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(The FASEB Journal. 2000;14:1996-2007.)
© 2000 FASEB

Mildly oxidized low density lipoprotein activates multiple apoptotic signaling pathways in human coronary cells

CLAUDIO NAPOLI*,{dagger}1, OSWALD QUEHENBERGER*, FILOMENA DE NIGRIS{dagger}, PASQUALE ABETE{dagger}, CHRISTOPHER K. GLASS* and WULF PALINSKI*1

* Department of Medicine-0682, University of California, San Diego, La Jolla, California 92093, USA; and
{dagger} Departments of Clinical and Experimental Medicine and Gerontology, Federico II University of Naples, 80131 Naples, Italy

1Correspondence: Department of Medicine, 0682, University of California, San Diego, 9500 Gilman Drive, MTF 110, La Jolla, CA 92093-0682, USA. E-mail: wpalinski{at}ucsd.edu/cnapoli{at}ucsd.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis of arterial cells induced by oxidized low density lipoproteins (OxLDL) is thought to contribute to the progression of atherosclerosis. However, most data on apoptotic effects and mechanisms of OxLDL were obtained with extensively oxidized LDL unlikely to occur in early stages of atherosclerotic lesions. We now demonstrate that mildly oxidized LDL generated by incubation with oxygen radical-producing xanthine/xanthine oxidase (X/XO) induces apoptosis in primary cultures of human coronary endothelial and SMC, as determined by TUNEL technique, DNA laddering, and FACS analysis. Apoptosis was markedly reduced when X/XO-LDL was generated in the presence of different oxygen radical scavengers. Apoptotic signals were mediated by intramembrane domains of both Fas and tumor necrosis factor (TNF) receptors I and II. Blocking of Fas ligand (FasL) reduced apoptosis by 50% and simultaneous blocking of FasL and TNF receptors by 70%. Activation of apoptotic receptors was accompanied by an increase of proapoptotic and a decrease in antiapoptotic proteins of the Bcl-2 family and resulted in marked activation of class I and II caspases. Mildly oxidized LDL also activated MAP and Jun kinases and increased p53 and other transcription factors (ATF-2, ELK-1, CREB, AP-1). Inhibitors of Map and Jun kinase significantly reduced apoptosis. Our results provide the first evidence that OxLDL-induced apoptosis involves TNF receptors and Jun activation. More important, they demonstrate that even mildly oxidized LDL formed in atherosclerotic lesions may activate a broad cascade of oxygen radical-sensitive signaling pathways affecting apoptosis and other processes influencing the evolution of plaques. Thus, we suggest that extensive oxidative modifications of LDL are not necessary to influence signal transduction and transcription in vivo.—Napoli, C., Quehenberger, O., de Nigris, F., Abete, P., Glass, C. K., Palinski, W. Mildly oxidized low density lipoprotein activates multiple apoptotic signaling pathways in human coronary cells.


Key Words: oxidized LDL • atherosclerosis • FasL • TNF receptors • caspases • MAPK • JunK


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DURING ATHEROGENESIS, INTIMAL macrophages and smooth muscle cells (SMC) take up large amounts of oxidized low density lipoproteins (OxLDL). Death of these cells then leads to the progressive formation of the necrotic core of atheromas (1) . Although necrosis induced by cytotoxicity accounts for some intimal cell death, programmed cell death may also play a role in the evolution of atherosclerosis (reviewed in ref 2 ). Electron microscopy revealed characteristic features of apoptosis in atherosclerotic lesions, such as shrinkage of cell membranes, nuclear chromatin condensation, and formation of apoptotic bodies. The more sensitive and specific terminal deoxynucleotidyl transferase (TdT) -mediated dUTP nick-end labeling (TUNEL) technique also indicated the presence of apoptotic cells in vivo (3 , 4) . Other evidence includes the colocalization of caspase 3 and apoptotic cells in advanced human atherosclerotic plaques (5) , the presence of the proapoptotic factor Bax in intimal SMC (6) , and the expression of p53 in lesions (7) .

Increasing evidence suggests that oxidation of LDL may be responsible for apoptosis in the arterial wall. OxLDL is prevalent in atherosclerotic lesions (8) and promotes atherogenesis by enhancing uptake of OxLDL by macrophage scavenger receptors, promoting recruitment of circulating monocytes (9 , 10) , and up-regulating chemotactic and growth-promoting factors in arterial cells (11) . OxLDL may also play an important role in initiating atherogenesis in human fetuses and in determining the rate of progression of atherosclerosis later in life (10 , 12 , 13) . Finally, OxLDL triggers humoral and cellular immune responses capable of modulating progression of atherosclerosis (14) .

OxLDL may induce apoptosis in all arterial cells (7 , 15 16 17 18 19 20 21) . Some of the apoptotic mechanisms triggered by OxLDL have also been investigated. In vitro studies suggest an involvement of Fas receptors and ligands (20) , activation of caspase 3 (18) , down-regulation of caspase inhibitors (21) , and activation of regulatory signal transducers, such as Bcl-2 (19 , 22) , STAT (23) , and NF{kappa}B (24 , 25) , which may promote apoptosis or growth. Nevertheless, the biological relevance of apoptotic signaling pathways induced by OxLDL for the evolution of atherosclerotic lesions remains unknown, because the majority of previous studies used extensively oxidized LDL generated by incubation with micromolar concentrations of copper. However, levels of copper found in plasma and arteries are a million-fold smaller than those used in vitro (26) and the mechanisms actually inducing LDL oxidation in vivo are unknown. Furthermore, little is known about the degree of LDL modification in the arterial wall. LDL extracted from advanced, necrotic lesions showed extensive apolipoprotein fragmentation (8) , but the onset of apoptosis appears to occur in earlier stages of lesions (2) . It is therefore more likely that cells in early atherosclerotic lesions are mainly exposed to less extensively oxidized LDL.

