Free Radical Research, March 2014; 48(3): 303–312 © 2014 Informa UK, Ltd. ISSN 1071-5762 print/ISSN 1029-2470 online DOI: 10.3109/10715762.2013.867484
ORIGINAL ARTICLE
Hypoxia induces cell damage via oxidative stress in retinal epithelial cells F. Cervellati1, C. Cervellati2, A. Romani2, E. Cremonini2, C. Sticozzi1, G. Belmonte3, F. Pessina4 & G. Valacchi1,5 of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy, 2Department of Biomedical and Specialist Surgical Sciences, Section of Medical Biochemistry, Molecular Biology and Genetics University of Ferrara, Ferrara, Italy, 3Department of Surgical and Medical Sciences and Neurosciences, University of Siena, Siena, Italy, 4Department of Development and Molecular Medicine, University of Siena, Siena, Italy, and 5Department of Food and Nutrition, Kyung Hee University, Seoul, South Korea Free Radic Res Downloaded from informahealthcare.com by University New South Wales on 03/30/15 For personal use only.
1Department
Abstract Retinal diseases (RD), including diabetic retinopathy, are among the most important eye diseases in industrialized countries. RD is characterized by abnormal angiogenesis associated with an increase in cell proliferation and apoptosis. Hypoxia could be one of the triggers of the pathogenic mechanism of this disease. A key regulatory component of the cell’s hypoxia response system is hypoxia-inducible factor 1 alpha (HIF-1α). It has been demonstrated that the induction of HIF-1α expression can be also achieved in vitro by exposure with cobalt chloride (CoCl2), leading to an intracellular hypoxia-like state. In this study we have investigated the effects of CoCl2 on human retinal epithelium cells (hRPE), which are an integral part of the blood–retinal barrier, with the aim to determine the possible role of oxidative stress in chemical hypoxia-induced damage in retinal epithelial cells. Our data showed that CoCl2 treatment is able to induce HIF-1α expression, that parallels with the formation of reactive oxygen species (ROS) and the increase of lipid 8-isoprostanes and 4-hydroxynonenal (4-HNE) protein adducts levels. In addition we observed the activation of the redox-sensitive transcription factor nuclear factor-kappaB (NFkB) by CoCl2 which can explain the increased levels of vascular endothelial growth factor (VEGF). The increased number of dead cells seems to be related to an apoptotic process. Taken together these evidences suggest that oxidative stress induced by hypoxia might be involved in RD development through the stimulation of two key-events of RD such as neo-angiogenesis and apoptosis. Keywords: hypoxia-inducible factor-1 alpha, reactive oxygen species, human retinal epithelium cells, 4-hydroxynonenal, 8-isoprostanes, apoptosis
Introduction Retinal diseases (RD), including diabetic retinopathy, are among the most important eye diseases in industrialized countries, and represent the leading cause of blindness among people of working age [1]. Typically, the symptoms associated with RD appear only when the lesions are in advanced state thereby limiting the effectiveness of the treatment. The pathogenic mechanism of this disease is still under investigation although it has been postulated that hypoxia could be one of the triggers of its onset [2]. Indeed, tissue hypoxia might develop from the exceptionally high oxygen consumption of rod receptors during dark adaptation that, in combination with impaired capillary function, has been widely observed in eyes of RD patients [3]. Besides, RD is characterized by abnormal angiogenesis (the new immature vessels play an important role in disease development) which, in turn, is often associated with an increase in endothelial cells number caused by an imbalance in cell proliferation and apoptosis [3]. Of note, these processes represent two of the most observed attempts to restore oxygen homeostasis in response to hypoxic stimuli in retinal epithelial cells [4].
The adaptive responses require the concerted activation of various transcription factors including hypoxia-inducible factor-1 alpha (HIF-1α) [4,5], which is generally considered the master regulator of events related to hypoxic state [6,7]. HIF-1α protein turnover in normoxia is very quick due to the action of prolyl hydroxylases [7]. These oxygen-dependent enzymes hydroxylate two conserved proline residues of HIF-1α, promoting the binding of the Von Hippel–Lindau protein, ubiquitination, and subsequent proteosomal degradation [8]. Under hypoxic conditions the prolyl hydroxylases are inhibited, thereby allowing stabilization and accumulation of the HIF-1α protein [6–10]. The stabilization of HIF1α can be also achieved in vitro by exposure with cobalt chloride (CoCl2) which blocks the catalysis of prolyl hydroxylases, thereby leading to an intracellular hypoxia-like state [11]. It is well-known that the stabilization of HIF-1α promotes the synthesis of reactive oxygen species (ROS), which, depending on their intracellular concentration, are able to modulate the transcription of genes involved in cell proliferation, differentiation, and death [5]. These various actions of ROS are, in several cases, exerted through their lipid peroxidation by-products, in primis F2-isoprostanes
Correspondence: Giuseppe Valacchi, PhD, Department of Life Sciences and Biotechnology, University of Ferrara, 44100 Ferrara, Italy. Office: (39) 0532 455 482. Lab: (39) 0532 455 477. Mobile: (39) 335 6722592. E-mail: [email protected] (Received date: 30 October 2013; Accepted date: 17 November 2013; Published online: 16 December 2013)
Free Radic Res Downloaded from informahealthcare.com by University New South Wales on 03/30/15 For personal use only.
304 F. Cervellati et al. and unsaturated aldehydes such as 4-hydroxynonenal (4-HNE). F2-isoprostanes, indeed, appeared to be mediators of important biological effects in retinal vessels’ contraction, and increase thromboxane production and apoptosis [12–15]. The concentration-dependent activity is more accentuated for 4-HNE which can promote cell proliferation at low doses and cell damage-apoptosis at higher dose [16,17]. The regulatory ability of these two products of ROS lipid damage seems to be exerted by the activation of the redox-sensitive transcription factor NF-kappaB, a key regulator of cell proliferation and apoptosis [17–18]. Taken together these evidences suggest that oxidative stress (OS) induced by hypoxia might be crucially involved in RD development through the stimulation of two key events of the disease such as neo-angiogenesis and apoptosis. Nevertheless, direct proofs of this association are still lacking. In the present study we investigated the effect of hypoxia chemically induced with 200 μM CoCl2, on human retinal epithelium cells (hRPE) which are an integral part of the blood–retinal barrier and particularly sensitive to changes in the concentration of O2. In particular, we aimed to evaluate the possible role of OS in the modulation of hRPE response to chemical hypoxia. Methods Cell culture Human retinal pigment epithelium (hRPE) cells provided by Dr. Pavan B., were routinely grown in 1:1 mixture of Dulbecco’s modified Eagle’s and Ham’s F12 media (Lonza Milan, Italy), supplemented with 10% FBS, 50 mg/mL streptomycin, and 50 U/mL penicillin (Lonza Milan, Italy) (complete medium) on tissue culture flasks until confluence (approximately 5 106) at 37°C in 5% CO2. CoCl2 exposure Prior to CoCl2 exposure the cells were trypsinized and then grown in 35 mm Petri dishes with complete medium until a
confluent monolayer was established (approximately 2 105 cells/well). Afterwards, media were removed and fresh serum-free medium was added with or without CoCl2 (final concentration in culture 200 μM). The cells were grown and collected at different time points for biochemical assays. Cellular viability Viability studies were performed after treatments at different time by Tripan Blue and LDH release assay. The LDH release was measured by enzymatic assay: in the first step NAD is reduced to NADH/H by the LDH-catalyzed conversion of lactate to pyruvate; in the second step the catalyst (diaphorase) transfers H/H from NADH/H to tetrazolium salt which is reduced to formazan. Prior to each assay, the cells were lysed with 2% (V/V) Triton X-100 in culture media for 30 min at 37°C to obtain a representative maximal LDH release as the positive control with 100% toxicity. The amounts of LDH in the supernatant were determined and calculated according to kit instructions (EuroClone Milan, Italy). All tests were performed in triplicate and assay was repeated three times independently with similar results. As for Trypan blue staining, cells are counted by cell counter (Invitrogen, Monza, Italy). Viable cells and nonviable cells were recorded separately, and the means of three independent counts were pooled for analysis and expressed as percent of living cells with respect to total cell number (viability in percentage-%). Quantitative real-time PCR Total RNA was extracted, using an AURUM total RNA Mini Kit with DNAse digestion (Bio-Rad, Laboratories Inc., Benicia, CA, USA), from 2 105 hRPE cells for each experimental condition, according to the manufacturer’s recommended procedure. First-strand cDNA was generated from 1 μg of total RNA using the iScript cDNA Synthesis Kit (Bio-Rad, Laboratories Inc., Benicia, CA, USA). As shown in Table I, primer pairs capable of hybridization with unique regions of the appropriate gene sequence were obtained
Table I. Primer sequences and PCR condition.
Gene HIF-1a VEGF Caspase3 RPL13A RPL11A GAPDH
QPCR Product Amplification length (bp) Efficiency* (%) n° of cycles
Primer sequence
Ta°C
F: 5′-tgcttggtgctgatttgtga-3′ R: 5′-ggtcagctgatcagcgtcca-3′ F: 5′-aggccagcacataggagaga-3′ R: 5′-acgcgagtctgtgtttttgc-3′ F: 5′-tttcggtgggtgtgccctgc-3′ R: 5′-ccctgaggtttgctgcatcgaca-3′ F: 5′-cctaagatgagcgcaagttgaa-3′ R: 5′-ccacaggactagaacacctgctaa-3′ F: 5′-tgcgggaacttcgcatccgc-3′ R: 5′-gggtctgccctgtgagctgc-3′ F: 5′-tgacgctggggctggcattg-3′ R: 5′-ggctggtggtccaggggtct-3′
60.1
210
96.2
39
GenBank accession NM 181054.2
60.4
175
98.4
39
GenBank accession NM 001025368
59.8
284
94.5
39
GenBank accession NM 005497
60.2
203
97.3
39
Pattyn et al. 2006 [19]
60.1
108
96.5
39
GenBank accession NM 000975.2
60
134
94.6
39
GenBank accession NM 002046.3
Data calculated by OpticonMonitor 3 Software (Bio-Rad).
Ref. primer bank
Free Radic Res Downloaded from informahealthcare.com by University New South Wales on 03/30/15 For personal use only.
