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Expression of Egr1 and p53 in human carotid plaques and apoptosis induced by 7-oxysterol or p53
Experimental and Toxicologic Pathology 65 (2013) 677–682
Contents lists available at SciVerse ScienceDirect
Experimental and Toxicologic Pathology journal homepage: www.elsevier.de/etp
Expression of Egr1 and p53 in human carotid plaques and apoptosis induced by 7-oxysterol or p53 Sayem Miah a,1 , Shahram Nour Mohammad Zadeh a , Xi-Ming Yuan a,b , Wei Li a,∗ a b
Division of Experimental Pathology, Department of Clinical and Experimental Medicine, Linköping University, Linköping SE-581 85, Sweden Division of Occupational and Environmental Medicine, University Hospital, Linköping SE-581 85, Sweden
a r t i c l e
i n f o
Article history: Received 2 February 2012 Accepted 19 August 2012 Keywords: Apoptosis Atherosclerosis p53 7-Oxysterols Immediate-early response protein (Egr-1) Nuclear fragmentation
a b s t r a c t Egr-1 and p53 are involved in pathology of both atherosclerosis and cancer. However, it is unknown whether p53 and Egr1 are interactively involved in apoptosis in atherosclerosis. We found that in human carotid plaques, the expression of p53 was inversely correlated with Egr1. In U937 cells, 7-hydroxycholesterol and 7-ketocholesterol induced production of reactive oxygen species (ROS), transient up-regulation of Egr1 followed by late induction of p53 and apoptosis. Cells with nuclear fragmentation induced by 7-oxysterol or p53 showed increased levels of p53, but decreased levels of Egr1. In conclusion, ROS induced by 7-oxysterols may function as an early initiator of Egr1 expression. The late induced p53 by 7-oxysterols contributes to apoptotic cell death and is linked to the reduction of Egr1 levels, which resembles the differential expression of p53 and Egr1 in human atheroma progression. © 2012 Elsevier GmbH. All rights reserved.
1. Introduction Apoptosis of arterial wall cells contribute to atherosclerotic plaque development, plaque rupture and atherothrombosis (Karaflou et al., 2008). 7-Oxyesterols, especially 7OH and 7keto, are the major toxic compounds found in oxidized low density lipoprotein (oxLDL) (Guyton et al., 1995) and induce apoptosis in vascular cells (Panini and Sinensky, 2001). Oxysterols can induce cellular reactive oxygen species (ROS), a key mediator in several signaling pathways to trigger vascular inflammation in the atherosclerotic process. We have previously reported that 7oxysterol mediated cell death is associated with increased levels of cellular ROS (Larsson et al., 2006). Recent data indicate that induction of ROS mediates expression of early growth response protein 1 (Egr1 protein) and apoptosis (Shin et al., 2009). Egr-1 plays regulatory roles in several cardiovascular diseases. Expression of Egr-1 was demonstrated in human atheroma lesions, mainly in smooth muscle cells that are important for plaque stability (McCaffrey et al., 2000). In apolipoprotein E knockout (apoE−/− ) mice, Egr1 expression was found in inflammatory cells surrounding regions of necrotic cores in atheroma (Karaflou et al., 2008; Khachigian, 2006). These indicate that Egr1 expression is involved
∗ Corresponding author. Tel.: +46 13 229602. E-mail address: [email protected] (W. Li). 1 Present address: Cell Biology and Cancer, Department of Biochemistry, College of Medicine, University of Saskatchewan, 107 Wiggins road, Saskatoon, SK S7NE5E, Canada. 0940-2993/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.etp.2012.08.002
in plaque progression. Shear stress stimulated Egr1 expression in response to the production of ROS (Yan et al., 2006). As a gene regulator, Egr1 controls a wide range of genes including p53 (Boone et al., 2011; Yu et al., 2007), which regulates signaling of cell cycle arrest and apoptosis (Krones-Herzig et al., 2003; Liu et al., 2001; Nair et al., 1997). Moreover, it has been suggested that signaling interaction in apoptosis between Egr1 and p53 may be bidirectional (Nair et al., 1997). In atheroma lesions, macrophages are the most prominent inflammatory cells to produce inflammatory cytokines, chemokines, and hydrolytic proteases. U937 cell line is a frequently used as macrophage reference model because of the similarity to human monocytes regarding cytotoxic and pro-inflammatory effects observed in these cells (Prunet et al., 2006). Tumor suppressor gene p53 has an important function in regulating apoptosis and cellularity in the advanced atherosclerotic plaques (Mercer and Bennett, 2006). We recently reported that p53 expression in human carotid atherosclerotic lesions (both mutant and wild type detected with p53 antibody clone DO-7) was significantly increased in plaques with necrotic core formation and significantly correlated with the expression of cell death related proteins and lysosomal cathepsin (Yuan et al., 2010). Although the results from in vivo and in vitro experiments suggest that both p53 and Egr1 are deregulated and involved in cell death in the progression of atherosclerotic plaques, possible interaction between them has not been investigated in a relevant model. In the present study, we aimed to determine whether apoptosis induced by 7-oxysterols is related to the production of ROS and induction or regulation of Egr1 and p53 in vitro and possible implications in human carotid atheroma.
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S. Miah et al. / Experimental and Toxicologic Pathology 65 (2013) 677–682
2. Materials and methods
2.5. Flow cytometry
2.1. Chemical reagents
Expression of Egr1 or p53, and production of ROS (DHE stained cells) were measured by flow cytometry using Facs Calibur (BD Biosciences, San José, CA, USA). For each sample 10,000 cells were collected for analysis. Data were analyzed using CELL Quest Prosoftware, Version 4.0.2 (BD Biosciences).