The goal of the present study was to determine whether mildly oxidized forms of LDL may also trigger apoptosis in human endothelial cells and SMC and to explore oxygen radical-sensitive apoptotic signaling pathways activated by mildly oxidized LDL. We initially focused on apoptotic mechanisms established for extensively oxidized LDL (e.g., the Fas receptor pathway and the activation of caspases) and then investigated pathways not previously linked to oxidized LDL, such as tumor necrosis factor (TNF) receptors and the activation of MAP and Jun kinase-dependent transcription factors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
LDL isolation and oxidation
Plasma was obtained from healthy nonsmoking males (n=6, age 23±5 years) and LDL was isolated by two rapid consecutive steps of ultracentrifugation in a KBr gradient, as described previously (27) . LDL (300 µg/ml) was incubated for 12 h at 37°C with xanthine (2 mM final concentration) and xanthine oxidase (100 mU/ml, salicylate-free, from bovine milk, specific activity 1 U/mg of protein) in 0.150 M NaCl-0.01 M sodium phosphate (pH 7.4), as described previously in detail (27 28 29 30) . The xanthine/xanthine oxidase (X/XO) reaction yields a short burst of superoxide radicals, singlet oxygen, and hydrogen peroxide, and therefore mimics oxygen radical generation in vivo. These radicals in turn generate hydroxyl radicals, which promote an oxidative chain reaction and further modification of LDL. Under the above conditions, only mildly oxidized LDL is generated (Table 1 and refs 27 28 29 30 ). This mildly oxidized LDL differs from ‘minimally’ oxidized LDL, extensively oxidized LDL generated by incubation with copper ions (5 µM for 24 h at 37°C), and other forms of chemically modified LDL in both the extent and the nature of modification. For example, when the incorporation of 14C oleic acid into cholesteryl esters in resident peritoneal macrophages after 12 h was used as the index of ‘bioactive’ modification, this was 2.1 ± 0.7* nmol/mg cell protein in native LDL, 12.3 ± 0.9* in X/XO-LDL, 56.8 ± 11.5**# in LDL oxidized for 24 h with 5 µM copper, and 575 ± 105 in acetylated LDL (*P<0.0001 and **P<0.001 vs. acetylated LDL; # P<0.001 vs. X/XO-LDL) (29) .


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  		Table 1. Degree of LDL oxidation induced by incubation of LDL with copper ions or xanthine/xanthine oxidase (X/XO) in the absence or presence of scavengersa

Actual superoxide radical production of the X/XO system was monitored in parallel experiments by following the reduction of cytochrome-c (1.2 mM) at 550 nm in a double-beam spectrophotometer (Uvikon 810, Kontron, Zurich, Switzerland). This system generates ~20 nmol x min/ml of superoxide radicals and ~40 nmol x min/ml of hydrogen peroxide at peak activity (i.e., at 1.5 min) and then progressively declines within 6 min (28 , 30) . An incubation time of 12 h was chosen because the peroxidative chain reaction initiated by X/XO continues to modify LDL long after the initial exposure to radicals generated by X/XO (28 29 30 31) . Oxygen radical scavengers were added to the LDL solution immediately before adding X/XO. Superoxide dismutase (SOD), a superoxide scavenger, was added to a final concentration of 330 U/ml (28 , 30) ; catalase, a hydrogen peroxide scavenger, was added to a final concentration of 1000 U/ml (28 , 30) , and histidine, a scavenger of the singlet oxygen radical (32) to 10 mM. In all experiments, LDL was first oxidized with X/XO in the absence or presence of scavengers. Because oxygen radicals and scavengers may have direct effects on oxidation-dependent intracellular processes that may confound the effects of mildly oxidized LDL, X/XO-LDL was extensively dialyzed against phosphate-buffered saline (PBS) at 4°C before being incubated for 24 h with cells.

Cell cultures
Primary human coronary endothelial cells and SMC were cultured as described previously (33) . For FACS analysis experiments, cells were scraped, transferred into 15 ml Falcon tubes, and centrifuged at 1500 rpm for 5 min. Cell pellets were then twice resuspended in 3 ml PBS and centrifuged again under the same conditions.

DNA laddering
DNA fragmentation was assessed on agarose electrophoresis gels, as described (34) . Adherent and nonadherent cells were collected, washed twice with cold PBS, and resuspended in lysis buffer containing 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 10 mM EDTA, 1% sodium dodecyl sulfate (SDS), and 0.1 mg/ml proteinase K. Cell suspensions were incubated at 4°C for 30 min and then centrifuged at 13,000 rpm for 10 min. Supernatants were extracted with 1:1 (v/v) phenol/chloroform (24:1 isoamylic) and precipitated with 2.5 volumes of pure ethanol. DNA pellets were resuspended in Tris-EDTA buffer and applied to a 1.2% agarose gel in TBE.

TUNEL assay
Apoptosis in cultured cells was also assessed by the TUNEL technique, using the ‘In situ cell death detection kit’ (Boehringer Mannheim, Mannheim, Germany), as described (34) .

Flow-cytometric analysis
Cells were prepared as described above, DNA stained with 50 µg/ml propidium iodide, and analyzed using a FACScan flow cytometer with fluorometric detection of both frontal and lateral diffusion (FACS VantageTM, Becton Dickinson, San Jose, Calif.) interfaced with a Hewlett Packard computer (34) . Cell cycle data analysis was performed with CELL-FIT software (Becton Dickinson). Cell distribution into different phases of the cell cycle was obtained by mathematical fitting of fluorometric histograms. Specifically, the first peak represents the fluorescence of cells in the G0/G1 phase (diploid DNA), the second represents G2 and M phases (premitotic and mitotic phases, and thus tetraploid DNA), and cells in the S phase are distributed between the two major peaks. Apoptotic cells (breaking DNA) represent the Sub-G1 phase and are localized before the first peak.

