Genistein Reverses Changes of the Proteome Induced by Oxidized-LDL in EA‚hy 926 Human Endothelial Cells Dagmar Fuchs,† Petra Erhard,† Rufus Turner,‡ Gerald Rimbach,‡,§ Hannelore Daniel,† and Uwe Wenzel*,† Department of Food and Nutrition, Molecular Nutrition Unit, Technical University of Munich, Hochfeldweg 2, D-85350 Freising, Germany, Hugh Sinclair Unit of Human Nutrition, School of Food Biosciences, University of Reading, Reading, United Kingdom Received October 4, 2004
Endothelial cells are primary targets for pro-atherosclerotic stressors such as oxidized LDL (ox-LDL). The isoflavone genistein, on the other hand, is suggested to prevent a variety of processes underlying atherosclerosis and cardiovascular diseases. By analyzing the proteome of EA‚hy 926 endothelial cells, here we show, that genistein reverses the ox-LDL-induced changes of the steady-state levels of several proteins involved in atherosclerosis. These alterations caused by genistein are functionally linked to the inhibition of ox-LDL induced apoptosis. Keywords: proteomics • atherosclerosis • ox-LDL • apoptosis
Introduction Epidemiological studies suggest that a diet rich in soy-based products contributes to the prevention of coronary heart disease,1,2 which causes over 40% of all deaths in industrialized countries.3 Although there is no final proof yet that the antiatherosclerotic effects of soy consumption are mediated by the isoflavone fraction,4 several studies have shown favorable effects of isoflavones on mechanisms underlying disease initiation and progression.5 Isoflavone effects encompass the reduction of plasma levels of pro-atherosclerotic oxidized low-density lipoproteins (ox-LDL),6 the prevention of LDL oxidation and lipid peroxidation,6-10 an improvement in vascular reactivity,11 the inhibition of secretion of pro-inflammatory cytokines, altered expression of cell adhesion proteins,12,13 reduced levels of reactive nitrogen species,14 and reduced platelet aggregation rates.15 Soy preparations without isoflavones in most cases failed to exert these effects in vivo.16 According to the ‘response to injury’ hypothesis the initial step in the pathogenesis is endothelial damage and dysfunction which results in increased platelet and leukocyte adhesion, thrombosis, smooth muscle cell proliferation, vasospasm, lipid accumulation and ultimately in atheromas.17,18 To assess whether isoflavones such as genistein besides their ability to lower the circulating levels of ox-LDL are also able to affect processes in endothelial cells as a prime target of the endothelial stressor, we analyzed the changes of the steady state levels of proteins from an endothelial cell culture exposed either to ox-LDL alone or in * To whom correspondence should be addressed. Phone: +49 8161/713997, Fax: +49 8161/71-3999. E-mail:
[email protected]. † Department of Food and Nutrition, Molecular Nutrition Unit, Technical University of Munich. ‡ Hugh Sinclair Unit of Human Nutrition, School of Food Biosciences, University of Reading. § Current address: Insitute of Human Nutrition and Food Science, Christian Albrechts University, D-24111 Kiel, Germany. 10.1021/pr049820r CCC: $30.25
2005 American Chemical Society
combination with genistein by using a proteomics approach. Methods for the large scale analysis of protein expression profiles are becoming important tools in cardiovascular research. For identification of the molecular targets of genistein action, we used EA‚hy 926 cells, which represent presently a well characterized macrovascular endothelial cell line.19 EA‚hy 926 cells were generated by fusion of human umbilical vein endothelial cells (HUVEC) with the human lung carcinoma cell line A549.20 Although most of the knowledge on endothelial cell functions is derived from in vitro studies with HUVEC, EA‚ hy 926 cells have the advantage of being more homogeneous than different HUVEC isolates which increases reliability.9 Moreover, there is need for laborious isolation procedures in case of HUVEC but not for EA‚hy 926 cells.9
