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Prevention of 7β-hydroxycholesterol-induced cell death by mangafodipir is mediated through lysosomal and mitochondrial pathways
European Journal of Pharmacology 640 (2010) 124–128
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Cardiovascular Pharmacology
Prevention of 7β-hydroxycholesterol-induced cell death by mangafodipir is mediated through lysosomal and mitochondrial pathways Amit Laskar, Sayem Miah, Rolf G.G. Andersson, Wei Li ⁎ Division of Drug Research/Pharmacology, Department of Medical and Health Sciences, Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden
a r t i c l e
i n f o
Article history: Received 12 October 2009 Received in revised form 5 April 2010 Accepted 24 April 2010 Available online 7 May 2010 Keywords: Atherosclerosis Apoptosis Mangafodipir Oxidized lipid Oxidative stress
a b s t r a c t Mangafodipir, a MRI contrast agent, has been used as a viability marker in patients with myocardial infarction and showed vascular relaxation effect. It confers myocardial protection against oxidative stress. However mechanisms underlying such protection have not yet been investigated. In this investigation we first studied whether mangafodipir inhibits apoptosis induced by 7β-hydroxycholesterol (7βOH), a cytotoxic cholesterol oxidation product found in atherosclerotic lesions in humans and in heart of ethanol-fed rats. We then focused on whether mangafodipir influences the production of reactive oxygen species, lysosomal and mitochondrial membrane permeabilities in the cell model. Our results revealed that pre-treatment with mangafodipir (400 µM) protected against cellular reactive oxygen species production, apoptosis, and permeabilization of lysosomal and mitochondrial membranes induced by 7βOH. In conclusion, a novel effect of mangafodipir on 7βOH-induced apoptosis is via reduction of cellular reactive oxygen species and stabilization of lysosomal and mitochondrial membranes. This is the first report to show the additional cytoprotective effect of mangafodipir, which may suggest possible use of the drug. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Atherosclerosis is a chronic inflammatory disease in which accumulation of inflammatory cells contribute to atheroma plaque development and vulnerability (Hansson, 2005). In advanced lesions, rupture of atherosclerotic plaques is the major pathogenic mechanism responsible for cardiovascular and cerebrovascular events, such as myocardial infarction and stroke. Tissue injury induced by myocardial ischemia involves apoptosis, attributing to cardiomyocyte death during acute and chronic myocardial infarctions (Baldi et al., 2002; Veinot et al., 1997). Very recent study demonstrated that macrophages are predominate fractions of apoptotic non-myocytes in myocardial biopsies from patients with ischemic cardiomyopathy (Park et al., 2009), which indicate that macrophage apoptosis not only contributes to atherosclerotic plaque rupture but also plays an important role in cardiac damage after infarction. Imaging techniques have been developed to permit a better visualization of components and severity of atherosclerotic plaques (Lerakis et al., 2008). Mangafodipir has long been used as an MRI contrast agent for liver imaging and its use in cardiac MRI has been proposed (Ni and Dymarkowski, 2006). Recently, it has been suggested that mangafodipir can be used as a viability marker in patients with myocardial infarction (Skjold et al., 2007) and could decrease myocardial damage in ischemic porcine myocardium
⁎ Corresponding author. Tel.: + 46 13 229602. E-mail address: [email protected] (W. Li). 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.04.046
(Brurok et al., 1999; Karlsson et al., 2001). It improved the contractile function and reduced enzyme release in rat heart tissues during reoxygenation (Brurok et al., 1999). However, the cellular mechanisms behind this protective effect are presently unknown. Oxysterols, cholesterol oxidation products, have been demonstrated at increased levels in both human atherosclerotic lesions and heart of ethanol-fed rats (Ohtsuka et al., 2006; Adachi et al., 2001). The increased oxysterols attribute to several diseases including atherosclerosis and Alzheimer's disease (Vejux and Lizard, 2009). Oxysterols, especially 7-oxysterols, induce oxidative and inflammatory processes and subsequent cell death. We and others have previously shown that 7-oxysterols are potent inducers of cell death in several cell models (Yuan et al., 2000; Larsson et al., 2006; Malvitte et al., 2008). In the present study we initially investigated whether mangafodipir could affect 7β-hydroxycholesterol (7βOH) induced cell death. We then studied the mechanisms that might be involved in the cell death model by focusing on mitochondrial and lysosomal pathways, and cellular oxidative stress. 2. Materials and methods 2.1. Cell culture and experimental conditions U-937 cells were maintained in RPMI culture medium (Gibco, Paisley, UK), supplemented with 10% FBS (Gibco), 2 mM L-glutamine, 100 µ/ml penicillin G and 100 µg/ml streptomycin (Gibco). The cells were cultured at 95% air and 5% CO2 in a humidified atmosphere at
A. Laskar et al. / European Journal of Pharmacology 640 (2010) 124–128
37 °C. The cells were grown in 25 cm2 culture flasks and subcultivated once every three days. For experiments, after subdividing, cells were pre-treated with different concentrations of mangafodipir (GE Health Care AS, Oslo, Norway) (15 µM to 800 µM) for 6–8 h and then exposed to (25 µm) 7β-hydroxycholesterol (7βOH, Sigma, St Louis, MO, USA) for another 18 h in the presence or absence of mangafodipir.