Culture media RPMI 1640 and DMEM, horse serum, fetal bovine serum, penicillin-G, and streptomycin were from Gibco (Paisley, UK). Cholesterol, 7-hydroxycholesterol (7OH) and 7ketocholesterol (7keto) were from Sigma (St. Louis, MO, USA). Dihydroethidium (DHE) was from Molecular Probes (Eugene, OR, USA). Hoechst dye and goat anti-rabbit-Alex488 and 546 were from Invitrogen (California, USA). Annexin V-PE (AV) was from Roche (BD, Germany). Rabbit anti-Egr-1 and p53 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
2.6. Linköping Carotid Study and human carotid atheroma The Linköping Carotid Study is a prospective clinical-pathology study in which atherosclerotic carotid arteries are collected from
2.2. Cells and culture conditions Human monocytic cell line U-937 cells maintained in RPMI1640 culture medium with glutamine supplemented with 10% FBS, 100 U/ml penicillin G and 100 g/ml streptomycin. The cells were cultured in 5% CO2 in humidified atmosphere at 37 ◦ C and subcultured once every three days. The sub-divided cells were untreated or exposed to 28 M of 7OH or 7keto for 3–70 h. Cells treated with cholesterol or ethanol were used as controls. M1 mouse myeloid leukemic cells that are p53 deficient were stably transfected with a plasmid containing a temperaturesensitive p53 mutant as we described previously [135 Ala → Val] (M1-t-p53 cells) (Yuan et al., 2002). The p53 protein in M1-t-p53 cells behaves like wild-type at 32 ◦ C and like mutant at 37 ◦ C. Cells were grown in DMEM with 10% horse serum in a humidified atmosphere (10% CO2 ) at 37 ◦ C, subcultured twice a week and subdivided cells were used for experiments in which they were maintained either at 37 ◦ C or at 32 ◦ C for different periods of time.
2.3. Determination of ROS Intracellular ROS was assessed using flow cytometry following DHE staining. After each time point of treatment, cells were collected, washed once with PBS. The cells were then stained with DHE for 15 min at 37 ◦ C (10 M in culture medium) and analyzed using flow cytometry. The percentage of cells with increased DHE red fluorescence was identified as the percentage of cells with increased ROS.
2.4. Immunocytochemistry of Egr1 and p53 After each time point of treatment, cells were collected, fixed with 2% paraformaldehyde (PFA), permeabilized with permeabilizing buffer (0.1% saponin in PBS containing 5% fetal bovine serum), and then incubated with polyclonal rabbit anti-Egr-1 (1:100) or rabbit anti-p53 (1:50) at 4 ◦ C overnight. Cells were then incubated with goat anti-rabbit Alex488 conjugated secondary antibody at 22 ◦ C for 1 h. In some experiments, M1-t-p53 cells were stained for 10 min with PE-conjugated Annexin V first and carefully washed. The Annexin V stained cells were then incubated with above anti-p53 or anti-Egr1 primary antibodies and followed by incubation with goat anti-rabbit Alex488 conjugated secondary antibody at 22 ◦ C for 1 h. Immuno-stained cells were analyzed using flow cytometry or fluorescence microscopy. For fluorescence microscopy, immunostained cells were counterstained with Hoechst nuclear stain. Controls without primary antibody or with isotype antibodies were used as negative controls, which showed consistently negative results.
Fig. 1. Egr1 levels are inversely correlated with p53 levels in human carotid plaques, especially in ruptured plaques with necrotic cores. Serial sections of human carotid plaques were immuno-stained with either Egr1 or p53 and immuno-positivity was measured separately. Correlation between Egr1 and p53 in immuno-positivity was determined by a Spearman correlation coefficient test in all plaques (A), in type I and II plaques (B) and in type III plaques (C).
S. Miah et al. / Experimental and Toxicologic Pathology 65 (2013) 677–682
patients who undergo carotid endarterectomy at Linköping University Hospital. The ethics committee of Linköping University Hospital has approved this study as described previously (Yuan et al., 2010). Forty-five atherosclerotic carotid samples obtained from consecutive patients were included in the present study. 4% formaldehyde fixed samples were cross-sectioned into three–five segments and embedded in paraffin. 78 sections were subjected to immunohistochemsitry of p53 or Egr1 by incubating with monoclonal mouse anti-human p53 (1:200, DAKO, Glostru, Denmark) or polyclonal anti-human Egr1 (1:100, Santa Cruz Biotechnology, Inc., Santa Cruz, U.S.A.). The immuno-reactions were visualized using the DAKO EnVisionTM +/HRP method. Controls without primary antibodies were run for each protocol, resulting in consistently negative results. Isotype controls were tested with normal serum from the same animal or species as the primary antibody or the same immunoglobulin isotype. 52 matched sections from p53 and Egr1 were applied for correlation test. All the carotid sections were classified into three groups (type I, II or III) based on their morphology and collagen staining, as described previously (Yuan et al., 2010). In brief, type I plaques were defined as intact plaques with a fibrous cap but no necrotic core formation; type II plaques were also intact plaques with necrotic core formation; and type III were ruptured plaques. All histological sections that had been stained for p53 or Egr1 were digitalized with the same setting for all the
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samples and analyzed with Adobe Photoshop as described previously (Yuan et al., 2010). 2.7. Statistics For statistical analyses, one-way ANOVA followed by a post hoc Newman–Keuls test was used for multiple comparisons. The results are given as the means ± SEM, n ≥ 3 unless otherwise stated. p ≤ 0.05 was considered statistically significant. Correlations between immuno-positivity for Egr1 and p53 was examined by the Spearman correlation test and presented as the Spearman correlation coefficient (r). 3. Results 3.1. Egr1 expression is inversely correlated with p53 in ruptured human carotid plaques Recently we have demonstrated that p53 expression in human carotid plaques was associated with lesion severity and plaque rupture. Here we further investigated the correlation between Egr1 and p53 in human carotid atheroma lesions. As shown in Fig. 1, there was a inverse correlation between Egr1 and p53 expression in all the plaques investigated (Fig. 1A). The correlation disappeared
Fig. 2. Exposure to 7OH or 7keto causes induction of cellular ROS, early induction of Egr1, and late induction of p53 in apoptotic cells. U937 cells were cultured for 1–70 h in the presence or absence of 7OH or 7keto. The cells were collected at each time point, stained with DHE or immuno-cytochemistry of Egr1 or p53 and analyzed by flow cytometry. (A) Production of cellular ROS. The percentages of cells with increased DHE red fluorescence were analyzed and the results were expressed as percentage of control. **p < 0.01 and ***p < 0.001 vs. controls from the same time point (n = 4–10). (B) Egr1 induction. *p < 0.05 vs. control cells (n = 4–6). (C and D) Late induction of p53 in apoptotic cells. (C) Representative photographs of p53 immuno-cytochemistry (left panel), nuclei stained by Hoechst (middle panel), and merged images of p53 and nuclei (right panel). Note: control cells showed faint cytosolic p53, while 7OH treated cells displayed enhanced p53 mainly in nuclei. (D) Quantification of p53 assayed by flow cytometry. **p < 0.01 and ***p < 0.001 vs. control cells (n = 4–10).