Experimental Procedures Materials. Media and supplements for cell culture were from Invitrogen. Cell culture plates were purchased from Renner. Genistein and the Hoechst dyes 33258 and 33342 were purchased from Sigma. The fluorogenic caspase-substrates acetylaspartyl-glutamyl-valyl-aspartyl-amino-4-methyl-coumarine (AcDEVD-AMC), acetyl-glutamyl-isoleucyl-aspartyl-amino-4-methylcoumarine (Ac-VKMD-AFC) and carboxybenzoxy-isoleucylglutamyl-threonyl-aspartyl-aminofluorocoumarine (Z-IETDAFC) were obtained from Calbiochem. Pharmalyte and immobilized pH-gradient (IPG) strips were from Amersham Biosciences and sequencing grade modified trypsin from Promega. Complete mini protease inhibitor cocktail was purchased from Roche, the protein assay from Bio-Rad and Coomassie brilliant blue G250 from Serva. Quadriperm wells were obtained from Merck. All other materials were from Sigma. Cell Culture. EA‚hy 926 cells were a generous gift from Prof. C. J. Edgell, University of North Carolina at Chapel Hill and Journal of Proteome Research 2005, 4, 369-376
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research articles were used at passage 28. Culturing of the cells was carried out in 75 cm2 flasks with DMEM supplemented with 25 mM HEPES, 10% fetal bovine serum, 12.5 g/mL gentamycin, 1× MEMamino acid-solution and 1× BME-vitamins. Antibiotics added were 400 U/mL penicillin and 400 µg/mL streptomycin. The cultures were maintained in a humidified atmosphere of 5% CO2 at 37 °C. Cells were passaged at preconfluent densities by the use of a solution containing 0.1% trypsin and 0.04 mM EDTA. Preparation of ox-LDL. Blood was collected in vacutainer tubes containing EDTA (1 mg/mL) from the antecubital vein of fasting healthy volunteers. The LDL fraction, corresponding to a density of 1.019-1.063 g/mL, was isolated from plasma by sequential ultracentrifugation in salt solutions, according to Havel et al.,21 using a Beckman T-100 benchtop ultracentrifuge (T-100.3 rotor). The LDL fraction was stored under nitrogen at 4 °C and used within 1 week of isolation. Lipoprotein concentration was expressed in terms of protein content. Protein was measured by the Bradford reaction using the BioRad protein assay. Prior to the experiments, LDL was dialyzed in the dark for 24 h at 4 °C against three changes of buffer (1 l each) containing 0.01 M phosphate-buffered saline, 2.7 mM KCl, and 138 mM NaCl, pH 7.4. Dialyzed LDL (200 µg of protein/mL) was oxidized with 5 µM CuCl2 in phosphatebuffered saline at 37 °C. Oxidation was followed by monitoring the increased formation of conjugated dienes at 234 nm using a Beckman DU 70 spectrophotometer and was stopped by using 6 µM EDTA to chelate all the metal ions. Upon chelation, the solution then does get dialyzed over 24 h using three changes of buffer. To standardize the level of oxidation, LDL was administered to EA‚hy 926 cells when its oxidation reached the midpoint of the propagation phase, at a final concentration of 5 µg/mL. To further assess the extent of oxidation of LDL in terms of the modified surface charge on the apolipoprotein B-100, lipoprotein electrophoresis was performed using a Beckman Paragon electrophoresis system. Apoptosis Assays. Caspase-3-, caspase-6-, and caspase-8like activities were measured as described previously,22 based on the method of Nicholson et al.23 In brief, EA‚hy 926 cells were seeded at a density of 5 × 105 per well onto 6-well plates and allowed to adhere for 24 h. Cells were then exposed for 24 h to the test compounds. Subsequently, cells were trypsinized, cell numbers were determined and then the cells were centrifuged at 2500 × g for 10 min. Cytosolic extracts were prepared by adding 750 µL of a buffer containing 2 mM EDTA, 0.1% CHAPS, 5 mM DTT, 1 mM PMSF, 10 µg/mL pepstatin A, 20 µg/mL leupeptin, 10 µg/mL aprotinin and 10 mM HEPES/KOH, pH 7.4 to each pellet and homogenizing by 10 strokes. The homogenate was centrifuged at 100.000 × g at 4 °C for 30 min and the cytosolic supernatants were incubated with the fluorogenic caspase substrates Ac-DEVD-AMC for caspase-3, AcVKMD-AFC for caspase-6 and Z-IETD-AFC for caspase-8 measurements at final concentrations of 20 µM. Cleavage of the caspase substrates was followed after excitation at 390 nm by determination of emission at 460 nm for the caspase-3 substrate and at 515 nm for the caspase-6 and caspase-8 substrates using a multiwell-plate reader (Fluoroskan Ascent,Thermo Electron). Changes in membrane permeability as an early apoptosis marker were assessed after 3 × 104 EA‚hy 926 cells were grown on glass slides placed into Quadriperm wells and then incubated with the test compounds for 24 h. Cells were stained with 1 µg/mL Hoechst 33342 and rate of accumulation of the dye 370
