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Hypoxia preconditioning protects corneal stromal cells against induced apoptosis
Experimental Eye Research 82 (2006) 780–787 www.elsevier.com/locate/yexer
Hypoxia preconditioning protects corneal stromal cells against induced apoptosis Dongmei Xing, Xingcai Sun, Jinhua Li, Miao Cui, Kah Tan-Allen, Joseph A. Bonanno * School of Optometry, Indiana University, 800 East Atwater Avenue, , Bloomington, IN 47405, USA Received 4 April 2005; accepted in revised form 27 September 2005 Available online 20 December 2005
Abstract The purpose of this study, was to determine whether hypoxia preconditioning can protect corneal stromal cells from UV stress and cytokine mediated apoptosis. Two models were implemented. First, primary cultured bovine corneal fibroblasts were preconditioned with 0.5–1.5% O2 for 4 hr and stressed with UV-irradiation or stimulation of Fas receptor. Second, bovine eyes were preconditioned with 0.5% O2 for 4 hr and stressed by epithelial scraping to induce anterior keratocyte apoptosis. Cell fate was analyzed at 4 hr after stress using quantitative TUNEL or condensed nuclei assays. Cell apoptotic rates in hypoxia preconditioned groups were significantly lower (50–80%) than that of normoxia control groups. Hypoxia prevented the degradation of the transcription factor HIF-1a. CoCl2 (100–200 mM), a chemical inducer of HIF-1a, also produced strong protection against UV and Fas induced apoptosis. Moreover, hypoxia preconditioned media protected cells against UV-induced apoptosis. These findings demonstrate that hypoxia preconditioning has a generalized protective effect against stromal fibroblast and keratocyte apoptosis and suggest that HIF-1a mediated expression and secretion of protective factors is involved. q 2005 Elsevier Ltd. All rights reserved. Keywords: cornea; keratocytes; apoptosis; hypoxia preconditioning
1. Introduction Apoptosis is a form of involutional cell death that occurs with limited inflammation and little release of cellular contents that would inflict collateral damage to surrounding tissue (Wyllie et al., 1980; Arends and Wyllie, 1991). This gentle mechanism for eliminating cells is of obvious significance to the maintenance of corneal clarity. Many types of epithelial injury can induce keratocyte apoptosis including mechanical scrape (Wilson et al., 1996), corneal refractive surgical procedures (Helena et al., 1998) and viral infection (Wilson et al., 1997). Keratocyte apoptosis is the earliest stromal event noted following epithelial injury and remains a likely target for modulation of the overall wound healing response (Wilson et al., 2001). Current evidence indicates that the etiology of keratocyte apoptosis is mediated by epithelial release of cytokines that could include IL-1 (Wilson et al., 1996),
* Corresponding author. Joseph A. Bonanno, School of Optometry, Indiana University, 800 East Atwater Avenue, Bloomington, IN 47405, USA. E-mail address: [email protected] (J.A. Bonanno).
0014-4835/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2005.09.020
tumor necrosis factor (TNF) (Mohan et al., 2000) and platelet activation factor (PAF) (Chandrasekher et al., 2002). Keratocyte apoptosis, along with subsequent repopulation by activated stromal keratocytes, has been hypothesized to be an important determinant of corneal wound healing associated with refractive variability and regression after photorefractive keratectomy and laser-assisted in situ keratomileusis (LASIK) (Wilson, 1997; Helena et al., 1998). Keratocyte loss promotes the activation and proliferation of surrounding keratocytes. Recent studies suggest that disruption of the basement membrane releases TGF-b (Transforming Growth Factor b), which can induce myofibroblast formation in vitro (Jester et al., 1999; Jester and Ho-Chang, 2003) and promotes differentiation of activated keratocytes into myofibroblasts in vivo (Stramer et al., 2003). Myofibroblasts are responsible for refractive variability and light scatter (Moller-Pedersen et al., 1998). These wound healing events are directly associated with the initial extent of apoptosis (Mohan et al., 2003) which have led to the notion that reducing keratocyte death by inhibiting the initial keratocyte apoptosis could also lessen the undesirable aspects of the wound healing response. Here, we examine the potential of using hypoxia preconditioning as a means to protect corneal stromal cells from lethal stresses. Hypoxia has been shown to be either antiapoptotic or pro-apoptotic, depending on the cell type
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and experimental conditions (Piret et al., 2002). Severe hypoxia impairs cellular energy production and ion homeostasis leading to cell injury and cell death. Interestingly, nonlethal hypoxia activates mechanisms that could produce a cellular phenotype more resistant to acute severe oxidative stress (Silverman et al., 1997). Hypoxic preconditioning attenuated cortical infarction in the rat brain induced by oxidative stress (Lin Anya et al., 2003), attenuated bladder over distension-induced oxidative injury (Yu et al., 2004), and protected retinal morphology and function against lightinduced apoptosis (Grimm et al., 2002). At the single cell level, isolated cardiomyocytes cultured at low oxygen tension acquire resistance against acute severe oxidative stress (Carmeliet et al., 1998). HepG2 cells preconditioned with hypoxia or CoCl2, a hypoxia mimetic, reduced serum deprivation and tert butyl hydrogen peroxide-induced apoptosis (Piret et al., 2004). The mechanisms for hypoxia preconditioning protection are incompletely understood, but often require activation of Hypoxia inducible factor-1a (HIF1a), the main transcriptional factor activated by hypoxia. Genes regulated by HIF-1a include erythropoietin (EPO) and VEGF (Vascular Endothelial Growth factor). These proteins have well described anti-apoptotic properties. For example, local production of EPO during retinal hypoxia protected photoreceptors from light induced cell death (Grimm et al., 2002). Hypoxia is also known to stimulate translocation of Hsp27 and aB-crystallin from diffuse locations to defined structures, which is associated with a decrease in caspase-3 activity (Sakamoto et al., 1998; Webster, 2003). Furthermore, Hsp27 is up-regulated by HIF1-a and is protective in a rat retinal ischemia model (Whitlock et al., 2005). These studies suggest that hypoxia preconditioning through HIF1-a mediated transcription can protect cells by multiple mechanisms. In the current study, we reasoned that corneal stromal cells exposed to significant but non-lethal hypoxia could show adaptations making them more tolerant of certain apoptotic stress. We asked whether hypoxia preconditioning could protect cells from UV-irradiation and Fas mediated apoptosis and whether this protection was associated with induction of HIF-1a. We also tested whether hypoxia preconditioning could protect keratocytes from epithelial scrape induced apoptosis in an ex vivo bovine cornea model. In all cases, we found that hypoxia preconditioning or induction of HIF-1a were strongly protective. 2. Materials and methods 2.1. Cell culture Blocks of stroma were cut from fresh bovine cornea and cultured in DMEM (GIBCO) supplemented with 10% fetal bovine serum (FBS), 100 units/ml of penicillin, 100 mg/ml of streptomycin and 0.25 mg/ml of amphotericin B. Corneal stromal fibroblasts migrated from the stromal explants and grew exponentially at densities below 5!105 cells/ml. Second generation fibroblasts were seeded onto coverslips or petridishes and used in all cell culture experiments.