CoCl2-induced oxidative stress in hRPE
from the Real-time PCR GenBank Primer and Probe Database Primer Bank, RT primerDB [19]. Quantitative realtime PCR (qPCR) was performed using SYBR Green on the iQ5 Multicolor Real-time PCR Detection System (Bio-Rad). The final reaction mixture contained 300 nM of each primer, 1 μL of cDNA and 7.5 μL of iQ SYBR Green Supermix (Bio-Rad), with RNAse-free water being used to bring the reaction mixture volume to 15 μL. All reactions were run in triplicate. Real-time PCR was initiated with a 3-min hotstart denaturation step at 95°C, and then performed for 40 cycles at 95°C for 10 s and at 60°C for 20 s. During the reaction, fluorescence, and therefore the quantity of PCR products, was continuously monitored by Opticon Monitor 3 software (Bio-Rad). Primers were initially used to generate a standard curve over a large dynamic range of starting cDNA quantities, permitting calculation of the amplification efficiency (a critical value for the correct quantification of expression data) for each of the primer pairs. Ribosomal proteins L13a (RPL13a) L11a (RPL11a), and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were employed as reference genes. As previously described [19], samples were compared using the relative cycle threshold (CT) method [20]. After normalization to more stable mRNA, RPL13a, RPL11a, and GAPDH, the fold increase or decrease was determined with respect to control, using the formula 2ΔΔCT, where ΔCT is (gene of interest CT)(reference gene CT), and ΔΔCT is (ΔCT experimental) (ΔCT control). Western blot analysis Proteins were extracted from cells as previously described [21]. Sample proteins were assessed according to Lowry et al. using bovine serum albumin as standard. Proteins (80 μg) were resolved in Bis-Tris–HCl polyacrylamide gel (4–12%), then separated by electrophoresis (SDS–PAGE) and transferred to a polyvinylidene fluoride membrane. Immunoblots were performed using anti-rabbit polyclonal antibodies (Cell signaling Technology Inc. Beverly, MA, USA) against CASP-3, PARP, 4HNE, and vascular endothelial growth factor (VEGF) (Cell signaling Technology Inc. Beverly, MA, USA) as primary antibody, in a primary antibody diluent kit and, after washings, with goat anti-rabbit IgG (1:2000) conjugated with horseradish peroxidase for 1 h (Cell signaling Technology Inc). Immunoblots were developed by enhanced chemioluminescence reagent, and a densitometric analysis of the films was performed by Image Master (Amersham-Pharmacia, Milan, Italy) equipped with Total Lab software. Values within each experiment were normalized to the control sample. NFkB immunofluorescence analysis hRPE cells were cultured on coverslips and stained using rabbit polyclonal antibodies (Cell signaling Technology Inc.) raised against the human nuclear factor-kappaB (NFkB) (working dilutions 1:200 in PBS containing 0.05% BSA and 0.1% sodium azide) as previously described [22]. Cells were incubated with the primary antibodies for 1 h at room temperature (RT), and with secondary FITC-labeled goat anti-rabbit IgG serum (Vector
305
Laboratories, Burlingame, CA, USA) diluted 1:100 in PBS, for 1 h at RT in the dark. Slides were mounted in Vectashield (Vector Laboratories) anti-fading and examined using an Epi-fluorescence microscope (Nikon Eclipse E800; Nikon Corporation, Surrey, UK) equipped with a plan apochromat 100 0.5–1.3 oil immersion objective and a mercury lamp source. Amplifier and detector optimizing parameters were maintained constant for all the experiments. 8-isoprostanes quantification The concentration of free 8-Isoprostanes (8-iso-prostaglandin F2α, 8-iso-PGF2α) which belong to F2-isoprostanes family, was assessed in culture medium by enzyme immunoassay (Cayman Chemical, Ann Arbor, MI) [23–25], following the manufacturer’s protocol. The detection limit of 8-iso-PGF2α of this assay is 5 pg/ml and the intra- and inter-assay coefficients of variation are less than 10%. The concentration of 8-iso-PGF2α cell was assessed in medium of CoCl2-treated and untreated hRPE collected at different time periods (0, 2, 6, 12, and 24 h) either with or without preincubation with Vitamin C or Vitamin E (Sigma-Aldrich, St. Louis, MO) for 24 h at 37° C (final concentration in culture medium 50 μM). To prevent any artefactual formation or degradation of 8-iso-PGF2α in every medium aliquot was added hydroxytoluene (final concentration 0.005% BHT) prior to the assay. ROS determination For ROS determination, after the CoCl2 treatment at different time-points (0, 5, and 20 min) the cells were incubated in fresh serum-free medium with 20 μM 2,7-dichlorofluorescein diacetate (DCFH-DA) (Sigma-Aldrich) for 30 min at 37°C in the dark. DCFH-DA diffuses through the cell membrane readily and enzymatically hydrolyzed by intracellular esterases to non-fluorescent dichlorofluorescein (DCFH), which is then rapidly oxidized to highly fluorescent DCF in the presence of ROS. Then the cells were washed with PBS, harvested and centrifuged at 8000 g for 10 min. The pellet was then resuspended in PBS and read by spectrofluorimeter (Tecan infinite m200, Tecan group Ltd, Switzerland). The ROS production was expressed as the mean DCFH-DA fluorescence intensity (arbitrary units). Furthermore, we also measured the superoxide production by using the indicator MitoSOX Red (Invitrogen), a mitochondrion-specific hydroethidine-derivative fluorescent dye, according to the manufacturer’s instructions. Briefly, for live cell imaging, hRPE cells were allowed to adhere on glass coverslips. After treatment, media was removed and cells were loaded with Mitosox Red (Invitrogen) (5 μM) in Hanks’ Balanced Salt Solution for 10 min at 37°C. Cells were then washed and imaged on a Leica inverted fluorescence microscope using a Rhodamine filter (MitoSOX was excited at 515 nm, and emitted light was measured from 520–620 nm). Morphology analysis Cells were scraped and collected in 0.1 M cacodylate buffer (pH 7.4), then spun in 1.5 ml tubes at 2.000 g for 5 min.
Free Radic Res Downloaded from informahealthcare.com by University New South Wales on 03/30/15 For personal use only.
306 F. Cervellati et al. Pellets were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 4 h at 4°C. They were then washed with 0.1 M cacodylate buffer (pH 7.4) three times and postfixed in 1% osmium tetroxide and 0.1 M cacodylate buffer at pH 7.4 for 1 h at RT. The specimens were dehydrated in graded concentrations of ethanol and embedded in epoxide resin (Agar Scientific, 66A Cambridge Road, Stanstead Essex, CM24 8DA, UK). Cells were then transferred to latex modules filled with resin and subsequently thermally cured at 60°C for 48 h. Semi-thin sections (0.5–1 μm thickness) were cut using an ultra-microtome (Reichard Ultracut S, Austria) stained with toluidine blue, and blocks were selected for thinning. Ultra-thin sections of about 40–60 nm were cut and mounted onto formvar-coated copper grids. These were then double-stained with 1% uranyl acetate and 0.1% lead citrate for 30 min each and examined under a transmission electron microscope, Hitachi H-800 (Tokyo, Japan), at an accelerating voltage of 100 KV. Statistical analysis Data were expressed as mean and standard deviation. For most experiments, comparison among the different groups were tested by unpaired 2-tailed Student’s t-test. A p-value inferior to 0.05 was regarded as significant. Results CoCl2 treatment induced HIF-1α gene expression As first step we wanted to evaluate whether in our system we were able to induce the hypoxia pathway as it has been reported in the literature [26] by measuring the expression of HIF-1. As showed in Figure 1, using qPCR, we demonstrated that hRPE cells treated with CoCl2 overexpressed HIF-1α at the early time points, confirming
Figure 1. Changes in hRPE cells of HIF1-α mRNA levels after CoCl2 treatment at different time points (0–24 h). Open bars: untreated cells; Close bars: cells treated with CoCl2. The results are expressed in % compared with the control value set equal to 100% of 2ΔΔCt values. The formula used to quantify the fold change with respect to control was 2ΔΔCT, where ΔCT is (gene of interest CT) (reference gene CT), and ΔΔCT is (ΔCT experimental) (ΔCT control). The data are the averages of the five different experiments. *p 0.05 versus control.
the typical cell response to hypoxia state. In particular, it was observed a sharp and significant increase of HIF-1α mRNA expression in the first 2 h (around 120%) followed by a decline to the control value after 24 h (Figure 1). Chemical hypoxia affects cell death As shown in Figure 2A, the treatment with CoCl2 resulted in a decreased cell viability. This decline started as early as 6 h reaching significance at later time points (12 and 24 h).
Figure 2. (A) Viability % after CoCl2-treatement at different time points. (B) LDH release after CoCl2-treatement at different time points. (C) Viability % after CoCl2-treatement at 0 and 24 h with or without Vitamin C or Vitamin E. The data are the averages of five different experiments. *p 0.05 versus control at time 0; § p 0.05 versus control at the same time point; #p 0.05 versus 2 h CoCl2.
Free Radic Res Downloaded from informahealthcare.com by University New South Wales on 03/30/15 For personal use only.
CoCl2-induced oxidative stress in hRPE
307
Figure 3. (A) Variation in ROS production measured by spectrometry with DCFH-DA staining in CoCl2-treated compared with non-treated (controls) hRPE cells. (B) Mitochondrial ROS production was evaluated by Mitosox fluorescence. Cells were loaded with Mitosox before and after CS exposure and subjected to live cell imaging. The data are the averages of five different experiments. *p 0.05 versus control at time 0; §versus control at the same time point.
The effect of CoCl2 on cell viability was confirmed by LDH release. As shown in Figure 2B, CoCl2 induced an increase in LDH release already after 2 h and this effect was noticeable up to 24 h after treatment Of note is that pretreatment with Vitamin C and Vitamin E prevented CoCl2-induced LDH release leading to the idea that CoCl2 exposure is linked to ROS production. This hypothesis was confirmed by the results showed in Panel C where pretreatement with Vitamin C and Vitamin E prevent CoCl2 decreased cell viability (Figure 2C), although this effect was more evident with Vitamin E.
production of 8-iso-PGF2α in culture medium of hRPE cells treated and untreated with CoCl2 (Figure 4). As displayed in Figure 4 there was an increase of 8-iso-PGF2α levels already after 2 h (25%) of treatment and it became more evident (around 75%) at 24 h. As depicted in Figure 5 preincubation of hRPE with Vitamin C or Vitamin E hampered CoCl2-induced F2-isoprostanes levels production at 24 h, thus confirming the association between chemical hypoxia and OS in hRPE cells.
ROS production
One of the consequences of lipid peroxidation is the formation of bioactive products such as unsaturated aldehydes like 4-HNE that are able to interact with cellular proteins. To evaluate whether chemical hypoxia-induced
As a consequence of Figure 2, we went to verify whether the induction of hypoxia by CoCl2 could also generate OS in hRPE cells. As shown in Figure 3A, cells treated with CoCl2 revealed a significant increase in ROS concentration after 20 min (circa 4 fold). Furthermore, mitochondrial ROS production was evaluated in hRPE cells after CoCl2 treatment by Mitosox assay. As shown in Figure 2B, cells exposed to CoCl2 showed an increased in fluorescence, characteristic of mitochondrial staining. This effect was detected already after 15 min of treatment and persist over 60 min. 8-iso-PGF2α concentration in medium cells is induced by CoCl2 treatment and hampered by antioxidant pretreatment To further verify the involvement of OS in cell response to hypoxia induced by CoCl2, we measured the time-course
4-HNE protein adducts formation after CoCl2 treatment expression
Figure 4. 8-iso-PGF2α levels present in hRPE cells medium after CoCl2 treatment. The results are expressed in percentage (%) compared with the control (not treated cells at time 0) value, set equal to 100%. The data are the averages of five different experiments. *p 0.001 versus control at time 0; §p 0.001 versus control at the same time point; #p 0.001 versus CoCl2 at time 24.
Free Radic Res Downloaded from informahealthcare.com by University New South Wales on 03/30/15 For personal use only.
308 F. Cervellati et al.
Figure 5. Effects of Vitamin C or Vitamin E preincubation on of 8-iso-PGF2α formation after 24 h treatment with CoCl2. The results are expressed in percentage (%) compared with the concentration of 8-iso-PGF2α detected in not treated hRPE (control), which was set equal to 100%. The data are the averages of five different experiments. *p 0.05 versus control; #p 0.05 versus CoCl2-treated cells without Vitamin C or Vitamin E preincubation.