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in early apoptotic cells24 was detected using an inverted fluorescence microscope (Leica DMIRBE) equipped with an band-pass excitation filter of 340-380 nm and a long-pass emission filter of 425 nm. Nuclear fragmentation as a late marker of apoptosis was determined by staining of DNA with Hoechst 33258. EA‚hy 926 cells (3 × 104) were seeded on glass slides placed into Quadriperm wells and then incubated with the test compounds for 24 h. Thereafter, cells were washed with PBS, allowed to air-dry for 30 min and then fixed with 2% paraformaldehyd prior to staining with 1 µg/mL Hoechst 33258 and visualization under the inverted fluorescence microscope. Sample Preparation for Two-Dimensional Gel Electrophoresis (2D-PAGE). Following 24 h incubation, cells were washed three times with ice cold 350 mM sucrose, containing Complete Mini proteinase inhibitor and then scraped off with a cell scraper. Cells of two flasks were combined in 6 mL of ice cold sucrose solution and subsequently centrifuged for 7 min at 2500 g. The supernatant was discarded and 200 µL of lysis buffer (7M urea, 2M thiourea, 2% CHAPS, 1% DTT, 0,8% Pharmalyte, CompleteMini) were added to the pellet. Homogenization of the cells was achieved by ultra sonification (10 strokes, low amplitude) on ice. Lysed cells were centrifuged for 30 min at 100 000 × g at 4 °C and the supernatant containing solubilized proteins was used immediately or stored at -80 °C. Protein concentration of samples was determined using the Bio-Rad protein assay. 2D-PAGE. 2D-PAGE, isoelectric focusing (IEF) in the first dimension and sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension, was performed as described by Go¨rg et al. with minor modifications.25 Briefly, IEF was performed on 18 cm pH 3-10 IPG strips using an Amersham IPGphor unit. Each strip was rehydrated for 12 h with 340 µL of rehydratation buffer (8 M urea, 0,5% CHAPS, 15 mM DTT, 0.5% IPG buffer). 500 µg of proteinsuspension was then loaded onto the strip by cuploading. IEF was performed under the following conditions: 500 V (10 min, gradient), 4000 V (1.5 h, gradient), 8000 V (25000 Vh, Step-nhold). After IEF, strips were incubated for 15 min in equilibration-buffer 1 (1.5 M TrisHCl, pH 8.8, 6 M urea, 26% glycerol, 2% SDS, 1% DTT) and then for another 15 min in equilibrationbuffer 2 (1.5 M TrisHCl, pH 8.8, 6 M urea, 26% glycerol, 2% SDS, 4% iodoacetamide) before loading onto SDS-PAGE gels. 1 mm-thick 12.5% SDS-polyacrylamide gels were cast according to the method of Laemmli26 and were run using an Amersham Biosciences Ettan-Dalt II System employing the following conditions: 4 mA per gel for 1 h, then 12 mA per gel. Staining of proteins ongels was performed by fixing in 40% ethanol and 10% acetic acid for 5 h. Gels were then stained overnight in Coomassie-solution containing 10% (NH4)2SO4, 2% phosphoric acid, 25% methanol and 0,625% Coomassie brilliant blue G250. Gels were destained in bi-distilled water until the background was completely clear. Analysis of Proteins using the ProteomWeaver Software. Gels stained with Coomassie were scanned using an ImageScanner (Amersham Biosciences) and spots detected by the ProteomWeaver software (Definiens). Background subtraction and volume normalization were made automatically by the software. After spot detection, all gels were matched to each other. Gels from at least three independent runs of cells derived from different treatments were compared to each other. Spots differing significantly (P < 0.05) by at least 2-fold in density were picked for MALDI-TOF MS analysis.
research articles
Effects of Genistein on Proteome of EA‚hy 926
Figure 1. 2D-PAGE of proteins from EA‚hy 926 cells incubated for 24 h with 5 µg/mL ox-LDL. Proteins were separated on a pH 3-10 IPG-strip in the first dimension and on a 12.5% SDS-polyacrylamide gel in the second dimension. The middle section shows a representative Coomassie-stained gel derived from EA‚hy 926 control cells. Around the typical control gel enlarged areas of gels derived from control cells and cells exposed to ox-LDL are shown. Only protein spots significantly affected by ox-LDL-treatment but not altered by genistein are marked by arrows.