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2.2. Induction of hypoxia Corneal fibroblasts (5!104 cells) were sub-cultured to 25 mm round coverslips in six-well plates with 2 ml DMEM supplemented with 10% FBS for 1 day. Media was changed immediately before experiments to DMEM with 0.5% FBS. This amount of serum was sufficient to prevent cell death, but does not promote proliferation. For hypoxia preconditioning, cells were placed in a hypoxia chamber (Coy Lab Products Inc., Grass Lake, Michigan) equilibrated with 5% CO2 and either 1.5 or 0.5% oxygen-balance nitrogen for at least 4 hr. 2.3. UV-irradiation A germicidal lamp (TUV/30W/G30 T8, Philips) that emitted radiation ranging from 230 to 400 nm was used as the UV source. The emission peak was 255 nm. UV dose was quantified using a radiometer (IL 1700, International Light Inc., Newburyport, MA, USA). To produce approximately 40% apoptotic cells 4 hr after irradiation, 2 min of UV exposure was required, which corresponded to 5.1 mJ/cm2. Cells were irradiated at 80% confluence. Culture media was removed and replaced with 2 ml of a balanced ringer solution to avoid variations in UV absorption from media components. After irradiation the ringer solution was discarded and replaced by fresh DMEM/0.5% FBS. 2.4. TUNEL assay Four hours after UV-irradiation cells on coverslips were fixed in the fresh 4% formaldehyde/PBS at 4 8C for 25 min. Following fixation the cells were rinsed twice with PBS and permeabilized with prechilled 0.2% Triton X-100/PBS on ice for 5 min. A fluorescence-based TUNEL assay was used according to the manufacturer’s instructions (ApoAlert, BD Biosciences, Palo Alto, CA, USA). Cells were counterstained with DAPI and mounted with prolong antifade reagent (Molecular Probe). Images were obtained with a fluorescence microscope (Nikon E600, Melville, NY, USA) equipped with charge coupled device camera with active cooling system. 2.5. Hypoxia preconditioned media Primary cultured cornea stromal fibroblasts (2!105) in 2 ml DMEM with 0.5% fetal bovine serum were incubated in 0.5% oxygen for 4 hr. Conditioned media was collected and stored at K808 until use. Fibroblasts (2!105) plated on glass coverslips were incubated in the hypoxia preconditioned media for 2 or 4 hr under normoxic conditions and then irradiated with 2 min of UV light. TUNEL staining was performed 4 hr after irradiation. 2.6. Cornea wound model Immediately after death at the abbatoir, paired bovine eyeballs from one animal were placed in plastic bags with PBS on ice and transported to the lab. Eyes were trimmed of
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connective tissues and stored at 4 8C overnight. The next day, the eyes were rinsed with PBS and placed in open dishes. One eye was incubated in a humidified chamber 0.5% O2/5% CO2 and the other eye was put into a standard 5% CO2/Air humidified tissue culture incubator for 4 hr and then the hypoxia preconditioned eye was placed into the standard incubator for 30 min. Using a scalpel blade, the entire epithelium of both eyes was removed by scraping while leaving the basement membrane intact. After injury, both eyes were placed in the standard incubator for 4 hr. 2.7. Tissue fixation, sectioning and staining Four hours after epithelial scraping, corneal tissue was dissected, frozen, sectioned and fixed for TUNEL assay as described previously (Mohan et al., 2003). Briefly the corneoscleral rims were removed with 0.12 forceps and sharp Westcott scissors. A central corneal block (10 mm!3 mm) was embedded in liquid OCT compound (Sakura FineTek, Torrance, CA, USA) within a 24 mm!24 mm!5 mm mold (Fisher, Pittsburgh, PA, USA). The tissue specimens were erected in the mold so that the whole cornea thickness could be cut transversely. The tissue was then rapidly frozen in 2-methyl butane within a stainless steel crucible suspended in liquid nitrogen. The frozen tissue blocks were stored at K80 8C until sectioning was performed. Corneal sections (7 mm thick) were cut with a cryostat (TISSUE-TEK II, Miles Scientific, Naperville, IL, USA). Sections were placed on 25 mm! 75 mm!1 mm microscope slides (Superfrost Plus, Fisher) and maintained frozen at K80 8C until staining was performed. For staining, tissue sections were fixed in acetone at K20 8C for 5 min, dried at room temperature for 5 min, placed in balanced salt solution, stained with TUNEL according to the manufacturer’s instructions (ApoAlert, Cat No; K2024-2; BD Biosciences, Palo Alto, CA, USA), counterstained with DAPI, and then mounted with prolong antifade reagent (Molecular Probe Cat: P-7481). 2.8. Cell counting for quantitation of apoptosis in cell culture and tissue sections For fibroblasts cultured on coverslips, five random distinct 200! microscopic fields were photographed on each coverslip. DAPI (C) cells were counted to obtain the total cell count. DAPI (C) and TUNEL (C) cells were counted as apoptotic cells. DAPI (K) and TUNEL (C) areas were considered artifacts and excluded from the count. Data was collected from about 750 cells for each condition in each experiment. Experiments were repeated at least three times giving a total of at least 2000 cells counted per condition. We found that nuclear condensation as viewed by DAPI staining perfectly correlated with TUNEL positive staining in cultures of corneal fibroblasts. In some of our experiments, we used nuclear condensation as an indicator of apoptosis. Using Metamorph Imaging Analysis software (Universal Imaging, West Chester, PA), those cells with condensed nuclei and with
DAPI fluorescence pixel values that were three times stronger than non-apoptotic nuclei were counted as apoptotic cells. For tissue sections, all of the cells from five consecutive non-overlapping columns of the anterior 300 mm of stroma in the central cornea were counted. The width of each column was that of a camera field using a 20! objective. The columns in which counts were performed were selected from the central cornea of each specimen. Data was collected from about 2000 cells for each condition using the same criteria as cultured fibroblasts stated above. 2.9. Western blot analysis Whole cell lysates were prepared as previously described (Zhang et al., 2004). Briefly, treated and untreated cells were extracted with lysis buffer (50 mmol/L Tris–HCl, pH 7.5, 5 mmol/L EDTA, 150 mmol/L NaCl, 0.5% Triton X-100, 10 mmol/L sodium fluoride, 20 mmol/L b-mercaptoethanol, 250 mmol/L sodium orthovanadate, 1 mmol/L PMSF and complete protease inhibitor cocktail (Sigma, St Louis, MO), and incubated at 4 8C for 30 min. The lysates were sonicated and centrifuged at 14,000g for 15 min. The supernatants were collected and stored at K80 8C. In separate experiments, nuclear protein was extracted according to the manufacturer’s instructions using a nuclear extract kit (Active Motif). Protein concentrations were determined by the BCA method. Protein (50 mg) was separated on 8% polyacrylamide-SDS gel and electroblotted onto nitrocellulose membranes (Bio-Rad laboratories, Hercules, CA). After blocking with TBS/5% skim milk, the membrane was incubated overnight at 4 8C with primary antibodies against HIF-1a (ABR, Cat: MA1-516), followed by peroxidase conjugated anti-mouse IgG for 1 hr at room temperature. Signals were detected with ECL. Data was analyzed using Un-scan-it gel analysis software (Silk Scientific, Orem, Utah, USA). Relative increase in HIF1a expression compared to its own control is calculated. 2.10. Statistical analysis Data is presented as the meanGSE for at least three separate experiments. Student’s t-test and one-way ANOVA were employed for statistical analysis, with significant differences determined as p!0.05. 3. Results 3.1. Hypoxic preconditioning can protect stromal cells against apoptosis In epithelial scrape models, peak keratocyte apoptosis occurs 4 hr following the epithelial wound (Mohan et al., 2003). Therefore, for the in vitro studies we examined apoptosis 4 hr after UV-irradiation. We chose a UV dose (2 min or 5.1 mJ/cm2) that produced approximately 30–40% apoptosis so that we could clearly test for protective effects. We examined the effect of 4 hr of hypoxia (1.5 or 0.5% O2) preconditioning on the apoptotic rate of fibroblasts induced by
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2 min of UV-irradiation using the TUNEL assay. Fig. 1 shows that the apoptotic rate in the UV-irradiated normoxic group (46.1G12.1%) was significantly higher than either 1.5% O2 (27.8G4.1%) or 0.5% O2 (7.2G2.0%) preconditioned groups. Also the 0.5% O2 group had a lower apoptotic rate than the 1.5% O2 group (p!0.05). There was a small level of background apoptosis in non-UV irradiated cells (1.5G0.2% for normoxia, 1.0G0.4% for 1.5% O2 and 2.8G2.1% for 0.5% O2), and these differences were not significant (pZ0.24), indicating that the corneal stromal fibroblasts could tolerate these levels of hypoxia. Our experiments suggest that hypoxia
Fig. 1. Hypoxic preconditioning shows protection against UV-irradiation induced corneal stromal fibroblast apoptosis. Images show representative TUNEL staining of normoxic control (A), hypoxic control (B), normoxia with 2 min of UV-irradiation (C), 0.5% O2 hypoxia preconditioned with 2 min of UV-irradiation (D) and 1.5% O2 hypoxia preconditioned with 2 min of UVirradiation (E). Bar graph summarizes these results (nZ4). Error bar shows standard error of the mean for each group. Approximately, 750 cells were counted for each experiment. Nuclei stain in blue by DAPI. Apoptotic cells stain in bright green by TUNEL. Magnification: 200!.
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preconditioning provides protection to stromal cells against UV-irradiation and the protection is dose dependent. 3.2. HIF-1a is associated with protection To begin to examine whether HIF-1a could be involved in the protection, we measured the HIF-1a levels in nuclei and whole cell lysates of primary cultured stromal fibroblasts following 4 hr treatment with normoxia, 1.5% O2, 0.5% O2, or 100–200 mM cobalt chloride (CoCl2), which interferes with prolyl hydroxylation of HIF-1a. Fig. 2(A) and (B) show western blots of a 116 kDa protein representing HIF-1a obtained from whole cell lysate and nuclear extract preparations, respectively. Both preparations showed a low background level of HIF-1a in normoxia. The level of nuclear HIF-1a increased 1.5 fold with 1.5% O2 incubation, 2.25 fold with 0.5% O2 and 1.9 fold with incubation in 0.2 mM CoCl2.
