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Inhibition of Oxidized Low-density Lipoprotein-induced Apoptosis in Endothelial Cells by Nitric Oxide

2001; Elsevier BV; Volume: 276; Issue: 20 Linguagem: Inglês

10.1074/jbc.m011731200

ISSN

1083-351X

Autores

Srigiridhar Kotamraju, Neil Hogg, Joy Joseph, Larry K. Keefer, Balaraman Kalyanaraman,

Tópico(s)

Vitamin C and Antioxidants Research

Resumo

Proatherogenic oxidized low-density lipoprotein (oxLDL) induces endothelial apoptosis. We investigated the anti-apoptotic effects of intracellular and extracellular nitric oxide (⋅NO) donors, iron chelators, cell-permeable superoxide dismutase (SOD), glutathione peroxidase mimetics, and nitrone spin traps. Peroxynitrite (ONOO−)-modified oxLDL induced endothelial apoptosis was measured by DNA fragmentation, TUNEL assay, and caspase-3 activation. Results indicated the following: (i) the lipid fraction of oxLDL was primarily responsible for endothelial apoptosis. (ii) Endothelial apoptosis was potently inhibited by ⋅NO donors and lipophilic phenolic antioxidants. OxLDL severely depleted Bcl-2 levels in endothelial cells and ⋅NO donors restored Bcl-2 protein in oxLDL-treated cells. (iii) The pretreatment of a lipid fraction derived from oxLDL with sodium borohydride or potassium iodide completely abrogated apoptosis in endothelial cells, suggesting that lipid hydroperoxides induce apoptosis. (iv) Metalloporphyrins dramatically inhibited oxLDL-induced apoptosis in endothelial cells. NeitherS-nitrosation of caspase-3 nor induction of Hsp70 appeared to play a significant role in the antiapoptotic mechanism of ⋅NO in oxLDL-induced endothelial apoptosis. We propose that cellular lipid peroxyl radicals or lipid hydroperoxides induce an apoptotic signaling cascade in endothelial cells exposed to oxLDL, and that⋅NO inhibits apoptosis by scavenging cellular lipid peroxyl radicals. Proatherogenic oxidized low-density lipoprotein (oxLDL) induces endothelial apoptosis. We investigated the anti-apoptotic effects of intracellular and extracellular nitric oxide (⋅NO) donors, iron chelators, cell-permeable superoxide dismutase (SOD), glutathione peroxidase mimetics, and nitrone spin traps. Peroxynitrite (ONOO−)-modified oxLDL induced endothelial apoptosis was measured by DNA fragmentation, TUNEL assay, and caspase-3 activation. Results indicated the following: (i) the lipid fraction of oxLDL was primarily responsible for endothelial apoptosis. (ii) Endothelial apoptosis was potently inhibited by ⋅NO donors and lipophilic phenolic antioxidants. OxLDL severely depleted Bcl-2 levels in endothelial cells and ⋅NO donors restored Bcl-2 protein in oxLDL-treated cells. (iii) The pretreatment of a lipid fraction derived from oxLDL with sodium borohydride or potassium iodide completely abrogated apoptosis in endothelial cells, suggesting that lipid hydroperoxides induce apoptosis. (iv) Metalloporphyrins dramatically inhibited oxLDL-induced apoptosis in endothelial cells. NeitherS-nitrosation of caspase-3 nor induction of Hsp70 appeared to play a significant role in the antiapoptotic mechanism of ⋅NO in oxLDL-induced endothelial apoptosis. We propose that cellular lipid peroxyl radicals or lipid hydroperoxides induce an apoptotic signaling cascade in endothelial cells exposed to oxLDL, and that⋅NO inhibits apoptosis by scavenging cellular lipid peroxyl radicals. oxidized low-density lipoprotein terminal deoxynucleotidyltransferase-mediated nick-end labeling (assay) α-phenyl-tert-butylnitrone bovine aortic endothelial cells nitric-oxide synthase nitric oxide low density lipoprotein diethylenetriamine NONOate S-nitrosoglutathione heat shock protein 70 butylated hydroxytoluene tetrakis(4-benzoic acid) porphyrin B-cell lymphoma-2 protein Mn(III) tetrakis-(4-benzoic acid)porphyrin Fe(III) tetrakis-(4-benzoic acid)porphyrin 7-ketocholesterol superoxide dismutase diethylenetriaminepentaacetic acid Ample evidence supports the notion that oxidatively modified low-density lipoprotein (oxLDL)1 plays a key role in the onset of atherogenic processes (1Steinberg D. Parthasarathy S. Carew T.E. Khoo J.C. Witztum J.L. N. Engl. J. 