4-HNE protein adducts formation, we performed Western blot analysis in cell lysates. As shown in Figure 6 CoCl2 induced a clear increase in 4-HNE protein adduct formation after 2 h of treatment ( 40%) and went back to the steady level after 12 h. This result is in line with the increased level of ROS and 8-iso-PGF2α observed in CoCl2 exposed cells. Effect of CoCl2 on NFkB nuclear translocation In CoCl2-treated cells there was a clear increase of NFkB nuclear translocation as displayed by the increment in punctate fluorescence (Figure 7). This effect was well evident already after 30 min of treatment and decremented in the following time point (1 h), showing that NFkB activation is an early event in CoCl2 induce cellular responses. CoCl2-induced VEGF gene and protein expression One of the main features of retina-related diseases is the presence of new blood vessels and since VEGF is the main inducer of neo-angiogenesis and its transcription is also regulated by NFkB we evaluated VEGF levels in hRPE cells exposed to CoCl2. As shown in Figure 8A, cells treated with 200 μM of CoCl2 showed a significant time-dependent increase in VEGF mRNA with and induction of circa 2.5 fold at 6 h and almost 4 fold at 24 h. In parallel also the protein levels showed a time-dependent increasing trend (Figure 8B). CoCl2 induced Caspase 3 expression As reported in Figure 2, treatment with CoCl2 decreased cell viability and this result prompted us to check if it was related to an induction of cellular apoptotosis. Clues of programmed cell death were obtained by the
Figure 6. Effect of CoCl2 treatment on 4-HNE protein adducts formation in hRPE cells. (A) a representative Western blot of five different experiments. (B) Quantification of 4-HNE protein adducts band. Data are expressed as Arbitrary Units (AU). *p 0.05 versus control.
determination Caspase 3 gene and protein expression. Indeed, the mRNA levels of the pro-apoptotic gene Caspase 3 increased significantly already at 6 h after the treatment with CoCl2 with respect to the control (Figure 9). This trend persisted also at later time points (24 h), with an overall up-regulation of circa 50% Figure 9A. This was also confirmed at the protein levels, as shown in Figure 9B, Western blot analysis demonstrated a clear increase in cleaved Caspase 3 protein levels starting at 6 h and reaching the maximum expression at 24 h. Of note, pretreatment with Vitamin C or Vitamin E prevented cleaved Caspase 3 increase, confirming the hypothesis that OS plays a role in CoCl2-induced cell death. Apoptosis was also evaluated by the detection of PARP cleavage and also in this case we have observed a similar trend noticed for Caspase 3. CoCl2 treatment affects cell morphology The electron microscopy analysis of hRPE before and after CoCl2 treatment showed an evident degeneration of cells’ ultra-structure. As shown in Figure 10, before
CoCl2-induced oxidative stress in hRPE
309
Free Radic Res Downloaded from informahealthcare.com by University New South Wales on 03/30/15 For personal use only.
Figure 7. NFkB staining in hRPE cells: (A) Control (not treated cells at t0); (B) CoCl2 for 30 min; (C) CoCl2 for 1 h. Scale bar 10 μm.
treatment, cells were tightly contacting each other (A). Mitochondria, endoplasmic reticulum, nuclear membranes and euchromatic nucleus with marginal heterochromatin showed normal morphological features (A); after 6 h of treatment with 200 μM CoCl2 the cells changed their morphology, showing vacuolation and condensation of the cytoplasm, nuclear bulge, and loss of intercellular adhesion (B); exposition for 24 h worsened the cell condition, leading to loss of mitochondria, degranulation of both the cytoplasm and the nucleoplasm (C). Discussion The human retinal pigment epithelial cell (hRPE) is a highly specialized retinal cell layer that plays important roles in the regulation and development of photoreceptors in the vertebrate retina [27]. In normal retina, hRPE cells are involved in transporting and stocking materials, for example retinaldehyde; phagocytosing detached photoreceptor outer segments; intercepting light; removing free radicals; synthesizing cytokines; and forming the blood– retinal barrier [28]. The integrity of the hRPE is of key importance for maintaining the integrity and proper functioning of the human retina [29]. Despite the central role of these cells in retinal physiology, there are few studies
where they are used as a model to investigate the pathogenic mechanisms of RD [30]. It has been shown that OS might play a role in the pathological changes, in particular apoptosis and neoangiogenesis processes, occurring in retinal pigment epithelium during the early stages of RD [31]. This postulated role for OS is confirmed with ability of reduced glutathione (GSH) [32], or compounds able to promote its synthesis [33], to protect hRPE cells against cell death induced by oxidants [34]. It is well known that hypoxia due to oxygen tension below the normal limits in a specific cell, tissue or organism is a common feature of the majority of RD. What has not been still clarified is whether OS could be the “armed hand” triggered by hypoxia in these diseases. In this study, we have demonstrated that chemical hypoxia induced by CoCl2 is able to mimic the hypoxic state as indicated by the rapid increase of mRNA HIF-1α expression and this has been also confirmed by other researchers using a similar approach [35]. The cause of loss of cell viability is most probably due to apoptotic process as highlighted by typical ultrastructural alterations and the consistent increase levels of the cleaved form of Caspase 3 and PARP cleavage. The CoCl2related damage of hRPE can be the result of a lipoperoxidative process as suggested by the increase of
Figure 8. VEGF expression in hRPE cells before and after CoCl2 treatment. (A) mRNA levels are expressed in % compared with the control value set equal to 100% of 2ΔΔCt values. The formula used to quantify the fold change with respect to control was 2ΔΔCT, where ΔCT is (gene of interest CT)(reference gene CT), and ΔΔCT is (ΔCT experimental) (ΔCT control) 2ΔΔCt values are the averages of five different experiments. (B) protein levels: upper panel is a representative Western blot of five independent experiments, the bands quantification are reported in the bottom panel. (A) *p 0.001 versus control; §p 0.001 versus t12. (B) *P 0.01 versus control.
Free Radic Res Downloaded from informahealthcare.com by University New South Wales on 03/30/15 For personal use only.
310 F. Cervellati et al.
Figure 9. Changes in Caspase3 mRNA (A) and protein (B) expression in hRPE after CoCl2 treatment. mRNA levels are expressed in % compared with the control value set equal to 100% of 2ΔΔCt values. 2ΔΔCT was the formula used to quantify the fold change with respect to control, where ΔCT is (gene of interest CT)(reference gene CT), and ΔΔCT is (ΔCT experimental) (ΔCT control) 2ΔΔCt values are the averages of five different experiments. (C) Changes in cleaved PARP protein levels in hRPE after CoCl2 treatment. (D) Changes in Caspase3 protein levels with or without Vitamin C or Vitamin E in hRPE after CoCl2 treatment. The Western blot shown is the representative of five independent experiments. Arbitrary units are the averages of the values from five different experiments. *p 0.001 versus control.
8-isoprostane release in medium and the detection of 4-HNE protein adducts in the cell lysates. The trigger of an OS condition in treated cells is further proved by the observed increase of intracellular ROS. It is true however that high levels of ROS can delay or block apoptosis since these species can readily inactivate the exposed –SH groups of caspase, but often cell death can continues by a necrotic or intermediate mechanism. The increase of ROS using CoCl2 has been shown in other experimental
approaches by several researcher and it has been postulated that the increased OS is a consequence of the enzyme xanthine oxidase activation [36] but it is also possible that the formation of 4-HNE, able to activate the enzyme NADPH oxidase (NOX), could also induce ROS formation, although this pathway was not investigated in the present work. [22]. Indeed it has been demonstrated that this lipoperoxidation by-product is able to directly activate NOX which in turn is responsible for the production of
Figure 10. Ultrastructural study: (A) Control cells tightly contacting each other, mitochondria, endoplasmic reticulum, nuclear membranes, and euchromatic nucleus with marginal heterochromatin show normal morphological features; (B) after 6 h 200 μM CoCl2-treated cells showing mitochondrial vacuolization (thick arrow), condensation of the cytoplasm and sharp decrease of intercellular contacts (thin arrow), as well as cytoplasmic and nuclear degranulation (arrows in C panel) after 24 h hypoxic treatment (C). Bars 2 μm.
Free Radic Res Downloaded from informahealthcare.com by University New South Wales on 03/30/15 For personal use only.
CoCl2-induced oxidative stress in hRPE
anion superoxide and hydrogen peroxide [22]. In addition, other studies indicate that 4-HNE promotes proliferative mechanisms and suggest that 4-HNE can induce VEGF secretion from hRPE cells acting in a paracrine fashion to induce angiogenic signaling mechanism in the endothelial cells [37]. This angiogenetic factor is involved in the repair processes of cellular damage induced by CoCl2. Then it is likely that OS induced by chelating CoCl2 and from the consequent accumulation lipid peroxidation products promotes the regulation of gene expression of the angiogenic factor VEGF. It should be mentioned that VEGF expression is controlled at both transcriptional and posttranscriptional (mRNA stabilization) levels via an array of signaling pathways [38] that leads to the activation of transcription factors, including both NFkB and HIF-1 to bind VEGF promoter at specific, functional DNA-binding sequences modulating therefore the hypoxic response [39]. Therefore it is possible that the increased levels of VEGF depends by ROS activates the transcription factor NFkB and also by the increased levels of lipid peroxidation products such as 4-HNE and this could one of the possible mechanisms involved in the new angiogenesis present in retinopathy [40]. Moreover, using Vitamin C and Vitamin E, we observed a decrease in lipid peroxidation products induced by CoCl2, confirming the hypothesis that the damage that derives from hypoxia treatment is related to OS [41–46]. The decrease of 8-isoprostanes in cells pretreated for 24 h with these antioxidants, is indicative of the decrease in the cellular damage observed also from images obtained by electron microscopy and LDH release, suggesting that in vitro, the angiogenic potential of cells is influenced in hRPE by hypoxiamimetic agent. The simultaneous activation of pro-apoptotic factors can be interpreted as a cellular defense mechanism to limit the damage caused by hypoxia. This result parallel with several other studies where it has been shown that the treatment with antioxidants such as retinoic acid, N-acetylcysteine and natural compounds protect cells against hypoxia mimetics [34,47,48]. Finally, the decreased cell viability could be a consequence of the induction of apoptosis as confirmed by the morphology results and the increased levels of Caspase 3 and PARP cleavage but also to necrosis as reported by LDH release levels. LDH is a constitutive enzyme present in the cytoplasm of all cell types and when the plasma membrane is damaged due to necrotic burst, LDH is released into the medium of cultured cells. In contrast, the cell membranes remain intact during apoptosis, preventing the release of LDH into the supernatant. Therefore this is an indirect method to show cell necrosis. This result is also in line with previous work performed in embrionic cell where it has been shown that CoCl2induced Caspases 3, 8, and 9 activation and upregulation of p53 level, with a downregulation of antiapoptotic genes such as Bcl-2. In the same study it has been suggested that CoCl2 is able to upregulate Fas and Fas-ligand, which are the death receptor assemblies [34]. Therefore it is possible that CoCl2 induces apoptosis through both mitochondria- and
311
death receptor-mediated pathways that are regulated by the Bcl-2 family [49]. In conclusion, the results herein presented showed that hypoxia induced by CoCl2, lead to increased ROS production, formation of lipid peroxidation products, activation of NFkB and the induction of VEGF. In parallel there is a decreased cell viability due also to the induction of an apoptotic pathway as shown by the increased levels of Caspase 3 and PARP. Therefore it is possible that treatments aimed to modulate retinal epithelial cells’ redox state could efficiently prevent the cascade of events that are activated by hypoxia-induced cell damage. Acknowledgments The authors are thankful to Dr. Pavan B. for the technical support in culturing the cells. Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper. References [1] Ciulla TA, Amador AG, Zinman B. Diabetic retinopathy and diabetic macular edema: pathophysiology, screening, and novel therapies. Diabetes Care 2003;26:2653–2664. [2] Gardner TW, Antonetti DA, Barber AJ, LaNoue KF, Levison SW. Diabetic retinopathy: more than meets the eye. Surv Ophthalmol 2002;47:253–262. [3] Arden GB, Sivaprasad S. The pathogenesis of early retinal changes of diabetic retinopathy. Doc Ophthalmol 2012;124: 15–26. [4] Walshe TE, D’Amore PA. The role of hypoxia in vascular injury and repair. Annu Rev Pathol 2008;3:615–643. [5] Jung SN, Yang WK, Kim J, Kim HS, Kim EJ, Yun H, et al. Reactive oxygen species stabilize hypoxia-inducible factor-1 alpha protein and stimulate transcriptional activity via AMPactivated protein kinase in DU145 human prostate cancer cells. Carcinogenesis 2008;29:713–721. [6] Berra E, Ginouvès A, Pouysségur J. The hypoxia-induciblefactor hydroxylases bring fresh air into hypoxia signalling. EMBO Rep 2006;7:41–45. [7] Ivan M, Kondo K, Yang H, Kim W, Valiando J, Ohh M, et al. HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science 2001;20: 464–468. [8] Ke Q, Costa M. Hypoxia-inducible factor-1 (HIF-1). Mol Pharmacol 2006;70:1469–1480. [9] Richard DE, Berra E, Pouyssegur J. Nonhypoxic pathway mediates the induction of hypoxia-inducible factor 1alpha in vascular smooth muscle cells. J Biol Chem 2000;1:26765–26771. [10] Görlach A, Diebold I, Schini-Kerth VB, Berchner-Pfannschmidt U, Roth U, Brandes RP, et al. Thrombin activates the hypoxiainducible factor-1 signaling pathway in vascular smooth muscle cells: role of the p22(phox)-containing NADPH oxidase. Circ Res 2001;89:47–54. [11] Yuan Y, Hilliard G, Ferguson T, Millhorn DE. Cobalt inhibits the interaction between hypoxia-inducible factor-alpha and von Hippel-Lindau protein by direct binding to hypoxiainducible factor-alpha. J Biol Chem 2003;278:15911–15916.
