upregulation of Apo J mRNA after treatment of cultured human RPE cells with H2O2 and TGF-β. Consistent with our results, a similar increase in Apo J expression after exposure to these stimuli has been reported in human fibroblasts. 15 20  
Previously, an increased expression of the profibrogenic CTGF in the RPE of donor eyes with the wet form of AMD has been demonstrated. 42 It has been put forward that CTGF may act as downstream effector for TGF-β promoting fibrosis of the macula. 43 The molecular basis is attributed largely to a unique TGF-β response element in the CTGF promoter. 44 Our results showed an induction of CTGF by H2O2 and TGF-β in cultured human RPE cells. Both stress stimuli are also able to trigger increased CTGF expression in senescent human fibroblasts. 16 Therefore, increased amounts of CTGF in human RPE cells may function as a profibrotic mediator and as a biomarker of SIPS. 
One of the major extracellular matrix (ECM) components in drusen of AMD patients is fibronectin. 41 45 In RPE cells of human AMD donor eyes, fibronectin is elevated by two to threefold compared with RPE cells of age-matched healthy donor eyes. 46 In our experiments, H2O2 and TGF-β1 and -β2 increased the expression of fibronectin by approximately three to fivefold. We were able to show in earlier work that TGF-β1 and -β2 are strong inducers of fibronectin in other ocular cell types. 47 48 Similarly, oxidative stress is capable of increasing the expression of fibronectin in senescent human fibroblasts. 47 49 Based on these results, increased fibronectin amounts in senescent RPE may contribute to the pathologic accumulation of ECM in the RPE and drusen of patients with AMD. 
Another protein involved in ECM turnover and senescence-associated changes is SM22. 15 50 Various studies showed an induction of SM22 by oxidative stress and TGF-β. 15 51 Until now, SM22 has not yet been detected in ocular tissues. We could show for the first time that SM22 is expressed and inducible by H2O2 and TGF-β1 and -β2 in human RPE cells. 
Frippiat et al. 14 18 demonstrated that oxidative stress-induced cellular senescence markers are detected up to 72 hours after H2O2 exposure. Our experiments with fluorescent 2′,7′-dichlorofluorescein (DCF) showed that there is no acute increase in intracellular ROS levels at the various time points up to 48 hours after stimulation with H2O2, TGF-β1, or TGF-β2. However, we observed elevated lipid peroxidation estimated by the loss of cis-parinaric acid (PNA) fluorescence, a known marker of late oxidative stress. 32 These results suggest that senescent cellular changes after prolonged periods of treatment may be attributable to the effects of ROS. 
Besides the paralleled senescence-associated changes induced by H2O2 and TGF-β1 and -β2, we demonstrated that H2O2 was also capable of increasing the TGF-β1 and -β2 mRNA and protein expression. Although TGF-β1 and -β2 mRNA levels were upregulated 12 hours after 50 μM H2O2 exposure, active TGF-β1 and -β2 protein secretion was only increased 24 hours after 100 and 150 μM H2O2 exposure. These observations suggest that a complex regulation may occur at prolonged treatment times, either at the level of RNA stability or posttranscriptionally. Furthermore, it is tempting to assume that oxidative stress-induced senescence in human RPE cells is triggered by release of TGF-β. In fact, we showed that oxidative stress-mediated elevation of senescence biomarkers could be blocked by simultaneous incubation with anti-TGF-β1, -β2, and -β3 or anti-TGF-βΙΙR antibodies. In previous studies, a similar mechanism has been observed with human fibroblasts 14 18 and in cultured human mesangial cells. 19 Based on our results, we assume that senescent cellular changes in cultured human RPE cells are induced by biological cascades attributable to both the effects of ROS and to subsequent pathophysiological changes in the cell. 
In our experiments, oxidative stress and TGF-β increased the expression of p21 but had no effect on p16. Parallel to the upregulation of p21, we showed a downregulation of the pRb protein. This finding suggests the involvement of the p21-pRb senescence-related pathway in the aging process of human RPE cells. In human fibroblasts, both p16 and p21 are upregulated after exposure to oxidative stress. 13 15 52 Both signaling pathways lead to a hypophosphorylation of pRb and thus induction of senescence. 53 54 Studies with various cellular systems have shown that the activation of the p21-pRb or p16- pRb or both pathways may be dependent on the specific combination of stresses and their severity in the various kinds of cell type. 12 17  
Low doses of oxidative stress as well as TGF-β can induce senescence-associated biomarkers in cultured human RPE cells. Oxidative stress–induced senescence may act via TGF-β upregulation, mediated via p21 and pRb as downstream regulators of the senescence-signaling pathway. Hence, it is tempting to speculate that reduction of oxidative stress exposure may help to block the senescent changes in the RPE as seen in AMD. 
 
Table 1.
 
Primers Used for Real-Time PCR
Table 1.
 
Primers Used for Real-Time PCR
Gene Target Gene Sequence Gene Position
Apo J 5′-ggacatccacttccacagc-3′ 692–710
5′-ggtcatcgtcgccttctc-3′ 745–762
CTGF 5′-ctgcaggctagagaagcagag-3′ 826–846
5′-gatgcactttttgcccttct-3′ 897–916
Fibronectin 5′-ctggccgaaaatacattgtaaa-3′ 2611–2632
5′-ccacagtcgggtcaggag-3′ 2707–2724
SM22 5′-agtgtggccctgatgtgg-3′ 185–202
5′-ctgttcaccagcttgctcag-3′ 259–278
TGF-β1 5′-gcacgtggagctgtacca-3′ 498–515
5′-cagccggttgctgaggta-3′ 541–558
TGF-β2 5′-caaagggtacaatgccaactt-3′ 1195–1215
5′-cagatgcttctggatttatggtatt-3′ 1283–1307
TGF-β3 5′-aagaagcgggctttggac-3′ 1154–1171
5′-cacacagcagttctcctcca-3′ 1194–1213
18S rRNA 5′-ctcaacacgggaaacctcac-3′ 1348–1367
5′-cgctccaccaactaagaacg-3′ 1438–1457
Figure 1.
 