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The cells were initially examined by morphological assessment by phase contrast microscopy directly on living cells or Giemsa stained cells. The numbers of apoptotic cells were further assayed by detection of phosphatidylserine exposure using flow cytometry following Annexin V (Roche (Mannheim, Germany)/PI (Sigma, St Louis, MO, USA) staining (Li et al., 2001). Cells scored as early apoptosis when they were positive to Annexin V, while when cells were positive to both Annexin V and PI, they were scored as post-apoptotic or necrotic cell death. In brief, control and treated cells were collected, washed once with PBS, and stained for 10 min on ice with Annexin V/PI and analyzed by flow cytometry.
tions between 100 and 400 μM inhibited 7βOH-induced cell death in a dose dependent manner, while it had no protective effect when the concentrations were less than 100 μM. Furthermore, there was no further protection on 7βOH-induced cell death when the concentrations of mangafodipir were more than 400 μM. Thus, in the following experiments, 400 μM mangafodipir was applied, which was within the dose range of clinical usage (Federle et al., 2000; Schalla et al., 2002) and mangafodipir alone had no cytotoxic effects. As shown in Fig. 1A, compared to control cells, 7βOH induced cell shrinkage and nuclei condensation, was remarkably prevented by mangafodipir pretreatment. The apoptotic and necrotic cell deaths were then further assayed by annexin V/PI staining. As shown in Fig. 1B, 7βOH induced remarkable exposure of phosphatidylserine in the cell membrane as viewed by annexin V staining and some post-apoptotic or necrotic cells were positive to both annexin V and PI, which was inhibited by treatment of mangafodipir. The quantified results of flow cytometry from the experiments showed that at 18 h 7βOH caused about 30% apoptosis and less than 10% necrosis (Fig. 1C and D), which was significantly protected by mangafodipir. Moreover, cell viability remained at more than 90% and was not significantly different in cells treated with equivalent concentrations of ethanol and cholesterol as compared to untreated control cells.
2.3. Lysosomal membrane permeabilization (LMP)
3.2. Mangafodipir prevents 7βOH-induced LMP
The integrity of the lysosomal membrane was assessed using the acridine orange (AO) uptake technique as established previously (Li et al., 2001). In brief, the cells following different treatments were collected, stained with 5 µg/ml AO (15 min, 37 °C), and analyzed with flow cytometry. Percentages of cells with decreased lysosomal AO red fluorescence were gated and considered as increased LMP.
To identify the potential cellular mechanisms of the protective effect of mangafodipir on 7βOH-induced cell injury, we examined the effect of mangafodipir on LMP using AO-uptake method. We found that 7βOH induced a significant reduction of AO red fluorescence indicating rupture of the lysosomes and redistribution of their contents to cytosol (Fig. 2). Mangafodipir pre-treated cells re-stored lysosomal AO red fluorescence suggesting a lysosomal protective effect (Fig. 2). As viewed by fluorescence microscopy, 7βOH induced remarkable decreases in AO red fluorescence in lysosomes while increases in AO green fluorescence in cytosol and nuclei as compared to control cells. Mangafodipir pre-treated cells showed similar AO staining patterns as control cells, which indicated that mangafodipir inhibited LMP and relocation of lysosomal contents induced by 7βOH. Equivalent concentrations of ethanol and cholesterol had no effect on LMP as compared to untreated control cells.
2.2. Apoptosis and necrosis
2.4. Mitochondrial membrane permeabilization (MMP) The mitochondrial potential (Δψm) or MMP was measured using the fluorescent probe JC-1 (Molecular Probes, Eugene, OR, USA) as described previously (Larsson et al., 2006). In brief, following different treatments control and treated cells were collected, incubated with JC-1 (5 µg/ml for 10 min at 37 °C) and analyzed with flow cytometry. The ratio of JC-1 red and green fluorescence intensities was used to calculate mitochondrial ΔΨm or MMP. 2.5. Reactive oxygen species
3.3. Mangafodipir prevents 7βOH-induced MMP
Intracellular reactive oxygen species was assayed by flow cytometry following dihydroethidium (DHE) (Molecular Probes, Eugene, OR, USA) staining. The cultured cells were collected, washed once with PBS, incubated for 15 min at 37 °C with 10 µM DHE and analyzed with flow cytometry. Percentages of cells with increased DHE red fluorescence were gated and considered as increased reactive oxygen species.
To determine whether the MMP was also involved in mangafodipir-mediated protection against 7βOH, we investigated the effect of mangafodipir on mitochondrial membrane permeability by using JC-1 fluorescent probe. As shown in Fig. 3, exposure to 7βOH for 18 h resulted in decreases in JC-1 induced orange/red fluorescence and increases in JC-1 green fluorescence indicating increased MMP in 7βOH induced apoptosis. Whereas mangafodipir pre-treatment restored mitochondrial potential up to the same level as seen in control cells, indicating mangafodipir pre-treatment prevents the MMP induced by 7βOH-treatment.