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in early lesions (type I) and intact lesions (type II) (Fig. 1B). However, a statistical significant inverse correlation was found between Egr1 and p53 expression in type III plaques. These lesions were ruptured plaques with necrotic cores and massive oxidized lipid accumulation including cholesterol oxidation products, oxysterols (Fig. 1C). 3.2. Exposure to 7ˇOH or 7keto causes early cellular ROS production, transient Egr1 induction, and late expression of p53 in apoptotic cells Earlier, we have demonstrated that 7-oxysterols mediate apoptotic cell death (Larsson et al., 2006). To explore whether Egr1 and p53 is interactively involved in cell death induced by 7-oxysterols we first examined whether Egr1 was induced in response to the exposure of 7-oxysterols. As compared to control cells, 7OH exposure induced slightly increase of ROS at 3 and 6 h and significant
increases of ROS after 16 and 24 h. In contrast, 7keto induced maximum production of cellular ROS at 3 and 6 h that was then gradually decreased (Fig. 2A). In the same model significantly increased Egr1 was observed following 6 h exposure to 7OH – that was drastically decreased to about control levels at 12 h (Fig. 2B). The exposure to 7keto caused a significant increase in Egr1 expression after 12 h as compared to untreated control cells (Fig. 2B). p53 expression in U937 cells following exposure to 7OH or 7keto was further examined (Fig. 2C and D). As shown in Fig. 2C, after 66 h incubation, control cells had faint p53 immuno-positivity mainly in the cytosol, these cells showed normal nuclear morphology (Fig. 2C). However, enhanced expression of p53 was observed in the 7OH treated cells, mostly in nuclei (Fig. 2C), these cells showed nuclear condensation and fragmentation. Colocalization of p53 with nuclear staining was exemplified in Fig. 2C. p53 expression was significantly induced after 70 h exposure to 7OH or 7keto (Fig. 2D).
Fig. 3. Exposure to 7OH or 7keto induces up-regulation of p53 and down-regulation of Egr1 in nuclear fragmented apoptotic cells. U937 cells were treated with or without 7OH or 7kseto for 70 h, subjected to double immunocytochemistry of p53 and Egr-1, and examined by fluorescence microscopy. (A–C) Representative photographs of the double immuno-stained cells with p53 (A), Egr-1 (B) and their corresponding nuclei stained with Hoechst (C). Note: triple stains show p53 immunofluorescence (arrow in A) and weak Egr1 immunofluorescence (arrow in B) in the same nuclear fragmented cells (arrows in C). (D and E) High power view of p53 (D) and Egr1 (E) merged with nuclear stains were taken from squared areas, indicating strong nuclear p53 expression and reduction of Egr1 in the same cells. (F and G) In the nuclear fragmented cells (>90 cells), immuno-reactivity of Egr1 or p53 were measured and expressed as arbitrary unit (a.u.) (F) or numbers of cells with nuclear positive p53 or Egr1 were counted (n = 8–12) and expressed as percentages (G). *p < 0.05, **p < 0.01 and ***p < 0.001 vs. Egr1 immuno-positive cells.
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3.3. Differential expression of p53 and Egr1 is significantly related to apoptosis induced by 7-oxysterols or p53 Next we examined the expression of both p53 and Egr1 in apoptotic cells induced by 7-oxysterols. U937 cells were incubated with 7OH or 7keto up to 70 h and then double stained with antibodies against both p53 and Egr1. As shown Fig. 3A, C and D, 7keto induced p53 nearly in all the cells including cells with nuclear fragmentation (arrows). However, in those nuclear fragmented cells (Fig. 3C), Egr1 levels decreased (Fig. 3B and E). Cellular immunoreactivity of p53 or Egr1 in the immuno-stained cells was measured and the results showed that p53 levels were significantly higher than Egr1 in the same nuclear fragmented cells (Fig. 3F). Cellular expression of p53 or Egr1 in those cells were further counted, which showed that about 70% cells (7OH-treated) and about 60% cells (7keto-treated) with nuclear fragmentation showed positive p53 expression (black bars), while only about 6% (7OH-treated) and 20% (7keto-treated) of them had positive Egr1 (white bars) (Fig. 3G). The results lead to a question whether the inversed expression of p53 and Egr1 in apoptotic cells is a general phenomenon in apoptosis. To answer the above question, we determined the expression levels of p53 and Egr1 in a p53 transfected cell line M1-t-p53 cells, which behaves as wild-type when the cells are maintained at 32 ◦ C
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but like mutant p53 when the cells are maintained at 37 ◦ C (Yuan et al., 2002). In this model, cells undergo apoptosis when they are cultured at 32 ◦ C (Yuan et al., 2002). In the present study, we first investigated apoptosis by annexin V staining. The results revealed that annexin V positive cells were about 4% in the cells kept at 37 ◦ C after 24 h, while about 35% cells showed annexin V positive staining when they were cultured at 32 ◦ C. The cell populations with annexin V positivity matched well with increases in small cell populations as exemplified in Fig. 4A. There was no apoptotic cell death (see R2 population) in the cells kept at 32 ◦ C for 6 h (Fig. 4B and C), while increased cell shrinkage (increased R2 and decreased R1 population) was observed after 16 h and the apoptotic cell death (R2 population) was more than 50% after 36 h in the cells maintained at 32 ◦ C (Fig. 4B and C). In these cells, immuno-fluorescence intensity (MFI) of either p53 or Egr1 was separately analyzed in normal (R1) or apoptotic (R2) cell populations. Normal cells (R1 population, empty bars) expressed basal levels of p53 in the cells maintained at 32 ◦ C for 6 and 16 h (MFI around 100), which was more than 2 fold increased after 24 h (Fig. 4D). In apoptotic cells (small cells, filled bars) p53 levels were more than 2.5 fold higher than normal cells as early at 6 h. The significant increase remained with 24 h. In contrast, Egr1 levels in apoptotic cells (small size cells) were significantly lower than normal cells at all time points (Fig. 4E).
