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Lipid peroxides induce expression of catalase in cultured vascular cells

2000; Elsevier BV; Volume: 41; Issue: 8 Linguagem: Inglês

10.1016/s0022-2275(20)33427-1

1539-7262

Olivier Meilhac, Mimi Zhou, Nalini Santanam, Sampath Parthasarathy,

Free Radicals and Antioxidants

Various forms of oxidized low-density lipoproteins (Ox-LDL) are thought to play a major role in the development of atherosclerosis. The lipid components of Ox-LDL present a plethora of proatherogenic effects in in vitro cell culture systems, suggesting that oxidative stress could be an important risk factor for coronary artery disease. However, buried among these effects are those that could be interpreted as antiatherogenic. The present study demonstrates that various oxidants, including oxidized fatty acids and mildly oxidized forms of LDL (MO-LDL), are able to induce catalase (an antioxidant enzyme) expression in rabbit femoral arterial smooth muscle cells (RFASMC), RAW cells (macrophages), and human umbilical vein endothelial cells (HUVEC). In RFASMC, catalase protein, mRNA, and the enzyme activity are increased in response to oxidized linoleic acid (13-hydroperoxy-9,11-octadecadienoic acid [13-HPODE] and 13-hydroxy-9,11-octadecadienoic acid [13-HODE]), MO-LDL, or hydrogen peroxide (H2O2). Such an increase in catalase gene expression cannot totally be attributed to the cellular response to an intracellular generation of H2O2 after the addition of 13-HPODE or 13-HODE because these agents induce a further increase of catalase as seen in catalase-transfected RFASMC. Taken together with the induction of heme oxygenase, NO synthase, manganese superoxide dismutase (Mn-SOD), and glutathione synthesis by oxidative stress, our results provide yet more evidence suggesting that a moderate oxidative stress can induce cellular antioxidant response in vascular cells, and thereby could be beneficial for preventing further oxidative stress. —Meilhac, O., M. Zhou, N. Santanam, and S. Parthasarathy. Lipid peroxides induce expression of catalase in cultured vascular cells. J. Lipid Res. 2000. 41: 1205–1213. Various forms of oxidized low-density lipoproteins (Ox-LDL) are thought to play a major role in the development of atherosclerosis. The lipid components of Ox-LDL present a plethora of proatherogenic effects in in vitro cell culture systems, suggesting that oxidative stress could be an important risk factor for coronary artery disease. However, buried among these effects are those that could be interpreted as antiatherogenic. The present study demonstrates that various oxidants, including oxidized fatty acids and mildly oxidized forms of LDL (MO-LDL), are able to induce catalase (an antioxidant enzyme) expression in rabbit femoral arterial smooth muscle cells (RFASMC), RAW cells (macrophages), and human umbilical vein endothelial cells (HUVEC). In RFASMC, catalase protein, mRNA, and the enzyme activity are increased in response to oxidized linoleic acid (13-hydroperoxy-9,11-octadecadienoic acid [13-HPODE] and 13-hydroxy-9,11-octadecadienoic acid [13-HODE]), MO-LDL, or hydrogen peroxide (H2O2). Such an increase in catalase gene expression cannot totally be attributed to the cellular response to an intracellular generation of H2O2 after the addition of 13-HPODE or 13-HODE because these agents induce a further increase of catalase as seen in catalase-transfected RFASMC. Taken together with the induction of heme oxygenase, NO synthase, manganese superoxide dismutase (Mn-SOD), and glutathione synthesis by oxidative stress, our results provide yet more evidence suggesting that a moderate oxidative stress can induce cellular antioxidant response in vascular cells, and thereby could be beneficial for preventing further oxidative stress. —Meilhac, O., M. Zhou, N. Santanam, and S. Parthasarathy. Lipid peroxides induce expression of catalase in cultured vascular cells. J. Lipid Res. 2000. 41: 1205–1213. The role of oxidized low density lipoproteins (Ox-LDL) in atherogenesis has been a topic of great interest for more than a decade. While the initial focus of the oxidation hypothesis centered on the uptake of Ox-LDL by macrophages and the development of foam cells, the cellular effects of oxidized lipids have more recently taken the center stage. It is now well established that oxidized lipids exhibit a wide variety of proatherogenic effects on cultured cells (1Parthasarathy S. Santanam N. Augé N. Oxidized low-density lipoprotein, a two-faced Janus in coronary artery disease?.Biochem. Pharmacol. 