2.6. Statistics For statistical analysis, one-way ANOVA followed by Bonferroni post test was used. Results are given as means ± S.E.M. P ≤ 0.05 was considered statistically significant. 3. Results 3.1. Mangafodipir inhibits 7βOH induced cell death A range of concentrations of mangafodipir was tested (15 to 800 μM), in which the drug was either simultaneously added to the cells together with 7βOH for 18 h or pre-incubated for 6 to 8 h and then exposed to 7βOH for another 18 h. Mangafodipir at concentra-
3.4. Mangafodipir decreases 7βOH-induced reactive oxygen species production Increased intracellular reactive oxygen species production has been suggested to play an important role in oxysterol-induced apoptosis. To determine the effect of the mangafodipir on the generation of reactive oxygen species induced by 7βOH the fluorescent probe DHE was used. As shown in Fig. 4, incubation of cells with 7βOH resulted in significant increase in cellular reactive oxygen species, which was significantly reduced by mangafodipir treatment. Equivalent concentrations of ethanol and cholesterol had
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A. Laskar et al. / European Journal of Pharmacology 640 (2010) 124–128
Fig. 1. Protection against 7βOH induced cell death by mangafodipir. U937 cells were pre-treated or not with 400 µM of mangafodipir for 8 h and then exposed to 7βOH (28 µM) for another 18 h in the presence of mangafodipir. A. Overview of morphological changes on Giemsa stained cells. B. Representative photographs of AVPI stained cells viewed with light (left panel) or fluorescence (right panel) microscopy. C. Summarized flow cytometric results. Data are means ± S.E.M. of five experiments. Upper panel: ***P b 0.001 vs. control and 400 µM mangafodipir pre-treated cells (M + 7βOH); Lower panel: ***P b 0.001 vs. control and **P b 0.01 vs. M + 7βOH treated cells.
no effect on reactive oxygen species production as compared to untreated control cells. 4. Discussion Apoptotic cell death, including cardiomyocytes and numbers of macrophages, play important roles in myocardial ischemia (Veinot et al., 1997), reperfusion injury (Kang et al., 2000), and heart failure
(Park et al., 2009). Mangafodipir, an imaging contrast agent, has used as an effective MRI marker in acute myocardial infarction in animal model (Skjold et al., 2007). However, despite the experimental results showing that mangafodipir could decrease ischemia and reperfusion induced myocardial damage (Brurok et al., 1999), the cellular mechanisms underlying this protection, remain poorly understood. In the present study, we for the first time demonstrate that mangafodipir significantly protect U937 cells against 7βOH induced
A. Laskar et al. / European Journal of Pharmacology 640 (2010) 124–128
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Fig. 2. Protective effect of mangafodipir on 7βOH induced LMP. Cells were treated the same as described in Fig. 1, stained with AO and analyzed with flow cytometry or fluorescence microscopy. A. Flow cytometry analysis data, means ± S.E.M. of seven experiments. **P b 0.01 vs. controls and *P b 0.05 vs. M + 7βOH treated cells. B. Representative photographs of AO stained cells. Note: control cells showed typical lysosomal red AO granular fluorescence and low levels of cytosolic and nuclear AO green fluorescence indicating cells with intact lysosomal membranes. 7βOH-treatment caused remarkable decreases in AO lysosomal red fluorescence and increases in cytosol and nuclear AO green fluorescence indicating cells with ruptured lysosomal membranes. Most cells with 400 µM mangafodipir pre-treatment showed similar AO staining patterns as seen in controls.
cell death through decreasing the production of cellular reactive oxygen species, lysosomal membrane permeabilization (LMP), and mitochondrial membrane permeabilization (MMP). Increased cellular oxidative stress (increased reactive oxygen species and decreased antioxidants) is involved in apoptosis and atherosclerosis (Bonomini et al., 2008). We previously showed that oxidative stress was involved in 7-oxysterols mediated cell death with increase of cellular reactive oxygen species and decrease of GSH (Larsson et al., 2006). In this study, we found that mangafodipir at
400 µM significantly decreased 7βOH induced reactive oxygen species production. We consider that the SOD mimetic activity of the mangafodipir is one of reasons for the decline in reactive oxygen species production in the cell model (Alexandre et al., 2006). In a previous study it has been shown that mangafodipir can prevent reactive oxygen species-induced mitochondrial damages and the subsequent cytochrome c release from acetaminophen-induced liver cell death, attributed to its superoxide-dismutase, catalase and glutathione reductase activities (Bedda et al., 2003).
Fig. 3. Protective effect of mangafodipir on 7βOH induced MMP. Cells were treated the same as described in Fig. 1, stained with JC-1 and analyzed by flow cytometry or fluorescence microscopy. Data are summarized flow cytometry results and expressed as means ± S.E.M. of six experiments. *P b 0.05 vs. control and M + 7βOH treated cells.
Fig. 4. Protective effect of mangafodipir on 7βOH induced reactive oxygen species production. Cells were treated the same as described in Fig. 1, stained with DHE and analyzed with flow cytometry. Data are means ± S.E.M. of seven experiments. *P b 0.05 vs. M + 7βOH treated cells and *** P b 0.001 vs. control cells.