Fig. 4. Expression levels of p53 and Egr1 were inversely correlated in apoptotic M1-t-p53 cells. M1-t-p53 cells were cultured at 32 ◦ C for 6–36 h, subjected to p53 or Egr-1 immunocytochemistry and examined using flow cytometry. (A) Cell population in annexin V stained cells cultured either at 37 ◦ C (left panels) or at 32 ◦ C (right panels). (B) Cell populations in forward and side scatter were gated regarding cell size and expressed as normal size cells (R1) and small size cells (R2, apoptotic cells). Note: Cells in R1 was 98% and in R2 was 2% after 6 h (left panel), while cells in R1 was 48% and in R2 was 52% after 36 h maintained at 32 ◦ C (right panel), indicating cell shrinkage (apoptosis). (C) Time dependent increases in small size cells following p53 activation (cells kept at 32 ◦ C). (D and E) Summarized mean fluorescence intensity of p53 (D) and Egr1 (E) in cells with normal size (white bars) or small size (black bars). *p < 0.05 and ***p < 0.001 (n = 4).
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4. Discussion
References
Cytotoxic components in OxLDL, 7OH and 7keto, are prominent constituents of atherosclerotic lesions and induce apoptosis (Li et al., 2001; Lizard et al., 1999). In the cell death process, several regulators and their interactions are critically involved including Egr1 and p53. In the present study, we demonstrated that in 7OH or 7keto induced apoptosis, Egr1 and p53 were differentially regulated in the apoptotic process. The level of ROS and Egr1were increased at early time points, while p53 was significantly upregulated at the later stages of the process. Egr-1, a proinflammatory gene, plays an important role in the initiation and progression of atherosclerotic lesions. It participates in signaling in the apoptotic process. In the present study, we found that 7-oxysterols induced early induction of ROS and transient induction of Egr1. Furthermore, the nuclear translocation of Egr1 induced by 7OH or 7keto may play important roles in arterial walls in response to cellular stress similarly seen in hemin mediated stressed model (Hasan and Schafer, 2008). Following cellular oxidative stress and apoptotic cell death, the induced Egr1 may lead to the expression of downstream proteins such as p53 (Park et al., 2008). Our findings of an increase of p53 and simultaneous reduction of Egr1 and increased levels of ROS in apoptotic cells suggest that Egr1 may be an early oxidative responsive protein in the apoptosis process, in which reduction of Egr1 is followed by induction of p53 and apoptosis. p53, a transcription factor, plays a pivotal role in cell proliferation, cell cycle arrest and apoptosis and its expression is associated with necrotic core formation and plaque rupture of atherosclerosis (Yuan et al., 2010). In the present study, a high level of p53 was induced after a longer time exposure to 7OH or 7keto, resulting in formation of condensation and fragmentation of nuclei. The finding indicates that late induced p53 by atheroma relevant oxysterols is of importance in atheroma progression because it decides the fate of the cell survival or apoptosis. The removal of counteraction of survival Egr1 signaling (de Belle et al., 1999) by the 7-oxysterols may accelerate apoptotic cell death. This notion is supported by our results that ruptured human carotid plaques with necrotic core formation have significantly inverse correlation in expression of p53 and Egr1. This finding suggests that differential regulation of Egr1 and p53 may be associated with cell death in the plaque progression. In conclusion, ROS induced by the 7-oxysterols may function as an early initiator of Egr1 expression. The late induced p53 by 7oxysterols contributes to apoptotic cell death and is linked to the reduction of Egr1 levels, which resembles the differential expression of p53 and Egr1 in human atheroma progression.
Boone DN, Qi Y, Li Z, Hann SR. Egr1 mediates p53-independent c-Myc-induced apoptosis via a noncanonical ARF-dependent transcriptional mechanism. Proceedings of the National Academy of Sciences of the United States of America 2011;108:632–7. de Belle I, Huang RP, Fan Y, Liu C, Mercola D. Adamson ED. p53 and Egr-1 additively suppress transformed growth in HT1080 cells but Egr-1 counteracts p53-dependent apoptosis. Oncogene 1999;18:3633–42. Guyton JR, Lenz ML, Mathews B, Hughes H, Karsan D, Selinger E, et al. Toxicity of oxidized low density lipoproteins for vascular smooth muscle cells and partial protection by antioxidants. Atherosclerosis 1995;118:237–49. Hasan RN, Schafer AI. Hemin upregulates Egr-1 expression in vascular smooth muscle cells via reactive oxygen species ERK-1/2-Elk-1 and NF-kappaB. Circulation Research 2008;102:42–50. Karaflou M, Lambrinoudaki I, Christodoulakos G. Apoptosis in atherosclerosis: a mini-review. Mini Reviews in Medicinal Chemistry 2008;8:912–8. Khachigian LM. Early growth response-1 in cardiovascular pathobiology. Circulation Research 2006;98:186–91. Krones-Herzig A, Adamson E, Mercola D. Early growth response 1 protein, an upstream gatekeeper of the p53 tumor suppressor, controls replicative senescence. Proceedings of the National Academy of Sciences of the United States of America 2003;100:3233–8. Larsson DA, Baird S, Nyhalah JD, Yuan XM, Li W. Oxysterol mixtures, in atheromarelevant proportions, display synergistic and proapoptotic effects. Free Radical Biology and Medicine 2006;41:902–10. Li W, Dalen H, Eaton JW, Yuan XM. Apoptotic death of inflammatory cells in human atheroma. Arteriosclerosis, Thrombosis, and Vascular Biology 2001;21: 1124–30. Liu J, Grogan L, Nau MM, Allegra CJ, Chu E, Wright JJ. Physical interaction between p53 and primary response gene Egr-1. International Journal of Oncology 2001;18:863–70. Lizard G, Monier S, Cordelet C, Gesquiere L, Deckert V, Gueldry S, et al. Characterization and comparison of the mode of cell death, apoptosis versus necrosis, induced by 7beta-hydroxycholesterol and 7-ketocholesterol in the cells of the vascular wall. Arteriosclerosis, Thrombosis, and Vascular Biology 1999;19:1190–200. McCaffrey TA, Fu C, Du B, Eksinar S, Kent KC, Bush Jr H, et al. High-level expression of Egr-1 and Egr-1-inducible genes in mouse and human atherosclerosis. Journal of Clinical Investigation 2000;105:653–62. Mercer J, Bennett M. The role of p53 in atherosclerosis. Cell Cycle 2006;5:1907–9. Nair P, Muthukkumar S, Sells SF, Han SS, Sukhatme VP, Rangnekar VM. Early growth response-1-dependent apoptosis is mediated by p53. Journal of Biological Chemistry 1997;272:20131–8. Panini SR, Sinensky MS. Mechanisms of oxysterol-induced apoptosis. Current Opinion in Lipidology 2001;12:529–33. Park SE, Lee SW, Hossain MA, Kim MY, Kim MN, Ahn EY, et al. A chenodeoxycholic derivative, HS-1200, induces apoptosis and cell cycle modulation via Egr-1 gene expression control on human hepatoma