1998; 56: 279-284Google Scholar). They affect every facet of atherogenesis, cellular accumulation of lipid (2Steinberg D. Lewis A. Conner Memorial Lecture. Oxidative modification of LDL and atherogenesis.Circulation. 1997; 95: 1062-1071Google Scholar), modulation of gene expression, proliferation of smooth muscle cells, alteration of the endothelial functions, cytotoxicity, fibrinolysis, and plaque disruption (3Steinberg D. Parthasarathy S. Carew T.E. Khoo J.C. Witztum J.L. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity [see comments].N. Engl. J. Med. 1989; 320: 915-924Google Scholar, 4Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s.Nature. 1993; 362: 801-809Google Scholar, 5Parthasarathy S. Modified lipoproteins in the pathogenesis of atherosclerosis. R.G. Landes, Austin, TX1994Google Scholar). The paradoxical beneficial effects of physical activity on cardiovascular disease, despite its potential to induce an oxidative stress, alerted us to the possibility that oxidative stress could, under certain circumstances, trigger an antioxidant response and be possibly antiatherogenic. Accordingly, we demonstrated a reduced susceptibility to oxidation of LDL isolated from chronic conditioned athletes as opposed to the greater propensity of LDL to undergo oxidation isolated from beginning exercisers (6Shern-Brewer R. Santanam N. Wetzstein C. White-Welkley J. Parthasarathy S. Exercise and cardiovascular disease: a new perspective.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1181-1187Google Scholar). We proposed that exposure to oxidative stress might lead to the induction of antioxidant defense by the arterial cells. Catalase is an antioxidant enzyme that is predominantly located in cellular peroxisomes, which catalyzes the dismutation of H2O2, forming O2 and H2O (7Aebi H. Catalase in vitro.Methods Enzymol. 1984; 105: 121-126Google Scholar). Previous study from our laboratory has demonstrated that oxidized linoleic acid (13-hydroperoxy-9,11-octadecadienoic acid, [13-HPODE]) could increase intracellular generation of H2O2 in smooth muscle cells, mediating cytotoxic effects (8Santanam N. Augé N. Zhou M. Keshava C. Parthasarathy S. Overexpression of human catalase gene decreases the injury due to oxidative stress in vascular smooth muscle cells.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1912-1917Google Scholar). More importantly, the overexpression of catalase in vascular smooth muscle cells not only abrogated the cytotoxic effects of H2O2 but also prevented the cytotoxic effects of 13-HPODE. On the basis of these findings we anticipated an induction of cellular catalase gene expression by oxidants. In the current study, we describe the induction of catalase expression by oxidized linoleic acids (13-HPODE and 13-hydroxy-9,11-octadecadienoic acid [13-HODE]), H2O2, and mildly oxidized forms of LDL (MO-LDL) in vascular cells. In light of earlier findings by others that the exposure of cultured cells to lipid peroxides results in the induction of heme oxygenase (an antioxidant enzyme) (9Agarwal A. Shiraishi F. Visner G.A. Nick H.S. Linoleyl hydroperoxide transcriptionally upregulates heme oxygenase-1 gene expression in human renal epithelial and aortic endothelial cells.J. Am. Soc. Nephrol. 1998; 9: 1990-1997Crossref Google Scholar), manganese superoxide dismutase (Mn-SOD) (10Kinscherf R. Deigner H.P. Usinger C. Pill J. Wagner M. Kamencic H. Hou D. Chen M. Schmiedt W. Schrader M. Kovacs G. Kato K. Metz J. Induction of mitochondrial manganese superoxide dismutase in macrophages by oxidized LDL: its relevance in atherosclerosis of humans and heritable hyperlipidemic rabbits.Faseb J. 1997; 11: 1317-1328Google Scholar), nitric oxide synthase (11Ramasamy S. Parthasarathy S. Harrison D.G. Regulation of endothelial nitric oxide synthase gene expression by oxidized linoleic acid.J. Lipid Res. 1998; 39: 268-276Google Scholar), and glutathione synthesis (12Darley-Usmar V.M. Severn A. O'Leary