Hydrogen peroxide induced SA-β-Gal activity in cultured human RPE cells. (A) Morphology and SA-β-Gal activity of untreated human RPE cells. Only single cells showed SA-β-Gal staining. (B) In contrast, RPE cells of the same passage exposed to 2 hours of 150 μM H2O2 showed a marked increase of SA-β-Gal activity after 48 hours. (C) Quantification of the number of SA-β-Gal-positive cells. The percentage of SA-β-Gal activity was analyzed 12, 24, and 48 hours after exposure to 50, 100, and 150 μM H2O2 for 1 or 2 hours and scored by counting at least 300 cells in phase contrast photomicrographs of representative fields. Data (mean ± SD) are based on the sampling of 6 to 10 photomicrographs per condition from nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control. Scale bar: (A, B) 100 μm.
Figure 1.
 
Hydrogen peroxide induced SA-β-Gal activity in cultured human RPE cells. (A) Morphology and SA-β-Gal activity of untreated human RPE cells. Only single cells showed SA-β-Gal staining. (B) In contrast, RPE cells of the same passage exposed to 2 hours of 150 μM H2O2 showed a marked increase of SA-β-Gal activity after 48 hours. (C) Quantification of the number of SA-β-Gal-positive cells. The percentage of SA-β-Gal activity was analyzed 12, 24, and 48 hours after exposure to 50, 100, and 150 μM H2O2 for 1 or 2 hours and scored by counting at least 300 cells in phase contrast photomicrographs of representative fields. Data (mean ± SD) are based on the sampling of 6 to 10 photomicrographs per condition from nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control. Scale bar: (A, B) 100 μm.
Figure 2.
 
Hydrogen peroxide increased the expression of senescence-associated genes. (A) Apo J, (B) CTGF, (C) fibronectin, and (D) SM22 mRNA expression was analyzed by real-time PCR 12, 24, and 48 hours after treatment of cultured human RPE cells with 150 μM H2O2 for 1 hour. Results were normalized to 18S rRNA as the reference. The steady state mRNA levels of these senescence-associated genes in untreated control cells after 12 hours were considered to be 100%. Results are given as the mean ± SD of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 2.
 
Hydrogen peroxide increased the expression of senescence-associated genes. (A) Apo J, (B) CTGF, (C) fibronectin, and (D) SM22 mRNA expression was analyzed by real-time PCR 12, 24, and 48 hours after treatment of cultured human RPE cells with 150 μM H2O2 for 1 hour. Results were normalized to 18S rRNA as the reference. The steady state mRNA levels of these senescence-associated genes in untreated control cells after 12 hours were considered to be 100%. Results are given as the mean ± SD of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 3.
 
Hydrogen peroxide increased the expression of TGF-β1 and -β2 in cultured human RPE cells. (A, B) Real-time PCR analyses of TGF-β1 and -β2 mRNA expressions 12 and 24 hours after exposure of cultured human RPE cells to 50, 100, and 150 μM H2O2 for 1 hour. Results were obtained with 18S rRNA as reference. The steady state mRNA levels of TGF-β1 and -β2 genes in untreated control cells after 12 hours were considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors. Quantification of the (C, D) active and (E, F) total TGF-β1 and -β2 proteins released into culture medium by ELISA assays after H2O2 treatment regimens as described in (A, B). Results are given as the mean ± SD of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 3.
 
Hydrogen peroxide increased the expression of TGF-β1 and -β2 in cultured human RPE cells. (A, B) Real-time PCR analyses of TGF-β1 and -β2 mRNA expressions 12 and 24 hours after exposure of cultured human RPE cells to 50, 100, and 150 μM H2O2 for 1 hour. Results were obtained with 18S rRNA as reference. The steady state mRNA levels of TGF-β1 and -β2 genes in untreated control cells after 12 hours were considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors. Quantification of the (C, D) active and (E, F) total TGF-β1 and -β2 proteins released into culture medium by ELISA assays after H2O2 treatment regimens as described in (A, B). Results are given as the mean ± SD of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 4.
 
TGF-β induced SA-β-Gal activity in cultured human RPE cells. (A) Morphology and SA-β-Gal activity of untreated cultured human RPE cells. Only single cells were stained blue indicating SA-β-Gal activity. (B) In contrast, RPE cells of the same passage exposed to 1.0 ng/mL TGF-β2 for 48 hours showed a marked increase of SA-β-Gal activity. (C) Quantification of SA-β-Gal-positive cells. The percentage of SA-β-Gal activity was analyzed after stimulation of the cells with 1.0 ng/mL TGF-β1 or -β2 for 12, 24, and 48 hours and scored by counting at least 300 cells in phase-contrast photomicrographs of representative fields. Data (mean ± SD) are based on the sampling of 6 to 10 photomicrographs per condition from nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control. Scale bar: (A, B) 100 μm.
Figure 4.
 
TGF-β induced SA-β-Gal activity in cultured human RPE cells. (A) Morphology and SA-β-Gal activity of untreated cultured human RPE cells. Only single cells were stained blue indicating SA-β-Gal activity. (B) In contrast, RPE cells of the same passage exposed to 1.0 ng/mL TGF-β2 for 48 hours showed a marked increase of SA-β-Gal activity. (C) Quantification of SA-β-Gal-positive cells. The percentage of SA-β-Gal activity was analyzed after stimulation of the cells with 1.0 ng/mL TGF-β1 or -β2 for 12, 24, and 48 hours and scored by counting at least 300 cells in phase-contrast photomicrographs of representative fields. Data (mean ± SD) are based on the sampling of 6 to 10 photomicrographs per condition from nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control. Scale bar: (A, B) 100 μm.
Figure 5.
 