Fas and TNF receptor-mediated apoptosis
The contribution of the intramembrane Fas receptor to X/XO-LDL-mediated apoptosis was investigated by using a combination of two blocking goat polyclonal antibodies against the carboxyl and amino-terminal ends of human Fas ligand (FasL) (C20-G and Q20, respectively; Santa Cruz Biotechnology, Santa Cruz, Calif.). In addition, competition assays were carried out by adding the blocking antibody to FasL together with an agonistic rabbit polyclonal antibody against full-length human Fas (FL-335, Santa Cruz Biotechnology). The latter recognizes an epitope spanning from amino acids 1 to 335 of Fas and acts as ligand, activating Fas. Antibodies were added to 70% confluent cells in 6-well cell culture slides at 10 µg/ml (C20 sc-957-G and Q20) or 100 µg/ml (FL-335). Twenty minutes after addition of the blocking antibodies, native LDL, X/XO-LDL, or X/XO-LDL generated in the presence of scavengers was added and cells were incubated for 24 h at 37°C. As a control, an irrelevant (nonspecific) human IgG was used. Cells were then paraformaldehyde-fixed and incubated with TUNEL solution (see below).

The contribution of the two apoptotic TNF receptors, TNFRI and TNFRII, in X/XO-LDL-induced apoptosis was assessed in an analogous fashion by simultaneous use of 10 µg/ml each of two blocking antibodies against TNFRI (C20 sc-1068) and TNFRII (L20, both from Santa Cruz Biotechnology). Since TNF may induce apoptosis by itself (35) , no competitive experiments with the ligand (TNF) were performed.

Western blot analysis
Whole-cell extracts were prepared as follows. Cells were scraped in cold PBS (4°C) and lysed in cold lysis buffer consisting of 0.5% Nonidet P-40 (Sigma, St. Louis, Mo.) in 50 mM HEPES (pH 7.5), 250 mM NaCl, 5 mM EDTA, 50 mM NaFl, 0.5 mM sodium orthovanadate, 0.5 mM phenylmethyl-sulfonyfluoride (PMSF), 5 µg/ml aprotinin, and 5 µg/ml leupeptin. Fifty micrograms of proteins separated by 12.5% SDS-polyacrylamide gel electrophoresis (PAGE) were transferred to Immobilon-P transfer membranes (Millipore) and Western blot analysis was performed according to standard procedures (36) , modified as described previously (37) . Antibodies to Bcl-2 (goat polyclonal IgG, # N-19), Bax (goat polyclonal IgG, # N-20), PARP (rabbit polyclonal IgG, # H250), caspase 1 (rabbit polyclonal IgG, # A-19), caspase 3 (CPP32) (rabbit polyclonal IgG, # H-277 or mouse monoclonal IgG, # E-8), caspase 6 (Mch2) (goat polyclonal IgG, # K-20), caspase 8 (Mch5) (goat polyclonal IgG, # C-20), p53 (mouse monoclonal IgG, # DO-1), c-Jun (rabbit polyclonal IgG, # H-79), CREB-1 (rabbit polyclonal IgG, # C-21), only the phosphorylated form of CREB-1 (p-CREB) (mouse monoclonal IgG, # Ser133), an antibody to the carboxyl terminus of human ATF-2 that recognizes both forms of ATF-2 (phosphorylated and unphosphorylated) (rabbit polyclonal IgG, # C-19), an antibody against the carboxyl terminus of ELK-1 that recognizes both forms of ELK-1 (rabbit polyclonal IgG, # I-20), an antibody against the carboxyl terminus of NF{kappa}B p65 (goat polyclonal IgG, # C-20), an antibody against amino terminus of I{kappa}B{alpha} (goat polyclonal IgG, # C-15), and an antibody to the carboxyl terminus of Bad that binds both forms of Bad (rabbit polyclonal IgG, # R-20) were purchased from Santa Cruz Biotechnology. An antibody against the phosphorylated form of Jun (p-Jun) (mouse monoclonal, # 420110-S) was purchased from Calbiochem Signal Transduction (San Diego, Calif.). Optimal antibody concentrations were determined in pilot assays (usually 1:1000 dilution). Antibodies bound to their respective antigen in the membrane were visualized using species-specific monoclonal second antibodies against the Fab region of the primary antibody labeled with horseradish peroxidase. After adding substrate, luminescence was determined in an ECL luminometer (Amersham, Milan, Italy) and exposed to autoradiograph film (X-ray, Kodak) for 2 min. To ascertain that blots were loaded with equal amounts of protein lysates, membranes were also incubated with a polyclonal antibody against {gamma} tubulin protein (Sigma).

Kinase assay
In contrast to all other experiments involving inhibitors or competitors, in the kinase assays cells were preincubated for 1 h with 50 µg/ml of an inhibitor of the map kinase MEK1 (PD98059, Calbiochem Signal Transduction) or a blocking peptide against the carboxyl-terminal end of Jun kinase JKK1 (Cat. # 347–363, Calbiochem-Novabiochem Corporation). The culture medium was then changed, and X/XO-LDL prepared in the absence or presence of scavengers was added to control or MEK1 inhibitor-treated cells. After 24 h of incubation at 37°C, cells were lysed with lysis buffer (PBS containing 1% Nonidet NP-40 (Sigma, Aldrich), 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml protease inhibitor PMSF, 30 µg/ml aprotinin A, and 100 mM sodium orthovanadate. Protein concentration was determined by Lowry assay. Kinase activity (38) was determined using the Kinase SPA Enzyme Assay system (Amersham, Little Chalfont, U.K.) as described (34) . Results were expressed as kinase activity. Zero activity was defined by cells treated with MEK1 inhibitor only.

Briefly, 400 µg of protein were immunoprecipitated by incubating them for 60 min at 4°C with 1–2 µg of the ERK1 and ERK2 antibodies for MAP-kinase activity and antibodies against the SAPK/JNK phospho-specific human domain for Jun-kinase activity (CALBIOCHEM). Immunoprecipitated proteins were collected and incubated overnight at 4°C with 20 µg protein A Sepharose (Pharmacia-Biotech, Sweden). Pellets were resuspended in 25 µl kinase buffer containing 50 mM HEPES (pH 7.5), 0.1 mM EDTA, 0.1 mg/ml BSA, 0.15 M NaCl, 0.1% mercaptoethanol. This was followed by addition of 5 µg myeloblastic basic protein as substrate for MAP kinase and 5 mg of GST fusion protein of c-Jun as substrate for Jun-kinase (CALBIOCHEM), and of 10 µl of ATP mix solution [930 µl kinase buffer, 6 µl 50 mM ATP (pH 7.0), 20 µl 2 mM MgCl2, 44 µl {gamma}P32-ATP (10 mCi/ml)]. After boiling for 5 min and SDS-PAGE, the incorporated radioactive phosphate was determined using a PhosphorImager (GS-525 Bio-Rad, Milan, Italy) interfaced with a Hewlett-Packard computer.