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Commun. 1993; 195: 539-544Crossref PubMed Scopus (278) Google Scholar). This rate constant is ∼107 times greater than that reported for the reaction between LOO⋅ and unsaturated lipid, and 104 times greater than the rate constant for the reaction of LOO⋅ with α-tocopherol (36Rubbo H. Radi R. Trujillo M. Telleri R. Kalyanaraman B. Barnes S. Kirk M. Freeman B.A. J. Biol. Chem. 1994; 269: 26066-26075Abstract Full Text PDF PubMed Google Scholar). Thus, ⋅NO can act as a potent chain-breaking antioxidant. Consequently, the reaction between⋅NO and O⨪2 has the combined effect of removing an antioxidant such as ⋅NO, and generating the prooxidant, ONOO−. The role of these reactions in regulating downstream apoptosis cell signaling has not been fully investigated. In vivo immunohistochemical studies in atherosclerotic tissues indicate the presence of oxidation and nitration marker products (i.e. lipid hydroperoxides and nitrotyrosine) that are diagnostic for reactive oxygen and nitrogen species (23Leeuwenburgh C. Hardy M.M. Hazen S.L. Wagner P. Oh-ishi S. Steinbrecher U.P. Heinecke J.W. J. Biol. Chem. 1997; 272: 1433-1436Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 37Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1624Crossref PubMed Scopus (6755) Google Scholar, 38Hui R.E. Padmaja S. Free Radic. Res. Commun. 1993; 18: 195-199Crossref PubMed Scopus (2036) Google Scholar, 39Padmaja S. Hui R.E. Biochem. Biophys. Res. Commun. 1993; 195: 539-544Crossref PubMed Scopus (278) Google Scholar, 40Beckman J.S. Ye Y.Z. Anderson P.G. Chein J. Accavitti M.A. Tarpey M.M. White C.R. Biol. Chem. Hoppe-Seyler. 1994; 375: 81-88Crossref PubMed Scopus (1082) Google Scholar). In this study we tested the hypothesis that the propagation of lipid peroxidation is primarily responsible for oxLDL-induced apoptosis. Therefore, we investigated the effects of extracellular and intracellular ⋅NO donors (NONOates), cell-permeable SOD mimetics and ONOO− scavengers (MnTBAP and FeTBAP), a cell-permeable mimetic of glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase (ebselen), lipophilic phenolic antioxidant (probucol), and a nitrone spin trap α-phenyl-tert-butyl nitrone (PBN) (Fig.1) on endothelial apoptosis induced by oxLDL. Results show that ⋅NO inhibits oxLDL-induced apoptosis as do MnTBAP/FeTBAP, ebselen, Probucol, and PBN. These structurally diverse antioxidants share a common mechanism that is their ability to inhibit the propagation of lipid peroxidation. Therefore, we propose that the major antiapoptotic mechanism of ⋅NO involves peroxyl radical scavenging. Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) and PBN were obtained from Sigma. PBN was also obtained from the Oklahoma Medical Research Foundation Spin Trap Source as a gift from Dr. Ronald Mason (NIEHS, National Institutes of Health, Research Triangle Park, NC). Mn(III)-tetrakis(4-benzoic acid) porphyrin (MnTBAP), FeTBAP, and S-nitrosoglutathione (GSNO) were synthesized according to the published methods (42Day B.J. Shawen S. Liochev S.I. Crapo J.D. J. Pharmacol. Exp. Ther. 1995; 275: 1227-1232PubMed Google Scholar, 43Field L. Dilts R.V. Ravichandran R. Lenhert P.G. Carnahan G.E. J. Chem. Soc. Chem. Commun. 