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The role of LMP and related cathepsins as apoptotic initiators has recently been the subject of interest in a number of study models and is now regarded as an important apoptotic pathway (Kirkegaard and Jäättelä, 2009; Boya and Kroemer, 2008). Here for the first time it is shown that mangafodipir protects the cell death via protection of LMP induced by 7βOH. The stabilization of lysosomal membranes prevents the release of lysosomal cathepsins into the cytosol or extracellular spaces, by which mangafodipir may improve mitochondrial function and regulate caspase activity. MMP and release of cytochrome c are involved in apoptosis and have been considered as promising therapeutic targets. Our present results clearly showed that mangafodipir significantly prevented MMP caused by 7βOH, which may prevent mitochondrial collapse and the consequent cell death. Our findings support the earlier in vivo results regarding protection by mangafodipir on cardiac dysfunction in reperfusion (Skjold et al., 2007) and liver injury (Bedda et al., 2003) through prevention of mitochondrial cytochrome c release and caspase-3 activation in mice (Bedda et al., 2003). One limitation of our study is that the protective effect of the drug has been studied only on a monocytic cell line U937 cells. It is warranted that further study to evaluate this protective action of the mangafodipir in different types of cells, like endothelial cells, macrophages and cardiomyocytes, will provide further information about the newer role of this drug. However, earlier studies have shown that mangafodipir protects leukocytes from apoptosis induced by H2O2 or chemotherapeutic agents like paclitaxel and oxaliplatin (Alexandre et al., 2006) and prevents death of hepatoma cell line HuH7 cells induced by H2O2 and superoxide anion (Bedda et al., 2003). In conclusion, mangafodipir confers protection against 7βOH induced apoptosis by reducing cellular reactive oxygen species, lysosomal and mitochondrial damages. The results suggest that in addition to its use in MRI, mangafodipir may have therapeutical potential to reduce cell death. Acknowledgments This work was supported by the Swedish Gamla Tjänarinnor Foundation, the Stroke Foundation, the Swedish Heart–Lung Foundation, Medical Research Council of Southeast Sweden, and the Cardiovascular Inflammatory Research Center at Linköping University. The authors would also like to thank Dr. Ximing Yuan for his critical reading of the manuscript. References Adachi, J., Kudo, R., Ueno, Y., Hunter, R., Rajendram, R., Want, E., Preedy, V.R., 2001. Heart 7-hydroperoxycholesterol and oxysterols are elevated in chronically ethanol-fed rats. J. Nutr. 131, 2916–2920. Alexandre, J., Nicco, C., Chereau, C., Laurent, A., Weill, B., Goldwasser, F., Batteux, F., 2006. Improvement of the therapeutic index of anticancer drugs by the superoxide dismutase mimic mangafodipir. J. Natl. Cancer Inst. 98, 236–244.
Baldi, A., Abbate, A., Bussani, R., Patti, G., Melfi, R., Angelini, A., Dobrina, A., Rossiello, R., Silvestri, F., Baldi, F., Di Sciascio, G., 2002. Apoptosis and post-infarction left ventricular remodeling. J. Mol. Cell. Cardiol. 34, 165–174. Bedda, S., Laurent, A., Conti, F., Chereau, C., Tran, A., Nhieu, T.V.J., Jaffray, P., Soubrane, O., Goulvestre, C., Calmus, Y., Weill, B., Batteux, F., 2003. Mangafodipir prevents liver injury induced by acetaminophen in the mouse. J. Hepatol. 39, 765–772. Bonomini, F., Tengattini, S., Fabiano, A., Bianchi, R., Rezzani, R., 2008. Atherosclerosis and oxidative stress. Histol. Histopathol. 23, 381–390. Boya, P., Kroemer, G., 2008. Lysosomal membrane permeabilization in cell death. Oncogene 27, 6434–6451. Brurok, H., Larsen, A.J.H., Hansson, G., Skarra, S., Berg, K., Karlsson, J.O., Laursen, I., Jynge, P., 1999. Manganese dipyridoxyl diphosphate: MRI contrast agent with antioxidative and cardioprotective properties? Biochem. Biophys. Res. Commun. 254, 768–772. Federle, M.P., Chezmar, J.L., Rubin, D.L., Weinreb, J.C., Freeny, P.C., Semelka, R.C., Brown, J.J., Borello, J.A., Lee, J.K., Mattrey, R., Dachman, A.H., Saini, S., Harmon, B., Fenstermacher, M., Pelsang, R.E., Harms, S.E., Mitchell, D.G., Halford, H.H., Anderson, M.W., Johnson, C.D., Francis, I.R., Bova, J.G., Kenney, P.J., Klippenstein, D.L., Foster, G.S., Turner, D.A., 2000. Safety and efficacy of mangafodipir trisodium (MnDPDP) injection for hepatic MRI in adults: results of the U.S. multicenter phase III clinical trials (safety). J. Magn. Reson. Imaging 12, 186–197. Hansson, G.K., 2005. Inflammation, atherosclerosis, and coronary artery disease. N. Engl. J. Med. 352, 1685–1695. Kang, P.M., Haunstetter, A., Aoki, H., Usheva, A., Izumo, S., 2000. Morphological and molecular characterization of adult cardiomyocyte apoptosis during hypoxia and reoxygenation. Circ. Res. 87, 118–125. Karlsson, J.O.G., Brurok, H., Eriksen, M., Towart, R., Toft, K.G., Moen, O., Engebresten, B., Jynge, P., Refsum, H., 2001. Cardio protective effects of the MR contrast agent MnDPDP and its metabolite MnPLED upon reperfusion of the ischemic porcine myocardium. Acta Radiol. 