Fig. 2. Western blots of HIF-1a. (A), whole cell lysates from primary cultured stromal fibroblast treated with normoxia, 0.5% O2, 0.1 or 0.2 mM cobalt chloride (a b-actin control for protein loading is included). (B), nuclear extracts from primary cultured stromal fibroblast. In the left hand blot, cells were treated with normoxia control, 0.2 mM cobalt chloride, or 1.5% O2. In the right hand blot cells were treated with 0.5% O2. For each blot there is a separate normoxic control (Con). Data were analyzed using gel imaging software. Relative increase of average pixel value from HIF-1a blot of each experiment group compared to its own normoxia control are represented in the bar graph (nZ4). Data are represented with meanGSE.
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Fig. 3. Cobalt chloride shows a dose-dependent protective effect against UVirradiation induced corneal stromal fibroblast apoptosis. Cells were treated with 0.1 or 0.2 mM cobalt chloride for 4 hr then irradiated with 2 min of UV. Cells were stained with TUNEL 4 hr after irradiation. Error bar represent the standard errors of the mean (nZ4). Approximately 750 cells were counted for each experiment.
In whole cell lysates, HIF-1a levels were increased 2.5 fold with 0.2 mM CoCl2 and two fold following 0.5% O2. Since cobalt chloride (CoCl2) is a strong inducer of HIF-1a in the corneal fibroblasts, we tested whether CoCl2 could also be protective. Fibroblasts were incubated with 0.1 or 0.2 mM CoCl2 in the standard 5% CO2/Air incubator for 4 hr, washed and incubated with fresh 0.5% FBS/DMEM for 1 hr and then irradiated with 2 min of UV. Cells were stained with TUNEL 4 hr after irradiation. Fig. 3 shows that the apoptotic rate for cells incubated in CoCl2 was greatly reduced compared to control irradiated cells. The apoptotic rate for cells incubated with 0.2 mM CoCl2 incubation (4.2G2.1%) was significantly lower than that with 0.1 mM CoCl2 incubation (20.5G9.8%). The apoptotic rates among non-irradiated groups were not significantly different. These experiments demonstrate that cobalt chloride as well as hypoxia provides protection against UV-induced apoptosis, and that the protection is associated with induction of HIF-1a. 3.3. Hypoxic preconditioned medium has a protective effect HIF-1a can induce transcription of various protective factors including VEGF, EPO and bFGF (Semenza, 2004). To explore whether protective factors could be released from hypoxia preconditioned cells, we tested whether preconditioned media could protect corneal fibroblasts. Following 2 or 4 hrs incubation in hypoxia preconditioned media, cells were UV-irradiated for 2 min. Fig. 4 shows that the apoptotic rates of cells following 2 hr incubation (8.4G3.4%) or 4 hr incubation (5.9G4.7%) in hypoxia conditioned medium were significantly lower than controls (24.9G3.0%). Although there was a slight drop in the apoptotic rate at the longer incubation time, the difference between 2 and 4 hr incubation in hypoxia conditioned medium was not significant. There was no significant difference in apoptotic rate among non-irradiated controls.
Fig. 4. Hypoxia preconditioned media shows protective effect against UVirradiation induced corneal fibroblast apoptosis. Cells were incubated with hypoxia preconditioned medium for 2 or 4 hr, then UV-irradiated for 2 min. Cells were stained with TUNEL 4 hr after irradiation. Error bar represent the standard errors of the mean. Approximately 750 cells were counted for each experiment.
3.4. Hypoxic preconditioning protects primary cultured cornea fibroblasts against Fas/FasL induced nuclear condensation UV-irradiation induces apoptosis via oxidative stress that ultimately involves the mitochondrial pathway of apoptosis induction and triggers the release of cytochrome c (Martin et al., 1995; Matsuda et al., 2002). Cytokine mediated apoptotic stress is generated through the activation of membrane receptor death domains. Previous studies have shown that activation of Fas receptor can induce apoptosis in keratocytes (Mohan et al., 1997). We asked if hypoxia preconditioning could protect corneal fibroblasts from Fas mediated nuclear condensation as well as from UV-irradiation. In this experiment, 24 hr of incubation with 100 ng/ml of anti-Fas antibody (Kamiya Biomedical Company) was used to induce apoptosis. In order to eliminate effects from serum containing cytokines, we used serum-free DMEM culture medium. Fig. 5 shows that nuclear condensation visualized by DAPI staining indicated that the proportion of condensed nuclei in the serum-free control group not exposed to anti-Fas, was 12.5G4.5%, which is significantly higher than in 0.5% serum DMEM (see Fig. 2). The proportion of condensed nuclei for anti-Fas treatment alone was 25.5G 4.1%, which is significantly higher than control. When anti-Fas was incubated with cells that had been preconditioned with 4 hr of 0.5% O2 hypoxia or with 0.1 M cobalt chloride, the proportion of condensed nuclei was significantly reduced to 4.8G2.9% and 9.2G5.4%, respectively, indicating that hypoxia preconditioning could provide a protective effect against Fas induced apoptosis. Interestingly, the proportion of condensed nuclei of hypoxia preconditioned cells was lower than control, suggesting that hypoxia preconditioning was also protective against serum starvation.
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Fig. 5. CoCl2 and hypoxia preconditioning has a protective effect against Fas/FasL induced nucelar condensation. Cells were stained by DAPI. Condensed nuclei were scored if fluorescence was at least three times brighter than control nuclei. Control cells were incubated in serum-free DMEM, cells were treated with anti-Fas alone, with hypoxia or 0.1 mM Cobalt chloride for 4 hr. Cells were fixed 4 hr after 2 min of UV-irradiation. Error bar represents standard error of mean (nZ3). Magnification: 200!. Approximately 750 cells were counted for each experiment.
3.5. Hypoxia preconditioning protects stromal cells in bovine cornea against epithelial scrape induced apoptosis Epithelial scrape has long been used as a model to induce keratocyte apoptosis in animal experiments. The disappearance of anterior stromal keratocytes in response to epithelial scrape was shown to be mediated by apoptosis (Wilson et al., 1996). To explore whether hypoxia preconditioning can protect keratocytes in situ, we performed ex vivo experiments using bovine eyes. Eyes were preconditioned for 4 hr with 0.5% O2 and placed in normoxia for 30 min. The epithelium was scraped and cornea was frozen 4 hr later. We quantified the apoptotic rate in the anterior portion of a 20! magnification camera field (300 mm) of stroma using the TUNEL assay. We found that all TUNEL positive cells were in the anterior half of this field. Fig. 6 shows representative tissue sections from control and hypoxia preconditioned corneas. The apoptotic rate in hypoxia preconditioned scraped corneas (14.2G2.5%) was
Fig. 6. TUNEL assay of bovine corneal sections and the apoptotic rates in anterior stroma. Cornea was frozen 4 hr after epithelial scraping. Cryosections were stained with TUNEL and counterstained with DAPI. Images represent normoxic-unscraped (A), normoxic-scraped (C, E), hypoxic-unscraped (B) and hypoxic-scraped (D, F) corneas. Nuclei stain blue by DAPI, apoptotic cells stain bright green by TUNEL. Bar graph summarizes these results, Error bar represents standard error of the mean (nZ3). Magnification: (A, B, C, D) 200!; (E, F) 100!.
significantly lower than that of scraped corneas that were kept in normoxia (26.3G1.4%). The apoptotic rates in unscraped controls for both hypoxic (7.6G4.6%) and normoxic groups (6.5G4.2%) were not significantly different. The background apoptosis is probably due to the ex vivo nature of this experiment.
