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Supplementation of Endothelial Cells with Mitochondria-targeted Antioxidants Inhibit Peroxide-induced Mitochondrial Iron Uptake, Oxidative Damage, and Apoptosis

2004; Elsevier BV; Volume: 279; Issue: 36 Linguagem: Inglês

10.1074/jbc.m404003200

1083-351X

Anuradha Dhanasekaran, Srigiridhar Kotamraju, Shasi V. Kalivendi, Toshiyuki Matsunaga, Tiesong Shang, Ágnes Keszler, Joy Joseph, B. Kalyanaraman,

ATP Synthase and ATPases Research

The mitochondria-targeted drugs mitoquinone (Mito-Q) and mitovitamin E (MitoVit-E) are a new class of antioxidants containing the triphenylphosphonium cation moiety that facilitates drug accumulation in mitochondria. In this study, Mito-Q (ubiquinone attached to a triphenylphosphonium cation) and MitoVit-E (vitamin E attached to a triphenylphosphonium cation) were used. The aim of this study was to test the hypothesis that mitochondria-targeted antioxidants inhibit peroxide-induced oxidative stress and apoptosis in bovine aortic endothelial cells (BAEC) through enhanced scavenging of mitochondrial reactive oxygen species, thereby blocking reactive oxygen species-induced transferrin receptor (TfR)-mediated iron uptake into mitochondria. Glucose/glucose oxidase-induced oxidative stress in BAECs was monitored by oxidation of dichlorodihydrofluorescein that was catalyzed by both intracellular H2O2 and transferrin iron transported into cells. Pretreatment of BAECs with Mito-Q (1 μm) and MitoVit-E (1 μm) but not untargeted antioxidants (e.g. vitamin E) significantly abrogated H2O2- and lipid peroxide-induced 2′,7′-dichlorofluorescein fluorescence and protein oxidation. Mitochondria-targeted antioxidants inhibit cytochrome c release, caspase-3 activation, and DNA fragmentation. Mito-Q and MitoVit-E inhibited H2O2- and lipid peroxide-induced inactivation of complex I and aconitase, TfR overexpression, and mitochondrial uptake of 55Fe, while restoring the mitochondrial membrane potential and proteasomal activity. We conclude that Mito-Q or MitoVit-E supplementation of endothelial cells mitigates peroxide-mediated oxidant stress and maintains proteasomal function, resulting in the overall inhibition of TfR-dependent iron uptake and apoptosis. The mitochondria-targeted drugs mitoquinone (Mito-Q) and mitovitamin E (MitoVit-E) are a new class of antioxidants containing the triphenylphosphonium cation moiety that facilitates drug accumulation in mitochondria. In this study, Mito-Q (ubiquinone attached to a triphenylphosphonium cation) and MitoVit-E (vitamin E attached to a triphenylphosphonium cation) were used. The aim of this study was to test the hypothesis that mitochondria-targeted antioxidants inhibit peroxide-induced oxidative stress and apoptosis in bovine aortic endothelial cells (BAEC) through enhanced scavenging of mitochondrial reactive oxygen species, thereby blocking reactive oxygen species-induced transferrin receptor (TfR)-mediated iron uptake into mitochondria. Glucose/glucose oxidase-induced oxidative stress in BAECs was monitored by oxidation of dichlorodihydrofluorescein that was catalyzed by both intracellular H2O2 and transferrin iron transported into cells. Pretreatment of BAECs with Mito-Q (1 μm) and MitoVit-E (1 μm) but not untargeted antioxidants (e.g. vitamin E) significantly abrogated H2O2- and lipid peroxide-induced 2′,7′-dichlorofluorescein fluorescence and protein oxidation. Mitochondria-targeted antioxidants inhibit cytochrome c release, caspase-3 activation, and DNA fragmentation. Mito-Q and MitoVit-E inhibited H2O2- and lipid peroxide-induced inactivation of complex I and aconitase, TfR overexpression, and mitochondrial uptake of 55Fe, while restoring the mitochondrial membrane potential and proteasomal activity. We conclude that Mito-Q or MitoVit-E supplementation of endothelial cells mitigates peroxide-mediated oxidant stress and maintains proteasomal function, resulting