TGF-β increased the expression of senescence-associated genes. Real-time PCR analyses of (A) Apo J, (B) CTGF, (C) fibronectin, and (D) SM22 mRNA expressions after stimulation of cultured human RPE cells with 1.0 ng/mL TGF-β1 or -β2 for 12 and 24 hours. Results were obtained with 18S rRNA as reference. The steady state mRNA levels of these senescence-associated genes in untreated control cells after 12 hours were considered to be 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 5.
 
TGF-β increased the expression of senescence-associated genes. Real-time PCR analyses of (A) Apo J, (B) CTGF, (C) fibronectin, and (D) SM22 mRNA expressions after stimulation of cultured human RPE cells with 1.0 ng/mL TGF-β1 or -β2 for 12 and 24 hours. Results were obtained with 18S rRNA as reference. The steady state mRNA levels of these senescence-associated genes in untreated control cells after 12 hours were considered to be 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 6.
 
Anti-TGF-β1, -β2, and -β3 and anti-TGF-βIIR neutralizing antibodies reduced the oxidative stress-mediated increase of SA-β-Gal-positive cells. Cells were exposed to 150 μM H2O2 for 2 hours in the presence or absence of anti-TGF-β1, -β2, or β3 or anti-TGF-βIIR neutralizing antibodies. Control cells were incubated with 10 μg/mL IgG (Co+IgG). Serum-free medium control (Co[SF]) showed results similar to Co+IgG. For comparison, cells were treated with 1.0 ng/mL TGF-β1 or -β2 for 48 hours. The percentage of SA-β-Gal activity was scored by counting at least 300 cells in phase-contrast photomicrographs of representative fields. Data (mean ± SD) are based on the sampling of 6 to 10 photomicrographs per condition from nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 6.
 
Anti-TGF-β1, -β2, and -β3 and anti-TGF-βIIR neutralizing antibodies reduced the oxidative stress-mediated increase of SA-β-Gal-positive cells. Cells were exposed to 150 μM H2O2 for 2 hours in the presence or absence of anti-TGF-β1, -β2, or β3 or anti-TGF-βIIR neutralizing antibodies. Control cells were incubated with 10 μg/mL IgG (Co+IgG). Serum-free medium control (Co[SF]) showed results similar to Co+IgG. For comparison, cells were treated with 1.0 ng/mL TGF-β1 or -β2 for 48 hours. The percentage of SA-β-Gal activity was scored by counting at least 300 cells in phase-contrast photomicrographs of representative fields. Data (mean ± SD) are based on the sampling of 6 to 10 photomicrographs per condition from nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 7.
 
Neutralizing antibodies against the TGF-β1, -β2, and -β3 isoforms and TGF-βIIR minimized the oxidative stress-mediated increase of senescence-associated genes in cultured human RPE cells. Cells were exposed to 150 μM H2O2 for 2 hours in the presence or absence of anti-TGF-β1, -β2, or -β3 or anti-TGF-βIIR neutralizing antibodies. Control cells were incubated with 10 μg/mL IgG (Co+IgG). Serum-free medium control (Co[SF]) showed similar results as Co+IgG. For comparison, cells were treated with 1.0 ng/mL TGF-β1 or -β2 for 48 hours. (A) Apo J, (B) CTGF, (C) fibronectin, and (D) SM22 mRNA expressions were analyzed by real-time PCR. Results were obtained with 18S rRNA as reference. The steady state mRNA levels of these senescence-associated genes in untreated control cells after 12 hours were considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 7.
 
Neutralizing antibodies against the TGF-β1, -β2, and -β3 isoforms and TGF-βIIR minimized the oxidative stress-mediated increase of senescence-associated genes in cultured human RPE cells. Cells were exposed to 150 μM H2O2 for 2 hours in the presence or absence of anti-TGF-β1, -β2, or -β3 or anti-TGF-βIIR neutralizing antibodies. Control cells were incubated with 10 μg/mL IgG (Co+IgG). Serum-free medium control (Co[SF]) showed similar results as Co+IgG. For comparison, cells were treated with 1.0 ng/mL TGF-β1 or -β2 for 48 hours. (A) Apo J, (B) CTGF, (C) fibronectin, and (D) SM22 mRNA expressions were analyzed by real-time PCR. Results were obtained with 18S rRNA as reference. The steady state mRNA levels of these senescence-associated genes in untreated control cells after 12 hours were considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 8.
 
Hydrogen peroxide and TGF-β1 and -β2 induced increased p21 and decreased pRb protein amounts. Western blot analyses of (A) p21, (B) p16, and (C) pRb protein expressions were performed 48 hours after exposure of cultured human RPE cells with 150 μM H2O2 for 2 hours or after 24 hours of treatment with 1.0 ng/mL TGF-β1 or -β2. Lysates containing approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of (A) p21, (B) p16, and (C) pRb content. Data are expressed as x-fold changes compared to untreated control cells kept for the same time periods and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 8.
 
Hydrogen peroxide and TGF-β1 and -β2 induced increased p21 and decreased pRb protein amounts. Western blot analyses of (A) p21, (B) p16, and (C) pRb protein expressions were performed 48 hours after exposure of cultured human RPE cells with 150 μM H2O2 for 2 hours or after 24 hours of treatment with 1.0 ng/mL TGF-β1 or -β2. Lysates containing approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of (A) p21, (B) p16, and (C) pRb content. Data are expressed as x-fold changes compared to untreated control cells kept for the same time periods and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 9.
 