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4. Discussion In this study we demonstrate that significant but non-lethal hypoxic preconditioning protects primary cultured stromal fibroblasts against UV-irradiation and Fas mediated apoptosis. Hypoxic preconditioning also protected corneal keratocytes, within ex vivo corneal tissue, against epithelial injury induced apoptosis. The degree of hypoxia protection was associated with the degree of HIF-1a expression. Hypoxia conditioned medium was also protective suggesting that protective factors had been released from hypoxic cells into the medium. We found that short-term treatment with 1.5% O2 or 0.5% O2 was non-lethal and protective for corneal fibroblasts. This is similar to hypoxia protection in other cell types including protection of HepG2 cells against serum deprivation and t-BHP induced apoptosis (Piret et al., 2004), protection of hepatocyte from cytokine induced apoptosis (Crenesse et al., 2001) and protection of myocytes against acute severe oxidative stress (Silverman et al., 1997). We explored whether hypoxia preconditioning was protective against apoptosis induced via a mitochondrial pathway and a death receptor pathway. UVirradiation is DNA damaging and produces oxidative stress, which activates caspase 9 via the mitochondrial pathway. AntiFas antibody induces apoptosis through a membrane receptor death domain to activate caspase 8. Both pathways finally converge to the activation of apoptosis executioner caspases 3, 6, and 7. We found that hypoxia preconditioning could protect fibroblasts from apoptosis induced by both types of stress factors including serum-deprivation suggesting that hypoxia preconditioning produces a broad generalized protection against apoptosis. HIF-1a, a master transcriptional factor of hypoxia-regulated genes, mediates a wide range of cellular and physiological adaptive responses to changes in oxygen tension. In normoxia, HIF-1a is modified by oxygen dependent prolyl hydroxylases, which allows association with the von Hippel-Lindau tumor suppressor protein (VHL) and targeting for proteasomal degradation, maintaining a low level of HIF-1a. Under hypoxic conditions, less HIF-1a protein is hydroxylated allowing translocation to the nucleus where it dimerizes with HIF-1b. The HIF complex can then bind to the Hypoxia Responsive Element (HRE) consensus sequence in the promoter of downstream target genes (Zhang et al., 2004). The issue of whether HIF-1a is a pro- or anti-apoptotic transcription factor is a matter of some debate (Piret et al., 2002). The current consensus is that HIF-1a participates in promoting apoptosis during severe or long-term hypoxia, while promoting adaptation to hypoxia when it is less severe. Using primary cultured corneal stromal cells, we demonstrated that the amount of HIF-1a in both nuclear extracts and whole cell lysates increased followed hypoxia. The increase in HIF-1a induced by CoCl2 was consistent with the degree of protection against UV-irradiation induced apoptosis. The protective effect of hypoxia preconditioned medium also suggests involvement of HIF-1a since the expression and secretion of protective cytokines can occur via HIF-1a mediated transcription of protective factors such as EPO (Semenza et al., 1994) and
VEGF (Liu et al., 1995). Additional experiments using pharmacological and/or genetic approaches to interfere with HIF-1a mediated activity will be needed to definitively establish the role of HIF-1a in hypoxia preconditioning protection of corneal stromal cells. Epithelial damage leads to keratocyte apoptosis probably mediated by diffusion of epithelial derived cytokines (Wilson et al., 2001). To test if hypoxia preconditioning could affect this apoptosis we used a simple ex vivo bovine model. We found that hypoxia preconditioning could also reduce keratocyte apoptosis induced by epithelial scraping. In this model, there was a background level of apoptosis probably related to the ex vivo nature of the experiment. More importantly there was no difference in background apoptotic rates between hypoxic and normoxic unscraped corneas, indicating that the hypoxia itself did not induce apoptosis. In a previous study, treatment of rabbit corneas with a general caspase inhibitor before epithelial scrape also reduced apoptosis, however cells apparently died instead by necrosis (Kim et al., 2000). We cannot exclude the possibility that the lower apoptotic rate in hypoxia preconditioned bovine corneas seen in our study is accompanied by an underlying necrosis. However, in other models like heart (Cai et al., 2003) and brain (Bergeron et al., 2000), hypoxia preconditioning protection is associated with both reduced apoptotic rates and reduced necrotic tissue. Further studies are needed using the rabbit in vivo model to test if hypoxia preconditioning can definitively reduce epithelial scrape-induced keratocyte apoptosis and prevent death by necrosis. In summary, our results indicate that non-lethal hypoxia preconditioning can protect corneal stromal cells against apoptosis induced by UV, Fas and epithelial scrape. This indicates that hypoxia preconditioning has broad protective properties and suggests that hypoxia or a hypoxia-like preconditioning could be used to lessen deleterious postsurgical healing. Further studies on the mechanism of hypoxia preconditioning protection and relevance to a surgical model are warranted. References Arends, M.J., Wyllie, A.H., 1991. Apoptosis: mechanisms and roles in pathology. Int. Rev. Exp. Pathol. 32, 223–254. Bergeron, M., Gidday, J.M., Yu, A.Y., Semenza, G.L., Ferriero, D.M., Sharp, F.R., 2000. Role of hypoxia-inducible factor-1 in hypoxia-induced ischemic tolerance in neonatal rat brain. Ann. Neurol. 48, 285–296. Cai, Z., Manalo, D.J., Wei, G., Rodriguez, E.R., Fox-Talbot, K., Lu, H., Zweier, J.L., Semenza, G.L., 2003. Hearts from rodents exposed to intermittent hypoxia or erythropoietin are protected against ischemiareperfusion injury. Circulation 108, 79–85. Carmeliet, P., Dor, Y., Herbert, J.-M., Fukumura, D., Brusselmans, K., Dewerchin, M., Neeman, M., Bono, F., Abramovitch, R., Maxwell, P., Koch, C.J., Ratcliffe, P., Moons, L., Jain, R.K., Collen, D., Keshet, E., 1998. Role of hif-1[alpha] in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature 394, 485–490. Chandrasekher, G., Ma, X., Lallier, T.E., Bazan, H.E., 2002. Delay of corneal epithelial wound healing and induction of keratocyte apoptosis by plateletactivating factor. Invest. Ophthalmol. Vis. Sci. 43, 1422–1428.
D. Xing et al. / Experimental Eye Research 82 (2006) 780–787 Crenesse, D., Laurens, M., Gugenheim, J., Heurteaux, C., Cursio, R., Rossi, B., Schmid-Alliana, A., 2001. Intermittent ischemia reduces warm hypoxiareoxygenation-induced jnk(1)/sapk(1) activation and apoptosis in rat hepatocytes. Hepatology 34, 972–978. Grimm, C., Wenzel, A., Groszer, M., Mayser, H., Seeliger, M., Samardzija, M., Bauer, C., Gassmann, M., Reme, C.E., 2002. Hif-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration. Nat Med 8, 718–724 (New York, NY, USA). Helena, M.C., Baerveldt, F., Kim, W.-J., Wilson, S.E., 1998. Keratocyte apoptosis after corneal surgery. Invest. Ophthalmol. Vis. Sci. 39, 276–283. Jester, J.V., Ho-Chang, J., 2003. Modulation of cultured corneal keratocyte phenotype by growth factors/cytokines control in vitro contractility and extracellular matrix contraction. Exp. Eye Res. 77, 581–592. Jester, J.V., Huang, J., Barry-Lane, P.A., Kao, W.W., Petroll, W.M., Cavanagh, H.D., 1999. Transforming growth factor(beta)-mediated corneal myofibroblast differentiation requires actin and fibronectin assembly. Invest. Ophthalmol. Vis. Sci. 40, 1959–1967. Kim, W.J., Mohan, R.R., Mohan, R.R., Wilson, S.E., 2000. Caspase inhibitor z-vad-fmk inhibits keratocyte apoptosis, but promotes keratocyte necrosis, after corneal epithelial scrape. Exp. Eye Res. 71, 225–232. Lin Anya, M.Y., Dung, S.-W., Chen, C.-F., Chen, W.-H., Ho, L.-T., 2003. Hypoxic preconditioning prevents cortical infarction by transient focal ischemia-reperfusion. Ann. NY Acad. Sci. 993, 168–178. [discussion 195–6]. Liu, Y., Cox, S.R., Morita, T., Kourembanas, S., 1995. Hypoxia regulates vascular endothelial growth factor gene expression in endothelial cells. Identification of a 5’ enhancer. Circ. Res. 77, 638–643. Martin, S.J., Newmeyer, D.D., Mathias, S., Farschon, D.M., Wang, H.G., Reed, J.C., Kolesnick, R.N., Green, D.R., 1995. Cell-free reconstitution of fas-, uv radiation- and ceramide-induced apoptosis. EMBO J. 14, 5191– 5200. Matsuda, K., Yoshida, K., Taya, Y., Nakamura, K., Nakamura, Y., Arakawa, H., 2002. P53aip1 regulates the mitochondrial apoptotic pathway. Cancer Res. 62, 2883–2889. Mohan, R.R., Liang, Q., Kim, W.J., Helena, M.C., Baerveldt, F., Wilson, S.E., 1997. Apoptosis in the cornea: further characterization of fas/fas ligand system. Exp. Eye Res. 65, 575–589. Mohan, R.R., Mohan, R.R., Kim, W.J., Wilson, S.E., 2000. Modulation of tnfalpha-induced apoptosis in corneal fibroblasts by transcription factor nfkappab. Invest. Ophthalmol. Vis. Sci. 41, 1327–1336. Mohan, R.R., Hutcheon, A.E.K., Choi, R., Hong, J., Lee, J., Mohan, R.R., Ambrosio, R., Zieske, J.D., Wilson, S.E., 2003. Apoptosis, necrosis, proliferation, and myofibroblast generation in the stroma following lasik and prk. Exp. Eye Res. 76, 71–87. Moller-Pedersen, T., Cavanagh, H.D., Petroll, W.M., Jester, J.V., 1998. Corneal haze development after prk is regulated by volume of stromal tissue removal. Cornea 17, 627–639. Piret, J.-P., Mottet, D., Raes, M., Michiels, C., 2002. Is hif-1[alpha] a pro- or an anti-apoptotic protein?*1. Biochem. Pharmacol. 64, 889–892.