V.J. Rogers M. Treatment of macrophages with oxidized low-density lipoprotein increases their intracellular glutathione content.Biochem. J. 1991; 278: 429-434Google Scholar), it appears likely that a moderate oxidative stress could, in general, induce cellular antioxidant responses and thus could be beneficial in the context of atherosclerosis and other chronic diseases in which oxidative stress has been implicated. Minimal essential medium (MEM), RPMI 1640, Medium 199, Dulbecco's modified Eagle's medium (DMEM)/F12 medium, penicillin, amphotericin, streptomycin, l-glutamine, trypsin–ethylenediaminetetraacetic acid (EDTA), and Hanks' balanced salt solution (HBSS) were purchased from Cellgro Mediatech (Herndon, VA). Fetal calf serum (FCS) was purchased from Atlanta Biologicals (Atlanta, GA). Endothelial mitogen growth factor (EMGF) was obtained from Biomedical Technologies (Stoughton, MA). Linoleic acid, oleic acid, thin-layer chromatography (TLC) plates, hydrogen peroxide, human kidney catalase, soybean lipoxygenase, anti-β-actin antibody, rabbit IgG, and peroxidase-conjugated secondary antibodies (anti-rabbit and anti-mouse IgG) were all obtained from Sigma (St. Louis, MO). Rabbit polyclonal anti-human erythrocyte catalase antibody was obtained from Athens Research and Technology (Athens, GA). 13-HODE was obtained from Cayman Chemicals (Ann Arbor, MI). [1-14C]Linoleic acid (53 mCi/mmol) was obtained from New England Nuclear (Boston, MA). Rabbit femoral arterial smooth muscle cells (RFASMC) (American Type Culture Collection [ATCC], Manassas, VA) were routinely cultured in MEM supplemented with 10% FCS, 2 mm l-glutamine, penicillin (100 U/mL), and streptomycin (100 μg/mL). A stably catalase-transfected RFASM cell line was established in our laboratory as previously described (8Santanam N. Augé N. Zhou M. Keshava C. Parthasarathy S. Overexpression of human catalase gene decreases the injury due to oxidative stress in vascular smooth muscle cells.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1912-1917Google Scholar). Human umbilical vein endothelial cells (HUVEC) were obtained from the Dermatology Core Cell Service Facility (Emory University, Atlanta, GA) and were grown on 0.1% gelatin-coated plates, in Medium 199 containing 20% heat-inactivated FCS, 2 mm l-glutamine, heparin (100 U/mL), EMGF (0.05 mg/mL), penicillin (100 U/mL), streptomycin (100 μg/mL), and amphotericin B (0.25 μg/mL). RAW macrophage cells (RAW 264.7 from the ATCC) were cultured in DMEM/F12 medium, 10% FCS, 2 mm l-glutamine, gentamicin (25 μg/mL). For Western blot and Northern blot studies, cells were grown in 25- and 175-cm2 cell culture flasks until 90% confluent. For other studies the cells were cultured in 6- or 24-well dishes. Before any experiment, cells were placed in their corresponding serum-free medium for 8–16 h. Cells were passaged with trypsin–EDTA. Cells were cultured to 90% confluence in 6-well dishes. Cellular lysates were prepared by sonication of scraped cells in phosphate-buffered saline (PBS), pH 7.4, containing 0.05% deoxycholate. An aliquot of cell lysate was used for protein estimation, by using a bicinchoninic acid (BCA) kit (Sigma), according to the procedure of Smith et al. (13Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Measurement of protein using bicinchoninic acid.Anal. Biochem. 1985; 150 ([published erratum appears in Anal Biochem 1987 May 15;163(1):279]): 76-85Google Scholar). Catalase activity was measured by the method of Aebi (7Aebi H. Catalase in vitro.Methods Enzymol. 1984; 105: 121-126Google Scholar), in which H2O2 was used as the substrate. The initial rate of disappearance of H2O2 (0 to 60 sec) was recorded spectrophotometrically at a wavelength of 240 nm. The catalase activity was expressed as units per milligram of protein, using a standard curve obtained with commercially available catalase. Catalase activity in control (untreated cells, 32 ± 5 units/mg of protein) is considered as 100% and results are expressed as a percentage of the control. Stock linoleic acid (C18:2) was prepared in absolute ethanol. The linoleic acid was oxidized to 13-HPODE, with immobilized soybean lipoxygenase (100 U/mL) at 37°C for 1 h. The formation of 13-HPODE was monitored spectrophotometrically by