Free Radic Res Downloaded from informahealthcare.com by University New South Wales on 03/30/15 For personal use only.
312 F. Cervellati et al. [12] Ruef J, Moser M, Bode C, Kübler W, Runge MS. 4-hydroxynonenal induces apoptosis, NF-kappaB-activation and formation of 8-isoprostane in vascular smooth muscle cells. Basic Res Cardiol 2001;96:143–150. [13] Scholz H, Aukrust P, Damås JK, Tonstad S, Sagen EL, Kolset SO, et al. 8-isoprostane increases scavenger receptor A and matrix metalloproteinase activity in THP-1 macrophages, resulting in long-lived foam cells. Eur J Clin Invest 2004; 34:451–458. [14] Benndorf RA, Schwedhelm E, Gnann A, Taheri R, Kom G, Didié M, et al. Isoprostanes inhibit vascular endothelial growth factor-induced endothelial cell migration, tube formation, and cardiac vessel sprouting in vitro, as well as angiogenesis in vivo via activation of the thromboxane A(2) receptor: a potential link between oxidative stress and impaired angiogenesis. Circ Res 2008;103:1037–1046. [15] Sun Y, Huang L, Mackenzie GG, Rigas B. Oxidative stress mediates through apoptosis the anticancer effect of phosphononsteroidal anti-inflammatory drugs: implications for the role of oxidative stress in the action of anticancer agents. J Pharmacol Exp Ther 2011;338:775–783. [16] Leonarduzzi G, Sottero B, Testa G, Biasi F, Poli G. Inflammation-related gene expression by lipid oxidation-derived products in the progression of atherosclerosis. Free Radic Biol Med 2012;52:19–34. [17] Sunjic SB, Cipak A, Rabuzin F, Wildburger R, Zarkovic N. The influence of 4-hydroxy-2-nonenal on proliferation, differentiation and apoptosis of human osteosarcoma cells. Biofactors 2005;24:141–148. [18] Mazière C, Mazière, JC. Activation of transcription factors and gene expression by oxidized low-density lipoprotein. Free Radic Biol Med 2009;46:127–137. [19] Pattyn F, Robbrecht P, De Paepe A, Speleman F, Vandesompele J. RTPrimerDB: the real-time PCR primer and probe database, major update 2006. Nucleic Acids Res 2006; 34:684–688. [20] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402–408. [21] Cervellati F, Franceschetti G, Lunghi L, Franzellitti S, Valbonesi P, Fabbri E, et al. Effect of high-frequency electromagnetic fields on trophoblastic connexins. Reprod Toxicol 2009;28:59–65. [22] Sticozzi C, Belmonte G, Pecorelli A, Arezzini B, Gardi C, Maioli E, et al. Cigarette smoke affects keratinocytes SRB1 expression and localization via H2O2 production and HNE protein adducts formation. PLoS One 2012; 7:e33592. [23] Morrow JD, Harris TM, Roberts LJ II. Noncyclooxygenase oxidative formation of a series of novel prostaglandins: analytical ramifications for measurement of eicosanoids. Anal Biochem 1990;184:1–10. [24] Banerjee M, Kang KH, Morrow JD, Roberts LJ, Newman JH. Effects of a novel prostaglandin, 8-epi-PGF2 alpha, in rabbit lung in situ. Am J Physiol 1992;263:660–663. [25] Vacchiano CA, Tempel GE. Role of nonenzymatically generated prostanoid, 8-iso-PGF2 alpha, in pulmonary oxygen toxicity. J Appl Physiol 1994;77:2912–2917. [26] Choi K, Kim J, Kim GW, Choi C. Oxidative stress-induced necrotic cell death via mitochondira-dependent burst of reactive oxygen species. Curr Neurovasc Res 2009;6:213–222. [27] Swaroop A, Kim D, Forrest D. Transcriptional regulation of photoreceptor development and homeostasis in the mammalian retina. Nat Rev Neurosci 2010;11:563–576. [28] Dunn KC, Aotaki-Keen AE, Putkey FR, Hjelmeland LM. ARPE-19, a human retinal pigment epithelial cell line with differentiated properties. Exp Eye Res 1996;62:155–170. [29] Xie P, Fujii I, Zhao J, Shinohara M, Matsukura M. A novel polysaccharide compound derived from algae extracts protects retinal pigment epithelial cells from high glucose-induced oxidative damage in vitro. Biol Pharm Bull 2012;35: 1447–1453.
[30] Rattner A, Sun H, Nathan J. Molecular genetics of human retinal disease. Annu Rev Genet 1999;33:89–131. [31] Cai J, Nelson KC, Wu M, Sternberg P Jr, Jones DP. Oxidative stress and protection of the RPE. Prog Retinal Eye Res 2000; 19:205–221. [32] Sternberg P Jr, Davidson PC, Jones DP, Hagen TM, Reed RL, Drews-Botsch C. Protection of retinal pigment epithelium from oxidative injury by glutathione and precursors. Invest Ophthalmol Vis Sci 1993;34:3661–3668. [33] Nelson KC, Armstrong JS, Moriarty S, Cai J, Wu MW, Sternberg P Jr, Jones DP. Protection of retinal pigment epithelial cells from oxidative damage by oltipraz, a cancer chemopreventive agent. Invest Ophthalmol Vis Sci 2002;43: 3550–3554. [34] Yang L, Tan P, Zhou W, Zhu X, Cui Y, Zhu L, et al. N-acetylcysteine protects against hypoxia mimetic-induced autophagy by targeting the HIF-1α pathway in retinal ganglion cells. Cell Mol Neurobiol 2012;32:1275–1285. [35] Wang Y, Tang Z, Xue R, Singh GK, Liu W, Lv Y, Yang L. Differential response to CoCl2-stimulated hypoxia on HIF-1α, VEGF, and MMP-2 expression in ligament cells. Mol Cell Biochem 2012;360:235–242. [36] Sumbayev VV, Yasinska IM. The effect of CoCl2 on xanthine oxidase, nitric oxide synthase, and protein kinase C activity as well as cytochrome P450 1A1, 1A2 and 1B1 quantities in rat liver. Ukr Biokhim Zh 2002;74:117–120. [37] Vatsyayan R, Lelsani PC, Chaudhary P, Kumar S, Awasthi S, Awasthi YC. The expression and function of vascular endothelial growth factor in retinal pigment epithelial (RPE) cells is regulated by 4-hydroxynonenal (HNE) and glutathione S-transferaseA4-4. Biochem Biophys Res Com 2012;417:346–351. [38] Liao D, Johnson RS. Hypoxia: a key regulator of angiogenesis in cancer. Cancer Metastasis Rev 2007;26:281–290. [39] Bian ZM, Elner SG, Elner VM. Thrombin-induced VEGF expression in human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 2007;48:2738–2746. [40] Wilkinson-Berka JL, Rana I, Armani R, Agrotis A. Reactive oxygen species, Nox and angiotensin II in angiogenesis: implications for retinopathy. Clin Sci (Lond) 2013;124: 597–615. [41] Meagher EA, Barry OP, Lawson JA, Rokach J, FitzGerald GA. Effects of vitamin E on lipid peroxidation in healthy persons. JAMA 2001;285:1178–1182. [42] Matés JM, Pérez-Gómez C, Núñez de Castro I. Antioxidant enzymes and human diseases. Clin Biochem 1999;32: 595–603. [43] Carr AC, Zhu BZ, Frei B. Potential antiatherogenic mechanisms of ascorbate (Vitamin C) and α-tocopherol (Vitamin E). Circ Res 2000;87:349–354. [44] Kennedy CJ, Rakoczy PE, Constable IJ. Lipofuscin of the retinal pigment epithelium: a review. Eye (Lond) 1995;9: 763–771. [45] Stoyanovsky DA, Goldman R, Darrow RM, Organisciak DT, Kagan VE. Endogenous ascorbate regenerates vitamin E in the retina directly and in combination with exogenous dihydrolipoic acid. Curr Eye Res 1995 14:181–189. [46] Pemp B, Polska E, Karl K, Lasta M, Minichmayr A, Garhofer G, et al. Effects of antioxidants (AREDS medication) on ocular blood flow and endothelial function in an endotoxininduced model of oxidative stress in humans. Invest Ophthalmol Vis Sci 2010;51:2–6. [47] Guo LX, Liu JH, Xia ZN. Geniposide inhibits CoCl2-induced PC12 cells death via the mitochondrial pathway. Chin Med J (Engl) 2009;122:2886–2892. [48] Chen JX, Zhao T, Huang DX. Protective effects of edaravone against cobalt chloride-induced apoptosis in PC12 cells. Neurosci Bull 2009;25:67–74. [49] Lee JH, Choi SH, Baek MW, Kim MH, Kim HJ, Kim SH, et al. CoCl2 induces apoptosis through the mitochondria- and death receptor-mediated pathway in the mouse embryonic stem cells. Mol Cell Biochem 2013;379:133–140.