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Piret, J.P., Lecocq, C., Toffoli, S., Ninane, N., Raes, M., Michiels, C., 2004. Hypoxia and cocl2 protect hepg2 cells against serum deprivation- and t-bhp-induced apoptosis: a possible anti-apoptotic role for hif-1. Exp. Cell Res. 295, 340–349. Sakamoto, K., Urushidani, T., Nagao, T., 1998. Translocation of hsp27 to cytoskeleton by repetitive hypoxia-reoxygenation in the rat myoblast cell line, h9c2. Biochem. Biophys. Res. Commun. 251, 576–579. Semenza, G.L., 2004. Intratumoral hypoxia, radiation resistance, and hif-1. Cancer Cell 5, 405–406. Semenza, G.L., Roth, P.H., Fang, H.M., Wang, G.L., 1994. Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J. Biol. Chem. 269, 23757–23763. Silverman, H.S., Wei, S.-k., Haigney, M.C.P., Ocampo, C.J., Stern, M.D., 1997. Myocyte adaptation to chronic hypoxia and development of tolerance to subsequent acute severe hypoxia. Circ. Res. 80, 699–707. Stramer, B.M., Zieske, J.D., Jung, J.C., Austin, J.S., Fini, M.E., 2003. Molecular mechanisms controlling the fibrotic repair phenotype in cornea: implications for surgical outcomes. Invest. Ophthalmol. Vis. Sci. 44, 4237– 4246. Webster, K.A., 2003. Serine phosphorylation and suppression of apoptosis by the small heat shock protein alphab-crystallin. Circ. Res. 92, 130–132. Whitlock, N.A., Agarwal, N., Ma, J.X., Crosson, C.E., 2005. Hsp27 upregulation by Hif-1 signaling offers protection against retinal ischemia in rats. Invest. Ophthalmol. Vis. Sci. 46, 1092–1098. Wilson, S.E., 1997. Molecular cell biology for the refractive corneal surgeon: programmed cell death and wound healing. J. Refract. Surg. 13, 171–175 (Thorofare, N.J.: 1995). Wilson, S.E., He, Y.-g., Weng, J., Li, Q., McDowall, A.W., Vital, M., Chwang, E.L., 1996. Epithelial injury induces keratocyte apoptosis: hypothesized role for the interleukin-1 system in the modulation of corneal tissue organization and wound healing. Exp. Eye Res. 62, 325–338. Wilson, S.E., Pedroza, L., Beuerman, R., Hill, J.M., 1997. Herpes simplex virus type-1 infection of corneal epithelial cells induces apoptosis of the underlying keratocytes. Exp. Eye Res. 64, 775–779. Wilson, S.E., Mohan, R.R., Mohan, R.R., Ambrosio, J., Renato, Hong, J., Lee, J., 2001. The corneal wound healing response: cytokine-mediated interaction of the epithelium, stroma, and inflammatory cells. Prog. Retin. Eye Res. 20, 625–637. Wyllie, A.H., Kerr, J.F., Currie, A.R., 1980. Cell death: the significance of apoptosis. Int. Rev. Cytol. 68, 251–306. Yu, H.J., Chien, C.T., Lai, Y.J., Lai, M.K., Chen, C.F., Levin, R.M., Hsu, S.M., 2004. Hypoxia preconditioning attenuates bladder overdistension-induced oxidative injury by up-regulation of bcl-2 in the rat. J. Physiol. 554, 815–828. Zhang, Q., Zhang, Z.F., Rao, J.Y., Sato, J.D., Brown, J., Messadi, D.V., Le A, D., 2004. Treatment with sirna and antisense oligonucleotides targeted to hif-1alpha induced apoptosis in human tongue squamous cell carcinomas. Int. J. Cancer 111, 849–857.
Hypoxia preconditioning protects corneal stromal cells against induced apoptosis Dongmei Xing, Xingcai Sun, Jinhua Li, Miao Cui, Kah Tan-Allen, Joseph A. Bonanno * School of Optometry, Indiana University, 800 East Atwater Avenue, , Bloomington, IN 47405, USA Received 4 April 2005; accepted in revised form 27 September 2005 Available online 20 December 2005
Abstract The purpose of this study, was to determine whether hypoxia preconditioning can protect corneal stromal cells from UV stress and cytokine mediated apoptosis. Two models were implemented. First, primary cultured bovine corneal fibroblasts were preconditioned with 0.5–1.5% O2 for 4 hr and stressed with UV-irradiation or stimulation of Fas receptor. Second, bovine eyes were preconditioned with 0.5% O2 for 4 hr and stressed by epithelial scraping to induce anterior keratocyte apoptosis. Cell fate was analyzed at 4 hr after stress using quantitative TUNEL or condensed nuclei assays. Cell apoptotic rates in hypoxia preconditioned groups were significantly lower (50–80%) than that of normoxia control groups. Hypoxia prevented the degradation of the transcription factor HIF-1a. CoCl2 (100–200 mM), a chemical inducer of HIF-1a, also produced strong protection against UV and Fas induced apoptosis. Moreover, hypoxia preconditioned media protected cells against UV-induced apoptosis. These findings demonstrate that hypoxia preconditioning has a generalized protective effect against stromal fibroblast and keratocyte apoptosis and suggest that HIF-1a mediated expression and secretion of protective factors is involved. q 2005 Elsevier Ltd. All rights reserved. Keywords: cornea; keratocytes; apoptosis; hypoxia preconditioning
1. Introduction Apoptosis is a form of involutional cell death that occurs with limited inflammation and little release of cellular contents that would inflict collateral damage to surrounding tissue (Wyllie et al., 1980; Arends and Wyllie, 1991). This gentle mechanism for eliminating cells is of obvious significance to the maintenance of corneal clarity. Many types of epithelial injury can induce keratocyte apoptosis including mechanical scrape (Wilson et al., 1996), corneal refractive surgical procedures (Helena et al., 1998) and viral infection (Wilson et al., 1997). Keratocyte apoptosis is the earliest stromal event noted following epithelial injury and remains a likely target for modulation of the overall wound healing response (Wilson et al., 2001). Current evidence indicates that the etiology of keratocyte apoptosis is mediated by epithelial release of cytokines that could include IL-1 (Wilson et al., 1996),
* Corresponding author. Joseph A. Bonanno, School of Optometry, Indiana University, 800 East Atwater Avenue, Bloomington, IN 47405, USA. E-mail address: [email protected] (J.A. Bonanno).