cells. Cancer Letters 2008;270: 77–86. Prunet C, Montange T, Véjux A, Laubriet A, Rohmer JF, Riedinger JM, et al. Multiplexed flow cytometric analyses of pro- and anti-inflammatory cytokines in the culture media of oxysterol-treated human monocytic cells and in the sera of atherosclerotic patients. Cytometry A 2006;69:359–73. Shin DY, Kim GY, Li W, Choi BT, Kim ND, Kang HS, et al. Implication of intracellular ROS formation, caspase-3 activation and Egr-1 induction in platycodon D-induced apoptosis of U937 human leukemia cells. Biomedicine and Pharmacotherapy 2009;63:86–94. Yan SF, Harja E, Andrassy M, Fujita T, Schmidt AM. Protein kinase C beta/early growth response-1 pathway: a key player in ischemia, atherosclerosis, and restenosis. Journal of the American College of Cardiology 2006;48:A47–55. Yu J, Baron V, Mercola D, Mustelin T, Adamson ED. A network of p73, p53 and Egr1 is required for efficient apoptosis in tumor cells. Cell Death and Differentiation 2007;14:436–46. Yuan XM, Li W, Dalen H, Lotem J, Kama R, Sachs L, et al. Lysosomal destabilization in p53-induced apoptosis. Proceedings of the National Academy of Sciences of the United States of America 2002;99:6286–91. Yuan XM, Osman E, Miah S, Zadeh SN, Xu L, Forssell C, et al. p53 expression in human carotid atheroma is significantly related to plaque instability and clinical manifestations. Atherosclerosis 2010;210:392–9.
Acknowledgements This work was supported by grants from the Swedish Heart Lung Foundation, the Torsten and Ragnar Söderbergs Foundation, the Stroke Foundation, the Olle Engkvist Foundation and the Swedish Gamla Tjänarinnor Foundation, the Linköping University and Linköping University Hospital Research Fund.
Contents lists available at SciVerse ScienceDirect
Experimental and Toxicologic Pathology journal homepage: www.elsevier.de/etp
Expression of Egr1 and p53 in human carotid plaques and apoptosis induced by 7-oxysterol or p53 Sayem Miah a,1 , Shahram Nour Mohammad Zadeh a , Xi-Ming Yuan a,b , Wei Li a,∗ a b
Division of Experimental Pathology, Department of Clinical and Experimental Medicine, Linköping University, Linköping SE-581 85, Sweden Division of Occupational and Environmental Medicine, University Hospital, Linköping SE-581 85, Sweden
a r t i c l e
i n f o
Article history: Received 2 February 2012 Accepted 19 August 2012 Keywords: Apoptosis Atherosclerosis p53 7-Oxysterols Immediate-early response protein (Egr-1) Nuclear fragmentation
a b s t r a c t Egr-1 and p53 are involved in pathology of both atherosclerosis and cancer. However, it is unknown whether p53 and Egr1 are interactively involved in apoptosis in atherosclerosis. We found that in human carotid plaques, the expression of p53 was inversely correlated with Egr1. In U937 cells, 7-hydroxycholesterol and 7-ketocholesterol induced production of reactive oxygen species (ROS), transient up-regulation of Egr1 followed by late induction of p53 and apoptosis. Cells with nuclear fragmentation induced by 7-oxysterol or p53 showed increased levels of p53, but decreased levels of Egr1. In conclusion, ROS induced by 7-oxysterols may function as an early initiator of Egr1 expression. The late induced p53 by 7-oxysterols contributes to apoptotic cell death and is linked to the reduction of Egr1 levels, which resembles the differential expression of p53 and Egr1 in human atheroma progression. © 2012 Elsevier GmbH. All rights reserved.
1. Introduction Apoptosis of arterial wall cells contribute to atherosclerotic plaque development, plaque rupture and atherothrombosis (Karaflou et al., 2008). 7-Oxyesterols, especially 7OH and 7keto, are the major toxic compounds found in oxidized low density lipoprotein (oxLDL) (Guyton et al., 1995) and induce apoptosis in vascular cells (Panini and Sinensky, 2001). Oxysterols can induce cellular reactive oxygen species (ROS), a key mediator in several signaling pathways to trigger vascular inflammation in the atherosclerotic process. We have previously reported that 7oxysterol mediated cell death is associated with increased levels of cellular ROS (Larsson et al., 2006). Recent data indicate that induction of ROS mediates expression of early growth response protein 1 (Egr1 protein) and apoptosis (Shin et al., 2009). Egr-1 plays regulatory roles in several cardiovascular diseases. Expression of Egr-1 was demonstrated in human atheroma lesions, mainly in smooth muscle cells that are important for plaque stability (McCaffrey et al., 2000). In apolipoprotein E knockout (apoE−/− ) mice, Egr1 expression was found in inflammatory cells surrounding regions of necrotic cores in atheroma (Karaflou et al., 2008; Khachigian, 2006). These indicate that Egr1 expression is involved
∗ Corresponding author. Tel.: +46 13 229602. E-mail address: [email protected] (W. Li). 1 Present address: Cell Biology and Cancer, Department of Biochemistry, College of Medicine, University of Saskatchewan, 107 Wiggins road, Saskatoon, SK S7NE5E, Canada. 0940-2993/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.etp.2012.08.002
in plaque progression. Shear stress stimulated Egr1 expression in response to the production of ROS (Yan et al., 2006). As a gene regulator, Egr1 controls a wide range of genes including p53 (Boone et al., 2011; Yu et al., 2007), which regulates signaling of cell cycle arrest and apoptosis (Krones-Herzig et al., 2003; Liu et al., 2001; Nair et al., 1997). Moreover, it has been suggested that signaling interaction in apoptosis between Egr1 and p53 may be bidirectional (Nair et al., 1997). In atheroma lesions, macrophages are the most prominent inflammatory cells to produce inflammatory cytokines, chemokines, and hydrolytic proteases. U937 cell line is a frequently used as macrophage reference model because of the similarity to human monocytes regarding cytotoxic and pro-inflammatory effects observed in these cells (Prunet et al., 2006). Tumor suppressor gene p53 has an important function in regulating apoptosis and cellularity in the advanced atherosclerotic plaques (Mercer and Bennett, 2006). We recently reported that p53 expression in human carotid atherosclerotic lesions (both mutant and wild type detected with p53 antibody clone DO-7) was significantly increased in plaques with necrotic core formation and significantly correlated with the expression of cell death related proteins and lysosomal cathepsin (Yuan et al., 2010). Although the results from in vivo and in vitro experiments suggest that both p53 and Egr1 are deregulated and involved in cell death in the progression of atherosclerotic plaques, possible interaction between them has not been investigated in a relevant model. In the present study, we aimed to determine whether apoptosis induced by 7-oxysterols is related to the production of ROS and induction or regulation of Egr1 and p53 in vitro and possible implications in human carotid atheroma.