Nuclear extract preparation
Nuclear extracts were prepared as described previously (34) . Briefly, cells were disrupted by forced passage through a 26-gauge needle and nuclei were collected by centrifugation at 1500 rpm and resuspended in 1.2 volumes of an extraction solution consisting of 10 mM HEPES (pH 7.9), 0.4 M NaCl, 1.5 mM MgCl2, 0.1 mM ß-aminoethyl-ether-N,N,N',N',-tetraacetic acid, 0.5 mM DTT, and 5% glycerol.

Electrophoretic mobility shift assay (EMSA)
Nuclear extracts (2.5–5 µg of proteins) were preincubated for 10 min at room temperature in 20 µl of a solution consisting of 20 mM HEPES (pH 7.5), 40 mM KCl, and 5% glycerol containing 1 µg poly(dI-dC) and 5 mM spermidine (34 , 39) . Binding reactions were incubated for an additional 15 min with the following probes: consensus binding site for NF{kappa}B (Cat. # sc-2511, Santa Cruz): 5'-AGTTGAGGGGACTTTCCCAGGC-3'; binding site for AP-1 (# sc-2514, Santa Cruz): 5'-CGCTTGATGACTCAGCCGGAA-3'. Samples were then separated on 8% native PAGE.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Apoptotic effects of mildly oxidized LDL on endothelial and SMC
The apoptotic effects of LDL mildly oxidized with xanthine/xanthine oxidase (X/XO-LDL) on cultured human coronary artery endothelial cells and SMC were first tested by determining the percentage of TUNEL-positive nuclei and the degree of DNA fragmentation by agarose gel electrophoresis. Results for endothelial cells (Fig. 1 , left) and SMC (not shown) were very similar. Incubation with either Cu2+-LDL (lane B)or X/XO-LDL (lane C) greatly increased the number of TUNEL-positive cells (upper panel) and resulted in extensive DNA laddering (lower panel), compared to native LDL (lane A). Both indicators of apoptosis were markedly reduced when cells were incubated with LDL oxidized by X/XO in the presence of SOD (lane D), catalase (lane E), or combined SOD, catalase, and histidine (lane F). When incubated with X/XO-LDL prepared in the combined presence of SOD, catalase, and histidine, TUNEL-positive cells and DNA fragmentation were reduced to almost control levels (lane F). Electron microscopy of cells incubated with X/XO-LDL also demonstrated the presence of typical signs of apoptosis such as DNA fragmentation, disruption of cytoplasmic organelles, and loss of membrane integrity. Figure 1B provides a typical example of nuclear chromatin condensation in a SMC incubated for 2 h with X/XO-LDL.



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  		Figure 1. Apoptotic effect of mildly oxidized LDL on cultured human coronary endothelial cells. Left panel: Cells were incubated with LDL that had been oxidized with copper ions or xanthine/xanthine oxidase (X/XO-LDL) in the absence or presence of oxygen radical scavengers, and apoptosis was assessed by TdT-mediated dUTP nick-end labeling (TUNEL) (top) and DNA laddering (bottom). A: Native LDL; B: Cu2+-LDL; C: X/XO-LDL; D: X/XO-LDL + SOD; E: X/XO-LDL + catalase (CAT); F: X/XO-LDL + SOD + CAT + histidine. TUNEL-positive nuclei are indicated by dark staining. Data shown are representative for 6 experiments. Right panel: Electron microscopic image of a smooth muscle cell after 2 h incubation with X/XO-LDL showing nuclear chromatin condensation (1.5 µm).

Involvement of Fas and TNF receptors
To investigate whether X/XO-LDL mediates cell death by ligand-mediated activation of apoptotic receptors of the TNF family (36) , we first analyzed the contribution of Fas, an intramembrane domain that may trigger an apoptotic signal when activated by FasL (40 , 41) .

Figure 2 shows the percent of TUNEL-positive cells induced by incubation of endothelial cells or SMC with Cu2+-LDL or with X/XO-LDL (or X/XO-LDL plus scavengers) in the absence or presence of blocking antibodies against FasL. As expected from the preceding results, incubation of endothelial cells and SMC with X/XO-LDL resulted in ~80% TUNEL-positive cells, slightly less than Cu2+-LDL. Similar degrees of apoptosis were induced by X/XO-LDL in cells preincubated with a nonspecific IgG. Preincubation with the anti-FasL antibody reduced TUNEL-positive cells induced by X/XO-LDL to about half, whereas a lesser degree of inhibition was seen for Cu2+-LDL. When cells were preincubated with a blocking antibody to FasL together with an agonistic antibody to Fas itself (which activates Fas), no inhibition of apoptosis was seen. A significant albeit relatively smaller antiapoptotic effect of the antibody to FasL was also seen in cells incubated with mildly oxidized LDL generated in the presence of SOD, catalase, or a combination thereof. Together, these data indicate that ~50% of the apoptotic effect of mildly oxidized LDL is mediated by the Fas receptor.



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  		Figure 2. Involvement of the Fas receptor in apoptosis induced by Cu2+-LDL and mildly oxidized LDL. Endothelial cells and SMC were preincubated for 20 min with 10 µg/ml of neutralizing antibodies to Fas ligand ({alpha}FasL) alone or in combination with 0.5 µg/ml of an antibody to the Fas receptor ({alpha}Fas), with an irrelevant nonspecific IgG, or with culture medium only. Cells were then incubated for 24 h with 200 µg/ml of Cu2+-LDL or with X/XO-LDL prepared with or without scavengers. Apoptosis was measured as the % of TUNEL-positive cells. Data are mean ± SD of 6 experiments. Med, cell culture medium without LDL; CAT, catalase; SOD, superoxide dismutase; HIS, histidine. *P < 0.01 vs. X/XO-LDL; **P < 0.001 and ***P < 0.01 vs. Cu2+-LDL without antibody; #P < 0.05 vs. the respective control without {alpha}Fas. In addition to significances indicated in the figure, all X/XO-LDL-treated cells were significantly different from LDL-treated cells (P < 0.001).