ORIGINAL ARTICLE
Hypoxia induces cell damage via oxidative stress in retinal epithelial cells F. Cervellati1, C. Cervellati2, A. Romani2, E. Cremonini2, C. Sticozzi1, G. Belmonte3, F. Pessina4 & G. Valacchi1,5 of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy, 2Department of Biomedical and Specialist Surgical Sciences, Section of Medical Biochemistry, Molecular Biology and Genetics University of Ferrara, Ferrara, Italy, 3Department of Surgical and Medical Sciences and Neurosciences, University of Siena, Siena, Italy, 4Department of Development and Molecular Medicine, University of Siena, Siena, Italy, and 5Department of Food and Nutrition, Kyung Hee University, Seoul, South Korea Free Radic Res Downloaded from informahealthcare.com by University New South Wales on 03/30/15 For personal use only.
1Department
Abstract Retinal diseases (RD), including diabetic retinopathy, are among the most important eye diseases in industrialized countries. RD is characterized by abnormal angiogenesis associated with an increase in cell proliferation and apoptosis. Hypoxia could be one of the triggers of the pathogenic mechanism of this disease. A key regulatory component of the cell’s hypoxia response system is hypoxia-inducible factor 1 alpha (HIF-1α). It has been demonstrated that the induction of HIF-1α expression can be also achieved in vitro by exposure with cobalt chloride (CoCl2), leading to an intracellular hypoxia-like state. In this study we have investigated the effects of CoCl2 on human retinal epithelium cells (hRPE), which are an integral part of the blood–retinal barrier, with the aim to determine the possible role of oxidative stress in chemical hypoxia-induced damage in retinal epithelial cells. Our data showed that CoCl2 treatment is able to induce HIF-1α expression, that parallels with the formation of reactive oxygen species (ROS) and the increase of lipid 8-isoprostanes and 4-hydroxynonenal (4-HNE) protein adducts levels. In addition we observed the activation of the redox-sensitive transcription factor nuclear factor-kappaB (NFkB) by CoCl2 which can explain the increased levels of vascular endothelial growth factor (VEGF). The increased number of dead cells seems to be related to an apoptotic process. Taken together these evidences suggest that oxidative stress induced by hypoxia might be involved in RD development through the stimulation of two key-events of RD such as neo-angiogenesis and apoptosis. Keywords: hypoxia-inducible factor-1 alpha, reactive oxygen species, human retinal epithelium cells, 4-hydroxynonenal, 8-isoprostanes, apoptosis
Introduction Retinal diseases (RD), including diabetic retinopathy, are among the most important eye diseases in industrialized countries, and represent the leading cause of blindness among people of working age [1]. Typically, the symptoms associated with RD appear only when the lesions are in advanced state thereby limiting the effectiveness of the treatment. The pathogenic mechanism of this disease is still under investigation although it has been postulated that hypoxia could be one of the triggers of its onset [2]. Indeed, tissue hypoxia might develop from the exceptionally high oxygen consumption of rod receptors during dark adaptation that, in combination with impaired capillary function, has been widely observed in eyes of RD patients [3]. Besides, RD is characterized by abnormal angiogenesis (the new immature vessels play an important role in disease development) which, in turn, is often associated with an increase in endothelial cells number caused by an imbalance in cell proliferation and apoptosis [3]. Of note, these processes represent two of the most observed attempts to restore oxygen homeostasis in response to hypoxic stimuli in retinal epithelial cells [4].
The adaptive responses require the concerted activation of various transcription factors including hypoxia-inducible factor-1 alpha (HIF-1α) [4,5], which is generally considered the master regulator of events related to hypoxic state [6,7]. HIF-1α protein turnover in normoxia is very quick due to the action of prolyl hydroxylases [7]. These oxygen-dependent enzymes hydroxylate two conserved proline residues of HIF-1α, promoting the binding of the Von Hippel–Lindau protein, ubiquitination, and subsequent proteosomal degradation [8]. Under hypoxic conditions the prolyl hydroxylases are inhibited, thereby allowing stabilization and accumulation of the HIF-1α protein [6–10]. The stabilization of HIF1α can be also achieved in vitro by exposure with cobalt chloride (CoCl2) which blocks the catalysis of prolyl hydroxylases, thereby leading to an intracellular hypoxia-like state [11]. It is well-known that the stabilization of HIF-1α promotes the synthesis of reactive oxygen species (ROS), which, depending on their intracellular concentration, are able to modulate the transcription of genes involved in cell proliferation, differentiation, and death [5]. These various actions of ROS are, in several cases, exerted through their lipid peroxidation by-products, in primis F2-isoprostanes
Correspondence: Giuseppe Valacchi, PhD, Department of Life Sciences and Biotechnology, University of Ferrara, 44100 Ferrara, Italy. Office: (39) 0532 455 482. Lab: (39) 0532 455 477. Mobile: (39) 335 6722592. E-mail: [email protected] (Received date: 30 October 2013; Accepted date: 17 November 2013; Published online: 16 December 2013)
Free Radic Res Downloaded from informahealthcare.com by University New South Wales on 03/30/15 For personal use only.
304 F. Cervellati et al. and unsaturated aldehydes such as 4-hydroxynonenal (4-HNE). F2-isoprostanes, indeed, appeared to be mediators of important biological effects in retinal vessels’ contraction, and increase thromboxane production and apoptosis [12–15]. The concentration-dependent activity is more accentuated for 4-HNE which can promote cell proliferation at low doses and cell damage-apoptosis at higher dose [16,17]. The regulatory ability of these two products of ROS lipid damage seems to be exerted by the activation of the redox-sensitive transcription factor NF-kappaB, a key regulator of cell proliferation and apoptosis [17–18]. Taken together these evidences suggest that oxidative stress (OS) induced by hypoxia might be crucially involved in RD development through the stimulation of two key events of the disease such as neo-angiogenesis and apoptosis. Nevertheless, direct proofs of this association are still lacking. In the present study we investigated the effect of hypoxia chemically induced with 200 μM CoCl2, on human retinal epithelium cells (hRPE) which are an integral part of the blood–retinal barrier and particularly sensitive to changes in the concentration of O2. In particular, we aimed to evaluate the possible role of OS in the modulation of hRPE response to chemical hypoxia. Methods Cell culture Human retinal pigment epithelium (hRPE) cells provided by Dr. Pavan B., were routinely grown in 1:1 mixture of Dulbecco’s modified Eagle’s and Ham’s F12 media (Lonza Milan, Italy), supplemented with 10% FBS, 50 mg/mL streptomycin, and 50 U/mL penicillin (Lonza Milan, Italy) (complete medium) on tissue culture flasks until confluence (approximately 5 106) at 37°C in 5% CO2. CoCl2 exposure Prior to CoCl2 exposure the cells were trypsinized and then grown in 35 mm Petri dishes with complete medium until a
confluent monolayer was established (approximately 2 105 cells/well). Afterwards, media were removed and fresh serum-free medium was added with or without CoCl2 (final concentration in culture 200 μM). The cells were grown and collected at different time points for biochemical assays. Cellular viability Viability studies were performed after treatments at different time by Tripan Blue and LDH release assay. The LDH release was measured by enzymatic assay: in the first step NAD is reduced to NADH/H by the LDH-catalyzed conversion of lactate to pyruvate; in the second step the catalyst (diaphorase) transfers H/H from NADH/H to tetrazolium salt which is reduced to formazan. Prior to each assay, the cells were lysed with 2% (V/V) Triton X-100 in culture media for 30 min at 37°C to obtain a representative maximal LDH release as the positive control with 100% toxicity. The amounts of LDH in the supernatant were determined and calculated according to kit instructions (EuroClone Milan, Italy). All tests were performed in triplicate and assay was repeated three times independently with similar results. As for Trypan blue staining, cells are counted by cell counter (Invitrogen, Monza, Italy). Viable cells and nonviable cells were recorded separately, and the means of three independent counts were pooled for analysis and expressed as percent of living cells with respect to total cell number (viability in percentage-%). Quantitative real-time PCR Total RNA was extracted, using an AURUM total RNA Mini Kit with DNAse digestion (Bio-Rad, Laboratories Inc., Benicia, CA, USA), from 2 105 hRPE cells for each experimental condition, according to the manufacturer’s recommended procedure. First-strand cDNA was generated from 1 μg of total RNA using the iScript cDNA Synthesis Kit (Bio-Rad, Laboratories Inc., Benicia, CA, USA). As shown in Table I, primer pairs capable of hybridization with unique regions of the appropriate gene sequence were obtained
Table I. Primer sequences and PCR condition.
Gene HIF-1a VEGF Caspase3 RPL13A RPL11A GAPDH
QPCR Product Amplification length (bp) Efficiency* (%) n° of cycles
Primer sequence
Ta°C
F: 5′-tgcttggtgctgatttgtga-3′ R: 5′-ggtcagctgatcagcgtcca-3′ F: 5′-aggccagcacataggagaga-3′ R: 5′-acgcgagtctgtgtttttgc-3′ F: 5′-tttcggtgggtgtgccctgc-3′ R: 5′-ccctgaggtttgctgcatcgaca-3′ F: 5′-cctaagatgagcgcaagttgaa-3′ R: 5′-ccacaggactagaacacctgctaa-3′ F: 5′-tgcgggaacttcgcatccgc-3′ R: 5′-gggtctgccctgtgagctgc-3′ F: 5′-tgacgctggggctggcattg-3′ R: 5′-ggctggtggtccaggggtct-3′
60.1
210
96.2
39
GenBank accession NM 181054.2
60.4
175
98.4
39
GenBank accession NM 001025368
59.8
284
94.5
39
GenBank accession NM 005497
60.2
203
97.3
39
Pattyn et al. 2006 [19]
60.1
108
96.5
39
GenBank accession NM 000975.2
60
134
94.6
39
GenBank accession NM 002046.3
Data calculated by OpticonMonitor 3 Software (Bio-Rad).
Ref. primer bank
Free Radic Res Downloaded from informahealthcare.com by University New South Wales on 03/30/15 For personal use only.