scanning the absorption between 200 and 300 nm (model DB-3500, SLM-AMINCO; Spectronic Instruments, Rochester, NY) using PBS as reference (14Fruebis J. Parthasarathy S. Steinberg D. Evidence for a concerted reaction between lipid hydroperoxides and polypeptides.Proc. Natl. Acad. Sci. USA. 1992; 89: 10588-10592Google Scholar). Under these conditions, the conversion into 13-HPODE is observed as an increase in absorbance at an optical density of 234 nm. Usually, more than 90% conversion of linoleic acid to 13-HPODE was achieved as determined by the molar extinction coefficient of the conjugated dienes, TLC, high-performance liquid chromatography (HPLC), or the leucomethylene blue (LMB) assay. The LMB assay, which is used to determine the actual peroxide content, provided a peroxide content of 90–94% (15Auerbach B.J. Kiely J.S. Cornicelli J.A. A spectrophotometric microtiter-based assay for the detection of hydroperoxy derivatives of linoleic acid.Anal. Biochem. 1992; 201: 375-380Google Scholar). Blood was collected from healthy donors, and LDL (d 1.019–1.063) was isolated by ultracentrifugation as previously described, using a TL-100 tabletop ultracentrifuge (16Santanam N. Parthasarathy S. Paradoxical actions of antioxidants in the oxidation of low density lipoprotein by peroxidases.J. Clin. Invest. 1995; 95: 2594-2600Google Scholar). The isolated LDL was dialyzed against PBS, pH 7.4, for 6 h. The concentration of apolipoprotein B (apoB) was determined by standard protein determination, using a BCA kit (Sigma) according to the procedure of Smith et al. (13Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Measurement of protein using bicinchoninic acid.Anal. Biochem. 1985; 150 ([published erratum appears in Anal Biochem 1987 May 15;163(1):279]): 76-85Google Scholar). MO-LDL was prepared by addition of 5 μm CuSO4 to a 2-g/L LDL solution in PBS at 37°C, and monitoring the formation of conjugated dienes at an optical density of 234 nm for about 1 h. The oxidation was stopped by the addition of 10 μm EDTA. MO-LDL contained 3–5 nmol of thiobarbituric acid-reactive substances (TBARS) per mg of apoB, as determined by the method of Yagi (17Yagi K. Lipid peroxides and human diseases.Chem. Phys. Lipids. 1987; 45: 337-351Google Scholar). Total RNA was isolated from RFASMC after various treatments, using TRI reagent (Sigma). Total RNA (30 μg) was separated on a 1.0% agarose–formaldehyde gel. The gel was stained with ethidium bromide in order to visualize the amount of RNA loaded in each lane. RNA was transferred to a nylon membrane and hybridized with [32P]dCTP random primer-labeled complementary DNA to human catalase, and the hybridization signal was visualized by exposing the membrane to Kodak film (Eastman Kodak, Rochester, NY). Quantification was performed with a densitometer (model GS-700; Bio-Rad, Hercules, CA), and results are expressed in arbitrary units, as the ratio between catalase signal and 18S RNA content for each lane. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was not used as internal control because its expression could vary in response to an oxidative stress (18Graven K.K. McDonald R.J. Farber H.W. Hypoxic regulation of endothelial glyceraldehyde-3-phosphate dehydrogenase.Am. J. Physiol. 1998; 274: C347-C355Google Scholar). Cell lysates were prepared by lysis and sonication in a hypotonic buffer (50 mm TRIS [pH 8], 150 mm NaCl, 1% Triton X-100, 1% sodium deoxycholate, 5 mm EDTA, containing 1 mm dithiothreitol, 10 mm β-glycerophosphate, aprotinin [10 μg/mL], trypsin inhibitor [10 μg/mL], leupeptin [2 μg/mL], and 0.1 mm phenylmethylsulfonyl fluoride). Samples were sonicated and centrifuged at 13,000 g for 10 min at 4°C. Protein extract (10–25 μg of RFASMC or HUVEC, or 75 μg of RAW cells) was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gel was transblotted onto a nitrocellulose membrane, blocked with 10% milk powder in TBS-T (TRIS-buffered saline [pH 7.4]–0.1% Tween 20) overnight, and then incubated with rabbit polyclonal anti-human catalase antibody (1:500 dilution) for 90 min, washed with TBS-T, and incubated with secondary antibody (anti-rabbit IgG conjugated to horseradish peroxidase, 1:1,500 dilution) for 1 h. After five washes, the signal was detected with a chemiluminescence