We then investigated the role of TNF receptors by using blocking antibodies against the two apoptotic members of the TNF receptor family, TNFRI and TNFRII (41 , 42) . As shown in Fig. 3 , only the simultaneous blockage of both TNFRI and TNFRII significantly inhibited the apoptotic effect of mildly oxidized LDL. Results for Cu2+-LDL were similar. This shows that both receptors are involved in apoptosis. However, antibody blocking only led to a 20% reduction of TUNEL-positive cells induced by both forms of oxidized LDL. Simultaneous addition of blocking antibodies to FasL, TNFRI, and TNFRII resulted in a 70% reduction of apoptosis (data not shown). Together, these experiments indicate that apoptosis of endothelial cells and SMC induced by Cu2+-LDL or mildly oxidized LDL is mediated by the Fas receptor and, to a lesser degree, receptors of the TNF receptor family.



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  		Figure 3. Analogous experiment as in Fig. 2 testing the involvement of TNF receptors in apoptosis induced by Cu2+-LDL or mildly oxidized LDL. Smooth muscle cell were preincubated with blocking antibodies to apoptotic TNF receptors I and II, or culture medium alone, and then incubated for 24 h with Cu2+-LDL or with X/XO-LDL prepared in the absence or presence of scavengers. Data are mean ± SD of 6 experiments. *P < 0.01 vs. X/XO-LDL; **P < 0.01 vs. Cu2+-LDL; #P < 0.05 vs. the respective control without anti-TNFRI and anti-TNFRII. In addition to significances indicated in the figure, all X/XO-LDL-treated cells and Cu2+-LDL were significantly different from LDL-treated cells (P<0.001).

The contribution of the Fas receptor was also investigated by FACS. Without blocking antibody to FasL, incubation of endothelial cells with X/XO-LDL resulted in ~95% apoptotic cells, i.e., cells in the Sub G-1 phase. In the presence of the antibody, apoptotic cells were reduced to 69%. Accumulation of apoptotic cells in the Sub-G1 fraction did not block cell cycle progression. The presence of scavengers during the generation of X/XO-LDL markedly decreased the percentage of apoptotic cells, and the protective effect of different scavengers was cumulative. When measured by FACS, the reduction of X/XO-LDL-induced apoptosis by the blocking antibodies was smaller than the reduction measured by TUNEL. For example, simultaneous addition of blocking antibodies to FasL, TNFRI, and TNFRII caused a 42% (range 27–56%) reduction of Sub-G1 cells but an ~70% reduction of apoptosis measured by TUNEL (data not shown). In most of the following experiments, results obtained by TUNEL were therefore verified by FACS. Very similar results were obtained with Cu2+-LDL (data not shown).

Activation of caspases
Upon ligand activation and oligomerization of the Fas and TNF receptors, their intracellular death domains interact with adaptor molecules containing death effector domains or caspase activation and recruitment domains, such as Fas-associated death domain (41 , 42) . These in turn trigger the activation of caspases, the major effectors of apoptosis. To determine whether X/XO-LDL-induced activation of apoptotic receptors leads to caspase activation, two class I caspases (caspases 2 and 8) and two class II caspases (caspases 3 and 6) were analyzed by Western blot (Fig. 4 ). Incubation with mildly oxidized LDL resulted in increased presence of active subunits of both class I caspases (thought to be the activators of class II caspases) and class II caspases (thought to be the main proteolytic effectors). The presence of scavengers during the incubation of LDL with X/XO markedly reduced caspase activation.



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  		Figure 4. Activation of class I caspases (caspases 2 and 8) and class II caspases (caspases 3 and 6) and presence of caspase substrate PARP in SMC incubated with native LDL or LDL oxidized by xanthine/xanthine oxidase (X/XO-LDL) in the absence or presence of scavengers. Cells were incubated for 24 h with native or oxidized LDL. Procaspases and active caspase subunits generated by proteolytic cleavage were determined by Western blot, as described in Materials and Methods. Active caspase 2 is indicated by p14 and p12 bands, active caspase 8 by p18, active class II caspases 3 by p17, and active caspase 6 by p16. A: LDL; B: X/XO-LDL; C: X/XO-LDL + SOD; D: X/XO-LDL + catalase (CAT); E: X/XO-LDL + SOD + CAT; F: X/XO-LDL + SOD + CAT + histidine. Data are representative for 6 experiments.

The activation of the caspase substrate poly (ADP-ribose) polymerase (PARP) was also determined as another indicator of the involvement of the caspase pathway in X/XO-LDL-induced apoptosis. PARP was markedly increased in cells exposed to X/XO-LDL; its level remained high even when X/XO-LDL had been generated in the presence of scavengers (Fig. 4 , bottom), suggesting the increased PARP expression is induced by minimum apoptotic stress. Very similar results were obtained with Cu2+-LDL (data not shown).

Pro- and antiapoptotic proteins of the Bcl-2 family
While caspase activation through recruitment of procaspases and dimerization constitutes a major pathway of apoptosis induced by Fas or TNF receptor activation, caspase activation is also induced by members of the Bcl-2 family of proteins. These proteins either promote (Bax) or inhibit (Bcl-2) apoptosis and work in conjunction with a distinct ATPase to activate specific caspases (42) . Pro- and antiapoptotic members of this family antagonize each other by forming heterodimers (41) . To determine whether mildly oxidized LDL may also influence apoptosis via Bax/Bcl-2-regulated mechanisms, we investigated the effects of X/XO-LDL and scavengers on Bax, Bcl-2 and Bad (Fig. 5 ). In endothelial cells, there was a marked decrease in the antiapoptotic Bcl-2 protein after incubation with X/XO-LDL, which was reduced by the presence of scavengers during LDL oxidation. No effect of X/XO-LDL on Bax was noticeable. In contrast, Bad, which also antagonizes Bcl-2 and induces apoptosis through cytochrome c-independent activation of caspases (42) , was markedly increased by X/XO-LDL and its up-regulation reduced by scavengers (Fig. 5) . No changes in Bcl-2 proteins were apparent in SMC. These results suggest that in endothelial cells, mildly oxidized LDL may also contribute to apoptosis by shifting the equilibrium between pro- and antiapoptotic members of the Bcl-2 family. Very similar results were obtained with Cu2+-LDL (data not shown).