CoCl2-induced oxidative stress in hRPE
from the Real-time PCR GenBank Primer and Probe Database Primer Bank, RT primerDB [19]. Quantitative realtime PCR (qPCR) was performed using SYBR Green on the iQ5 Multicolor Real-time PCR Detection System (Bio-Rad). The final reaction mixture contained 300 nM of each primer, 1 μL of cDNA and 7.5 μL of iQ SYBR Green Supermix (Bio-Rad), with RNAse-free water being used to bring the reaction mixture volume to 15 μL. All reactions were run in triplicate. Real-time PCR was initiated with a 3-min hotstart denaturation step at 95°C, and then performed for 40 cycles at 95°C for 10 s and at 60°C for 20 s. During the reaction, fluorescence, and therefore the quantity of PCR products, was continuously monitored by Opticon Monitor 3 software (Bio-Rad). Primers were initially used to generate a standard curve over a large dynamic range of starting cDNA quantities, permitting calculation of the amplification efficiency (a critical value for the correct quantification of expression data) for each of the primer pairs. Ribosomal proteins L13a (RPL13a) L11a (RPL11a), and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were employed as reference genes. As previously described [19], samples were compared using the relative cycle threshold (CT) method [20]. After normalization to more stable mRNA, RPL13a, RPL11a, and GAPDH, the fold increase or decrease was determined with respect to control, using the formula 2ΔΔCT, where ΔCT is (gene of interest CT)(reference gene CT), and ΔΔCT is (ΔCT experimental) (ΔCT control). Western blot analysis Proteins were extracted from cells as previously described [21]. Sample proteins were assessed according to Lowry et al. using bovine serum albumin as standard. Proteins (80 μg) were resolved in Bis-Tris–HCl polyacrylamide gel (4–12%), then separated by electrophoresis (SDS–PAGE) and transferred to a polyvinylidene fluoride membrane. Immunoblots were performed using anti-rabbit polyclonal antibodies (Cell signaling Technology Inc. Beverly, MA, USA) against CASP-3, PARP, 4HNE, and vascular endothelial growth factor (VEGF) (Cell signaling Technology Inc. Beverly, MA, USA) as primary antibody, in a primary antibody diluent kit and, after washings, with goat anti-rabbit IgG (1:2000) conjugated with horseradish peroxidase for 1 h (Cell signaling Technology Inc). Immunoblots were developed by enhanced chemioluminescence reagent, and a densitometric analysis of the films was performed by Image Master (Amersham-Pharmacia, Milan, Italy) equipped with Total Lab software. Values within each experiment were normalized to the control sample. NFkB immunofluorescence analysis hRPE cells were cultured on coverslips and stained using rabbit polyclonal antibodies (Cell signaling Technology Inc.) raised against the human nuclear factor-kappaB (NFkB) (working dilutions 1:200 in PBS containing 0.05% BSA and 0.1% sodium azide) as previously described [22]. Cells were incubated with the primary antibodies for 1 h at room temperature (RT), and with secondary FITC-labeled goat anti-rabbit IgG serum (Vector
305
Laboratories, Burlingame, CA, USA) diluted 1:100 in PBS, for 1 h at RT in the dark. Slides were mounted in Vectashield (Vector Laboratories) anti-fading and examined using an Epi-fluorescence microscope (Nikon Eclipse E800; Nikon Corporation, Surrey, UK) equipped with a plan apochromat 100 0.5–1.3 oil immersion objective and a mercury lamp source. Amplifier and detector optimizing parameters were maintained constant for all the experiments. 8-isoprostanes quantification The concentration of free 8-Isoprostanes (8-iso-prostaglandin F2α, 8-iso-PGF2α) which belong to F2-isoprostanes family, was assessed in culture medium by enzyme immunoassay (Cayman Chemical, Ann Arbor, MI) [23–25], following the manufacturer’s protocol. The detection limit of 8-iso-PGF2α of this assay is 5 pg/ml and the intra- and inter-assay coefficients of variation are less than 10%. The concentration of 8-iso-PGF2α cell was assessed in medium of CoCl2-treated and untreated hRPE collected at different time periods (0, 2, 6, 12, and 24 h) either with or without preincubation with Vitamin C or Vitamin E (Sigma-Aldrich, St. Louis, MO) for 24 h at 37° C (final concentration in culture medium 50 μM). To prevent any artefactual formation or degradation of 8-iso-PGF2α in every medium aliquot was added hydroxytoluene (final concentration 0.005% BHT) prior to the assay. ROS determination For ROS determination, after the CoCl2 treatment at different time-points (0, 5, and 20 min) the cells were incubated in fresh serum-free medium with 20 μM 2,7-dichlorofluorescein diacetate (DCFH-DA) (Sigma-Aldrich) for 30 min at 37°C in the dark. DCFH-DA diffuses through the cell membrane readily and enzymatically hydrolyzed by intracellular esterases to non-fluorescent dichlorofluorescein (DCFH), which is then rapidly oxidized to highly fluorescent DCF in the presence of ROS. Then the cells were washed with PBS, harvested and centrifuged at 8000 g for 10 min. The pellet was then resuspended in PBS and read by spectrofluorimeter (Tecan infinite m200, Tecan group Ltd, Switzerland). The ROS production was expressed as the mean DCFH-DA fluorescence intensity (arbitrary units). Furthermore, we also measured the superoxide production by using the indicator MitoSOX Red (Invitrogen), a mitochondrion-specific hydroethidine-derivative fluorescent dye, according to the manufacturer’s instructions. Briefly, for live cell imaging, hRPE cells were allowed to adhere on glass coverslips. After treatment, media was removed and cells were loaded with Mitosox Red (Invitrogen) (5 μM) in Hanks’ Balanced Salt Solution for 10 min at 37°C. Cells were then washed and imaged on a Leica inverted fluorescence microscope using a Rhodamine filter (MitoSOX was excited at 515 nm, and emitted light was measured from 520–620 nm). Morphology analysis Cells were scraped and collected in 0.1 M cacodylate buffer (pH 7.4), then spun in 1.5 ml tubes at 2.000 g for 5 min.
Free Radic Res Downloaded from informahealthcare.com by University New South Wales on 03/30/15 For personal use only.
306 F. Cervellati et al. Pellets were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 4 h at 4°C. They were then washed with 0.1 M cacodylate buffer (pH 7.4) three times and postfixed in 1% osmium tetroxide and 0.1 M cacodylate buffer at pH 7.4 for 1 h at RT. The specimens were dehydrated in graded concentrations of ethanol and embedded in epoxide resin (Agar Scientific, 66A Cambridge Road, Stanstead Essex, CM24 8DA, UK). Cells were then transferred to latex modules filled with resin and subsequently thermally cured at 60°C for 48 h. Semi-thin sections (0.5–1 μm thickness) were cut using an ultra-microtome (Reichard Ultracut S, Austria) stained with toluidine blue, and blocks were selected for thinning. Ultra-thin sections of about 40–60 nm were cut and mounted onto formvar-coated copper grids. These were then double-stained with 1% uranyl acetate and 0.1% lead citrate for 30 min each and examined under a transmission electron microscope, Hitachi H-800 (Tokyo, Japan), at an accelerating voltage of 100 KV. Statistical analysis Data were expressed as mean and standard deviation. For most experiments, comparison among the different groups were tested by unpaired 2-tailed Student’s t-test. A p-value inferior to 0.05 was regarded as significant. Results CoCl2 treatment induced HIF-1α gene expression As first step we wanted to evaluate whether in our system we were able to induce the hypoxia pathway as it has been reported in the literature [26] by measuring the expression of HIF-1. As showed in Figure 1, using qPCR, we demonstrated that hRPE cells treated with CoCl2 overexpressed HIF-1α at the early time points, confirming
Figure 1. Changes in hRPE cells of HIF1-α mRNA levels after CoCl2 treatment at different time points (0–24 h). Open bars: untreated cells; Close bars: cells treated with CoCl2. The results are expressed in % compared with the control value set equal to 100% of 2ΔΔCt values. The formula used to quantify the fold change with respect to control was 2ΔΔCT, where ΔCT is (gene of interest CT) (reference gene CT), and ΔΔCT is (ΔCT experimental) (ΔCT control). The data are the averages of the five different experiments. *p 0.05 versus control.
the typical cell response to hypoxia state. In particular, it was observed a sharp and significant increase of HIF-1α mRNA expression in the first 2 h (around 120%) followed by a decline to the control value after 24 h (Figure 1). Chemical hypoxia affects cell death As shown in Figure 2A, the treatment with CoCl2 resulted in a decreased cell viability. This decline started as early as 6 h reaching significance at later time points (12 and 24 h).
Figure 2. (A) Viability % after CoCl2-treatement at different time points. (B) LDH release after CoCl2-treatement at different time points. (C) Viability % after CoCl2-treatement at 0 and 24 h with or without Vitamin C or Vitamin E. The data are the averages of five different experiments. *p 0.05 versus control at time 0; § p 0.05 versus control at the same time point; #p 0.05 versus 2 h CoCl2.
Free Radic Res Downloaded from informahealthcare.com by University New South Wales on 03/30/15 For personal use only.
CoCl2-induced oxidative stress in hRPE
307
Figure 3. (A) Variation in ROS production measured by spectrometry with DCFH-DA staining in CoCl2-treated compared with non-treated (controls) hRPE cells. (B) Mitochondrial ROS production was evaluated by Mitosox fluorescence. Cells were loaded with Mitosox before and after CS exposure and subjected to live cell imaging. The data are the averages of five different experiments. *p 0.05 versus control at time 0; §versus control at the same time point.
The effect of CoCl2 on cell viability was confirmed by LDH release. As shown in Figure 2B, CoCl2 induced an increase in LDH release already after 2 h and this effect was noticeable up to 24 h after treatment Of note is that pretreatment with Vitamin C and Vitamin E prevented CoCl2-induced LDH release leading to the idea that CoCl2 exposure is linked to ROS production. This hypothesis was confirmed by the results showed in Panel C where pretreatement with Vitamin C and Vitamin E prevent CoCl2 decreased cell viability (Figure 2C), although this effect was more evident with Vitamin E.
production of 8-iso-PGF2α in culture medium of hRPE cells treated and untreated with CoCl2 (Figure 4). As displayed in Figure 4 there was an increase of 8-iso-PGF2α levels already after 2 h (25%) of treatment and it became more evident (around 75%) at 24 h. As depicted in Figure 5 preincubation of hRPE with Vitamin C or Vitamin E hampered CoCl2-induced F2-isoprostanes levels production at 24 h, thus confirming the association between chemical hypoxia and OS in hRPE cells.
ROS production
One of the consequences of lipid peroxidation is the formation of bioactive products such as unsaturated aldehydes like 4-HNE that are able to interact with cellular proteins. To evaluate whether chemical hypoxia-induced
As a consequence of Figure 2, we went to verify whether the induction of hypoxia by CoCl2 could also generate OS in hRPE cells. As shown in Figure 3A, cells treated with CoCl2 revealed a significant increase in ROS concentration after 20 min (circa 4 fold). Furthermore, mitochondrial ROS production was evaluated in hRPE cells after CoCl2 treatment by Mitosox assay. As shown in Figure 2B, cells exposed to CoCl2 showed an increased in fluorescence, characteristic of mitochondrial staining. This effect was detected already after 15 min of treatment and persist over 60 min. 8-iso-PGF2α concentration in medium cells is induced by CoCl2 treatment and hampered by antioxidant pretreatment To further verify the involvement of OS in cell response to hypoxia induced by CoCl2, we measured the time-course
4-HNE protein adducts formation after CoCl2 treatment expression
Figure 4. 8-iso-PGF2α levels present in hRPE cells medium after CoCl2 treatment. The results are expressed in percentage (%) compared with the control (not treated cells at time 0) value, set equal to 100%. The data are the averages of five different experiments. *p 0.001 versus control at time 0; §p 0.001 versus control at the same time point; #p 0.001 versus CoCl2 at time 24.
Free Radic Res Downloaded from informahealthcare.com by University New South Wales on 03/30/15 For personal use only.
308 F. Cervellati et al.
Figure 5. Effects of Vitamin C or Vitamin E preincubation on of 8-iso-PGF2α formation after 24 h treatment with CoCl2. The results are expressed in percentage (%) compared with the concentration of 8-iso-PGF2α detected in not treated hRPE (control), which was set equal to 100%. The data are the averages of five different experiments. *p 0.05 versus control; #p 0.05 versus CoCl2-treated cells without Vitamin C or Vitamin E preincubation.