42, 540–547. Kirkegaard, T., Jäättelä, M., 2009. Lysosomal involvement in cell death and cancer. Biochim. Biophys. Acta 1793, 746–754. Larsson, D.A., Baird, S., Nyhalah, J.D., Yuan, X.M., Li, W., 2006. Oxysterol mixtures, in atheroma-relevant proportions, display synergistic and proapoptotic effects. Free Radic. Biol. Med. 41, 902–910. Lerakis, S., Synetos, A., Toutouzas, K., Vavuranakis, M., Tsiamis, E., Stefanadis, C., 2008. Imaging of the vulnerable plaque: noninvasive and invasive techniques. Am. J. Med. Sci. 336, 342–348. Li, W., Dalen, H., Eaton, J.W., Yuan, X.M., 2001. Apoptotic death of inflammatory cells in human atheroma. Arterioscler. Thromb. Vasc. Biol. 21, 1124–1130. Malvitte, L., Montange, T., Vejux, A., Joffre, C., Bron, A., Creuzot-Garcher, C., Lizard, G., 2008. Activation of a caspase-3-independent mode of cell death associated with lysosomal destabilization in cultured human retinal pigment epithelial cells (ARPE-19) exposed to 7beta-hydroxycholesterol. Curr. Eye Res. 33, 769–781. Ni, Y., Dymarkowski, S., 2006. Contrast agents for cardiac MRI. In: Bogaert, J., ymarkowski, S., Taylor, A.M. (Eds.), Clinical Cardiac MRI. Springer, Berlin, pp. 33–50. Ohtsuka, M., Miyashita, Y., Shirai, K., 2006. Lipids deposited in human atheromatous lesions induce apoptosis of human vascular smooth muscle cells. J. Atheroscler. Thromb. 13, 256–262. Park, M., Shen, Y.T., Gaussin, V., Heyndrickx, G.R., Bartunek, J., Resuello, R.G., Natividad, F.F., Kitsis, R.N., Vatner, D.E., Vatner, S.F., 2009. Apoptosis predominates in nonmyocytes in heart failure. Am. J. Physiol. Heart Circ. Physiol. 297, H785–H791. Schalla, S., Higgins, C.B., Saeed, M., 2002. Contrast agents for cardiovascular magnetic resonance imaging. Current status and future directions. Drugs R. D. 3, 285–302. Skjold, A., Amundsen, B.H., Wiseth, R., Stoylen, A., Haraldseth, O., Larsson, H.B., Jynge, P., 2007. Manganese dipyridoxyl-diphosphate (MnDPDP) as a viability marker in patients with myocardial infarction. J. Magn. Reson. Imaging 26, 720–727. Veinot, J.P., Gattinger, D.A., Fliss, H., 1997. Early apoptosis in human myocardial infarcts. Hum. Pathol. 28, 485–492. Vejux, A., Lizard, G., 2009. Cytotoxic effects of oxysterols associated with human diseases: induction of cell death (apoptosis and/or oncosis), oxidative and inflammatory activities, and phospholipidosis. Mol. Aspects Med. 30, 153–170. Yuan, X.M., Li, W., Brunk, U.T., Dalen, H., Chang, Y.H., Sevanian, A., 2000. Lysosomal destabilization during macrophage damage induced by cholesterol oxidation products. Free Radic. Biol. Med. 28, 208–218.
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Cardiovascular Pharmacology
Prevention of 7β-hydroxycholesterol-induced cell death by mangafodipir is mediated through lysosomal and mitochondrial pathways Amit Laskar, Sayem Miah, Rolf G.G. Andersson, Wei Li ⁎ Division of Drug Research/Pharmacology, Department of Medical and Health Sciences, Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden
a r t i c l e
i n f o
Article history: Received 12 October 2009 Received in revised form 5 April 2010 Accepted 24 April 2010 Available online 7 May 2010 Keywords: Atherosclerosis Apoptosis Mangafodipir Oxidized lipid Oxidative stress
a b s t r a c t Mangafodipir, a MRI contrast agent, has been used as a viability marker in patients with myocardial infarction and showed vascular relaxation effect. It confers myocardial protection against oxidative stress. However mechanisms underlying such protection have not yet been investigated. In this investigation we first studied whether mangafodipir inhibits apoptosis induced by 7β-hydroxycholesterol (7βOH), a cytotoxic cholesterol oxidation product found in atherosclerotic lesions in humans and in heart of ethanol-fed rats. We then focused on whether mangafodipir influences the production of reactive oxygen species, lysosomal and mitochondrial membrane permeabilities in the cell model. Our results revealed that pre-treatment with mangafodipir (400 µM) protected against cellular reactive oxygen species production, apoptosis, and permeabilization of lysosomal and mitochondrial membranes induced by 7βOH. In conclusion, a novel effect of mangafodipir on 7βOH-induced apoptosis is via reduction of cellular reactive oxygen species and stabilization of lysosomal and mitochondrial membranes. This is the first report to show the additional cytoprotective effect of mangafodipir, which may suggest possible use of the drug. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Atherosclerosis is a chronic inflammatory disease in which accumulation of inflammatory cells contribute to atheroma plaque development and vulnerability (Hansson, 2005). In advanced lesions, rupture of atherosclerotic plaques is the major pathogenic mechanism responsible for cardiovascular and cerebrovascular events, such as myocardial infarction and stroke. Tissue injury induced by myocardial ischemia involves apoptosis, attributing to cardiomyocyte death during acute and chronic myocardial infarctions (Baldi et al., 2002; Veinot et al., 1997). Very recent study demonstrated that macrophages are predominate fractions of apoptotic non-myocytes in myocardial biopsies from patients with ischemic cardiomyopathy (Park et al., 2009), which indicate that macrophage apoptosis not only contributes to atherosclerotic plaque rupture but also plays an important role in cardiac damage after infarction. Imaging techniques have been developed to permit a better visualization of components and severity of atherosclerotic plaques (Lerakis et al., 2008). Mangafodipir has long been used as an MRI contrast agent for liver imaging and its use in cardiac MRI has been proposed (Ni and Dymarkowski, 2006). Recently, it has been suggested that mangafodipir can be used as a viability marker in patients with myocardial infarction (Skjold et al., 2007) and could decrease myocardial damage in ischemic porcine myocardium