1978; : 249-250Crossref Google Scholar). Probucol was purchased from Sigma. Diethylenetriamine NONOate (DETA/NO) was obtained from Cayman Chemical Co. Anti-Hsp70 antibody and hamster anti-human Bcl-2 antibody and diethylenetriaminepentaacetic acid (DTPA) were purchased from Pharmingen. Bovine aortic endothelial cells (BAEC) harvested from thoracic aortas were maintained (37 °C, 5% CO2) in Dulbecco's modified eagles medium (1 g/liter of glucose, Life Technologies, Inc.) containing 15% fetal bovine serum (Sigma) with antibiotics. Cells used in this study were between passages 5 and 10. Human umbilical vein endothelial cells were obtained from Clonetics and cultured in endothelial basal medium (Clonetics) containing 2% fetal bovine serum, 10 ng/ml human recombinant epidermal growth factor, 1 μg/ml hydrocortisone, 50 μg/ml gentamycin, 50 ng/ml amphotericin-B, and 3 mg/ml bovine brain extract. Cells cultured between passages 2 and 4 were used in this study. LDL was isolated by sequential ultracentrifugation (d = 1.019–1.063) from freshly drawn, normolipidemic human plasma to which EDTA was added (44Hatch F.T. Adv. Lipid Res. 1968; 6: 1-68Crossref PubMed Google Scholar). LDL was oxidized by adding ONOO− (1 mm). In control experiments, LDL was added to a phosphate buffer (pH 7.4, 100 mm) containing pre-decomposed ONOO−. Medium containing 150 μg of the modified LDL was extracted by adding 2 volumes of ice-cold methanol followed by 2 volumes of chloroform (45Kotamraju, S., Konorev, E. A., Joseph, B., and Kalyanaraman, B. (2000) J. Biol. Chem. 275, 33585-33592Google Scholar). The mixture was centrifuged at 1800 ×g for 10 min to separate the phases. The lipid phase was carefully removed and evaporated under a stream of nitrogen and re-dissolved in a minimum volume of methanol. For all treatments, cells were washed twice with Dulbecco's phosphate-buffered saline and incubated with serum-free Dulbecco's modified Eagle's medium in the presence or absence of reagents. Either native or oxidized LDL was added to a final concentration of 150 μg of LDL protein/ml. DNA was isolated from BAEC. Culture medium was removed and centrifuged at 3000 × g for 5 min to collect any detached cells. Adherent cells were lysed with a hypotonic lysis buffer (10 mm Tris-HCl, 10 mm EDTA, 0.5% Triton X-100) and then pooled with the pellet made up of detached cells. After incubation at 4 °C for 15 min, lysates were incubated with 10 μl of 10 mg/ml RNase A for 1 h at 37 °C followed by 10 μl of 20 mg/ml proteinase K for 2 h at 50 °C. DNA was extracted using chloroform:phenol:isoamyl alcohol (25:24:1). It was then precipitated overnight with 1 volume of isopropyl alcohol at −20 °C, electrophoresed on 2% agarose gel, and then visualized under UV light after staining with ethidium bromide. Apoptosis was detected in BAEC's using terminal deoxynucleotidyltransferase-mediated nick-end labeling (TUNEL) assay (46Kotamraju S. Konorev E.A. Joseph B. Kalyanaraman B. J. Biol. Chem. 2000; 275: 33585-33592Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). Labeling of 3′ free hydroxyl ends of the fragmented DNA with fluorescein-dUTP, was catalyzed by terminal deoxynucleotidyltransferase (TdT) using a commercially available kit (ApoAlert,CLONTECH) following the manufacturer's directions. The areas of apoptotic cells were then detected by fluorescence microscopy equipped with rhodamine (for propidium iodide staining) and fluorescein isothiocyanate filters. The quantification of apoptosis was performed using Sigma Scan 5.0 Image Analysis package. Propidium iodide-stained cells (which represents the total number) were counted under rhodamine filter and the apoptotic (TUNEL positive) cells were counted under fluorescein isothiocyanate filter. Percentage of apoptosis was calculated from the ratio of these two measurements. Cells were washed after treating with appropriate drugs with phosphate-buffered saline and resuspended in 50 μl of chilled lysis buffer (Caspase-3 assay kit,CLONTECH) and incubated on ice for 10 min. The cell lysates were centrifuged in a microcentrifuge at 12,000 rpm for 3 min at 4 °C to precipitate cellular