in the overall inhibition of TfR-dependent iron uptake and apoptosis. Enhanced mitochondrial oxidative damage is a prominent feature of most age-related human diseases including neurode-generative disorders. Aberrant electron leakage from mitochondria in the respiratory chain in oxidant-stressed cells triggers the formation of reactive oxygen species (ROS), 1The abbreviations used are: ROS, reactive oxygen species; BAEC, bovine aortic endothelial cells; CSS, control salt solution; DCF, 2′,7′-dichlorofluorescein; DCFH-DA, 2′,7′-dichlorodihydrofluorescein diacetate; DMEM, Dulbecco's modified Eagle's medium; Lac, clasto-lactacystin-β-lactone; MG-132, N-carbobenzoxyl-l-leucinyl-l-leucinyl-l-norleucinal; Mito-Q, ubiquinone conjugated to triphenyl phosphonium cation; MitoVit-E, vitamin E conjugated to triphenyl phosphonium cation; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TBS, Tris-buffered saline; TfR, transferrin receptor; TUNEL, terminal deoxynucleotidyltransferase-mediated nick-end labeling; TfFe, transferrin iron; FBS, fetal bovine serum; PBS, phosphate-buffered saline; DPBS, Dulbecco's PBS; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; pNA, para-nitroanilide; Glu/GO, glucose/glucose oxidase; 13-HpODE, 13-hydroperoxyoctadecadienoic acid; FACS, fluorescence-activated cell sorter. 1The abbreviations used are: ROS, reactive oxygen species; BAEC, bovine aortic endothelial cells; CSS, control salt solution; DCF, 2′,7′-dichlorofluorescein; DCFH-DA, 2′,7′-dichlorodihydrofluorescein diacetate; DMEM, Dulbecco's modified Eagle's medium; Lac, clasto-lactacystin-β-lactone; MG-132, N-carbobenzoxyl-l-leucinyl-l-leucinyl-l-norleucinal; Mito-Q, ubiquinone conjugated to triphenyl phosphonium cation; MitoVit-E, vitamin E conjugated to triphenyl phosphonium cation; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TBS, Tris-buffered saline; TfR, transferrin receptor; TUNEL, terminal deoxynucleotidyltransferase-mediated nick-end labeling; TfFe, transferrin iron; FBS, fetal bovine serum; PBS, phosphate-buffered saline; DPBS, Dulbecco's PBS; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; pNA, para-nitroanilide; Glu/GO, glucose/glucose oxidase; 13-HpODE, 13-hydroperoxyoctadecadienoic acid; FACS, fluorescence-activated cell sorter. leading to enhanced oxidative damage in mitochondria (1Beckman K.B. 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Sohal R.S. Arch. Biochem. Biophys. 1998; 352: 229-236Crossref PubMed Scopus (145) Google Scholar, 21Maguire J.J. Wilson D.S. Packer L. J. Biol. Chem. 1989; 264: 21462-21465Abstract Full Text PDF PubMed Google Scholar, 23Ingold K.U. Bowry V.W. Stocker R. Walling C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 45-49Crossref PubMed Scopus (309) Google Scholar, 25Mukai K. Kikuchi S. Urano S. Biochim. Biophys. Acta. 1990; 1035: 77-82Crossref PubMed Scopus (109) Google Scholar). Previously, it was reported (26Atamna H. Robinson C. Ingersoll R. Ames B.N. FASEB J. 2001; 15: 2196-2204Crossref PubMed Scopus (39) Google Scholar) that N-tert-butyl hydroxylamine reversed age-related changes in mitochondria by undergoing redox cycling in the mitochondrial electron transport chain. In this study, we tested the efficacy of two mitochondria-targeted antioxidants in preventing mitochondrial oxidative damage and apoptosis in bovine aortic endothelial cells (BAEC) treated with glucose/glucose oxidase and lipid hydroperoxide. Results suggest that mitochondria-targeted antioxidants are more effective inhibitors of mitochondrial damage and apoptosis than the corresponding "untargeted" counterparts (i.e. Vit-E). Glucose oxidase was obtained from Sigma. 13-Hydroperoxyoctadecadienoic acid (13-HpODE) and 13-hydroxyoctadecadienoic acid were purchased from Cayman Chemical Co. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) was purchased from Molecular Probes. N-Carbobenzoxyl-l-leucinyl-l-leucinyl-l-norleucinal (MG-132) was obtained from Biomol, and clasto-lactacystin-β-lactone (Lac) was purchased from Sigma. Mito-Q and MitoVit-E were synthesized as follows. MitoVit-E—The method of Murphy and co-workers (27Smith R.A.J. Porteous C.M. Coulter C.V. Murphy M.P. Eur. J. Biochem. 1999; 263: 709-716Crossref PubMed Scopus (405) Google Scholar) was used with some modifications. 6-Hydroxy-2-methoxy-2, 5,7,8-tetramethylchroman was synthesized from trimethylhydroquinone, trimethyl ortho-formate, and methyl vinyl ketone. This chroman was converted to the corresponding nitrile which, upon hydrolysis with aqueous ethylene glycol in KOH, gave the corresponding acid. The acid was reduced to the alcohol that was converted to the bromo derivative by using carbon tetrabromide and triphenylphosphine in dimethyl formamide. The bromo derivative was heated with triphenylphosphine in dioxane for 10 days. The solid was separated and purified by repeated precipitation from a dichloromethane solution by addition of ether. The overall yield was about 25%. Mito-Q—Mitoquinone was synthesized according to the published method (15Kelso G.F. Porteous C.M. Coulter C.V. Hughes G. Porteous W.K. Ledgerwood E.C. Smith R.A.J. Murphy M.P. J. Biol. Chem. 2001; 276: 4588-4596Abstract Full Text Full Text PDF PubMed Scopus (851) Google Scholar). Briefly, 11-bromoperoxyundecanoic acid prepared from 11-bromoundecanoic acid was coupled with 2,3-dimethoxy-5-methyl-1,4-benzoquinone to yield the 6-(10-bromodecyl) ubiquinone. The quinone was reduced to the quinol using sodium borohydride and heated with triphenylphosphine in dioxane for 4 days. The oily product separated from the reaction medium was purified and analyzed by mass spectroscopy. Endothelial Cell Culture—The cells were grown to confluence in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), insulin (10 μg/ml), transferrin (5 μg/ml), glutamine (4 mm), penicillin (100 units/ml), and streptomycin (100 μg/ml) and incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Cells were used between passages 4 and 12, as described by Balla et al. (28Balla G. Jacob H.S. Balla J. Rosenberg M. Nath K. Apple F. Eaton J.W. Vercellotti G.M. J. Biol. Chem. 1992; 267: 18148-18153Abstract Full Text PDF PubMed Google Scholar). On the day of the treatment, the medium was replaced with DMEM containing 2% FBS. Mito-Q (1 μm) or MitoVit-E (1 μm) was added to the cells in the medium that contained 25 mm glucose as a substrate for glucose oxidase. Cell Viability—Intracellular conversion of MTT to formazan was used as an indicator of cell viability (λmax = 562 nm). After treatment with Glu/GO in the presence and absence of antioxidants, the culture media were removed, and the cells were washed three times with control salt solution (CSS) (120 mm NaCl, 25 mm HEPES, pH 7.4, 25 mm KCl, 1.8 mm CaCl2, 4 mm MgCl2, and 15 mm glucose). Cells were incubated in CSS buffer containing 0.25 mg/ml MTT for 2 h at 37 °C. CSS buffer was removed, and cells were solubilized and mixed thoroughly in isopropyl alcohol, 0.08 n HCl (1:1) and the concentration of formazan measured optically as described previously (29Mosmann T. J. Immunol. Methods. 1983; 65: 55-63Crossref PubMed Scopus (45064) Google Scholar). Caspase Activity—After treatment with glucose/glucose oxidase (Glu/GO) and antioxidants, cells were washed twice with DPBS and lysed with cell lysis buffer (caspase-3 assay kit, Clontech). The caspase activities were measured as described previously (30Kotamraju S. Konorev E.A. Joseph J. Kalyanaraman B. J. Biol. Chem. 2000; 275: 33585-33592Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). Cells were washed twice with DPBS following treatment with the aforementioned antioxidants and then lysed with 50 mm HEPES buffer, pH 7.4, containing 5 mm CHAPS and 5 mm dithiothreitol. After the cytosolic fraction was taken by centrifugation at 12,000 × g for 30 min, the caspase activities were measured in the supernatant using DEVD-pNA (acetyl-Asp-Glu-Val-Asp para-nitroanilide), acetyl-LEHD-pNA, and acetyl-IETD-pNA as substrates. The absorbance at 405 nm of the released pNA was monitored in a spectrophotometer and quantitated by using pNA as standard. TUNEL Measurements—The terminal deoxynucleotidyltransferase-mediated nick-end labeling (TUNEL) assay was used for microscopic detection of apoptosis (30Kotamraju S. Konorev E.A. Joseph J. Kalyanaraman B. J. Biol. Chem. 2000; 275: 33585-33592Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). This assay was based on labeling of 3′-free hydroxyl ends of the fragmented DNA with fluorescein-dUTP catalyzed by terminal deoxynucleotidyltransferase. Procedures were followed according to the commercially available kit (ApoAlert) from Clontech. Apoptotic cells exhibit a strong nuclear green fluorescence that can be detected by using a standard fluorescein filter (520 nm). All cells stained with propidium iodide exhibit a strong red cytoplasmic fluorescence at 620 nm. The areas of apoptotic cells were detected by fluorescence microscopy equipped with rhodamine and fluorescein isothiocyanate filters. Detection of Cytochrome c Release into Cytosol—The release of mitochondrial cytochrome c into the cytosol was measured according to methods described previously (31Tampo Y. Kotamraju S. Chitambar C.R. Kalivendi S.V. Keszler A. Joseph J. Kalyanaraman B. Circ. Res. 2003; 92: 56-63Crossref PubMed Scopus (133) Google Scholar, 32Heinloth A. Brune B. Fischer B. Galle J. Atherosclerosis. 2002; 162: 93-101Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Briefly, BAEC were washed with DPBS and homogenized in PBS supplemented with 40 μg/ml saponin. Lysate was centrifuged at 12,000 × g for 20 min. The supernatant was used as the cytosolic fraction to measure the released cytochrome c into the cytosol by Western blot analysis using a mouse anti-cytochrome c antibody (Pharmingen). Detection was by horseradish peroxidase-conjugated goat anti-mouse antibody using the ECL method. Aconitase Activity—BAECs were washed three times with cold PBS and lysed with a lysing buffer containing 0.2% Triton X-100, 100 μm DTPA, and 5 mm citrate in PBS. The activity of aconitase in cell lysates was measured in 100 mm Tris-HCl, pH 8.0, containing 20 mm dl-trisodium isocitrate. An extinction coefficient for cis-aconitate of 3.6 mm–1 at 240 nm was used (33Kennedy M.C. Emptage M.H. Dreyer J.L. Beinert H. J. Biol. Chem. 1983; 258: 11098-11105Abstract Full Text PDF PubMed Google Scholar). Complex I Activity—The complex I activity was measured as described previously (34Yen H.C. Oberley T.D. Gairola G. Szweda L. Calir D.K. Arch. Biochem. Biophys. 1999; 362: 59-66Crossref PubMed Scopus (120) Google Scholar). Briefly, cells were pelleted at 500 × g at 4 °C for 10 min and resuspended in 2.5 ml of TES buffer (0.25 m sucrose, 1 mm EGTA and 10 mm triethanolamine acetate, pH 7.0) after treatment. Cells were homogenized with a Dounce homogenizer with 15 strokes. Resulting material was transferred to a 2-ml tube and centrifuged at 1,500 × g. Postnuclear supernatant was centrifuged at 10,000 × g at 4 °C for 10 min to obtain a mitochondria-enriched pellet. The pellet was washed twice with 1 ml of homogenization buffer. The protein content of the pellet was assayed by the Bradford method. The mitochondrial pellet was subjected to "freeze-thawing" three times. Twenty microliters (0.3 mg of protein) of mitochondrial homogenate was mixed with 930 μl of 10 mm potassium phosphate buffer, pH 8.0, in a 1-ml cuvette containing 50 μl of 100 μm NADH. The rate of NADH oxidation was monitored at 340 nm for 2 min in a UV spectrophotometer. Then 5 μlof 10 mm ubiquinone-1 was added, and the stimulated rate of NADH oxidation was measured as complex I activity, using an extinction coefficient of 6.81 mm–1 cm–1 at 340 nm. DCFH Staining—The determination of intracellular oxidant production is based on the oxidation of 2′,7′-dichlorodihydrofluorescein (DCFH) to a fluorescent 2′,7′-dichlorofluorescein (DCF). Following treatment of BAEC with Glu/GO and other targeted antioxidants, the medium was aspirated, and cells were washed twice with DPBS and incubated in 1 ml of medium without FBS. DCFH was added at a final concentration of 10 μm and incubated for 20 min. The cells were then washed once with DPBS and maintained in a 1-ml culture medium. The fluorescence was monitored after 30 min by using a Nikon fluorescence microscope equipped with a fluorescein isothiocyanate filter. Flow Cytometry—Following different treatments, BAECs were washed in PBS three times and incubated with DCFH-DA or Mitosensor reagent for 15 min at 37 °C. The fluorescence was measured using a BD Biosciences FACScan flow cytometer with excitation and emission setting at 488 and 530 nm, respectively (35Senturker S. Tschirret-Guth R. Morrow J. Levine R. Shacter E. Arch. Biochem. Biophys. 2002; 397: 262-272Crossref PubMed Scopus (55) Google Scholar). Glutathione Measurement—GSH levels were measured by high pressure liquid chromatography as the o-phthalaldehyde (OPA) adduct at pH 8.0 (36Tietze F. Anal. Biochem. 1969; 27: 502-522Crossref PubMed Scopus (5488) Google Scholar). Cells were washed twice with PBS, suspended in 250 μl of PBS, and lysed by sonication. After centrifugation at 10,000 × g for 2 min, 200 μl of the clear supernatant was derivatized by incubating with OPA for 30 min at room temperature. An aliquot of sample was injected into a column (Kramasil C-18) and eluted isocratically with a mobile phase consisting of 150 mm sodium acetate/methanol (91.5:8.5). The OPA-GSH adduct was monitored using a fluorescence detector operating at excitation and emission wavelengths at 250 and 410 nm, respectively. The levels of intracellular GSH were detected using a GSH solution as a standard. Measurement of 55Fe Uptake in Endothelial Cells—BAECs were grown in DMEM containing 10% FBS until confluence. On the day of treatment the medium was replaced with DMEM containing 2% FBS, and the cells were allowed to adjust to the medium conditions. 0.2 μCi of 55Fe (as ferric chloride) was added to the medium, and its levels were measured after 4 h. Cells were washed with DPBS and lysed with PBS containing 0.1% Triton X-100, and the radioactivity was counted in a beta counter (37Chitambar C.R. Seligman P.A. J. Clin. Investig. 1986; 78: 1538-1546Crossref PubMed Scopus (137) Google Scholar, 38Kotamraju S. Chitambar C.R. Kalivendi S.V. Joseph J. Kalyanaraman B. J. Biol. Chem. 2002; 277: 17179-17187Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Measurement of55Fe Uptake into Mitochondria of Endothelial Cells—BAECs were grown in DMEM containing 10% FBS until confluence. On the day of treatment the medium was replaced with DMEM containing 2% FBS, and the cells were allowed to adjust to the medium conditions. 0.2 μCi of 55Fe (ferric chloride) was added to the medium. Following treatment, cells were washed twice with an isolation medium (320 mm sucrose, 1 mm potassium EDTA, 10 mm Tris-HCl, pH 7.4). The cells were then scraped by using 1 ml of isolation medium and spun at 500 × g for 5 min at 4 °C. Cell pellets were resuspended in a 4-ml isolation medium and spun at 1500 × g. The supernatants were combined and spun at 1500 × g for 10 min at 4 °C. The supernatant was spun at 17,000 × g for 11 min at 4 °C. Final pellets were resuspended in a 200 μl of isolation medium. An aliquot was taken for protein estimation, and the remaining suspension was used for counting in a beta counter. Detection of Transferrin Receptor Levels—BAECs were washed with ice-cold phosphate-buffered saline and resuspended in 100 μl of RIPA buffer (20 mm Tris-HCl, pH 7.4, 2.5 mm EDTA, 1% Triton X-100, 1% sodium deoxycholate, 1% SDS, 100 mm NaCl, 100 mm sodium fluoride). To a 10-ml solution of the above, the following agents were added: 1 mm sodium vanadate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 