Enzymatic Digestion of Protein Spots for Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry (MALDITOF MS). Coomassie-stained spots were picked with a 2 mm or 3 mm “skin-picker”. The destaining of spots occurred with alternating washing procedures in pure 50 mM NH4HCO3 and acetonitrile/pure 50 mM NH4HCO3 1/1. After the blue color was fully removed, a last washing step with pure acetonitrile followed and the spots were dried in a SpeedVac. The dry spots were rehydrated for 1 h at 4 °C with 5 µL of 0,02 µg/µL sequencing grade modified trypsin (Promega, Mannheim, Germany) for 60 min on ice. The trypsin-supernatant was removed and proteins in the soaked gel spots were digested by overnight incubation at 37 °C. 7 µL of 1% trifluoracetic acid (TFA) were added to each spot and peptide fragments were extracted by ultra sonification for 10 min. The supernatants derived from each spot were collected and used for mass spectrometry. MALDI-TOF MS Analysis of Tryptic Peptides. Peptide mass analysis was performed using the Autoflex mass spectrometer of Bruker Daltonics. The dried protein sample was resuspended in 7 µL of 1% TFA and 2-4 µL were spotted onto alpha-Cyano4-HydroxycinnamicAcid (HCCA) AnchorChip targets using the double-layer method from Bruker Daltonics. Detection was performed in the positive ion reflector mode and a peptide calibration standard (Bruker Daltonics) was used for external calibration. Proteins were identified by the use of the Mascot Server 1.9 (Bruker Daltonics) based on mass searches within human sequences only. The search parameters allowed for carboxyamidomethylation of cysteine and one missing cleavage. The criteria for positive identification of proteins were set as follows: (i) a minimum score of 63; (ii) a mass accuracy of (0.01%; (iii) at least a 2-fold analysis from two independent
gels, and (iv) that the protein exhibits a significant difference in the number of matched peptides to the next potential hit.
Results More than 700 proteins spots were resolved by 2 D-gel based separation and thereof 47 spots were found to differ at least 2-fold in the steady-state level in EA‚hy 926 cells treated with 5 µg/mL ox-LDL when compared to nontreated cells. MALDITOF analysis based on the peptide mass fingerprints allowed these proteins to be identified (Figures 1 and 2; Tables 1 and 2). Ox-LDL treatment caused a reduction in various proteins with putative anti-atherosclerotic properties such as lamin C or serine/cysteine proteinase inhibitor clade H, whereas those with proposed pro-atherosclerotic functions such as ubiquinolcytochrome c-reductase, showed increased levels. A combined treatment with ox-LDL and genistein reversed in case of 29 of the 47 proteins the alterations induced by the stressor (Figure 2; Table 2). Among those, proteins involved in gene regulation, signal transduction, protein folding, metabolism and detoxification were identified. A number of cytoskeletal proteins and annexins also responded with altered expression levels to the treatment with ox-LDL and were reversed by co-exposure of cells to genistein (Figure 2; Table 2). Those observed changes in steady-state levels of the various cytoskeletal proteins which are known to be cleaved when cells undergo apoptosis, suggested that ox-LDL could have induced apoptosis in EA‚hy 926 cells. We therefore determined the activities of caspases 3, 6, and 8 as well as plasma membrane disintegration and nuclear fragmentation in cells exposed to the stressor for 24 h. None of the caspases showed significant alterations in activity after exposure of cells to ox-LDL (Figure 3A) and campothecin, an apoptosis inducing chemotherapeutic drug serving as a Journal of Proteome Research • Vol. 4, No. 2, 2005 371
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Figure 2. Comparative analysis of relative spot intensities of a subset of identified proteins from EA‚hy 926 cells that showed a significant change in steady-state level upon treatment of cells with 5 µg/mL ox-LDL versus control cells (BLACK BARS) or the same proteins in cells exposed to 5 µg/mL ox-LDL and 2.5 µM genistein (GREY BARS) in comparison to cells treated with ox-LDL alone.
control, also failed to increase caspase activities (Figure 3A). Despite a lack in activation of effector proteases, camptothecin but also ox-LDL induced apoptosis in EA‚hy 926 cells as indicated by advanced plasma membrane disintegration (Figure 3B) and increased nuclear fragmentation and chromatin condensation (Figure 3C). Whereas genistein alone did also not affect caspase activities (data not shown), it was able to prevent the ox-LDL mediated disintegration of the plasma membranes (Figure 3B) and nuclear fragmentation (Figure 3C).
Discussion Elevated serum levels of oxidized LDL are a recognized risk factor in the development of cardiovascular diseases.27,28 OxLDL can exert pro-atherogenic effects by increasing the interactions between endothelial cells and leukocytes and has been shown to affect inflammatory gene expression in primary human endothelial cells.29 An increased interaction between endothelial cells and leukocytes as caused by ox-LDL is found to be due to altered expression of cell adhesion molecules that enable the infiltration of monocytes into the intima as an initiating step in atherosclerosis.30,31 The much lower prevalence of coronary heart disease in Eastern societies than in the West may be related to the higher consumption of soy products in the East.2 The FDA has recently approved a health claim for soy since laboratory investigations, preclinical trials and epidemiological data all suggest that a high consumption is associated with a lower risk of coronary disease.4,5,32 Soy products contain a group of compounds called isoflavones, genistein and daidzein (and their corresponding glycosides) being the main components. Substantial evidence indicates that it is the isoflavone fraction that provides the beneficial cardiovascular effects of soy.4,5 Their average dietary intake in European countries is