kit (ECL kit; Amersham, Arlington Heights, IL). The membrane was then stripped in 62.5 mm TRIS (pH 6.7), 2% SDS, and 0.75% 2-mercaptoethanol for 30 min at 50°C. After 3 washes in TBS-T, the membrane was reprobed as described above with an anti-β-actin primary antibody (1:2,000 dilution). Films were analyzed by densitometry (Bio-Rad model GS-700). The OD catalase/OD β-actin ratio was calculated and the fold increase is reported for each figure, considering untreated control cells as 1. After various treatments, cells grown on cover slides at 50–60% confluence were washed twice with PBS, then fixed for 15 min with 3.7% paraformaldehyde in PBS. Cells were washed twice and then incubated with the primary antibody against catalase (1:50 dilution in 3% bovine serum albumin, BSA) for 1 h, at room temperature. After 2 washes with PBS, cells were incubated with a horseradish peroxidase-conjugated secondary antibody (1:100 dilution in 3% BSA). Cells were washed three times and then incubated with peroxidase substrate diaminobenzidine (DAB), according to the manufacturer's instructions (Sigma); cover slides were then washed, mounted on an aqueous medium, and observed under a microscope. A negative control (replacing primary antibody with 3% BSA–PBS or with the same concentration of rabbit IgG during the first incubation) was done for each condition, and did not show any nonspecific staining. The MTT assay was also used to measure cell viability (19Carmichael J. DeGraff W.G. Gazdar A.F. Minna J.D. Mitchell J.B. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing.Cancer Res. 1987; 47: 936-942Google Scholar). The principle of this assay is that the compound 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) undergoes cellular reduction by the mitochondrial dehydrogenase of viable cells into a blue formazan that can be measured spectrophotometrically. Briefly, cells were grown in a 6-well plate and the medium was replaced overnight by serum-free medium, before incubation with various concentrations of the oxidants for 16 or 24 h. At the end of incubation, MTT at 0.1 mg/mL was added to each well and incubated at 37°C for a further 30 min. After 30 min, the medium was removed carefully. Dimethylsulfoxide (DMSO) was added to each well in order to solubilize the formazan crystals. The solubilized blue formazan in DMSO was quantified with a spectrophotometer at wavelength 540 nm. There is a linear relationship between the formazan generated and the number of viable cells present. RFASMC were seeded in 6-well plates and grown until they reached 90% confluence. Experiments were performed in the absence of serum by using a 25 μm concentration (5,000 dpm/nmol) of a labeled solution of linoleic acid. After various times of incubation, the medium was removed and the cells were washed with PBS. Radioactivity in aliquots of the medium and washings was determined in order to estimate the unincorporated fraction of linoleic acid. The cells were scraped into 2 mL of 0.05% deoxycholate-containing PBS, and the radioactivity was determined in 100 μl of the cell lysate. After acidification by adding 20 μl of 6 n HCl, 4 mL of chloroform–methanol 1:1 (v/v) was added to the 1.9 mL of cell lysate to extract the cellular lipids (20Bligh L.H. Dyer W.J. Rapid method to total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar). After centrifugation (10 min, 3,000 rpm), the lower chloroform phase was gently dried (37°C, under nitrogen), dissolved in 100 μL of chloroform for loading on a TLC silica plate. Neutral lipids were separated with a solvent system containing n-hexane–diethyl ether–acetic acid 90:20:1.5 (v/v/v), and identified by iodine in the presence of standards. Spots were scraped off and the radioactivity was determined. Results are expressed as a percentage of total radioactivity. RFASMC were grown in 6-well dishes until subconfluent. The cells were then transferred to serum-free medium. After 16 h, cells were incubated with 13-HPODE or unoxidized linoleic acid (C18:2) (Fig. 1A), 13-HODE (Fig. 1B), MO-LDL or native LDL (Fig. 1C), or H2O2 (Fig. 1D) for 16 h. Catalase activity was determined in cell homogenates as described in Materials and Methods, using H2O2 as the substrate. Under the conditions used, none of the