4-HNE protein adducts formation, we performed Western blot analysis in cell lysates. As shown in Figure 6 CoCl2 induced a clear increase in 4-HNE protein adduct formation after 2 h of treatment ( 40%) and went back to the steady level after 12 h. This result is in line with the increased level of ROS and 8-iso-PGF2α observed in CoCl2 exposed cells. Effect of CoCl2 on NFkB nuclear translocation In CoCl2-treated cells there was a clear increase of NFkB nuclear translocation as displayed by the increment in punctate fluorescence (Figure 7). This effect was well evident already after 30 min of treatment and decremented in the following time point (1 h), showing that NFkB activation is an early event in CoCl2 induce cellular responses. CoCl2-induced VEGF gene and protein expression One of the main features of retina-related diseases is the presence of new blood vessels and since VEGF is the main inducer of neo-angiogenesis and its transcription is also regulated by NFkB we evaluated VEGF levels in hRPE cells exposed to CoCl2. As shown in Figure 8A, cells treated with 200 μM of CoCl2 showed a significant time-dependent increase in VEGF mRNA with and induction of circa 2.5 fold at 6 h and almost 4 fold at 24 h. In parallel also the protein levels showed a time-dependent increasing trend (Figure 8B). CoCl2 induced Caspase 3 expression As reported in Figure 2, treatment with CoCl2 decreased cell viability and this result prompted us to check if it was related to an induction of cellular apoptotosis. Clues of programmed cell death were obtained by the
Figure 6. Effect of CoCl2 treatment on 4-HNE protein adducts formation in hRPE cells. (A) a representative Western blot of five different experiments. (B) Quantification of 4-HNE protein adducts band. Data are expressed as Arbitrary Units (AU). *p 0.05 versus control.
determination Caspase 3 gene and protein expression. Indeed, the mRNA levels of the pro-apoptotic gene Caspase 3 increased significantly already at 6 h after the treatment with CoCl2 with respect to the control (Figure 9). This trend persisted also at later time points (24 h), with an overall up-regulation of circa 50% Figure 9A. This was also confirmed at the protein levels, as shown in Figure 9B, Western blot analysis demonstrated a clear increase in cleaved Caspase 3 protein levels starting at 6 h and reaching the maximum expression at 24 h. Of note, pretreatment with Vitamin C or Vitamin E prevented cleaved Caspase 3 increase, confirming the hypothesis that OS plays a role in CoCl2-induced cell death. Apoptosis was also evaluated by the detection of PARP cleavage and also in this case we have observed a similar trend noticed for Caspase 3. CoCl2 treatment affects cell morphology The electron microscopy analysis of hRPE before and after CoCl2 treatment showed an evident degeneration of cells’ ultra-structure. As shown in Figure 10, before
CoCl2-induced oxidative stress in hRPE
309
Free Radic Res Downloaded from informahealthcare.com by University New South Wales on 03/30/15 For personal use only.
Figure 7. NFkB staining in hRPE cells: (A) Control (not treated cells at t0); (B) CoCl2 for 30 min; (C) CoCl2 for 1 h. Scale bar 10 μm.
treatment, cells were tightly contacting each other (A). Mitochondria, endoplasmic reticulum, nuclear membranes and euchromatic nucleus with marginal heterochromatin showed normal morphological features (A); after 6 h of treatment with 200 μM CoCl2 the cells changed their morphology, showing vacuolation and condensation of the cytoplasm, nuclear bulge, and loss of intercellular adhesion (B); exposition for 24 h worsened the cell condition, leading to loss of mitochondria, degranulation of both the cytoplasm and the nucleoplasm (C). Discussion The human retinal pigment epithelial cell (hRPE) is a highly specialized retinal cell layer that plays important roles in the regulation and development of photoreceptors in the vertebrate retina [27]. In normal retina, hRPE cells are involved in transporting and stocking materials, for example retinaldehyde; phagocytosing detached photoreceptor outer segments; intercepting light; removing free radicals; synthesizing cytokines; and forming the blood– retinal barrier [28]. The integrity of the hRPE is of key importance for maintaining the integrity and proper functioning of the human retina [29]. Despite the central role of these cells in retinal physiology, there are few studies
where they are used as a model to investigate the pathogenic mechanisms of RD [30]. It has been shown that OS might play a role in the pathological changes, in particular apoptosis and neoangiogenesis processes, occurring in retinal pigment epithelium during the early stages of RD [31]. This postulated role for OS is confirmed with ability of reduced glutathione (GSH) [32], or compounds able to promote its synthesis [33], to protect hRPE cells against cell death induced by oxidants [34]. It is well known that hypoxia due to oxygen tension below the normal limits in a specific cell, tissue or organism is a common feature of the majority of RD. What has not been still clarified is whether OS could be the “armed hand” triggered by hypoxia in these diseases. In this study, we have demonstrated that chemical hypoxia induced by CoCl2 is able to mimic the hypoxic state as indicated by the rapid increase of mRNA HIF-1α expression and this has been also confirmed by other researchers using a similar approach [35]. The cause of loss of cell viability is most probably due to apoptotic process as highlighted by typical ultrastructural alterations and the consistent increase levels of the cleaved form of Caspase 3 and PARP cleavage. The CoCl2related damage of hRPE can be the result of a lipoperoxidative process as suggested by the increase of
Figure 8. VEGF expression in hRPE cells before and after CoCl2 treatment. (A) mRNA levels are expressed in % compared with the control value set equal to 100% of 2ΔΔCt values. The formula used to quantify the fold change with respect to control was 2ΔΔCT, where ΔCT is (gene of interest CT)(reference gene CT), and ΔΔCT is (ΔCT experimental) (ΔCT control) 2ΔΔCt values are the averages of five different experiments. (B) protein levels: upper panel is a representative Western blot of five independent experiments, the bands quantification are reported in the bottom panel. (A) *p 0.001 versus control; §p 0.001 versus t12. (B) *P 0.01 versus control.
Free Radic Res Downloaded from informahealthcare.com by University New South Wales on 03/30/15 For personal use only.
310 F. Cervellati et al.
Figure 9. Changes in Caspase3 mRNA (A) and protein (B) expression in hRPE after CoCl2 treatment. mRNA levels are expressed in % compared with the control value set equal to 100% of 2ΔΔCt values. 2ΔΔCT was the formula used to quantify the fold change with respect to control, where ΔCT is (gene of interest CT)(reference gene CT), and ΔΔCT is (ΔCT experimental) (ΔCT control) 2ΔΔCt values are the averages of five different experiments. (C) Changes in cleaved PARP protein levels in hRPE after CoCl2 treatment. (D) Changes in Caspase3 protein levels with or without Vitamin C or Vitamin E in hRPE after CoCl2 treatment. The Western blot shown is the representative of five independent experiments. Arbitrary units are the averages of the values from five different experiments. *p 0.001 versus control.
8-isoprostane release in medium and the detection of 4-HNE protein adducts in the cell lysates. The trigger of an OS condition in treated cells is further proved by the observed increase of intracellular ROS. It is true however that high levels of ROS can delay or block apoptosis since these species can readily inactivate the exposed –SH groups of caspase, but often cell death can continues by a necrotic or intermediate mechanism. The increase of ROS using CoCl2 has been shown in other experimental
approaches by several researcher and it has been postulated that the increased OS is a consequence of the enzyme xanthine oxidase activation [36] but it is also possible that the formation of 4-HNE, able to activate the enzyme NADPH oxidase (NOX), could also induce ROS formation, although this pathway was not investigated in the present work. [22]. Indeed it has been demonstrated that this lipoperoxidation by-product is able to directly activate NOX which in turn is responsible for the production of
Figure 10. Ultrastructural study: (A) Control cells tightly contacting each other, mitochondria, endoplasmic reticulum, nuclear membranes, and euchromatic nucleus with marginal heterochromatin show normal morphological features; (B) after 6 h 200 μM CoCl2-treated cells showing mitochondrial vacuolization (thick arrow), condensation of the cytoplasm and sharp decrease of intercellular contacts (thin arrow), as well as cytoplasmic and nuclear degranulation (arrows in C panel) after 24 h hypoxic treatment (C). Bars 2 μm.
Free Radic Res Downloaded from informahealthcare.com by University New South Wales on 03/30/15 For personal use only.
CoCl2-induced oxidative stress in hRPE
anion superoxide and hydrogen peroxide [22]. In addition, other studies indicate that 4-HNE promotes proliferative mechanisms and suggest that 4-HNE can induce VEGF secretion from hRPE cells acting in a paracrine fashion to induce angiogenic signaling mechanism in the endothelial cells [37]. This angiogenetic factor is involved in the repair processes of cellular damage induced by CoCl2. Then it is likely that OS induced by chelating CoCl2 and from the consequent accumulation lipid peroxidation products promotes the regulation of gene expression of the angiogenic factor VEGF. It should be mentioned that VEGF expression is controlled at both transcriptional and posttranscriptional (mRNA stabilization) levels via an array of signaling pathways [38] that leads to the activation of transcription factors, including both NFkB and HIF-1 to bind VEGF promoter at specific, functional DNA-binding sequences modulating therefore the hypoxic response [39]. Therefore it is possible that the increased levels of VEGF depends by ROS activates the transcription factor NFkB and also by the increased levels of lipid peroxidation products such as 4-HNE and this could one of the possible mechanisms involved in the new angiogenesis present in retinopathy [40]. Moreover, using Vitamin C and Vitamin E, we observed a decrease in lipid peroxidation products induced by CoCl2, confirming the hypothesis that the damage that derives from hypoxia treatment is related to OS [41–46]. The decrease of 8-isoprostanes in cells pretreated for 24 h with these antioxidants, is indicative of the decrease in the cellular damage observed also from images obtained by electron microscopy and LDH release, suggesting that in vitro, the angiogenic potential of cells is influenced in hRPE by hypoxiamimetic agent. The simultaneous activation of pro-apoptotic factors can be interpreted as a cellular defense mechanism to limit the damage caused by hypoxia. This result parallel with several other studies where it has been shown that the treatment with antioxidants such as retinoic acid, N-acetylcysteine and natural compounds protect cells against hypoxia mimetics [34,47,48]. Finally, the decreased cell viability could be a consequence of the induction of apoptosis as confirmed by the morphology results and the increased levels of Caspase 3 and PARP cleavage but also to necrosis as reported by LDH release levels. LDH is a constitutive enzyme present in the cytoplasm of all cell types and when the plasma membrane is damaged due to necrotic burst, LDH is released into the medium of cultured cells. In contrast, the cell membranes remain intact during apoptosis, preventing the release of LDH into the supernatant. Therefore this is an indirect method to show cell necrosis. This result is also in line with previous work performed in embrionic cell where it has been shown that CoCl2induced Caspases 3, 8, and 9 activation and upregulation of p53 level, with a downregulation of antiapoptotic genes such as Bcl-2. In the same study it has been suggested that CoCl2 is able to upregulate Fas and Fas-ligand, which are the death receptor assemblies [34]. Therefore it is possible that CoCl2 induces apoptosis through both mitochondria- and
311
death receptor-mediated pathways that are regulated by the Bcl-2 family [49]. In conclusion, the results herein presented showed that hypoxia induced by CoCl2, lead to increased ROS production, formation of lipid peroxidation products, activation of NFkB and the induction of VEGF. In parallel there is a decreased cell viability due also to the induction of an apoptotic pathway as shown by the increased levels of Caspase 3 and PARP. Therefore it is possible that treatments aimed to modulate retinal epithelial cells’ redox state could efficiently prevent the cascade of events that are activated by hypoxia-induced cell damage. Acknowledgments The authors are thankful to Dr. Pavan B. for the technical support in culturing the cells. Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper. References [1] Ciulla TA, Amador AG, Zinman B. Diabetic retinopathy and diabetic macular edema: pathophysiology, screening, and novel therapies. Diabetes Care 2003;26:2653–2664. [2] Gardner TW, Antonetti DA, Barber AJ, LaNoue KF, Levison SW. Diabetic retinopathy: more than meets the eye. Surv Ophthalmol 2002;47:253–262. [3] Arden GB, Sivaprasad S. The pathogenesis of early retinal changes of diabetic retinopathy. Doc Ophthalmol 2012;124: 15–26. [4] Walshe TE, D’Amore PA. The role of hypoxia in vascular injury and repair. Annu Rev Pathol 2008;3:615–643. [5] Jung SN, Yang WK, Kim J, Kim HS, Kim EJ, Yun H, et al. Reactive oxygen species stabilize hypoxia-inducible factor-1 alpha protein and stimulate transcriptional activity via AMPactivated protein kinase in DU145 human prostate cancer cells. Carcinogenesis 2008;29:713–721. [6] Berra E, Ginouvès A, Pouysségur J. The hypoxia-induciblefactor hydroxylases bring fresh air into hypoxia signalling. EMBO Rep 2006;7:41–45. [7] Ivan M, Kondo K, Yang H, Kim W, Valiando J, Ohh M, et al. HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science 2001;20: 464–468. [8] Ke Q, Costa M. Hypoxia-inducible factor-1 (HIF-1). Mol Pharmacol 2006;70:1469–1480. [9] Richard DE, Berra E, Pouyssegur J. Nonhypoxic pathway mediates the induction of hypoxia-inducible factor 1alpha in vascular smooth muscle cells. J Biol Chem 2000;1:26765–26771. [10] Görlach A, Diebold I, Schini-Kerth VB, Berchner-Pfannschmidt U, Roth U, Brandes RP, et al. Thrombin activates the hypoxiainducible factor-1 signaling pathway in vascular smooth muscle cells: role of the p22(phox)-containing NADPH oxidase. Circ Res 2001;89:47–54. [11] Yuan Y, Hilliard G, Ferguson T, Millhorn DE. Cobalt inhibits the interaction between hypoxia-inducible factor-alpha and von Hippel-Lindau protein by direct binding to hypoxiainducible factor-alpha. J Biol Chem 2003;278:15911–15916.