(A) Hydrogen peroxide, TGF-β1, and TGF-β2 did not increase intracellular ROS after 48 hours. Fluorescent DCF was measured 48 hours after 150 μM H2O2 exposure and after 48 hours’ of TGF-β1 and -β2 treatment. (B) Hydrogen peroxide, TGF-β1, and TGF-β2 increased lipid peroxidation after 48 hours. cis-Parinaric acid (PNA) fluorescence was analyzed 48 hours after 150 μM H2O2 exposure and after 48 hours’ of TGF-β1 and -β2 treatment. (A, B) Data are expressed as x-fold changes compared with untreated control cells kept for the same time periods and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 9.
 
(A) Hydrogen peroxide, TGF-β1, and TGF-β2 did not increase intracellular ROS after 48 hours. Fluorescent DCF was measured 48 hours after 150 μM H2O2 exposure and after 48 hours’ of TGF-β1 and -β2 treatment. (B) Hydrogen peroxide, TGF-β1, and TGF-β2 increased lipid peroxidation after 48 hours. cis-Parinaric acid (PNA) fluorescence was analyzed 48 hours after 150 μM H2O2 exposure and after 48 hours’ of TGF-β1 and -β2 treatment. (A, B) Data are expressed as x-fold changes compared with untreated control cells kept for the same time periods and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
The authors thank Jerome Moriniere and Katja Obholzer for excellent technical assistance. 
HawkinsBS, BirdA, KleinR, WestSK. Epidemiology of age-related macular degeneration. Mol Vis. 1999;5:26. [PubMed]
VingerlingJR, KlaverCC, HofmanA, de JongPT. Epidemiology of age-related maculopathy. Epidemiol Rev. 1995;17:347–360. [PubMed]
BeattyS, KohH, PhilM, HensonD, BoultonM. The role of oxidative stress in the pathogenesis of age-related macular degeneration. Surv Ophthalmol. 2000;45:115–134. [CrossRef] [PubMed]
EvansJR. Risk factors for age-related macular degeneration. Prog Retin Eye Res. 2001;20:227–253. [CrossRef] [PubMed]
SeddonJM, GeorgeS, RosnerB. Cigarette smoking, fish consumption, omega-3 fatty acid intake, and associations with age-related macular degeneration: the US Twin Study of Age-Related Macular Degeneration. Arch Ophthalmol. 2006;124:995–1001. [CrossRef] [PubMed]
KleinR, PetoT, BirdA, VannewkirkMR. The epidemiology of age-related macular degeneration. Am J Ophthalmol. 2004;137:486–495. [CrossRef] [PubMed]
Age-Related Eye Disease Study Research Group. A randomized, placebo-controlled, clinical trial of high-dose supplementation with vitamins C and E, beta carotene, and zinc for age-related macular degeneration and vision loss: AREDS report no. 8. Arch Ophthalmol. 2001;119:1417–1436. [CrossRef] [PubMed]
YangP, PeairsJJ, TanoR, JaffeGJ. Oxidant-mediated Akt activation in human RPE cells. Invest Ophthalmol Vis Sci. 2006;47:4598–4606. [CrossRef] [PubMed]
FinkelT, HolbrookNJ. Oxidants, oxidative stress and the biology of aging. Nature. 2000;408:239–247. [CrossRef] [PubMed]
SerranoM, BlascoMA. Putting the stress on senescence. Curr Opin Cell Biol. 2001;13:748–753. [CrossRef] [PubMed]
ToussaintO, MedranoEE, von ZglinickiT. Cellular and molecular mechanisms of stress-induced premature senescence (SIPS) of human diploid fibroblasts and melanocytes. Exp Gerontol. 2000;35:927–945. [CrossRef] [PubMed]
ChenJ, GoligorskyMS. Premature senescence of endothelial cells: Methusaleh’s dilemma. Am J Physiol Heart Circ Physiol. 2006;290:1729–1739. [CrossRef]
ChenQM, BartholomewJC, CampisiJ, AcostaM, ReaganJD, AmesBN. Molecular analysis of H2O2-induced senescent-like growth arrest in normal human fibroblasts: p53 and Rb control G1 arrest but not cell replication. Biochem J. 1998;332:43–50. [PubMed]
FrippiatC, ChenQM, ZdanovS, MagalhaesJP, RemacleJ, ToussaintO. Subcytotoxic H2O2 stress triggers a release of transforming growth factor-beta1, which induces biomarkers of cellular senescence of human diploid fibroblasts. J Biol Chem. 2001;276:2531–2537. [CrossRef] [PubMed]
DumontP, BurtonM, ChenQM, et al. Induction of replicative senescence biomarkers by sublethal oxidative stresses in normal human fibroblast. Free Radic Biol Med. 2000;28:361–373. [CrossRef] [PubMed]
KimKH, ParkGT, LimYB, et al. Expression of connective tissue growth factor, a biomarker in senescence of human diploid fibroblasts, is up-regulated by a transforming growth factor-beta-mediated signaling pathway. Biochem Biophys Res Commun. 2004;318:819–825. [CrossRef] [PubMed]
Ben-PorathI, WeinbergRA. The signals and pathways activating cellular senescence. Int J Biochem Cell Biol. 2005;37:961–976. [CrossRef] [PubMed]
FrippiatC, DewelleJ, RemacleJ, ToussaintO. Signal transduction in H2O2-induced senescence-like phenotype in human diploid fibroblasts. Free Radic Biol Med. 2002;33:1334–1346. [CrossRef] [PubMed]
Iglesias-De La CruzMC, Ruiz-TorresP, AlcamiJ, et al. Hydrogen peroxide increases extracellular matrix mRNA through TGF-beta in human mesangial cells. Kidney Int. 2001;59:87–95. [CrossRef] [PubMed]
Debacq-ChainiauxF, BorlonC, PascalT, et al. Repeated exposure of human skin fibroblasts to UVB at subcytotoxic level triggers premature senescence through the TGF-beta1 signaling pathway. J Cell Sci. 2005;118:743–758. [CrossRef] [PubMed]
GurjalaAN, LiuWR, MogfordJE, ProcacciniPS, MustoeTA. Age-dependent response of primary human dermal fibroblasts to oxidative stress: cell survival, pro-survival kinases, and entrance into cellular senescence. Wound Repair Regen. 2005;13:565–575. [CrossRef] [PubMed]
MishimaK, HandaJT, Aotaki-KeenA, LuttyGA, MorseLS, HjelmelandLM. Senescence-associated beta-galactosidase histochemistry for the primate eye. Invest Ophthalmol Vis Sci. 1999;40:1590–1593. [PubMed]
HondaS, HjelmelandLM, HandaJT. Senescence associated beta galactosidase activity in human retinal pigment epithelial cells exposed to mild hyperoxia in vitro. Br J Ophthalmol. 2002;86:159–162. [CrossRef] [PubMed]
KliffenM, SharmaHS, MooyCM, KerkvlietS, de JongPT. Increased expression of angiogenic growth factors in age-related maculopathy. Br J Ophthalmol. 1997;81:154–162. [CrossRef] [PubMed]
CampochiaroPA, JerdonJA, GlaserBM. The extracellular matrix of human retinal pigment epithelial cells in vivo and its synthesis in vitro. Invest Ophthalmol Vis Sci. 1986;27:1615–1621. [PubMed]