debris. To confirm the correlation between protease activity and signal detection, we performed a control reaction as follows. An induced sample was incubated with 0.5 μl of 1 mm DEVD-fmk (caspase inhibitor) at 37 °C for 30 min during which time the other tubes consisting of lysis buffer were kept on ice. Subsequently, 50 μl of reaction buffer (containing 7 μl of 1 mmdithiothreitol/ml of reaction buffer) was added, followed by the addition of 5 μl of 1 mm conjugated substrate (DEVD-p-nitroanilide, 50 μm final concentration) to each sample. After incubating at 37 °C for 1 h, absorptions were monitored at 405 nm in a spectrophotometer (46Kotamraju S. Konorev E.A. Joseph B. Kalyanaraman B. J. Biol. Chem. 2000; 275: 33585-33592Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). BAEC 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 mmEDTA, 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 (10 strokes). The lysate was centrifuged at 750 × g for 10 min at 4 °C to pellet out the nuclei. The remaining supernatant was centrifuged for 15 min at 12,000 × g. Protein was determined with the Lowry method (47Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar), and 25 μg were used for the Western blot analysis. Proteins were resolved on polyacrylamide gels (8% for Hsp70 and 18% for Bcl-2) and blotted onto nitrocellulose membranes. Sheets were washed twice with TBS (140 mm NaCl, 50 mm Tris-HCl, pH 7.2) containing 0.1% Tween 20 before blocking the nonspecific binding with TBS, 5% skim milk, 1% fetal calf serum. Filters were incubated with either mouse anti-Hsp70 antibody (Pharmingen) or hamster anti-human Bcl-2 antibody (Pharmingen) 1 μg/ml in TBS, 2% skim milk, 0.7% fetal calf serum for 2 h at room temperature. Sheets were washed 5 times and detected by horseradish peroxidase-conjugated goat anti-mouse monoclonal antibody (1:1000) for Hsp70 and anti-hamster IgG (1:1000) for Bcl-2 for 1.5 h at room temperature using the ECL method (Amersham Pharmacia Biotech). After incubating BAEC for 24 h with native LDL, no DNA fragmentation was observed (Fig.2 A, lane 2). However, if the LDL was pre-oxidized, either with copper (II) sulfate (100 μm) or with ONOO− (1 mm), significant DNA laddering occurred (Fig. 2 A, lanes 3 and4). DNA laddering did not occur in the presence of decomposed ONOO− (not shown). In addition, the dialysis of LDL after treatment with ONOO− did not alter the ability of LDL to fragment cellular DNA (not shown). This indicates that ONOO− caused the oxidation of a component of LDL to an intermediate or product that stimulated DNA fragmentation, and that this intermediate remained associated with the LDL particle during dialysis. This suggests that apoptosis is not caused by a decomposition product of ONOO−, nor by a low-molecular weight, soluble, LDL oxidation product, such as malondialdehyde or 4-hydroxynonenal in cells exposed to ONOO−-modified LDL. OxLDL-induced DNA laddering was significantly inhibited in the presence of Probucol (Fig.2 B, lane 4), nitrone trap, PBN (Fig. 2 B, lane 6), and metalloporphyrin antioxidant, FeTBAP (Fig. 2 B, lane 7). In addition to DNA fragmentation, the effect of oxLDL on the activation of caspase-3 was also investigated. Caspase-3, one of the downstream members of this enzyme family, is activated by proteolysis and is considered to be a committed step in several apoptotic pathways (46Kotamraju S. Konorev E.A. Joseph B. Kalyanaraman B. J. Biol. Chem. 2000; 275: 33585-33592Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). As shown in Table I, native LDL did not enhance caspase-3 activity, whereas oxLDL stimulated caspase-3 activity by ∼5-fold. This suggests that the oxLDL-mediated apoptosis acts through the activation of caspase-3.Table IEffect of antioxidants and antioxidant enzymes on oxLDL-induced caspase-3 activationTreatmentCaspase activitynmol