⁎ Corresponding author. Tel.: + 46 13 229602. E-mail address: [email protected] (W. Li). 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.04.046
(Brurok et al., 1999; Karlsson et al., 2001). It improved the contractile function and reduced enzyme release in rat heart tissues during reoxygenation (Brurok et al., 1999). However, the cellular mechanisms behind this protective effect are presently unknown. Oxysterols, cholesterol oxidation products, have been demonstrated at increased levels in both human atherosclerotic lesions and heart of ethanol-fed rats (Ohtsuka et al., 2006; Adachi et al., 2001). The increased oxysterols attribute to several diseases including atherosclerosis and Alzheimer's disease (Vejux and Lizard, 2009). Oxysterols, especially 7-oxysterols, induce oxidative and inflammatory processes and subsequent cell death. We and others have previously shown that 7-oxysterols are potent inducers of cell death in several cell models (Yuan et al., 2000; Larsson et al., 2006; Malvitte et al., 2008). In the present study we initially investigated whether mangafodipir could affect 7β-hydroxycholesterol (7βOH) induced cell death. We then studied the mechanisms that might be involved in the cell death model by focusing on mitochondrial and lysosomal pathways, and cellular oxidative stress. 2. Materials and methods 2.1. Cell culture and experimental conditions U-937 cells were maintained in RPMI culture medium (Gibco, Paisley, UK), supplemented with 10% FBS (Gibco), 2 mM L-glutamine, 100 µ/ml penicillin G and 100 µg/ml streptomycin (Gibco). The cells were cultured at 95% air and 5% CO2 in a humidified atmosphere at
A. Laskar et al. / European Journal of Pharmacology 640 (2010) 124–128
37 °C. The cells were grown in 25 cm2 culture flasks and subcultivated once every three days. For experiments, after subdividing, cells were pre-treated with different concentrations of mangafodipir (GE Health Care AS, Oslo, Norway) (15 µM to 800 µM) for 6–8 h and then exposed to (25 µm) 7β-hydroxycholesterol (7βOH, Sigma, St Louis, MO, USA) for another 18 h in the presence or absence of mangafodipir.
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The cells were initially examined by morphological assessment by phase contrast microscopy directly on living cells or Giemsa stained cells. The numbers of apoptotic cells were further assayed by detection of phosphatidylserine exposure using flow cytometry following Annexin V (Roche (Mannheim, Germany)/PI (Sigma, St Louis, MO, USA) staining (Li et al., 2001). Cells scored as early apoptosis when they were positive to Annexin V, while when cells were positive to both Annexin V and PI, they were scored as post-apoptotic or necrotic cell death. In brief, control and treated cells were collected, washed once with PBS, and stained for 10 min on ice with Annexin V/PI and analyzed by flow cytometry.
tions between 100 and 400 μM inhibited 7βOH-induced cell death in a dose dependent manner, while it had no protective effect when the concentrations were less than 100 μM. Furthermore, there was no further protection on 7βOH-induced cell death when the concentrations of mangafodipir were more than 400 μM. Thus, in the following experiments, 400 μM mangafodipir was applied, which was within the dose range of clinical usage (Federle et al., 2000; Schalla et al., 2002) and mangafodipir alone had no cytotoxic effects. As shown in Fig. 1A, compared to control cells, 7βOH induced cell shrinkage and nuclei condensation, was remarkably prevented by mangafodipir pretreatment. The apoptotic and necrotic cell deaths were then further assayed by annexin V/PI staining. As shown in Fig. 1B, 7βOH induced remarkable exposure of phosphatidylserine in the cell membrane as viewed by annexin V staining and some post-apoptotic or necrotic cells were positive to both annexin V and PI, which was inhibited by treatment of mangafodipir. The quantified results of flow cytometry from the experiments showed that at 18 h 7βOH caused about 30% apoptosis and less than 10% necrosis (Fig. 1C and D), which was significantly protected by mangafodipir. Moreover, cell viability remained at more than 90% and was not significantly different in cells treated with equivalent concentrations of ethanol and cholesterol as compared to untreated control cells.
2.3. Lysosomal membrane permeabilization (LMP)
3.2. Mangafodipir prevents 7βOH-induced LMP
The integrity of the lysosomal membrane was assessed using the acridine orange (AO) uptake technique as established previously (Li et al., 2001). In brief, the cells following different treatments were collected, stained with 5 µg/ml AO (15 min, 37 °C), and analyzed with flow cytometry. Percentages of cells with decreased lysosomal AO red fluorescence were gated and considered as increased LMP.