LescheyKH, HackettSF, SingerJH, CampochiaroPA. Growth factor responsiveness of human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 1990;31:839–846. [PubMed]
DimriGP, LeeX, BasileG, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA. 1995;92:9363–9367. [CrossRef] [PubMed]
Welge-LüssenU, MayCA, EichhornM, BloemendalH, Lütjen-DrecollE. AlphaB-crystallin in the trabecular meshwork is inducible by transforming growth factor-beta. Invest Ophthalmol Vis Sci. 1999;40:2235–2241. [PubMed]
HodgesNJ, GreenRM, ChipmanJK, GrahamM. Induction of DNA strand breaks and oxidative stress in HeLa cells by ethanol is dependent on CYP2E1 expression. Mutagenesis. 2007;22:189–194. [CrossRef] [PubMed]
VolobouevaLA, LiuJ, SuhJH, AmesBN, MillerSS. (R)-α-lipoic acid protects retinal pigment epithelial cells from oxidative damage. Invest Ophthalmol Vis Sci. 2005;46:4302–4310. [CrossRef] [PubMed]
WangX, SimpkinsJW, DykensJA, CammarataPR. Oxidative damage to human lens epithelial cells in culture: estrogen protection of mitochondrial potential, ATP, and cell viability. Invest Ophthalmol Vis Sci. 2003;44:2067–2075. [CrossRef] [PubMed]
CariniM, AldiniG, PicconeM, FacinoRM. Fluorescent probes as markers of oxidative stress in keratinocyte cell lines following UVB exposure. Farmaco. 2000;55:526–534. [CrossRef] [PubMed]
MartinJA, BuckwalterJA. Aging, articular cartilage chondrocyte senescence and osteoarthritis. Biogerontology. 2002;3:257–264. [CrossRef] [PubMed]
MatsunagaH, HandaJT, Aotaki-KeenA, SherwoodSW, WestMD, HjelmelandLM. Beta-galactosidase histochemistry and telomere loss in senescent retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 1999;40:197–202. [PubMed]
WangXF, CuiJZ, NieW, PrasadSS, MatsubaraJA. Differential gene expression of early and late passage retinal pigment epithelial cells. Exp Eye Res. 2004;79:209–221. [CrossRef] [PubMed]
KwakDJ, KwakSD, GaudaEB. The effect of hyperoxia on reactive oxygen species (ROS) in rat petrosal ganglion neurons during development using organotypic slices. Pediatr Res. 2006;60:371–376. [CrossRef] [PubMed]
DumontP, ChainiauxF, EliaersF, et al. Overexpression of apolipoprotein J in human fibroblasts protects against cytotoxicity and premature senescence induced by ethanol and tert-butylhydroperoxide. Cell Stress Chaperones. 2002;7:23–35. [CrossRef] [PubMed]
TrougakosIP, SoA, JansenB, GleaveME, GonosES. Silencing expression of the clusterin/apolipoprotein j gene in human cancer cells using small interfering RNA induces spontaneous apoptosis, reduced growth ability, and cell sensitization to genotoxic and oxidative stress. Cancer Res. 2004;64:1834–1842. [CrossRef] [PubMed]
PoonS, RybchynMS, Easterbrook-SmithSB, CarverJA, PankhurstGJ, WilsonMR. Mildly acidic pH activates the extracellular molecular chaperone clusterin. J Biol Chem. 2002;277:39532–39540. [CrossRef] [PubMed]
SakaguchiH, MiyagiM, ShadrachKG, RaybornME, CrabbJW, HollyfieldJG. Clusterin is present in drusen in age-related macular degeneration. Exp Eye Res. 2002;74:547–549. [CrossRef] [PubMed]
CrabbJW, MiyagiM, GuX, et al. Drusen proteome analysis: an approach to the etiology of age-related macular degeneration. Proc Natl Acad Sci USA. 2002;99:14682–14687. [CrossRef] [PubMed]
HeS, JinML, WorpelV, HintonDR. A role for connective tissue growth factor in the pathogenesis of choroidal neovascularization. Arch Ophthalmol. 2003;121:1283–1288. [CrossRef] [PubMed]
WatanabeD, TakagiH, SuzumaK, OhH, OhashiH, HondaY. Expression of connective tissue growth factor and its potential role in choroidal neovascularization. Retina. 2005;25:911–918. [CrossRef] [PubMed]
IgarashiA, OkochiH, BradhamDM, GrotendorstGR. Regulation of connective growth factor gene expression in human skin fibroblasts and during wound repair. Mol Biol Cell. 1993;4:637–645. [CrossRef] [PubMed]
JohnsonLV, LeitnerWP, StaplesMK, AndersonDH. Complement activation and inflammatory processes in Drusen formation and age related macular degeneration. Exp Eye Res. 2001;73:887–896. [CrossRef] [PubMed]
AnE, LuX, FlippinJ, et al. Secreted proteome profiling in human RPE cell cultures derived from donors with age related macular degeneration and age matched healthy donors. J Proteome Res. 2006;5:2599–2610. [CrossRef] [PubMed]
Welge-LüssenU, MayCA, Lütjen-DrecollE. Induction of tissue transglutaminase in the trabecular meshwork by TGF-beta1 and TGF-beta2. Invest Ophthalmol Vis Sci. 2000;41:2229–2238. [PubMed]
FuchshoferR, BirkeM, Welge-LüssenU, KookD, Lütjen-DrecollE. Transforming growth factor-beta 2 modulated extracellular matrix component expression in cultured human optic nerve head astrocytes. Invest Ophthalmol Vis Sci. 2005;46:568–578. [CrossRef] [PubMed]
SiwikDA, PaganoPJ, ColucciWS. Oxidative stress regulates collagen synthesis and matrix metalloproteinase activity in cardiac fibroblasts. Am J Physiol Cell Physiol. 2001;280:53–60.
NairRR, SolwayJ, BoydDD. Expression cloning identifies transgelin (SM22) as a novel repressor of 92-kDa type IV collagenase (MMP-9) expression. J Biol Chem. 2006;281:26424–26436. [CrossRef] [PubMed]
QiuP, RitchieRP, GongXQ, HamamoriY, LiL. Dynamic changes in chromatin acetylation and the expression of histone acetyltransferases and histone deacetylases regulate the SM22alpha transcription in response to Smad3-mediated TGFbeta1 signaling. Biochem Biophys Res Commun. 2006;348:351–358. [CrossRef] [PubMed]
DengQ, LiaoR, WuBL, SunP. High intensity ras signalling induces premature senescence by activating p38 pathway in primary human fibroblasts. J Biol Chem. 2004;279:1050–1059. [CrossRef] [PubMed]
KrishnamurthyJ, RamseyMR, LigonKL, et al. p16INK4a induces an age-dependent decline in islet regenerative potential. Nature. 2006;443:453–457. [CrossRef] [PubMed]
TakahashiA, OhtaniN, YamakoshiK, et al. Mitogenic signalling and the p16(INK4a)-Rb pathway cooperate to enforce irreversible cellular senescence. Nat Cell Biol. 2006;8:1291–1297. [CrossRef] [PubMed]
Figure 1.
 