0014-4835/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2005.09.020
tumor necrosis factor (TNF) (Mohan et al., 2000) and platelet activation factor (PAF) (Chandrasekher et al., 2002). Keratocyte apoptosis, along with subsequent repopulation by activated stromal keratocytes, has been hypothesized to be an important determinant of corneal wound healing associated with refractive variability and regression after photorefractive keratectomy and laser-assisted in situ keratomileusis (LASIK) (Wilson, 1997; Helena et al., 1998). Keratocyte loss promotes the activation and proliferation of surrounding keratocytes. Recent studies suggest that disruption of the basement membrane releases TGF-b (Transforming Growth Factor b), which can induce myofibroblast formation in vitro (Jester et al., 1999; Jester and Ho-Chang, 2003) and promotes differentiation of activated keratocytes into myofibroblasts in vivo (Stramer et al., 2003). Myofibroblasts are responsible for refractive variability and light scatter (Moller-Pedersen et al., 1998). These wound healing events are directly associated with the initial extent of apoptosis (Mohan et al., 2003) which have led to the notion that reducing keratocyte death by inhibiting the initial keratocyte apoptosis could also lessen the undesirable aspects of the wound healing response. Here, we examine the potential of using hypoxia preconditioning as a means to protect corneal stromal cells from lethal stresses. Hypoxia has been shown to be either antiapoptotic or pro-apoptotic, depending on the cell type
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and experimental conditions (Piret et al., 2002). Severe hypoxia impairs cellular energy production and ion homeostasis leading to cell injury and cell death. Interestingly, nonlethal hypoxia activates mechanisms that could produce a cellular phenotype more resistant to acute severe oxidative stress (Silverman et al., 1997). Hypoxic preconditioning attenuated cortical infarction in the rat brain induced by oxidative stress (Lin Anya et al., 2003), attenuated bladder over distension-induced oxidative injury (Yu et al., 2004), and protected retinal morphology and function against lightinduced apoptosis (Grimm et al., 2002). At the single cell level, isolated cardiomyocytes cultured at low oxygen tension acquire resistance against acute severe oxidative stress (Carmeliet et al., 1998). HepG2 cells preconditioned with hypoxia or CoCl2, a hypoxia mimetic, reduced serum deprivation and tert butyl hydrogen peroxide-induced apoptosis (Piret et al., 2004). The mechanisms for hypoxia preconditioning protection are incompletely understood, but often require activation of Hypoxia inducible factor-1a (HIF1a), the main transcriptional factor activated by hypoxia. Genes regulated by HIF-1a include erythropoietin (EPO) and VEGF (Vascular Endothelial Growth factor). These proteins have well described anti-apoptotic properties. For example, local production of EPO during retinal hypoxia protected photoreceptors from light induced cell death (Grimm et al., 2002). Hypoxia is also known to stimulate translocation of Hsp27 and aB-crystallin from diffuse locations to defined structures, which is associated with a decrease in caspase-3 activity (Sakamoto et al., 1998; Webster, 2003). Furthermore, Hsp27 is up-regulated by HIF1-a and is protective in a rat retinal ischemia model (Whitlock et al., 2005). These studies suggest that hypoxia preconditioning through HIF1-a mediated transcription can protect cells by multiple mechanisms. In the current study, we reasoned that corneal stromal cells exposed to significant but non-lethal hypoxia could show adaptations making them more tolerant of certain apoptotic stress. We asked whether hypoxia preconditioning could protect cells from UV-irradiation and Fas mediated apoptosis and whether this protection was associated with induction of HIF-1a. We also tested whether hypoxia preconditioning could protect keratocytes from epithelial scrape induced apoptosis in an ex vivo bovine cornea model. In all cases, we found that hypoxia preconditioning or induction of HIF-1a were strongly protective. 2. Materials and methods 2.1. Cell culture Blocks of stroma were cut from fresh bovine cornea and cultured in DMEM (GIBCO) supplemented with 10% fetal bovine serum (FBS), 100 units/ml of penicillin, 100 mg/ml of streptomycin and 0.25 mg/ml of amphotericin B. Corneal stromal fibroblasts migrated from the stromal explants and grew exponentially at densities below 5!105 cells/ml. Second generation fibroblasts were seeded onto coverslips or petridishes and used in all cell culture experiments.
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2.2. Induction of hypoxia Corneal fibroblasts (5!104 cells) were sub-cultured to 25 mm round coverslips in six-well plates with 2 ml DMEM supplemented with 10% FBS for 1 day. Media was changed immediately before experiments to DMEM with 0.5% FBS. This amount of serum was sufficient to prevent cell death, but does not promote proliferation. For hypoxia preconditioning, cells were placed in a hypoxia chamber (Coy Lab Products Inc., Grass Lake, Michigan) equilibrated with 5% CO2 and either 1.5 or 0.5% oxygen-balance nitrogen for at least 4 hr. 2.3. UV-irradiation A germicidal lamp (TUV/30W/G30 T8, Philips) that emitted radiation ranging from 230 to 400 nm was used as the UV source. The emission peak was 255 nm. UV dose was quantified using a radiometer (IL 1700, International Light Inc., Newburyport, MA, USA). To produce approximately 40% apoptotic cells 4 hr after irradiation, 2 min of UV exposure was required, which corresponded to 5.1 mJ/cm2. Cells were irradiated at 80% confluence. Culture media was removed and replaced with 2 ml of a balanced ringer solution to avoid variations in UV absorption from media components. After irradiation the ringer solution was discarded and replaced by fresh DMEM/0.5% FBS. 2.4. TUNEL assay Four hours after UV-irradiation cells on coverslips were fixed in the fresh 4% formaldehyde/PBS at 4 8C for 25 min. Following fixation the cells were rinsed twice with PBS and permeabilized with prechilled 0.2% Triton X-100/PBS on ice for 5 min. A fluorescence-based TUNEL assay was used according to the manufacturer’s instructions (ApoAlert, BD Biosciences, Palo Alto, CA, USA). Cells were counterstained with DAPI and mounted with prolong antifade reagent (Molecular Probe). Images were obtained with a fluorescence microscope (Nikon E600, Melville, NY, USA) equipped with charge coupled device camera with active cooling system. 2.5. Hypoxia preconditioned media Primary cultured cornea stromal fibroblasts (2!105) in 2 ml DMEM with 0.5% fetal bovine serum were incubated in 0.5% oxygen for 4 hr. Conditioned media was collected and stored at K808 until use. Fibroblasts (2!105) plated on glass coverslips were incubated in the hypoxia preconditioned media for 2 or 4 hr under normoxic conditions and then irradiated with 2 min of UV light. TUNEL staining was performed 4 hr after irradiation. 2.6. Cornea wound model Immediately after death at the abbatoir, paired bovine eyeballs from one animal were placed in plastic bags with PBS on ice and transported to the lab. Eyes were trimmed of
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connective tissues and stored at 4 8C overnight. The next day, the eyes were rinsed with PBS and placed in open dishes. One eye was incubated in a humidified chamber 0.5% O2/5% CO2 and the other eye was put into a standard 5% CO2/Air humidified tissue culture incubator for 4 hr and then the hypoxia preconditioned eye was placed into the standard incubator for 30 min. Using a scalpel blade, the entire epithelium of both eyes was removed by scraping while leaving the basement membrane intact. After injury, both eyes were placed in the standard incubator for 4 hr. 2.7. Tissue fixation, sectioning and staining Four hours after epithelial scraping, corneal tissue was dissected, frozen, sectioned and fixed for TUNEL assay as described previously (Mohan et al., 2003). Briefly the corneoscleral rims were removed with 0.12 forceps and sharp Westcott scissors. A central corneal block (10 mm!3 mm) was embedded in liquid OCT compound (Sakura FineTek, Torrance, CA, USA) within a 24 mm!24 mm!5 mm mold (Fisher, Pittsburgh, PA, USA). The tissue specimens were erected in the mold so that the whole cornea thickness could be cut transversely. The tissue was then rapidly frozen in 2-methyl butane within a stainless steel crucible suspended in liquid nitrogen. The frozen tissue blocks were stored at K80 8C until sectioning was performed. Corneal sections (7 mm thick) were cut with a cryostat (TISSUE-TEK II, Miles Scientific, Naperville, IL, USA). Sections were placed on 25 mm! 75 mm!1 mm microscope slides (Superfrost Plus, Fisher) and maintained frozen at K80 8C until staining was performed. For staining, tissue sections were fixed in acetone at K20 8C for 5 min, dried at room temperature for 5 min, placed in balanced salt solution, stained with TUNEL according to the manufacturer’s instructions (ApoAlert, Cat No; K2024-2; BD Biosciences, Palo Alto, CA, USA), counterstained with DAPI, and then mounted with prolong antifade reagent (Molecular Probe Cat: P-7481). 2.8. Cell counting for quantitation of apoptosis in cell culture and tissue sections For fibroblasts cultured on coverslips, five random distinct 200! microscopic fields were photographed on each coverslip. DAPI (C) cells were counted to obtain the total cell count. DAPI (C) and TUNEL (C) cells were counted as apoptotic cells. DAPI (K) and TUNEL (C) areas were considered artifacts and excluded from the count. Data was collected from about 750 cells for each condition in each experiment. Experiments were repeated at least three times giving a total of at least 2000 cells counted per condition. We found that nuclear condensation as viewed by DAPI staining perfectly correlated with TUNEL positive staining in cultures of corneal fibroblasts. In some of our experiments, we used nuclear condensation as an indicator of apoptosis. Using Metamorph Imaging Analysis software (Universal Imaging, West Chester, PA), those cells with condensed nuclei and with