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S. Miah et al. / Experimental and Toxicologic Pathology 65 (2013) 677–682
2. Materials and methods
2.5. Flow cytometry
2.1. Chemical reagents
Expression of Egr1 or p53, and production of ROS (DHE stained cells) were measured by flow cytometry using Facs Calibur (BD Biosciences, San José, CA, USA). For each sample 10,000 cells were collected for analysis. Data were analyzed using CELL Quest Prosoftware, Version 4.0.2 (BD Biosciences).
Culture media RPMI 1640 and DMEM, horse serum, fetal bovine serum, penicillin-G, and streptomycin were from Gibco (Paisley, UK). Cholesterol, 7-hydroxycholesterol (7OH) and 7ketocholesterol (7keto) were from Sigma (St. Louis, MO, USA). Dihydroethidium (DHE) was from Molecular Probes (Eugene, OR, USA). Hoechst dye and goat anti-rabbit-Alex488 and 546 were from Invitrogen (California, USA). Annexin V-PE (AV) was from Roche (BD, Germany). Rabbit anti-Egr-1 and p53 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
2.6. Linköping Carotid Study and human carotid atheroma The Linköping Carotid Study is a prospective clinical-pathology study in which atherosclerotic carotid arteries are collected from
2.2. Cells and culture conditions Human monocytic cell line U-937 cells maintained in RPMI1640 culture medium with glutamine supplemented with 10% FBS, 100 U/ml penicillin G and 100 g/ml streptomycin. The cells were cultured in 5% CO2 in humidified atmosphere at 37 ◦ C and subcultured once every three days. The sub-divided cells were untreated or exposed to 28 M of 7OH or 7keto for 3–70 h. Cells treated with cholesterol or ethanol were used as controls. M1 mouse myeloid leukemic cells that are p53 deficient were stably transfected with a plasmid containing a temperaturesensitive p53 mutant as we described previously [135 Ala → Val] (M1-t-p53 cells) (Yuan et al., 2002). The p53 protein in M1-t-p53 cells behaves like wild-type at 32 ◦ C and like mutant at 37 ◦ C. Cells were grown in DMEM with 10% horse serum in a humidified atmosphere (10% CO2 ) at 37 ◦ C, subcultured twice a week and subdivided cells were used for experiments in which they were maintained either at 37 ◦ C or at 32 ◦ C for different periods of time.
2.3. Determination of ROS Intracellular ROS was assessed using flow cytometry following DHE staining. After each time point of treatment, cells were collected, washed once with PBS. The cells were then stained with DHE for 15 min at 37 ◦ C (10 M in culture medium) and analyzed using flow cytometry. The percentage of cells with increased DHE red fluorescence was identified as the percentage of cells with increased ROS.
2.4. Immunocytochemistry of Egr1 and p53 After each time point of treatment, cells were collected, fixed with 2% paraformaldehyde (PFA), permeabilized with permeabilizing buffer (0.1% saponin in PBS containing 5% fetal bovine serum), and then incubated with polyclonal rabbit anti-Egr-1 (1:100) or rabbit anti-p53 (1:50) at 4 ◦ C overnight. Cells were then incubated with goat anti-rabbit Alex488 conjugated secondary antibody at 22 ◦ C for 1 h. In some experiments, M1-t-p53 cells were stained for 10 min with PE-conjugated Annexin V first and carefully washed. The Annexin V stained cells were then incubated with above anti-p53 or anti-Egr1 primary antibodies and followed by incubation with goat anti-rabbit Alex488 conjugated secondary antibody at 22 ◦ C for 1 h. Immuno-stained cells were analyzed using flow cytometry or fluorescence microscopy. For fluorescence microscopy, immunostained cells were counterstained with Hoechst nuclear stain. Controls without primary antibody or with isotype antibodies were used as negative controls, which showed consistently negative results.
Fig. 1. Egr1 levels are inversely correlated with p53 levels in human carotid plaques, especially in ruptured plaques with necrotic cores. Serial sections of human carotid plaques were immuno-stained with either Egr1 or p53 and immuno-positivity was measured separately. Correlation between Egr1 and p53 in immuno-positivity was determined by a Spearman correlation coefficient test in all plaques (A), in type I and II plaques (B) and in type III plaques (C).