Hydrogen peroxide induced SA-β-Gal activity in cultured human RPE cells. (A) Morphology and SA-β-Gal activity of untreated human RPE cells. Only single cells showed SA-β-Gal staining. (B) In contrast, RPE cells of the same passage exposed to 2 hours of 150 μM H2O2 showed a marked increase of SA-β-Gal activity after 48 hours. (C) Quantification of the number of SA-β-Gal-positive cells. The percentage of SA-β-Gal activity was analyzed 12, 24, and 48 hours after exposure to 50, 100, and 150 μM H2O2 for 1 or 2 hours and scored by counting at least 300 cells in phase contrast photomicrographs of representative fields. Data (mean ± SD) are based on the sampling of 6 to 10 photomicrographs per condition from nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control. Scale bar: (A, B) 100 μm.
Figure 1.
 
Hydrogen peroxide induced SA-β-Gal activity in cultured human RPE cells. (A) Morphology and SA-β-Gal activity of untreated human RPE cells. Only single cells showed SA-β-Gal staining. (B) In contrast, RPE cells of the same passage exposed to 2 hours of 150 μM H2O2 showed a marked increase of SA-β-Gal activity after 48 hours. (C) Quantification of the number of SA-β-Gal-positive cells. The percentage of SA-β-Gal activity was analyzed 12, 24, and 48 hours after exposure to 50, 100, and 150 μM H2O2 for 1 or 2 hours and scored by counting at least 300 cells in phase contrast photomicrographs of representative fields. Data (mean ± SD) are based on the sampling of 6 to 10 photomicrographs per condition from nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control. Scale bar: (A, B) 100 μm.
Figure 2.
 
Hydrogen peroxide increased the expression of senescence-associated genes. (A) Apo J, (B) CTGF, (C) fibronectin, and (D) SM22 mRNA expression was analyzed by real-time PCR 12, 24, and 48 hours after treatment of cultured human RPE cells with 150 μM H2O2 for 1 hour. Results were normalized to 18S rRNA as the reference. The steady state mRNA levels of these senescence-associated genes in untreated control cells after 12 hours were considered to be 100%. Results are given as the mean ± SD of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 2.
 
Hydrogen peroxide increased the expression of senescence-associated genes. (A) Apo J, (B) CTGF, (C) fibronectin, and (D) SM22 mRNA expression was analyzed by real-time PCR 12, 24, and 48 hours after treatment of cultured human RPE cells with 150 μM H2O2 for 1 hour. Results were normalized to 18S rRNA as the reference. The steady state mRNA levels of these senescence-associated genes in untreated control cells after 12 hours were considered to be 100%. Results are given as the mean ± SD of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 3.
 