DAPI fluorescence pixel values that were three times stronger than non-apoptotic nuclei were counted as apoptotic cells. For tissue sections, all of the cells from five consecutive non-overlapping columns of the anterior 300 mm of stroma in the central cornea were counted. The width of each column was that of a camera field using a 20! objective. The columns in which counts were performed were selected from the central cornea of each specimen. Data was collected from about 2000 cells for each condition using the same criteria as cultured fibroblasts stated above. 2.9. Western blot analysis Whole cell lysates were prepared as previously described (Zhang et al., 2004). Briefly, treated and untreated cells were extracted with lysis buffer (50 mmol/L Tris–HCl, pH 7.5, 5 mmol/L EDTA, 150 mmol/L NaCl, 0.5% Triton X-100, 10 mmol/L sodium fluoride, 20 mmol/L b-mercaptoethanol, 250 mmol/L sodium orthovanadate, 1 mmol/L PMSF and complete protease inhibitor cocktail (Sigma, St Louis, MO), and incubated at 4 8C for 30 min. The lysates were sonicated and centrifuged at 14,000g for 15 min. The supernatants were collected and stored at K80 8C. In separate experiments, nuclear protein was extracted according to the manufacturer’s instructions using a nuclear extract kit (Active Motif). Protein concentrations were determined by the BCA method. Protein (50 mg) was separated on 8% polyacrylamide-SDS gel and electroblotted onto nitrocellulose membranes (Bio-Rad laboratories, Hercules, CA). After blocking with TBS/5% skim milk, the membrane was incubated overnight at 4 8C with primary antibodies against HIF-1a (ABR, Cat: MA1-516), followed by peroxidase conjugated anti-mouse IgG for 1 hr at room temperature. Signals were detected with ECL. Data was analyzed using Un-scan-it gel analysis software (Silk Scientific, Orem, Utah, USA). Relative increase in HIF1a expression compared to its own control is calculated. 2.10. Statistical analysis Data is presented as the meanGSE for at least three separate experiments. Student’s t-test and one-way ANOVA were employed for statistical analysis, with significant differences determined as p!0.05. 3. Results 3.1. Hypoxic preconditioning can protect stromal cells against apoptosis In epithelial scrape models, peak keratocyte apoptosis occurs 4 hr following the epithelial wound (Mohan et al., 2003). Therefore, for the in vitro studies we examined apoptosis 4 hr after UV-irradiation. We chose a UV dose (2 min or 5.1 mJ/cm2) that produced approximately 30–40% apoptosis so that we could clearly test for protective effects. We examined the effect of 4 hr of hypoxia (1.5 or 0.5% O2) preconditioning on the apoptotic rate of fibroblasts induced by
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2 min of UV-irradiation using the TUNEL assay. Fig. 1 shows that the apoptotic rate in the UV-irradiated normoxic group (46.1G12.1%) was significantly higher than either 1.5% O2 (27.8G4.1%) or 0.5% O2 (7.2G2.0%) preconditioned groups. Also the 0.5% O2 group had a lower apoptotic rate than the 1.5% O2 group (p!0.05). There was a small level of background apoptosis in non-UV irradiated cells (1.5G0.2% for normoxia, 1.0G0.4% for 1.5% O2 and 2.8G2.1% for 0.5% O2), and these differences were not significant (pZ0.24), indicating that the corneal stromal fibroblasts could tolerate these levels of hypoxia. Our experiments suggest that hypoxia
Fig. 1. Hypoxic preconditioning shows protection against UV-irradiation induced corneal stromal fibroblast apoptosis. Images show representative TUNEL staining of normoxic control (A), hypoxic control (B), normoxia with 2 min of UV-irradiation (C), 0.5% O2 hypoxia preconditioned with 2 min of UV-irradiation (D) and 1.5% O2 hypoxia preconditioned with 2 min of UVirradiation (E). Bar graph summarizes these results (nZ4). Error bar shows standard error of the mean for each group. Approximately, 750 cells were counted for each experiment. Nuclei stain in blue by DAPI. Apoptotic cells stain in bright green by TUNEL. Magnification: 200!.
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preconditioning provides protection to stromal cells against UV-irradiation and the protection is dose dependent. 3.2. HIF-1a is associated with protection To begin to examine whether HIF-1a could be involved in the protection, we measured the HIF-1a levels in nuclei and whole cell lysates of primary cultured stromal fibroblasts following 4 hr treatment with normoxia, 1.5% O2, 0.5% O2, or 100–200 mM cobalt chloride (CoCl2), which interferes with prolyl hydroxylation of HIF-1a. Fig. 2(A) and (B) show western blots of a 116 kDa protein representing HIF-1a obtained from whole cell lysate and nuclear extract preparations, respectively. Both preparations showed a low background level of HIF-1a in normoxia. The level of nuclear HIF-1a increased 1.5 fold with 1.5% O2 incubation, 2.25 fold with 0.5% O2 and 1.9 fold with incubation in 0.2 mM CoCl2.
Fig. 2. Western blots of HIF-1a. (A), whole cell lysates from primary cultured stromal fibroblast treated with normoxia, 0.5% O2, 0.1 or 0.2 mM cobalt chloride (a b-actin control for protein loading is included). (B), nuclear extracts from primary cultured stromal fibroblast. In the left hand blot, cells were treated with normoxia control, 0.2 mM cobalt chloride, or 1.5% O2. In the right hand blot cells were treated with 0.5% O2. For each blot there is a separate normoxic control (Con). Data were analyzed using gel imaging software. Relative increase of average pixel value from HIF-1a blot of each experiment group compared to its own normoxia control are represented in the bar graph (nZ4). Data are represented with meanGSE.
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Fig. 3. Cobalt chloride shows a dose-dependent protective effect against UVirradiation induced corneal stromal fibroblast apoptosis. Cells were treated with 0.1 or 0.2 mM cobalt chloride for 4 hr then irradiated with 2 min of UV. Cells were stained with TUNEL 4 hr after irradiation. Error bar represent the standard errors of the mean (nZ4). Approximately 750 cells were counted for each experiment.
In whole cell lysates, HIF-1a levels were increased 2.5 fold with 0.2 mM CoCl2 and two fold following 0.5% O2. Since cobalt chloride (CoCl2) is a strong inducer of HIF-1a in the corneal fibroblasts, we tested whether CoCl2 could also be protective. Fibroblasts were incubated with 0.1 or 0.2 mM CoCl2 in the standard 5% CO2/Air incubator for 4 hr, washed and incubated with fresh 0.5% FBS/DMEM for 1 hr and then irradiated with 2 min of UV. Cells were stained with TUNEL 4 hr after irradiation. Fig. 3 shows that the apoptotic rate for cells incubated in CoCl2 was greatly reduced compared to control irradiated cells. The apoptotic rate for cells incubated with 0.2 mM CoCl2 incubation (4.2G2.1%) was significantly lower than that with 0.1 mM CoCl2 incubation (20.5G9.8%). The apoptotic rates among non-irradiated groups were not significantly different. These experiments demonstrate that cobalt chloride as well as hypoxia provides protection against UV-induced apoptosis, and that the protection is associated with induction of HIF-1a. 3.3. Hypoxic preconditioned medium has a protective effect HIF-1a can induce transcription of various protective factors including VEGF, EPO and bFGF (Semenza, 2004). To explore whether protective factors could be released from hypoxia preconditioned cells, we tested whether preconditioned media could protect corneal fibroblasts. Following 2 or 4 hrs incubation in hypoxia preconditioned media, cells were UV-irradiated for 2 min. Fig. 4 shows that the apoptotic rates of cells following 2 hr incubation (8.4G3.4%) or 4 hr incubation (5.9G4.7%) in hypoxia conditioned medium were significantly lower than controls (24.9G3.0%). Although there was a slight drop in the apoptotic rate at the longer incubation time, the difference between 2 and 4 hr incubation in hypoxia conditioned medium was not significant. There was no significant difference in apoptotic rate among non-irradiated controls.
Fig. 4. Hypoxia preconditioned media shows protective effect against UVirradiation induced corneal fibroblast apoptosis. Cells were incubated with hypoxia preconditioned medium for 2 or 4 hr, then UV-irradiated for 2 min. Cells were stained with TUNEL 4 hr after irradiation. Error bar represent the standard errors of the mean. Approximately 750 cells were counted for each experiment.
3.4. Hypoxic preconditioning protects primary cultured cornea fibroblasts against Fas/FasL induced nuclear condensation UV-irradiation induces apoptosis via oxidative stress that ultimately involves the mitochondrial pathway of apoptosis induction and triggers the release of cytochrome c (Martin et al., 1995; Matsuda et al., 2002). Cytokine mediated apoptotic stress is generated through the activation of membrane receptor death domains. Previous studies have shown that activation of Fas receptor can induce apoptosis in keratocytes (Mohan et al., 1997). We asked if hypoxia preconditioning could protect corneal fibroblasts from Fas mediated nuclear condensation as well as from UV-irradiation. In this experiment, 24 hr of incubation with 100 ng/ml of anti-Fas antibody (Kamiya Biomedical Company) was used to induce apoptosis. In order to eliminate effects from serum containing cytokines, we used serum-free DMEM culture medium. Fig. 5 shows that nuclear condensation visualized by DAPI staining indicated that the proportion of condensed nuclei in the serum-free control group not exposed to anti-Fas, was 12.5G4.5%, which is significantly higher than in 0.5% serum DMEM (see Fig. 2). The proportion of condensed nuclei for anti-Fas treatment alone was 25.5G 4.1%, which is significantly higher than control. When anti-Fas was incubated with cells that had been preconditioned with 4 hr of 0.5% O2 hypoxia or with 0.1 M cobalt chloride, the proportion of condensed nuclei was significantly reduced to 4.8G2.9% and 9.2G5.4%, respectively, indicating that hypoxia preconditioning could provide a protective effect against Fas induced apoptosis. Interestingly, the proportion of condensed nuclei of hypoxia preconditioned cells was lower than control, suggesting that hypoxia preconditioning was also protective against serum starvation.