oxidants used was cytotoxic for RFASMC, except for H2O2 used at 50 μm (MTT viability test, data not shown). As seen in Fig. 1A–D, catalase activity increased by 1.5- to 2-fold in cells treated with 13-HPODE, 13-HODE, MO-LDL, or H2O2 in a concentration-dependent manner, except when cells were treated with 50 μm H2O2 (likely due to the cytotoxicity). Neither C18:2 nor native LDL increased catalase activity (Fig. 1A and C). To test whether the increased activity involved an activation of the catalase gene and synthesis of protein, we first investigated the catalase protein level in RFASMC by both Western blot and immunocytochemistry. Western blot analysis of RFASMC with anti-human catalase antibody was performed under denaturing conditions (SDS-PAGE). The 240-kDa tetramer characterizing catalase was recognized by the antibody as a single 60-kDa band, corresponding to the monomer. As seen in Fig. 2A, treatment of RFASMC with 13-HPODE and 13-HODE at various concentrations for 16 h was able to induce at least a 1.5-fold increase in catalase protein levels. The membrane was then stripped and reprobed with an anti-β-actin antibody. Ox-LDL is known to play a central role in atherosclerosis. When LDL undergoes mild oxidation (MO-LDL), its lipid peroxide content increases (21Steinbrecher U.P. Parthasarathy S. Leake D.S. Witztum J.L. Steinberg D. Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids.Proc. Natl. Acad. Sci. USA. 1984; 81: 3883-3887Google Scholar, 22Benz D.J. Mol M. Ezaki M. Mori-Ito N. Zelan I. Miyanohara A. Friedmann T. Parthasarathy S. Steinberg D. Witztum J.L. Enhanced levels of lipoperoxides in low density lipoprotein incubated with murine fibroblast expressing high levels of human 15-lipoxygenase.J. Biol. Chem. 1995; 270: 5191-5197Google Scholar). Lipids containing oxidized linoleic acids (e.g., 13-HPODE and 13-HODE) have been observed in LDL extracted from atherosclerotic patients (23Jira W. Spiteller G. Carson W. Schramm A. Strong increase in hydroxy fatty acids derived from linoleic acid in human low density lipoproteins of atherosclerotic patients.Chem. Phys. Lipids. 1998; 91: 1-11Google Scholar). These lipids are suggested to be responsible for some of the proatherogenic effects attributed to Ox-LDL (24Khan B.V. Parthasarathy S.S. Alexander R.W. Medford R.M. Modified low density lipoprotein and its constituents augment cytokine-activated vascular cell adhesion molecule-1 gene expression in human vascular endothelial cells.J. Clin. Invest. 1995; 95: 1262-1270Google Scholar, 25Nagy L. Tontonoz P. Alvarez J.G. Chen H. Evans R.M. Oxidized LDL regulates macrophage gene expression through ligand activation of PPARgamma.Cell. 1998; 93: 229-240Google Scholar). Results presented in Fig. 2B show that MO-LDL and 13-HPODE were able to induce a dose-dependent increase in catalase protein after 16 h of treatment. It may be noted that higher concentrations of either 13-HPODE or MO-LDL were toxic for RFASMC under these conditions (MTT assay, data not shown). Linoleic acid (C18:2,50 μm), used as a control, poorly stimulated the expression of catalase protein, suggesting the effect is specific to oxidized products. Native LDL at 50 μg/mL showed a slight increase in catalase protein, probably because of their moderate oxidation during the process of isolation and/or the incubation period with RFASMC. An increase in catalase protein could be seen after 4 to 16 h of treatment of cells with 13-HPODE (10 μm). However, no significant increase in catalase protein could be noticed after 24 h of stimulation, suggesting a transient activation of the protein (data not shown). To confirm the results from the Western blot analysis, we stained the treated RFASM cells for the immunodetection of catalase, using a peroxidase–DAB detection system, after 16 h of treatment with various oxidants. Figure 3 shows a specific cytoplasmic staining for catalase in RFASMC treated with 13-HPODE (Fig. 3B and C, 10 and 50 μm), 13-HODE (Fig. 3D, 25 μm), and MO-LDL (Fig. 3E, 25 μg/mL) in comparison with the control (Fig. 3A, nontreated cells). A positive control of RFASM cells stably transfected with catalase gene (8Santanam N. Augé N. Zhou M. Keshava C. Parthasarathy S. Overexpression of human catalase gene decreases the injury due to oxidative stress in vascular smooth muscle cells.