Hydrogen peroxide increased the expression of TGF-β1 and -β2 in cultured human RPE cells. (A, B) Real-time PCR analyses of TGF-β1 and -β2 mRNA expressions 12 and 24 hours after exposure of cultured human RPE cells to 50, 100, and 150 μM H2O2 for 1 hour. Results were obtained with 18S rRNA as reference. The steady state mRNA levels of TGF-β1 and -β2 genes in untreated control cells after 12 hours were considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors. Quantification of the (C, D) active and (E, F) total TGF-β1 and -β2 proteins released into culture medium by ELISA assays after H2O2 treatment regimens as described in (A, B). Results are given as the mean ± SD of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 3.
 
Hydrogen peroxide increased the expression of TGF-β1 and -β2 in cultured human RPE cells. (A, B) Real-time PCR analyses of TGF-β1 and -β2 mRNA expressions 12 and 24 hours after exposure of cultured human RPE cells to 50, 100, and 150 μM H2O2 for 1 hour. Results were obtained with 18S rRNA as reference. The steady state mRNA levels of TGF-β1 and -β2 genes in untreated control cells after 12 hours were considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors. Quantification of the (C, D) active and (E, F) total TGF-β1 and -β2 proteins released into culture medium by ELISA assays after H2O2 treatment regimens as described in (A, B). Results are given as the mean ± SD of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 4.
 
TGF-β induced SA-β-Gal activity in cultured human RPE cells. (A) Morphology and SA-β-Gal activity of untreated cultured human RPE cells. Only single cells were stained blue indicating SA-β-Gal activity. (B) In contrast, RPE cells of the same passage exposed to 1.0 ng/mL TGF-β2 for 48 hours showed a marked increase of SA-β-Gal activity. (C) Quantification of SA-β-Gal-positive cells. The percentage of SA-β-Gal activity was analyzed after stimulation of the cells with 1.0 ng/mL TGF-β1 or -β2 for 12, 24, and 48 hours and scored by counting at least 300 cells in phase-contrast photomicrographs of representative fields. Data (mean ± SD) are based on the sampling of 6 to 10 photomicrographs per condition from nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control. Scale bar: (A, B) 100 μm.
Figure 4.
 
TGF-β induced SA-β-Gal activity in cultured human RPE cells. (A) Morphology and SA-β-Gal activity of untreated cultured human RPE cells. Only single cells were stained blue indicating SA-β-Gal activity. (B) In contrast, RPE cells of the same passage exposed to 1.0 ng/mL TGF-β2 for 48 hours showed a marked increase of SA-β-Gal activity. (C) Quantification of SA-β-Gal-positive cells. The percentage of SA-β-Gal activity was analyzed after stimulation of the cells with 1.0 ng/mL TGF-β1 or -β2 for 12, 24, and 48 hours and scored by counting at least 300 cells in phase-contrast photomicrographs of representative fields. Data (mean ± SD) are based on the sampling of 6 to 10 photomicrographs per condition from nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control. Scale bar: (A, B) 100 μm.
Figure 5.
 
TGF-β increased the expression of senescence-associated genes. Real-time PCR analyses of (A) Apo J, (B) CTGF, (C) fibronectin, and (D) SM22 mRNA expressions after stimulation of cultured human RPE cells with 1.0 ng/mL TGF-β1 or -β2 for 12 and 24 hours. Results were obtained with 18S rRNA as reference. The steady state mRNA levels of these senescence-associated genes in untreated control cells after 12 hours were considered to be 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 5.
 
TGF-β increased the expression of senescence-associated genes. Real-time PCR analyses of (A) Apo J, (B) CTGF, (C) fibronectin, and (D) SM22 mRNA expressions after stimulation of cultured human RPE cells with 1.0 ng/mL TGF-β1 or -β2 for 12 and 24 hours. Results were obtained with 18S rRNA as reference. The steady state mRNA levels of these senescence-associated genes in untreated control cells after 12 hours were considered to be 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 6.
 
Anti-TGF-β1, -β2, and -β3 and anti-TGF-βIIR neutralizing antibodies reduced the oxidative stress-mediated increase of SA-β-Gal-positive cells. Cells were exposed to 150 μM H2O2 for 2 hours in the presence or absence of anti-TGF-β1, -β2, or β3 or anti-TGF-βIIR neutralizing antibodies. Control cells were incubated with 10 μg/mL IgG (Co+IgG). Serum-free medium control (Co[SF]) showed results similar to Co+IgG. For comparison, cells were treated with 1.0 ng/mL TGF-β1 or -β2 for 48 hours. The percentage of SA-β-Gal activity was scored by counting at least 300 cells in phase-contrast photomicrographs of representative fields. Data (mean ± SD) are based on the sampling of 6 to 10 photomicrographs per condition from nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 6.
 
Anti-TGF-β1, -β2, and -β3 and anti-TGF-βIIR neutralizing antibodies reduced the oxidative stress-mediated increase of SA-β-Gal-positive cells. Cells were exposed to 150 μM H2O2 for 2 hours in the presence or absence of anti-TGF-β1, -β2, or β3 or anti-TGF-βIIR neutralizing antibodies. Control cells were incubated with 10 μg/mL IgG (Co+IgG). Serum-free medium control (Co[SF]) showed results similar to Co+IgG. For comparison, cells were treated with 1.0 ng/mL TGF-β1 or -β2 for 48 hours. The percentage of SA-β-Gal activity was scored by counting at least 300 cells in phase-contrast photomicrographs of representative fields. Data (mean ± SD) are based on the sampling of 6 to 10 photomicrographs per condition from nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 7.
 