To identify the potential cellular mechanisms of the protective effect of mangafodipir on 7βOH-induced cell injury, we examined the effect of mangafodipir on LMP using AO-uptake method. We found that 7βOH induced a significant reduction of AO red fluorescence indicating rupture of the lysosomes and redistribution of their contents to cytosol (Fig. 2). Mangafodipir pre-treated cells re-stored lysosomal AO red fluorescence suggesting a lysosomal protective effect (Fig. 2). As viewed by fluorescence microscopy, 7βOH induced remarkable decreases in AO red fluorescence in lysosomes while increases in AO green fluorescence in cytosol and nuclei as compared to control cells. Mangafodipir pre-treated cells showed similar AO staining patterns as control cells, which indicated that mangafodipir inhibited LMP and relocation of lysosomal contents induced by 7βOH. Equivalent concentrations of ethanol and cholesterol had no effect on LMP as compared to untreated control cells.
2.2. Apoptosis and necrosis
2.4. Mitochondrial membrane permeabilization (MMP) The mitochondrial potential (Δψm) or MMP was measured using the fluorescent probe JC-1 (Molecular Probes, Eugene, OR, USA) as described previously (Larsson et al., 2006). In brief, following different treatments control and treated cells were collected, incubated with JC-1 (5 µg/ml for 10 min at 37 °C) and analyzed with flow cytometry. The ratio of JC-1 red and green fluorescence intensities was used to calculate mitochondrial ΔΨm or MMP. 2.5. Reactive oxygen species
3.3. Mangafodipir prevents 7βOH-induced MMP
Intracellular reactive oxygen species was assayed by flow cytometry following dihydroethidium (DHE) (Molecular Probes, Eugene, OR, USA) staining. The cultured cells were collected, washed once with PBS, incubated for 15 min at 37 °C with 10 µM DHE and analyzed with flow cytometry. Percentages of cells with increased DHE red fluorescence were gated and considered as increased reactive oxygen species.
To determine whether the MMP was also involved in mangafodipir-mediated protection against 7βOH, we investigated the effect of mangafodipir on mitochondrial membrane permeability by using JC-1 fluorescent probe. As shown in Fig. 3, exposure to 7βOH for 18 h resulted in decreases in JC-1 induced orange/red fluorescence and increases in JC-1 green fluorescence indicating increased MMP in 7βOH induced apoptosis. Whereas mangafodipir pre-treatment restored mitochondrial potential up to the same level as seen in control cells, indicating mangafodipir pre-treatment prevents the MMP induced by 7βOH-treatment.
2.6. Statistics For statistical analysis, one-way ANOVA followed by Bonferroni post test was used. Results are given as means ± S.E.M. P ≤ 0.05 was considered statistically significant. 3. Results 3.1. Mangafodipir inhibits 7βOH induced cell death A range of concentrations of mangafodipir was tested (15 to 800 μM), in which the drug was either simultaneously added to the cells together with 7βOH for 18 h or pre-incubated for 6 to 8 h and then exposed to 7βOH for another 18 h. Mangafodipir at concentra-
3.4. Mangafodipir decreases 7βOH-induced reactive oxygen species production Increased intracellular reactive oxygen species production has been suggested to play an important role in oxysterol-induced apoptosis. To determine the effect of the mangafodipir on the generation of reactive oxygen species induced by 7βOH the fluorescent probe DHE was used. As shown in Fig. 4, incubation of cells with 7βOH resulted in significant increase in cellular reactive oxygen species, which was significantly reduced by mangafodipir treatment. Equivalent concentrations of ethanol and cholesterol had
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Fig. 1. Protection against 7βOH induced cell death by mangafodipir. U937 cells were pre-treated or not with 400 µM of mangafodipir for 8 h and then exposed to 7βOH (28 µM) for another 18 h in the presence of mangafodipir. A. Overview of morphological changes on Giemsa stained cells. B. Representative photographs of AVPI stained cells viewed with light (left panel) or fluorescence (right panel) microscopy. C. Summarized flow cytometric results. Data are means ± S.E.M. of five experiments. Upper panel: ***P b 0.001 vs. control and 400 µM mangafodipir pre-treated cells (M + 7βOH); Lower panel: ***P b 0.001 vs. control and **P b 0.01 vs. M + 7βOH treated cells.