Free Radic Res Downloaded from informahealthcare.com by University New South Wales on 03/30/15 For personal use only.
312 F. Cervellati et al. [12] Ruef J, Moser M, Bode C, Kübler W, Runge MS. 4-hydroxynonenal induces apoptosis, NF-kappaB-activation and formation of 8-isoprostane in vascular smooth muscle cells. Basic Res Cardiol 2001;96:143–150. [13] Scholz H, Aukrust P, Damås JK, Tonstad S, Sagen EL, Kolset SO, et al. 8-isoprostane increases scavenger receptor A and matrix metalloproteinase activity in THP-1 macrophages, resulting in long-lived foam cells. Eur J Clin Invest 2004; 34:451–458. [14] Benndorf RA, Schwedhelm E, Gnann A, Taheri R, Kom G, Didié M, et al. Isoprostanes inhibit vascular endothelial growth factor-induced endothelial cell migration, tube formation, and cardiac vessel sprouting in vitro, as well as angiogenesis in vivo via activation of the thromboxane A(2) receptor: a potential link between oxidative stress and impaired angiogenesis. Circ Res 2008;103:1037–1046. [15] Sun Y, Huang L, Mackenzie GG, Rigas B. Oxidative stress mediates through apoptosis the anticancer effect of phosphononsteroidal anti-inflammatory drugs: implications for the role of oxidative stress in the action of anticancer agents. J Pharmacol Exp Ther 2011;338:775–783. [16] Leonarduzzi G, Sottero B, Testa G, Biasi F, Poli G. Inflammation-related gene expression by lipid oxidation-derived products in the progression of atherosclerosis. Free Radic Biol Med 2012;52:19–34. [17] Sunjic SB, Cipak A, Rabuzin F, Wildburger R, Zarkovic N. The influence of 4-hydroxy-2-nonenal on proliferation, differentiation and apoptosis of human osteosarcoma cells. Biofactors 2005;24:141–148. [18] Mazière C, Mazière, JC. Activation of transcription factors and gene expression by oxidized low-density lipoprotein. Free Radic Biol Med 2009;46:127–137. [19] Pattyn F, Robbrecht P, De Paepe A, Speleman F, Vandesompele J. RTPrimerDB: the real-time PCR primer and probe database, major update 2006. Nucleic Acids Res 2006; 34:684–688. [20] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402–408. [21] Cervellati F, Franceschetti G, Lunghi L, Franzellitti S, Valbonesi P, Fabbri E, et al. Effect of high-frequency electromagnetic fields on trophoblastic connexins. Reprod Toxicol 2009;28:59–65. [22] Sticozzi C, Belmonte G, Pecorelli A, Arezzini B, Gardi C, Maioli E, et al. Cigarette smoke affects keratinocytes SRB1 expression and localization via H2O2 production and HNE protein adducts formation. PLoS One 2012; 7:e33592. [23] Morrow JD, Harris TM, Roberts LJ II. Noncyclooxygenase oxidative formation of a series of novel prostaglandins: analytical ramifications for measurement of eicosanoids. Anal Biochem 1990;184:1–10. [24] Banerjee M, Kang KH, Morrow JD, Roberts LJ, Newman JH. Effects of a novel prostaglandin, 8-epi-PGF2 alpha, in rabbit lung in situ. Am J Physiol 1992;263:660–663. [25] Vacchiano CA, Tempel GE. Role of nonenzymatically generated prostanoid, 8-iso-PGF2 alpha, in pulmonary oxygen toxicity. J Appl Physiol 1994;77:2912–2917. [26] Choi K, Kim J, Kim GW, Choi C. Oxidative stress-induced necrotic cell death via mitochondira-dependent burst of reactive oxygen species. Curr Neurovasc Res 2009;6:213–222. [27] Swaroop A, Kim D, Forrest D. Transcriptional regulation of photoreceptor development and homeostasis in the mammalian retina. Nat Rev Neurosci 2010;11:563–576. [28] Dunn KC, Aotaki-Keen AE, Putkey FR, Hjelmeland LM. ARPE-19, a human retinal pigment epithelial cell line with differentiated properties. Exp Eye Res 1996;62:155–170. [29] Xie P, Fujii I, Zhao J, Shinohara M, Matsukura M. A novel polysaccharide compound derived from algae extracts protects retinal pigment epithelial cells from high glucose-induced oxidative damage in vitro. Biol Pharm Bull 2012;35: 1447–1453.
[30] Rattner A, Sun H, Nathan J. Molecular genetics of human retinal disease. Annu Rev Genet 1999;33:89–131. [31] Cai J, Nelson KC, Wu M, Sternberg P Jr, Jones DP. Oxidative stress and protection of the RPE. Prog Retinal Eye Res 2000; 19:205–221. [32] Sternberg P Jr, Davidson PC, Jones DP, Hagen TM, Reed RL, Drews-Botsch C. Protection of retinal pigment epithelium from oxidative injury by glutathione and precursors. Invest Ophthalmol Vis Sci 1993;34:3661–3668. [33] Nelson KC, Armstrong JS, Moriarty S, Cai J, Wu MW, Sternberg P Jr, Jones DP. Protection of retinal pigment epithelial cells from oxidative damage by oltipraz, a cancer chemopreventive agent. Invest Ophthalmol Vis Sci 2002;43: 3550–3554. [34] Yang L, Tan P, Zhou W, Zhu X, Cui Y, Zhu L, et al. N-acetylcysteine protects against hypoxia mimetic-induced autophagy by targeting the HIF-1α pathway in retinal ganglion cells. Cell Mol Neurobiol 2012;32:1275–1285. [35] Wang Y, Tang Z, Xue R, Singh GK, Liu W, Lv Y, Yang L. Differential response to CoCl2-stimulated hypoxia on HIF-1α, VEGF, and MMP-2 expression in ligament cells. Mol Cell Biochem 2012;360:235–242. [36] Sumbayev VV, Yasinska IM. The effect of CoCl2 on xanthine oxidase, nitric oxide synthase, and protein kinase C activity as well as cytochrome P450 1A1, 1A2 and 1B1 quantities in rat liver. Ukr Biokhim Zh 2002;74:117–120. [37] Vatsyayan R, Lelsani PC, Chaudhary P, Kumar S, Awasthi S, Awasthi YC. The expression and function of vascular endothelial growth factor in retinal pigment epithelial (RPE) cells is regulated by 4-hydroxynonenal (HNE) and glutathione S-transferaseA4-4. Biochem Biophys Res Com 2012;417:346–351. [38] Liao D, Johnson RS. Hypoxia: a key regulator of angiogenesis in cancer. Cancer Metastasis Rev 2007;26:281–290. [39] Bian ZM, Elner SG, Elner VM. Thrombin-induced VEGF expression in human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 2007;48:2738–2746. [40] Wilkinson-Berka JL, Rana I, Armani R, Agrotis A. Reactive oxygen species, Nox and angiotensin II in angiogenesis: implications for retinopathy. Clin Sci (Lond) 2013;124: 597–615. [41] Meagher EA, Barry OP, Lawson JA, Rokach J, FitzGerald GA. Effects of vitamin E on lipid peroxidation in healthy persons. JAMA 2001;285:1178–1182. [42] Matés JM, Pérez-Gómez C, Núñez de Castro I. Antioxidant enzymes and human diseases. Clin Biochem 1999;32: 595–603. [43] Carr AC, Zhu BZ, Frei B. Potential antiatherogenic mechanisms of ascorbate (Vitamin C) and α-tocopherol (Vitamin E). Circ Res 2000;87:349–354. [44] Kennedy CJ, Rakoczy PE, Constable IJ. Lipofuscin of the retinal pigment epithelium: a review. Eye (Lond) 1995;9: 763–771. [45] Stoyanovsky DA, Goldman R, Darrow RM, Organisciak DT, Kagan VE. Endogenous ascorbate regenerates vitamin E in the retina directly and in combination with exogenous dihydrolipoic acid. Curr Eye Res 1995 14:181–189. [46] Pemp B, Polska E, Karl K, Lasta M, Minichmayr A, Garhofer G, et al. Effects of antioxidants (AREDS medication) on ocular blood flow and endothelial function in an endotoxininduced model of oxidative stress in humans. Invest Ophthalmol Vis Sci 2010;51:2–6. [47] Guo LX, Liu JH, Xia ZN. Geniposide inhibits CoCl2-induced PC12 cells death via the mitochondrial pathway. Chin Med J (Engl) 2009;122:2886–2892. [48] Chen JX, Zhao T, Huang DX. Protective effects of edaravone against cobalt chloride-induced apoptosis in PC12 cells. Neurosci Bull 2009;25:67–74. [49] Lee JH, Choi SH, Baek MW, Kim MH, Kim HJ, Kim SH, et al. CoCl2 induces apoptosis through the mitochondria- and death receptor-mediated pathway in the mouse embryonic stem cells. Mol Cell Biochem 2013;379:133–140.