Neutralizing antibodies against the TGF-β1, -β2, and -β3 isoforms and TGF-βIIR minimized the oxidative stress-mediated increase of senescence-associated genes in cultured human RPE cells. Cells were exposed to 150 μM H2O2 for 2 hours in the presence or absence of anti-TGF-β1, -β2, or -β3 or anti-TGF-βIIR neutralizing antibodies. Control cells were incubated with 10 μg/mL IgG (Co+IgG). Serum-free medium control (Co[SF]) showed similar results as Co+IgG. For comparison, cells were treated with 1.0 ng/mL TGF-β1 or -β2 for 48 hours. (A) Apo J, (B) CTGF, (C) fibronectin, and (D) SM22 mRNA expressions were analyzed by real-time PCR. Results were obtained with 18S rRNA as reference. The steady state mRNA levels of these senescence-associated genes in untreated control cells after 12 hours were considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 7.
 
Neutralizing antibodies against the TGF-β1, -β2, and -β3 isoforms and TGF-βIIR minimized the oxidative stress-mediated increase of senescence-associated genes in cultured human RPE cells. Cells were exposed to 150 μM H2O2 for 2 hours in the presence or absence of anti-TGF-β1, -β2, or -β3 or anti-TGF-βIIR neutralizing antibodies. Control cells were incubated with 10 μg/mL IgG (Co+IgG). Serum-free medium control (Co[SF]) showed similar results as Co+IgG. For comparison, cells were treated with 1.0 ng/mL TGF-β1 or -β2 for 48 hours. (A) Apo J, (B) CTGF, (C) fibronectin, and (D) SM22 mRNA expressions were analyzed by real-time PCR. Results were obtained with 18S rRNA as reference. The steady state mRNA levels of these senescence-associated genes in untreated control cells after 12 hours were considered as 100%. Results are given as mean ± SD of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 8.
 
Hydrogen peroxide and TGF-β1 and -β2 induced increased p21 and decreased pRb protein amounts. Western blot analyses of (A) p21, (B) p16, and (C) pRb protein expressions were performed 48 hours after exposure of cultured human RPE cells with 150 μM H2O2 for 2 hours or after 24 hours of treatment with 1.0 ng/mL TGF-β1 or -β2. Lysates containing approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of (A) p21, (B) p16, and (C) pRb content. Data are expressed as x-fold changes compared to untreated control cells kept for the same time periods and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 8.
 
Hydrogen peroxide and TGF-β1 and -β2 induced increased p21 and decreased pRb protein amounts. Western blot analyses of (A) p21, (B) p16, and (C) pRb protein expressions were performed 48 hours after exposure of cultured human RPE cells with 150 μM H2O2 for 2 hours or after 24 hours of treatment with 1.0 ng/mL TGF-β1 or -β2. Lysates containing approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of (A) p21, (B) p16, and (C) pRb content. Data are expressed as x-fold changes compared to untreated control cells kept for the same time periods and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 9.
 
(A) Hydrogen peroxide, TGF-β1, and TGF-β2 did not increase intracellular ROS after 48 hours. Fluorescent DCF was measured 48 hours after 150 μM H2O2 exposure and after 48 hours’ of TGF-β1 and -β2 treatment. (B) Hydrogen peroxide, TGF-β1, and TGF-β2 increased lipid peroxidation after 48 hours. cis-Parinaric acid (PNA) fluorescence was analyzed 48 hours after 150 μM H2O2 exposure and after 48 hours’ of TGF-β1 and -β2 treatment. (A, B) Data are expressed as x-fold changes compared with untreated control cells kept for the same time periods and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Figure 9.
 
(A) Hydrogen peroxide, TGF-β1, and TGF-β2 did not increase intracellular ROS after 48 hours. Fluorescent DCF was measured 48 hours after 150 μM H2O2 exposure and after 48 hours’ of TGF-β1 and -β2 treatment. (B) Hydrogen peroxide, TGF-β1, and TGF-β2 increased lipid peroxidation after 48 hours. cis-Parinaric acid (PNA) fluorescence was analyzed 48 hours after 150 μM H2O2 exposure and after 48 hours’ of TGF-β1 and -β2 treatment. (A, B) Data are expressed as x-fold changes compared with untreated control cells kept for the same time periods and represent the mean ± SD of results of nine experiments with three different cell cultures from different donors (*P < 0.05). Co, control.
Table 1.
 
Primers Used for Real-Time PCR
Table 1.
 
Primers Used for Real-Time PCR
Gene Target Gene Sequence Gene Position
Apo J 5′-ggacatccacttccacagc-3′ 692–710
5′-ggtcatcgtcgccttctc-3′ 745–762
CTGF 5′-ctgcaggctagagaagcagag-3′ 826–846
5′-gatgcactttttgcccttct-3′ 897–916
Fibronectin 5′-ctggccgaaaatacattgtaaa-3′ 2611–2632
5′-ccacagtcgggtcaggag-3′ 2707–2724
SM22 5′-agtgtggccctgatgtgg-3′ 185–202
5′-ctgttcaccagcttgctcag-3′ 259–278
TGF-β1 5′-gcacgtggagctgtacca-3′ 498–515
5′-cagccggttgctgaggta-3′ 541–558
TGF-β2 5′-caaagggtacaatgccaactt-3′ 1195–1215
5′-cagatgcttctggatttatggtatt-3′ 1283–1307
TGF-β3 5′-aagaagcgggctttggac-3′ 1154–1171
5′-cacacagcagttctcctcca-3′ 1194–1213
18S rRNA 5′-ctcaacacgggaaacctcac-3′ 1348–1367
5′-cgctccaccaactaagaacg-3′ 1438–1457
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×