pNA/mg protein1None9.67 ± 1.2Native LDL11.6 ± 2.3OxLDL50.2 ± 6.3+ Ebselen (50 μm)12.6 ± 2.1+ SOD (500 units)40.3 ± 5.9+ Catalase (500 units)42.6 ± 5.0+ MnTBAP (100 μm)14.9 ± 1.3+ FeTBAP (10 μm)16.4 ± 1.9+ TBAP (100 μm)43.9 ± 5.2+ DTPA (10 μm)16.3 ± 2.4+ Desferral (10 μm)17.2 ± 2.6+ BHT (10 μm)11.9 ± 1.9+ PBN (1 mm)16.4 ± 3.4OxLDL was prepared by adding ONOO− (100 μm) to native LDL (150 μg/ml) in 50 mm phosphate buffer after 2 h, the LDL particle was extensively dialyzed to remove excess ONOO− and other oxidants. BAEC were treated with oxLDL for 16 h. Values are mean ± S.D. of three independent experiments. Open table in a new tab OxLDL was prepared by adding ONOO− (100 μm) to native LDL (150 μg/ml) in 50 mm phosphate buffer after 2 h, the LDL particle was extensively dialyzed to remove excess ONOO− and other oxidants. BAEC were treated with oxLDL for 16 h. Values are mean ± S.D. of three independent experiments. In order to examine the mechanism of oxLDL-induced apoptosis, the effects of a range of antioxidants were examined (Table I). These compounds were chosen based on their wide range of targets and on their differential compartmentalization. BHT, a peroxyl radical scavenger, and DTPA, a metal chelator, both inhibited caspase-3 activation. This suggests that metal ion-dependent lipid peroxidation propagation reactions, involving the breakdown of LOOH and the formation of LO⋅/LOO⋅ are important steps in mediating the apoptotic cascade. Ebselen, a selenium-containing glutathione peroxidase mimetic abolished the activation of oxLDL-mediated caspase-3. Ebselen is able to access both the intracellular and extracellular compartments and cause degradation of both intracellular and extracellular hydroperoxides (48Sies H. Parnham M. Expert Opin. Investig. Drugs. 2000; 9: 607-619Crossref PubMed Scopus (254) Google Scholar). However, the availability of thiols (which are required to mediate ebselen-dependent peroxide decomposition) may be limited in the extracellular environment. The antioxidant enzymes, SOD and catalase, had little effect on caspase-3 activation. As the actions of these enzymes are limited to the extracellular environment, this observation implies that neither superoxide nor hydrogen peroxide present in the extracellular compartment is involved in the initiation of apoptosis. In contrast, the cell-permeable metalloporphyrin SOD mimetics, MnTBAP and FeTBAP, inhibited caspase-3 activation to almost control levels (49Faulkner K.M. Liochev S.I. Fridovich I. J. Biol. Chem. 1994; 269: 23471-23476Abstract Full Text PDF PubMed Google Scholar, 50Lee J. Hunt J.A. Groves J.T. J. Am. Chem. Soc. 1998; 120: 7493-7501Crossref Scopus (163) Google Scholar, 51Konorev E.A. Kennedy M.C. Kalyanaraman B. Arch. Biochem. Biophys. 1999; 368: 421-428Crossref PubMed Scopus (115) Google Scholar). It is noteworthy that FeTBAP was effective at a 10-fold lower concentration than MnTBAP. Although the exact reasons for this differential effect are not understood, it is consistent with the increased activity of FeTBAP as a catalyst of superoxide dismutation or ONOO−decomposition. The metal-free porphyrin, TBAP, had little effect on caspase-3 activation. Nitrone trap caused a 70% inhibition in caspase-3 activation as did desferral, a chelator of redox-active iron (Table I). The present data suggest that oxLDL-induced intracellular superoxide or chain-propagating lipid peroxyl radicals mediate the intracellular signal transduction mechanism leading to caspase-3 activation. We previously reported that ⋅NO donor compounds and S-nitrosothiols will inhibit the toxicity of oxLDL to endothelial cells in culture (52Struck A.T. Hogg N. Thomas J.P. Kalyanaraman B. FEBS Lett. 1995; 361: 291-294Crossref PubMed Scopus (62) Google Scholar). We ascribed this effect to the ability of ⋅NO to scavenge LOO⋅radicals and so to