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patients who undergo carotid endarterectomy at Linköping University Hospital. The ethics committee of Linköping University Hospital has approved this study as described previously (Yuan et al., 2010). Forty-five atherosclerotic carotid samples obtained from consecutive patients were included in the present study. 4% formaldehyde fixed samples were cross-sectioned into three–five segments and embedded in paraffin. 78 sections were subjected to immunohistochemsitry of p53 or Egr1 by incubating with monoclonal mouse anti-human p53 (1:200, DAKO, Glostru, Denmark) or polyclonal anti-human Egr1 (1:100, Santa Cruz Biotechnology, Inc., Santa Cruz, U.S.A.). The immuno-reactions were visualized using the DAKO EnVisionTM +/HRP method. Controls without primary antibodies were run for each protocol, resulting in consistently negative results. Isotype controls were tested with normal serum from the same animal or species as the primary antibody or the same immunoglobulin isotype. 52 matched sections from p53 and Egr1 were applied for correlation test. All the carotid sections were classified into three groups (type I, II or III) based on their morphology and collagen staining, as described previously (Yuan et al., 2010). In brief, type I plaques were defined as intact plaques with a fibrous cap but no necrotic core formation; type II plaques were also intact plaques with necrotic core formation; and type III were ruptured plaques. All histological sections that had been stained for p53 or Egr1 were digitalized with the same setting for all the
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samples and analyzed with Adobe Photoshop as described previously (Yuan et al., 2010). 2.7. Statistics For statistical analyses, one-way ANOVA followed by a post hoc Newman–Keuls test was used for multiple comparisons. The results are given as the means ± SEM, n ≥ 3 unless otherwise stated. p ≤ 0.05 was considered statistically significant. Correlations between immuno-positivity for Egr1 and p53 was examined by the Spearman correlation test and presented as the Spearman correlation coefficient (r). 3. Results 3.1. Egr1 expression is inversely correlated with p53 in ruptured human carotid plaques Recently we have demonstrated that p53 expression in human carotid plaques was associated with lesion severity and plaque rupture. Here we further investigated the correlation between Egr1 and p53 in human carotid atheroma lesions. As shown in Fig. 1, there was a inverse correlation between Egr1 and p53 expression in all the plaques investigated (Fig. 1A). The correlation disappeared
Fig. 2. Exposure to 7OH or 7keto causes induction of cellular ROS, early induction of Egr1, and late induction of p53 in apoptotic cells. U937 cells were cultured for 1–70 h in the presence or absence of 7OH or 7keto. The cells were collected at each time point, stained with DHE or immuno-cytochemistry of Egr1 or p53 and analyzed by flow cytometry. (A) Production of cellular ROS. The percentages of cells with increased DHE red fluorescence were analyzed and the results were expressed as percentage of control. **p < 0.01 and ***p < 0.001 vs. controls from the same time point (n = 4–10). (B) Egr1 induction. *p < 0.05 vs. control cells (n = 4–6). (C and D) Late induction of p53 in apoptotic cells. (C) Representative photographs of p53 immuno-cytochemistry (left panel), nuclei stained by Hoechst (middle panel), and merged images of p53 and nuclei (right panel). Note: control cells showed faint cytosolic p53, while 7OH treated cells displayed enhanced p53 mainly in nuclei. (D) Quantification of p53 assayed by flow cytometry. **p < 0.01 and ***p < 0.001 vs. control cells (n = 4–10).
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in early lesions (type I) and intact lesions (type II) (Fig. 1B). However, a statistical significant inverse correlation was found between Egr1 and p53 expression in type III plaques. These lesions were ruptured plaques with necrotic cores and massive oxidized lipid accumulation including cholesterol oxidation products, oxysterols (Fig. 1C). 3.2. Exposure to 7ˇOH or 7keto causes early cellular ROS production, transient Egr1 induction, and late expression of p53 in apoptotic cells Earlier, we have demonstrated that 7-oxysterols mediate apoptotic cell death (Larsson et al., 2006). To explore whether Egr1 and p53 is interactively involved in cell death induced by 7-oxysterols we first examined whether Egr1 was induced in response to the exposure of 7-oxysterols. As compared to control cells, 7OH exposure induced slightly increase of ROS at 3 and 6 h and significant
increases of ROS after 16 and 24 h. In contrast, 7keto induced maximum production of cellular ROS at 3 and 6 h that was then gradually decreased (Fig. 2A). In the same model significantly increased Egr1 was observed following 6 h exposure to 7OH – that was drastically decreased to about control levels at 12 h (Fig. 2B). The exposure to 7keto caused a significant increase in Egr1 expression after 12 h as compared to untreated control cells (Fig. 2B). p53 expression in U937 cells following exposure to 7OH or 7keto was further examined (Fig. 2C and D). As shown in Fig. 2C, after 66 h incubation, control cells had faint p53 immuno-positivity mainly in the cytosol, these cells showed normal nuclear morphology (Fig. 2C). However, enhanced expression of p53 was observed in the 7OH treated cells, mostly in nuclei (Fig. 2C), these cells showed nuclear condensation and fragmentation. Colocalization of p53 with nuclear staining was exemplified in Fig. 2C. p53 expression was significantly induced after 70 h exposure to 7OH or 7keto (Fig. 2D).
Fig. 3. Exposure to 7OH or 7keto induces up-regulation of p53 and down-regulation of Egr1 in nuclear fragmented apoptotic cells. U937 cells were treated with or without 7OH or 7kseto for 70 h, subjected to double immunocytochemistry of p53 and Egr-1, and examined by fluorescence microscopy. (A–C) Representative photographs of the double immuno-stained cells with p53 (A), Egr-1 (B) and their corresponding nuclei stained with Hoechst (C). Note: triple stains show p53 immunofluorescence (arrow in A) and weak Egr1 immunofluorescence (arrow in B) in the same nuclear fragmented cells (arrows in C). (D and E) High power view of p53 (D) and Egr1 (E) merged with nuclear stains were taken from squared areas, indicating strong nuclear p53 expression and reduction of Egr1 in the same cells. (F and G) In the nuclear fragmented cells (>90 cells), immuno-reactivity of Egr1 or p53 were measured and expressed as arbitrary unit (a.u.) (F) or numbers of cells with nuclear positive p53 or Egr1 were counted (n = 8–12) and expressed as percentages (G). *p < 0.05, **p < 0.01 and ***p < 0.001 vs. Egr1 immuno-positive cells.