10 μg/ml pepstatin inhibitors. Cells were homogenized by passing the suspension through a 25-gauge needle (15 strokes). The lysate was centrifuged for 15 min at 12,000 × g. Protein was determined by the Bradford method, and 20 μg were used for the Western blot analysis. Proteins were resolved on polyacrylamide gels and blotted onto nitrocellulose membranes. Membrane was washed twice with TBST (140 mm NaCl, 50 mm Tris-HCl, pH 7.2) containing 0.1% Tween 20 before blocking the nonspecific binding with TBS containing 5% skim milk. Membrane was incubated with mouse anti-human transferrin receptor monoclonal antibody (1 μg/ml in TBS) (Zymed Laboratories Inc.) and 2% skim milk for 2 h at room temperature. Membrane was washed 5 times and detected with horseradish peroxidase-conjugated rabbit anti-mouse IgG (1:5000) for 1.5 h at room temperature. The bands were detected by using the ECL method (Amersham Biosciences). Detection of Mitochondrial Transmembrane Potential Changes— BAECs were treated in 6-well plates with Glu/GO with or without the targeted antioxidants. After treatment the cells were washed with serum-free media and incubated with the BD MitoSensor dye (Clontech) and incubated for 20 min according to the manufacturer's instructions. This dye is sensitive to changes in mitochondrial transmembrane potential changes. The fluorescence was monitored by using a fluorescein isothiocyanate and a rhodamine filter. Proteasome Activity—26 S proteasome. Proteasomal activity was measured as reported earlier (39Coux O. Tanaka K. Goldberg A.L. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2215) Google Scholar, 40Pajonk F. Riess K. Sommer A. Mc Bride W.H. Free Radic. Biol. Med. 2002; 32: 536-543Crossref PubMed Scopus (65) Google Scholar). Briefly, BAECs were washed with buffer I (50 mm Tris, pH 7.4, 2 mm dithiothreitol, 5 mm MgCl2, 2 mm ATP) and homogenized with buffer I containing 250 mm sucrose. Twenty micrograms of 10,000 × g supernatant were diluted with buffer I to a final volume of 1 ml. The fluorogenic proteasome substrates N-succinyl-LLVY-AMC (chymotrypsin-like) and benzyloxycarbonyl-Leu-Leu-Lys-amino-4-methylcoumarin (trypsin-like) were added in a final concentration of 100 and 80 μm, respectively. Proteolytic activity was measured by monitoring the release of the fluorescent group 7-amino-4-methylcoumarin (excitation 380 nm, emission 460 nm). Measurements of Cell Viability—BAECs were incubated with Glu/GO for various times with or without Mito-Q (1 μm) and MitoVit-E (1 μm), and the cell viability was estimated by using the MTT assay. Glu/GO decreased the cell viability to 18% which was restored to 64 and 51%, respectively, in the presence of Mito-Q and MitoVit-E. Similar results with HpODE were observed, where treatment with Mito-Q and MitoVit-E restored the cell viability to 59 and 48%, respectively (data not shown). Mitochondria-targeted Antioxidants Inhibit Apoptosis in BAEC Treated with Hydroperoxides—BAECs were incubated with Glu/GO (generator of H2O2) or 13-HpODE (lipoxygenase-catalyzed oxidative metabolite of linoleic acid) for different times, and caspase-3, -9, and -8 activities were measured (Figs. 2 and 3). The caspase-3 and -9 activities were markedly elevated after a 4-h treatment in Glu/GO-treated cells (Fig. 2, A–D). Unlike caspase-3 and caspase-9 activities, the caspase-8 activity increased only slightly in Glu/GO-treated cells (Fig. 2E). This is consistent with an intrinsic pathway involving mitochondria-mediated apoptosis. Exposure of BAEC to Glu/GO increased the percentage of TUNEL-positive cells (Fig. 2F). Pretreatment with mitochondria-targeted antioxidants, Mito-Q and MitoVit-E, inhibited the caspase-3 and caspase-9 proteolytic activation induced by Glu/GO. As shown in Fig. 2, A and C, Mito-Q-pretreated cells were markedly resistant to H2O2-induced caspase activation. Pretreatment with MitoVit-E also inhibited H2O2-mediated caspase activa