prevent lipid hydroperoxide-mediated oxidation of the membranes (35Hogg N. Kalyanaraman B. Joseph J. Struck A. Parthasarathy S. FEBS Lett. 1993; 334: 170-174Crossref PubMed Scopus (368) Google Scholar, 36Rubbo H. Radi R. Trujillo M. Telleri R. Kalyanaraman B. Barnes S. Kirk M. Freeman B.A. J. Biol. Chem. 1994; 269: 26066-26075Abstract Full Text PDF PubMed Google Scholar, 53Goss S.P.A. Hogg N. Kalyanaraman B. J. Biol. Chem. 1997; 272: 21647-21653Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). In the present study we examined whether nitric oxide donors and S-nitrosothiols could inhibit oxLDL-mediated apoptosis. Fig. 3 confirms the pro-apoptotic effect of oxLDL on BAEC, as treatment of cells with ONOO−-modified oxLDL for 18 h results in significant TUNEL positive staining (Fig. 3 A). Both the nitric oxide donor, DETA/NO, and the S-nitrosothiol, GSNO, significantly prevented the TUNEL positive staining (Fig. 3, C andD), suggesting that these compounds inhibited DNA fragmentation. These results are shown quantitatively in Fig.3 E. Caspase-3 is a cysteine protease that is synthesized in an inactive "pro" form. The apoptotic cascade results in the proteolytic activation of pro-caspase to generate active caspase-3. It has been demonstrated that caspase-3 can be inactivated byS-nitrosation of the active site thiol (54Mannick J.B. Hausladen A. Liu L. Hess D.T. Zeng M. Miao Q.X. Kane L.S. Gow A.J. Stamler J.S. Science. 1999; 284: 651-654Crossref PubMed Scopus (710) Google Scholar, 55Rossig L. Fichtlscherer B. Breitschopf K. Haendeler J. Zeiher A.M. Mulsch A. Dimmeler S. J. Biol. Chem. 1997; 274: 6823-6826Abstract Full Text Full Text PDF Scopus (384) Google Scholar). In addition to DNA fragmentation, ONOO−-treated LDL caused an almost 5-fold induction in caspase-3 activity, as measured by following the formation of p-nitroanilide (Table I). Both DETA/NO and GSNO substantially inhibited caspase-3 activation (Fig.4 A). This inhibition of caspase activity was not due to S-nitrosation as the addition of dithiothreitol, which will remove anyS-nitrosothiols, did not alter the caspase activity (data not shown). The time course of caspase activation by oxLDL is shown in Fig.4 B. Caspase-3 activity was stimulated between 4 and 8 h after adding oxLDL, and remained high for up to 24 h. In the presence of an ⋅NO donor, DETA/NO, only a slight increase in caspase-3 activity was observed over 24 h, but the large increase in activity between 4 and 8 h was abolished. DETA/NO, an extracellular ⋅NO donor, spontaneously decays within the cell culture medium. Consequently, ⋅NO is generated in the solution above the cells. To examine the effect of intracellular ⋅NO production, we used a novel set of compounds that pass through the cell membrane and are de-esterified inside the cell to give the active⋅NO donor (56Saavedra J.E. Shami P.J. Wang L.Y. Davies K.M. Booth M.N. Citro M.L. Keefer L.K. J. Med. Chem. 2000; 43: 261-269Crossref PubMed Scopus (114) Google Scholar). Consequently, ⋅NO released from these compounds is generated within the intracellular environment. As shown in Fig. 5, these compounds were able to suppress oxLDL-mediated caspase-3 activation at concentrations that were 50–100-fold lower than the extracellular ⋅NO donor, DETA/NO. With 1 μm of these esterase-sensitive intracellular ⋅NO donors, robust inhibition was observed, whereas 5 μm AcOM-DEA/NO or AcOM-PYRRO/NO completely suppressed caspase-3 activation (Fig. 5). To determine the component of the LDL particle that was responsible for apoptosis, we isolated the lipid component of oxidized and native LDL and examined the effects of the extracts on caspase-3 activation. As shown in Fig. 6, exposure of cells to the lipid extract of native LDL resulted in only a minor increase in caspase activity,

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