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  		Figure 5. Western blot analysis of apoptotic factors Bcl-2, Bax, and Bad in endothelial cells and SMC. Cells were incubated for 24 h with LDL oxidized with xanthine/xanthine oxidase (X/XO-LDL) in the absence or presence of scavengers, and proteins of the Bcl-2 family were determined with specific antibodies, as described in Materials and Methods. Tubulin served as reference protein. A: LDL; B: X/XO-LDL; C: X/XO-LDL + SOD; D: X/XO-LDL + catalase (CAT); E: X/XO-LDL + SOD + CAT; F: X/XO-LDL + SOD + CAT + histidine. Data are representative for 6 (Bcl-2 and Bax) or 4 (Bad) experiments.

Activation of MAP and Jun kinase pathways
The transduction of apoptotic signals to the nucleus may also be mediated by activation of MAP kinases, which in turn may activate Jun kinase (43) . Together, MAP and Jun kinases form a complex regulatory system interposed between cytokine-induced activation of cell membrane receptors and downstream signaling factors (38 , 44) . As shown in Fig. 6 , MAP-kinase activity in SMC incubated with Cu2+-LDL and X/XO-LDL was about 12-fold and 9-fold greater than that in cells incubated with native LDL, respectively. Addition of X/XO-LDL prepared in the presence of scavengers resulted in lesser increases (approximately half for X/XO-LDL plus SOD or CAT) or no increase of MAP kinase activity (for X/XO-LDL plus SOD, CAT, and histidine). In each case, preincubation of cells with a MEK1 inhibitor significantly reduced MAP kinase activity induced by Cu2+-LDL or X/XO-LDL ± scavengers, compared to the respective control. More important, TUNEL analysis indicated that the MEK1 inhibitor significantly reduced SMC apoptosis (e.g., by 67.6±6.8% in cells incubated with X/XO-LDL and 52.4% in cells incubated with Cu2+-LDL). Similar results were obtained in endothelial cells (not shown). These data indicate that the MAP kinase pathway plays an important role in mediating apoptosis induced by both forms of oxidized LDL.



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  		Figure 6. Effect of Cu2+-LDL and mildly oxidized LDL on MAP-kinase activity in control cells and cells pretreated with an inhibitor of MEK1. SMC were incubated for 1 h with or without 50 µg/ml MEK1 inhibitor and then for 24 h with cell culture medium, native LDL, Cu2+-LDL, or xanthine/xanthine oxidase LDL (X/XO-LDL) prepared in the absence or presence of scavengers. MAP kinase activity was determined as described in Materials and Methods. Data are the mean ± SD of 6 experiments. Control (culture medium only); SOD, superoxide dismutase; CAT, catalase; HIS, histidine. #P < 0.001 vs. LDL; *P < 0.01 vs. X/XO-LDL; **P < 0.001 vs. X/XO-LDL (or Cu2+-LDL); ***P < 0.0001 vs. X/XO-LDL; +P < 0.001 vs. X/XO-LDL + MEK1 inhibitor; ++P < 0.0001 vs. X/XO-LDL + MEK1 inhibitor. P values for cells incubated with MEK1 inhibitor vs. the respective controls are indicated below the x axis.

Jun, one of the downstream effectors of MAP kinase (43 , 44) should also be activated by X/XO-LDL. Indeed, Western blot analysis revealed a marked increase in the active, phosphorylated form of Jun protein (pc-Jun) in endothelial cells, compared to controls incubated with native LDL (Fig. 7A ). X/XO-LDL prepared in the presence of scavengers induced markedly less Jun activation. The amount of c-Jun protein also appeared increased in cells incubated with X/XO-LDL. Blocking of the Jun kinase JKK1 significantly reduced apoptosis (Fig. 7B ). The degree of reduction was limited, perhaps because of limited cell penetration of the blocking peptide, but inhibition of Jun kinase was dose dependent (data not shown). Direct measurements of Jun kinase activity also indicated that X/XO-LDL significantly increased Jun kinase activity, compared to native LDL (3.5±0.8 arbitrary units vs. 1.1±0.5; P<0.001), and that scavengers progressively reduced it (Fig. 7C ). Similar results were obtained with Cu2+-LDL (Fig. 7A , B , C ).



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  		Figure 7. Effect of Cu2+-LDL or mildly oxidized LDL on Jun. A) Activation of c-Jun. Endothelial cells were incubated for 24 h with Cu2+-LDL or X/XO-LDL prepared in the absence or presence of scavengers. The c-Jun protein and the active (phosphorylated) form of Jun (p-c-Jun) were detected in Western blots of whole-cell lysates using specific monoclonal antibodies. Data are representative for 6 experiments. A: LDL; B: Cu2+-LDL; C: X/XO-LDL; D: X/XO-LDL + SOD; E: X/XO-LDL + catalase (CAT); F: X/XO-LDL + SOD + CAT; G: X/XO-LDL + SOD + CAT + histidine. B) Effect of Jun-kinase inhibition on apoptosis. SMC were incubated for 1 h with or without a blocking peptide against JKK1 (50 µM) and then for 24 h with native LDL, Cu2+-LDL, or xanthine/xanthine oxidase-LDL (X/XO-LDL). Apoptosis was determined as the percentage of TUNEL-positive cells. Data are the mean ± SD of 4 experiments. *P < 0.05 vs. X/XO-LDL. C) Effect of Cu2+-LDL or mildly oxidized LDL on Jun-kinase activity in SMC. Activity was determined as described in Materials and Methods, using an antibody against the SAPK/JNK phospho-specific human domain. **P < 0.01 vs. X/XO-LDL; #P < 0.001 vs. LDL; +P < 0.05 vs. Cu2+-LDL.