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3.3. Differential expression of p53 and Egr1 is significantly related to apoptosis induced by 7-oxysterols or p53 Next we examined the expression of both p53 and Egr1 in apoptotic cells induced by 7-oxysterols. U937 cells were incubated with 7OH or 7keto up to 70 h and then double stained with antibodies against both p53 and Egr1. As shown Fig. 3A, C and D, 7keto induced p53 nearly in all the cells including cells with nuclear fragmentation (arrows). However, in those nuclear fragmented cells (Fig. 3C), Egr1 levels decreased (Fig. 3B and E). Cellular immunoreactivity of p53 or Egr1 in the immuno-stained cells was measured and the results showed that p53 levels were significantly higher than Egr1 in the same nuclear fragmented cells (Fig. 3F). Cellular expression of p53 or Egr1 in those cells were further counted, which showed that about 70% cells (7OH-treated) and about 60% cells (7keto-treated) with nuclear fragmentation showed positive p53 expression (black bars), while only about 6% (7OH-treated) and 20% (7keto-treated) of them had positive Egr1 (white bars) (Fig. 3G). The results lead to a question whether the inversed expression of p53 and Egr1 in apoptotic cells is a general phenomenon in apoptosis. To answer the above question, we determined the expression levels of p53 and Egr1 in a p53 transfected cell line M1-t-p53 cells, which behaves as wild-type when the cells are maintained at 32 ◦ C
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but like mutant p53 when the cells are maintained at 37 ◦ C (Yuan et al., 2002). In this model, cells undergo apoptosis when they are cultured at 32 ◦ C (Yuan et al., 2002). In the present study, we first investigated apoptosis by annexin V staining. The results revealed that annexin V positive cells were about 4% in the cells kept at 37 ◦ C after 24 h, while about 35% cells showed annexin V positive staining when they were cultured at 32 ◦ C. The cell populations with annexin V positivity matched well with increases in small cell populations as exemplified in Fig. 4A. There was no apoptotic cell death (see R2 population) in the cells kept at 32 ◦ C for 6 h (Fig. 4B and C), while increased cell shrinkage (increased R2 and decreased R1 population) was observed after 16 h and the apoptotic cell death (R2 population) was more than 50% after 36 h in the cells maintained at 32 ◦ C (Fig. 4B and C). In these cells, immuno-fluorescence intensity (MFI) of either p53 or Egr1 was separately analyzed in normal (R1) or apoptotic (R2) cell populations. Normal cells (R1 population, empty bars) expressed basal levels of p53 in the cells maintained at 32 ◦ C for 6 and 16 h (MFI around 100), which was more than 2 fold increased after 24 h (Fig. 4D). In apoptotic cells (small cells, filled bars) p53 levels were more than 2.5 fold higher than normal cells as early at 6 h. The significant increase remained with 24 h. In contrast, Egr1 levels in apoptotic cells (small size cells) were significantly lower than normal cells at all time points (Fig. 4E).
Fig. 4. Expression levels of p53 and Egr1 were inversely correlated in apoptotic M1-t-p53 cells. M1-t-p53 cells were cultured at 32 ◦ C for 6–36 h, subjected to p53 or Egr-1 immunocytochemistry and examined using flow cytometry. (A) Cell population in annexin V stained cells cultured either at 37 ◦ C (left panels) or at 32 ◦ C (right panels). (B) Cell populations in forward and side scatter were gated regarding cell size and expressed as normal size cells (R1) and small size cells (R2, apoptotic cells). Note: Cells in R1 was 98% and in R2 was 2% after 6 h (left panel), while cells in R1 was 48% and in R2 was 52% after 36 h maintained at 32 ◦ C (right panel), indicating cell shrinkage (apoptosis). (C) Time dependent increases in small size cells following p53 activation (cells kept at 32 ◦ C). (D and E) Summarized mean fluorescence intensity of p53 (D) and Egr1 (E) in cells with normal size (white bars) or small size (black bars). *p < 0.05 and ***p < 0.001 (n = 4).
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4. Discussion
References
Cytotoxic components in OxLDL, 7OH and 7keto, are prominent constituents of atherosclerotic lesions and induce apoptosis (Li et al., 2001; Lizard et al., 1999). In the cell death process, several regulators and their interactions are critically involved including Egr1 and p53. In the present study, we demonstrated that in 7OH or 7keto induced apoptosis, Egr1 and p53 were differentially regulated in the apoptotic process. The level of ROS and Egr1were increased at early time points, while p53 was significantly upregulated at the later stages of the process. Egr-1, a proinflammatory gene, plays an important role in the initiation and progression of atherosclerotic lesions. It participates in signaling in the apoptotic process. In the present study, we found that 7-oxysterols induced early induction of ROS and transient induction of Egr1. Furthermore, the nuclear translocation of Egr1 induced by 7OH or 7keto may play important roles in arterial walls in response to cellular stress similarly seen in hemin mediated stressed model (Hasan and Schafer, 2008). Following cellular oxidative stress and apoptotic cell death, the induced Egr1 may lead to the expression of downstream proteins such as p53 (Park et al., 2008). Our findings of an increase of p53 and simultaneous reduction of Egr1 and increased levels of ROS in apoptotic cells suggest that Egr1 may be an early oxidative responsive protein in the apoptosis process, in which reduction of Egr1 is followed by induction of p53 and apoptosis. p53, a transcription factor, plays a pivotal role in cell proliferation, cell cycle arrest and apoptosis and its expression is associated with necrotic core formation and plaque rupture of atherosclerosis (Yuan et al., 2010). In the present study, a high level of p53 was induced after a longer time exposure to 7OH or 7keto, resulting in formation of condensation and fragmentation of nuclei. The finding indicates that late induced p53 by atheroma relevant oxysterols is of importance in atheroma progression because it decides the fate of the cell survival or apoptosis. The removal of counteraction of survival Egr1 signaling (de Belle et al., 1999) by the 7-oxysterols may accelerate apoptotic cell death. This notion is supported by our results that ruptured human carotid plaques with necrotic core formation have significantly inverse correlation in expression of p53 and Egr1. This finding suggests that differential regulation of Egr1 and p53 may be associated with cell death in the plaque progression. In conclusion, ROS induced by the 7-oxysterols may function as an early initiator of Egr1 expression. The late induced p53 by 7oxysterols contributes to apoptotic cell death and is linked to the reduction of Egr1 levels, which resembles the differential expression of p53 and Egr1 in human atheroma progression.
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Acknowledgements This work was supported by grants from the Swedish Heart Lung Foundation, the Torsten and Ragnar Söderbergs Foundation, the Stroke Foundation, the Olle Engkvist Foundation and the Swedish Gamla Tjänarinnor Foundation, the Linköping University and Linköping University Hospital Research Fund.