no effect on reactive oxygen species production as compared to untreated control cells. 4. Discussion Apoptotic cell death, including cardiomyocytes and numbers of macrophages, play important roles in myocardial ischemia (Veinot et al., 1997), reperfusion injury (Kang et al., 2000), and heart failure
(Park et al., 2009). Mangafodipir, an imaging contrast agent, has used as an effective MRI marker in acute myocardial infarction in animal model (Skjold et al., 2007). However, despite the experimental results showing that mangafodipir could decrease ischemia and reperfusion induced myocardial damage (Brurok et al., 1999), the cellular mechanisms underlying this protection, remain poorly understood. In the present study, we for the first time demonstrate that mangafodipir significantly protect U937 cells against 7βOH induced
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Fig. 2. Protective effect of mangafodipir on 7βOH induced LMP. Cells were treated the same as described in Fig. 1, stained with AO and analyzed with flow cytometry or fluorescence microscopy. A. Flow cytometry analysis data, means ± S.E.M. of seven experiments. **P b 0.01 vs. controls and *P b 0.05 vs. M + 7βOH treated cells. B. Representative photographs of AO stained cells. Note: control cells showed typical lysosomal red AO granular fluorescence and low levels of cytosolic and nuclear AO green fluorescence indicating cells with intact lysosomal membranes. 7βOH-treatment caused remarkable decreases in AO lysosomal red fluorescence and increases in cytosol and nuclear AO green fluorescence indicating cells with ruptured lysosomal membranes. Most cells with 400 µM mangafodipir pre-treatment showed similar AO staining patterns as seen in controls.
cell death through decreasing the production of cellular reactive oxygen species, lysosomal membrane permeabilization (LMP), and mitochondrial membrane permeabilization (MMP). Increased cellular oxidative stress (increased reactive oxygen species and decreased antioxidants) is involved in apoptosis and atherosclerosis (Bonomini et al., 2008). We previously showed that oxidative stress was involved in 7-oxysterols mediated cell death with increase of cellular reactive oxygen species and decrease of GSH (Larsson et al., 2006). In this study, we found that mangafodipir at
400 µM significantly decreased 7βOH induced reactive oxygen species production. We consider that the SOD mimetic activity of the mangafodipir is one of reasons for the decline in reactive oxygen species production in the cell model (Alexandre et al., 2006). In a previous study it has been shown that mangafodipir can prevent reactive oxygen species-induced mitochondrial damages and the subsequent cytochrome c release from acetaminophen-induced liver cell death, attributed to its superoxide-dismutase, catalase and glutathione reductase activities (Bedda et al., 2003).
Fig. 3. Protective effect of mangafodipir on 7βOH induced MMP. Cells were treated the same as described in Fig. 1, stained with JC-1 and analyzed by flow cytometry or fluorescence microscopy. Data are summarized flow cytometry results and expressed as means ± S.E.M. of six experiments. *P b 0.05 vs. control and M + 7βOH treated cells.
Fig. 4. Protective effect of mangafodipir on 7βOH induced reactive oxygen species production. Cells were treated the same as described in Fig. 1, stained with DHE and analyzed with flow cytometry. Data are means ± S.E.M. of seven experiments. *P b 0.05 vs. M + 7βOH treated cells and *** P b 0.001 vs. control cells.
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The role of LMP and related cathepsins as apoptotic initiators has recently been the subject of interest in a number of study models and is now regarded as an important apoptotic pathway (Kirkegaard and Jäättelä, 2009; Boya and Kroemer, 2008). Here for the first time it is shown that mangafodipir protects the cell death via protection of LMP induced by 7βOH. The stabilization of lysosomal membranes prevents the release of lysosomal cathepsins into the cytosol or extracellular spaces, by which mangafodipir may improve mitochondrial function and regulate caspase activity. MMP and release of cytochrome c are involved in apoptosis and have been considered as promising therapeutic targets. Our present results clearly showed that mangafodipir significantly prevented MMP caused by 7βOH, which may prevent mitochondrial collapse and the consequent cell death. Our findings support the earlier in vivo results regarding protection by mangafodipir on cardiac dysfunction in reperfusion (Skjold et al., 2007) and liver injury (Bedda et al., 2003) through prevention of mitochondrial cytochrome c release and caspase-3 activation in mice (Bedda et al., 2003). One limitation of our study is that the protective effect of the drug has been studied only on a monocytic cell line U937 cells. It is warranted that further study to evaluate this protective action of the mangafodipir in different types of cells, like endothelial cells, macrophages and cardiomyocytes, will provide further information about the newer role of this drug. However, earlier studies have shown that mangafodipir protects leukocytes from apoptosis induced by H2O2 or chemotherapeutic agents like paclitaxel and oxaliplatin (Alexandre et al., 2006) and prevents death of hepatoma cell line HuH7 cells induced by H2O2 and superoxide anion (Bedda et al., 2003). In conclusion, mangafodipir confers protection against 7βOH induced apoptosis by reducing cellular reactive oxygen species, lysosomal and mitochondrial damages. The results suggest that in addition to its use in MRI, mangafodipir may have therapeutical potential to reduce cell death. Acknowledgments This work was supported by the Swedish Gamla Tjänarinnor Foundation, the Stroke Foundation, the Swedish Heart–Lung Foundation, Medical Research Council of Southeast Sweden, and the Cardiovascular Inflammatory Research Center at Linköping University. The authors would also like to thank Dr. Ximing Yuan for his critical reading of the manuscript. References Adachi, J., Kudo, R., Ueno, Y., Hunter, R., Rajendram, R., Want, E., Preedy, V.R., 2001. Heart 7-hydroperoxycholesterol and oxysterols are elevated in chronically ethanol-fed rats. J. Nutr. 131, 2916–2920. Alexandre, J., Nicco, C., Chereau, C., Laurent, A., Weill, B., Goldwasser, F., Batteux, F., 2006. Improvement of the therapeutic index of anticancer drugs by the superoxide dismutase mimic mangafodipir. J. Natl. Cancer Inst. 98, 236–244.
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