Nuclear presence of MAP and Jun dependent transcription factors
To further study the involvement of the kinase pathway in apoptosis induced by mildly oxidized LDL, several kinase-dependent factors were investigated in nuclear extracts. These included the apoptotic factor p53 (activated by the MKK4-JNK1 pathway), ELK-1 (activated by either the MAP-MEK1-ERK-1 or MKK3-p38 pathway), and the ATF-2/CREB family (activated by either the MKK4-JNK1–3 or MEK3-p38 pathways) (44) . As shown in Fig. 8 , the active (phosphorylated) forms of p53, ATF-2, ELK-1 and p-CREB were all markedly increased by X/XO-LDL and their up-regulation was progressively reduced by scavengers. Virtually identical results as for X/XO-LDL were obtained with Cu2+-LDL (data not shown).



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  		Figure 8. Western blot analysis of Jun-dependent factors p53, ATF-2, ELK-1, and CREB-1. SMC were incubated with LDL oxidized with xanthine/xanthine oxidase (X/XO-LDL) in the absence or presence of scavengers, and factors potentially involved in mediating apoptosis were determined with specific antibodies. The upper ATF-2 band represents the phosphorylated, active form. Of the two bands recognized by the antibody to ELK-1, the upper band probably represents the active form. Results shown for CREB were obtained with an antibody specific for the phosphorylated form only (p-CREB). A: LDL; B: X/XO-LDL; C: X/XO-LDL + SOD; D: X/XO-LDL + catalase (CAT); E: X/XO-LDL + SOD + CAT; F: X/XO-LDL + SOD + CAT + histidine. Data are representative for 4 experiments.

Activation of NF{kappa}B and AP-1 complex
Extensively oxidized LDL has previously been shown to enhance phosphorylation of I{kappa}B{alpha} in mononuclear phagocytes (24) . The effect of X/XO-LDL and oxygen radical scavengers on NF{kappa}B was investigated by EMSA of nuclear extracts (Fig. 9A ). In both SMC and endothelial cells, X/XO-LDL induced increased NF{kappa}B activity. Western blot analysis of nuclear extracts also showed a direct effect of mildly OxLDL on I{kappa}B{alpha}/NF{kappa}B proteins (Fig. 9B ). NF{kappa}B was increased in cells exposed to X/XO-LDL. In parallel, I{kappa}B{alpha} was markedly reduced in cells incubated with X/XO-LDL. These effects were partially prevented by scavengers. Similar results as for X/XO-LDL were obtained with Cu2+-LDL (data not shown). Because AP-1 is selectively activated by the JNK1–3 pathway, this provides additional evidence for Jun kinase activation by mildly oxidized LDL or Cu2+-LDL (Fig. 9C ).



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  		Figure 9. Effects of mildly oxidized LDL on nuclear transcription factors NF{kappa}B and I{kappa}B{alpha}. A) Binding shift analysis of the activity of NF{kappa}B in nuclear extracts from smooth muscle and endothelial cells. A: Free oligonucleotides; B: LDL; C: xanthine/xanthine oxidase-LDL (X/XO-LDL); D: X/XO-LDL + SOD; E: X/XO-LDL + catalase (CAT); F: X/XO-LDL + SOD + CAT; G: X/XO-LDL + SOD + CAT + histidine (HIS). B) Western blot analysis of I{kappa}B{alpha} (p42) and NF{kappa}B (p65). A: LDL; B: X/XO-LDL; C: X/XO-LDL + SOD; D: X/XO-LDL + CAT; E: X/XO-LDL + SOD + CAT + HIS. C) Binding shift assay showing the effect of native, Cu2+-LDL, and mildly oxidized LDL on the activity of AP-1 in nuclear extracts of SMC. A: Free oligonucleotides; B: LDL; C: Cu2+-LDL; D: X/XO-LDL; E: X/XO-LDL + SOD; F: X/XO-LDL + CAT; G: X/XO-LDL + SOD + CAT + HIS.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our study provides the most extensive investigation of the effects of oxidized LDL on apoptotic signaling pathways to date and indicates for the first time that OxLDL-induced apoptosis involves TNF receptors and Jun activation. More important, it demonstrates that mildly oxidized LDL likely to be prevalent in earlier stages of atherosclerotic lesions is capable of inducing apoptosis in human coronary artery endothelial cells and SMC by activating multiple oxygen radical-sensitive signaling pathways.

Extensively oxidized LDL has previously been shown to trigger apoptosis of endothelial cells (18 19 20 21) , SMC (17) , macrophages (7 , 15) , and lymphocytes (22) . However, the pathophysiological relevance of OxLDL-induced apoptosis in the arterial wall remained uncertain, because extensively oxidized LDL is unlikely to be formed in transitional lesions, where apoptosis begins (2) .

Our results demonstrated that up to 70% of apoptosis induced by mildly oxidized LDL was mediated by receptors of the TNF receptor family and that both Fas and TNF receptors I and II were involved. To date, only the involvement of the Fas pathway in OxLDL-induced apoptosis had been reported. In fact, copper-oxidized LDL up-regulated endothelial expression of FasL (but not Fas) and apoptosis was reduced in endothelial cells from knockout mice lacking either FasL or Fas (20) . Our experiments with blocking antibodies to FasL and Fas suggest that mildly oxidized LDL also acts mainly by up-regulating expression of FasL. Activation of the Fas pathway results in oligomerization of Fas (41) and recruitment of FADD and FADD homologous, ICE-like protease (FLICE), which then activate caspases. The observation that the FLICE inhibitory protein (which inhibits caspase activation by binding to FLICE) is down-regulated by OxLDL further supports the involvement of the Fas pathway in OxLDL-induced apoptosis (21) . As shown in Fig. 3 , TNF receptors I and II are also involved in OxLDL-induced apoptosis. Although the TNF receptors seemed to play a lesser role than Fas/FasL, our findings are consistent with the immunohistochemical observation that apoptotic cells, OxLDL, and TNF-{alpha} colocalize in atherosclerotic lesions (17) and that TNF-{alpha} induces apoptosis in SMC (17) and macrophages (7) .