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Fig. 5. CoCl2 and hypoxia preconditioning has a protective effect against Fas/FasL induced nucelar condensation. Cells were stained by DAPI. Condensed nuclei were scored if fluorescence was at least three times brighter than control nuclei. Control cells were incubated in serum-free DMEM, cells were treated with anti-Fas alone, with hypoxia or 0.1 mM Cobalt chloride for 4 hr. Cells were fixed 4 hr after 2 min of UV-irradiation. Error bar represents standard error of mean (nZ3). Magnification: 200!. Approximately 750 cells were counted for each experiment.
3.5. Hypoxia preconditioning protects stromal cells in bovine cornea against epithelial scrape induced apoptosis Epithelial scrape has long been used as a model to induce keratocyte apoptosis in animal experiments. The disappearance of anterior stromal keratocytes in response to epithelial scrape was shown to be mediated by apoptosis (Wilson et al., 1996). To explore whether hypoxia preconditioning can protect keratocytes in situ, we performed ex vivo experiments using bovine eyes. Eyes were preconditioned for 4 hr with 0.5% O2 and placed in normoxia for 30 min. The epithelium was scraped and cornea was frozen 4 hr later. We quantified the apoptotic rate in the anterior portion of a 20! magnification camera field (300 mm) of stroma using the TUNEL assay. We found that all TUNEL positive cells were in the anterior half of this field. Fig. 6 shows representative tissue sections from control and hypoxia preconditioned corneas. The apoptotic rate in hypoxia preconditioned scraped corneas (14.2G2.5%) was
Fig. 6. TUNEL assay of bovine corneal sections and the apoptotic rates in anterior stroma. Cornea was frozen 4 hr after epithelial scraping. Cryosections were stained with TUNEL and counterstained with DAPI. Images represent normoxic-unscraped (A), normoxic-scraped (C, E), hypoxic-unscraped (B) and hypoxic-scraped (D, F) corneas. Nuclei stain blue by DAPI, apoptotic cells stain bright green by TUNEL. Bar graph summarizes these results, Error bar represents standard error of the mean (nZ3). Magnification: (A, B, C, D) 200!; (E, F) 100!.
significantly lower than that of scraped corneas that were kept in normoxia (26.3G1.4%). The apoptotic rates in unscraped controls for both hypoxic (7.6G4.6%) and normoxic groups (6.5G4.2%) were not significantly different. The background apoptosis is probably due to the ex vivo nature of this experiment.
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4. Discussion In this study we demonstrate that significant but non-lethal hypoxic preconditioning protects primary cultured stromal fibroblasts against UV-irradiation and Fas mediated apoptosis. Hypoxic preconditioning also protected corneal keratocytes, within ex vivo corneal tissue, against epithelial injury induced apoptosis. The degree of hypoxia protection was associated with the degree of HIF-1a expression. Hypoxia conditioned medium was also protective suggesting that protective factors had been released from hypoxic cells into the medium. We found that short-term treatment with 1.5% O2 or 0.5% O2 was non-lethal and protective for corneal fibroblasts. This is similar to hypoxia protection in other cell types including protection of HepG2 cells against serum deprivation and t-BHP induced apoptosis (Piret et al., 2004), protection of hepatocyte from cytokine induced apoptosis (Crenesse et al., 2001) and protection of myocytes against acute severe oxidative stress (Silverman et al., 1997). We explored whether hypoxia preconditioning was protective against apoptosis induced via a mitochondrial pathway and a death receptor pathway. UVirradiation is DNA damaging and produces oxidative stress, which activates caspase 9 via the mitochondrial pathway. AntiFas antibody induces apoptosis through a membrane receptor death domain to activate caspase 8. Both pathways finally converge to the activation of apoptosis executioner caspases 3, 6, and 7. We found that hypoxia preconditioning could protect fibroblasts from apoptosis induced by both types of stress factors including serum-deprivation suggesting that hypoxia preconditioning produces a broad generalized protection against apoptosis. HIF-1a, a master transcriptional factor of hypoxia-regulated genes, mediates a wide range of cellular and physiological adaptive responses to changes in oxygen tension. In normoxia, HIF-1a is modified by oxygen dependent prolyl hydroxylases, which allows association with the von Hippel-Lindau tumor suppressor protein (VHL) and targeting for proteasomal degradation, maintaining a low level of HIF-1a. Under hypoxic conditions, less HIF-1a protein is hydroxylated allowing translocation to the nucleus where it dimerizes with HIF-1b. The HIF complex can then bind to the Hypoxia Responsive Element (HRE) consensus sequence in the promoter of downstream target genes (Zhang et al., 2004). The issue of whether HIF-1a is a pro- or anti-apoptotic transcription factor is a matter of some debate (Piret et al., 2002). The current consensus is that HIF-1a participates in promoting apoptosis during severe or long-term hypoxia, while promoting adaptation to hypoxia when it is less severe. Using primary cultured corneal stromal cells, we demonstrated that the amount of HIF-1a in both nuclear extracts and whole cell lysates increased followed hypoxia. The increase in HIF-1a induced by CoCl2 was consistent with the degree of protection against UV-irradiation induced apoptosis. The protective effect of hypoxia preconditioned medium also suggests involvement of HIF-1a since the expression and secretion of protective cytokines can occur via HIF-1a mediated transcription of protective factors such as EPO (Semenza et al., 1994) and
VEGF (Liu et al., 1995). Additional experiments using pharmacological and/or genetic approaches to interfere with HIF-1a mediated activity will be needed to definitively establish the role of HIF-1a in hypoxia preconditioning protection of corneal stromal cells. Epithelial damage leads to keratocyte apoptosis probably mediated by diffusion of epithelial derived cytokines (Wilson et al., 2001). To test if hypoxia preconditioning could affect this apoptosis we used a simple ex vivo bovine model. We found that hypoxia preconditioning could also reduce keratocyte apoptosis induced by epithelial scraping. In this model, there was a background level of apoptosis probably related to the ex vivo nature of the experiment. More importantly there was no difference in background apoptotic rates between hypoxic and normoxic unscraped corneas, indicating that the hypoxia itself did not induce apoptosis. In a previous study, treatment of rabbit corneas with a general caspase inhibitor before epithelial scrape also reduced apoptosis, however cells apparently died instead by necrosis (Kim et al., 2000). We cannot exclude the possibility that the lower apoptotic rate in hypoxia preconditioned bovine corneas seen in our study is accompanied by an underlying necrosis. However, in other models like heart (Cai et al., 2003) and brain (Bergeron et al., 2000), hypoxia preconditioning protection is associated with both reduced apoptotic rates and reduced necrotic tissue. Further studies are needed using the rabbit in vivo model to test if hypoxia preconditioning can definitively reduce epithelial scrape-induced keratocyte apoptosis and prevent death by necrosis. In summary, our results indicate that non-lethal hypoxia preconditioning can protect corneal stromal cells against apoptosis induced by UV, Fas and epithelial scrape. This indicates that hypoxia preconditioning has broad protective properties and suggests that hypoxia or a hypoxia-like preconditioning could be used to lessen deleterious postsurgical healing. Further studies on the mechanism of hypoxia preconditioning protection and relevance to a surgical model are warranted. References Arends, M.J., Wyllie, A.H., 1991. Apoptosis: mechanisms and roles in pathology. Int. Rev. Exp. Pathol. 32, 223–254. Bergeron, M., Gidday, J.M., Yu, A.Y., Semenza, G.L., Ferriero, D.M., Sharp, F.R., 2000. Role of hypoxia-inducible factor-1 in hypoxia-induced ischemic tolerance in neonatal rat brain. Ann. Neurol. 48, 285–296. Cai, Z., Manalo, D.J., Wei, G., Rodriguez, E.R., Fox-Talbot, K., Lu, H., Zweier, J.L., Semenza, G.L., 2003. Hearts from rodents exposed to intermittent hypoxia or erythropoietin are protected against ischemiareperfusion injury. Circulation 108, 79–85. Carmeliet, P., Dor, Y., Herbert, J.-M., Fukumura, D., Brusselmans, K., Dewerchin, M., Neeman, M., Bono, F., Abramovitch, R., Maxwell, P., Koch, C.J., Ratcliffe, P., Moons, L., Jain, R.K., Collen, D., Keshet, E., 1998. Role of hif-1[alpha] in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature 394, 485–490. Chandrasekher, G., Ma, X., Lallier, T.E., Bazan, H.E., 2002. Delay of corneal epithelial wound healing and induction of keratocyte apoptosis by plateletactivating factor. Invest. Ophthalmol. Vis. Sci. 43, 1422–1428.
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