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1912-1917Google Scholar) showed a strong cytoplasmic staining (Fig. 3F). To determine the effect of oxidants on catalase mRNA, we performed a Northern blot analysis using total RNA isolated from treated cells. Because Western blot analysis showed a sustained overexpression of the protein for up to 16 h of stimulation, we treated the RFASMC for 8 h. Figure 4 shows that catalase mRNA increased when cells were treated with 13-HODE, 13-HPODE (10, 25, and 50 μm), and MO-LDL (25 and 50 μg/mL) for 8 h. It may be noted that native LDL induced catalase gene expression by almost 2-fold, probably due to a slight oxidation occurring during the process of isolation. Catalase-transfected cellsare used as a positive control and show a 10-fold increase in mRNA. These results suggest that the upregulation of catalase by 13-HPODE, HODE, and MO-LDL is at least in part due to transcriptional activation and/or increased catalase mRNA stability. Smooth muscle cells possess 12-lipoxygenase, which could, under certain circumstances (e.g., on addition of angiotensin II), convert linoleic acid to its oxidized form (26Kim J.A. Gu J.L. Natarajan R. Berliner J.A. Nadler J.L. A leukocyte type of 12-lipoxygenase is expressed in human vascular and mononuclear cells. Evidence for upregulation by angiotensin II.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 942-948Google Scholar). To determine whether RFASMC could convert C18:2 into 13-HPODE and stimulate catalase gene expression, we determined the conversion of C18:2 in RFASMC after 4, 8, or 16 h of incubation by using [1-14C]linoleic acid. Figure 5 shows that linoleic acid is readily incorporated into triglycerides (Fig. 5, hatched columns) and phospholipids (Fig. 5, gray columns), and free linoleic acid content decreased during the incubation time (Fig. 5, solid columns). Oxidized linoleic acid has a different migration profile in the TLC system that we used (n-hexane–ethyl ether–acetic acid, 90:20:1.5 [v/v/v]) and a small amount of it could be detected in the cells (Fig. 5, open columns) (the stock solution of C18:2 contained about 5% of the oxidized form). No oxidized linoleic acid was released into the medium during the incubation. This was no surprise, as the incubation medium did not contain any stimulation of lipoxygenase pathway that would generate oxidized products. Because H2O2 induces catalase expression (Figs. Fig. 1., Fig. 2.) in RFASMC, it is possible that the peroxisomal degradation of HPODE might generate H2O2, which might be responsible for the activation of catalase gene. To test this, we incubated catalase-transfected RFASMC (CAT-RFASMC) with 13-HPODE and looked for further activation of catalase. As shown in Fig. 6, oxidized linoleic acid stimulates further the expression of catalase in CAT-RFASMC. However, only a 1.5-fold induction was observed, suggesting that the intracellular generation of H2O2 was not the only mechanism by which HPODE might activate catalase. Induction of catalase by oxidative stress could be of a great importance in the vascular wall and especially in the prevention of atherosclerosis. To investigate if oxidative stress could induce catalase in other cell types present in the arterial wall, we looked for catalase expression in response to HPODE in HUVEC and RAW cells (macrophages). Figure 7A shows the induction of catalase in RAW cells by 13-HPODE (25 and 50 μm) and H2O2 (50 μm). As compared with the basal level, induction of catalase by the same concentration of 13-HPODE (50 μm) is stronger in macrophages than in smooth muscle cells (7-fold induction versus ~3-fold). We included oleic acid (an oxidation-resistant fatty acid) as a control because macrophages can convert linoleic acid to its oxidized form (27Rankin S.M. Parthasarathy S. Steinberg D. Evidence for a dominant role of lipoxygenase(s) in the oxidation of LDL by mouse peritoneal macrophages.J. Lipid Res. 1991; 32: 449-456Google Scholar); however, neither linoleic acid (C18:2) nor oleic acid (C18:1) was able to induce catalase. Incubation of a primary culture (passage 2) of HUVEC with 13-HPODE and 13-HODE for 16 h (Fig. 7B) resulted in a 1.3- to 1.7-fold increase in cata