As shown in Fig. 4 , mildly oxidized LDL induced activation of two class I caspases (caspases 2 and 8), as well as two class II caspases (3 and 6). To date, an effect of extensively oxidized LDL had only been reported for caspases 1 and 3 (18 , 46) . PARP, a caspase substrate, also was increased by mildly oxidized LDL. This suggests that caspase activation plays a major role in oxidation-induced apoptosis, a conclusion also supported by the fact that inhibitors of caspases 1 and 3 reduced apoptosis induced by extensively oxidized LDL in human umbilical vein endothelial cells (46) .

Proteins of the Bcl-2 family are important regulatory elements for caspase activation and apoptosis (19 , 41 , 46) . Incubation of endothelial cells (but not SMC) with X/XO-LDL increased proapoptotic Bad (Fig. 5) and decreased antiapoptotic Bcl-2 protein. Thus, mildly oxidized LDL seems to induce ‘dynamic movements’ in these regulatory proteins. Extensively oxidized LDL appeared not to affect Bax (21) . However, both the absolute amount of proteins of the Bcl-2 family and their intracellular location may be as important for apoptosis as their overall cellular content. Cultured cardiomyocytes exposed to oxygen radicals showed increased Bad and unchanged Bax and Bcl-2 expression, but superoxide treatment elicited translocation of Bax and Bad from the cytosol into mitochondria (47) .

Another apoptotic mechanism induced by mildly oxidized LDL was the activation of MAP and Jun kinases (for background, see ref 44 ). MAP kinase activity induced by mildly oxidized LDL was significantly decreased by a MEK1 inhibitor, consistent with a previous report on the effects of extensively oxidized LDL on MAP kinases (48) . In addition, our results show that mildly oxidized LDL also activates Jun and that Jun activation contributes to programmed cell death (Fig. 7) . The fact that scavengers greatly reduced activation of MAP and Jun kinases indicates the involvement of oxygen radicals in these processes. Mildly oxidized LDL also increased expression of factors p53, the ATF-2/CREB family, ELK-1, and AP-1. This may merely reflect activation of MAP and Jun kinases. On the other hand, some of the above factors may actively promote apoptosis. For example, phosphorylated forms of ATF-2 and c-Jun (via the MEKK1 pathway) induce FasL expression (44 , 49) . It is therefore possible that activation of ATF-2 contributes to the up-regulation of FasL discussed above, but other proapoptotic effects cannot be ruled out. For example, the DNA binding protein p53 also triggers apoptosis (50) , but no effect of p53 on FasL has been reported.

Together, our data indicate that mildly oxidized LDL up-regulates FasL and TNF receptors I and II and induces cell death by activation of the caspase cascade, which may be increased by a shift toward proapoptotic proteins of the Bcl-2 family. However, we do not know whether the enhanced expression of apoptotic receptors in cultured cells is a direct effect of mildly oxidized LDL or whether it is secondary to cytokine responses triggered by effects of OxLDL on the cell membrane or intracellular effects of oxygen radicals.

Although our results and those of others indicate that OxLDL also induces activation of NF{kappa}B, the role of this nuclear transcription factor in apoptosis is not well understood. The observation that antioxidants inhibit expression of NF{kappa}B-regulated cytokines, such as VCAM-1, in vivo (51) supports the role of oxidation in the up-regulation of NF{kappa}B. If one accepts an antiapoptotic role of NF{kappa}B, its activation by mildly oxidized LDL may indicate that OxLDL can both promote and inhibit apoptosis by different pathways (52) . It may also indicate that proapoptotic effects trigger a protective reaction via NF{kappa}B activation.

Mildly oxidized LDL generally had similar apoptotic effects on SMC and endothelial cells. However, cell culture studies may not necessarily reflect apoptotic mechanisms in humans (2 , 53) , much less actual cell death by apoptosis in the arterial wall. It can safely be assumed that LDL oxidized to a similar mild degree as X/XO-LDL (or X/XO-LDL prepared in the presence of scavengers) is present in both early and transitional atherosclerotic lesions. Indeed, oxidation-specific epitopes are already present in the earliest fatty streaks of premature human fetuses and children (10 , 12 , 13) , as well as in animal models. In contrast, apoptosis seems to occur only in transitional and more advanced lesions. Our results should therefore be considered as evidence for the interference of mildly oxidized LDL in a broad range of intracellular signaling processes, rather than induction of actual cell death. In most cases, we found only quantitative differences in the apoptotic mechanisms induced by Cu2+-LDL and X/XO-LDL, even though the difference in the degree of ex vivo oxidative modification was substantial. Our data support the concept that extensive oxidative modifications of LDL are not necessary to influence a cascade of signaling events in vivo.

The biological importance of LDL oxidation is not limited to the induction of apoptosis. It is increasingly recognized that OxLDL may also modulate the evolution of atherosclerosis by enhancing expression of cytokines and growth factors via NF{kappa}B by down-regulating proinflammatory genes through the peroxisome proliferator-activated receptor {gamma} (54) , altering the activity of the proteasome pathway (55) , and by inducing persistent changes in the arterial wall during fetal development that determine the rate of lesion progression later in life (13) . Oxygen radicals may interfere with many of these mechanisms (52) , and LDL oxidation is not the sole source of peroxidative compounds. Increased formation of oxygen radicals and oxidative compounds in the arterial wall also can result from myocardial reperfusion injury or other inflammatory conditions (30 , 37) . The observation that the scavengers used in the present study had qualitatively similar synergistic effects indicates that different oxygen radicals have analogous effects on signaling pathways. Antioxidants such as vitamin E (34) and C (56) may reduce the degree of apoptosis induced by oxidized LDL. It is therefore likely that even mild shifts in oxidation within the arterial wall may profoundly influence the evolution of atherosclerosis by activating multiple regulatory pathways and nuclear transcription factors.


   ACKNOWLEDGMENTS
 
C.N. would like to dedicate this study to the memory of Dr. Gaetano Salvatore. We regret that for reasons of brevity many relevant original papers could not be quoted. The studies were supported by NHLBI grant HL56989 (La Jolla SCOR in Molecular Medicine and Atherosclerosis), CNR grant 99.00198, MURST grant 96/40%, and ISNIH grant 56980/99.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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