© 1997 American Heart Association, Inc.
Articles |
Oxidized LDLs Induce Massive Apoptosis of Cultured Human Endothelial Cells Through a Calcium-Dependent Pathway
Prevention by Aurintricarboxylic Acid
the Laboratory of Biochemistry, INSERM Unit 466, and the Laboratory of Physiology, INSERM Unit 397 (J.-F.A.), Faculty of Medicine in Rangueil, University Paul Sabatier, Toulouse, France.
Correspondence to Dr A. Negre-Salvayre or Prof R. Salvayre, Laboratoire de Biochimie Maladies Metaboliques, INSERM U-466, CHU Rangueil, 1 Ave J. Poulhes, 31054 Toulouse Cedex, France.
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Abstract |
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Oxidized LDLs are thought to play a central role in atherogenesis. Among their wide variety of biological properties, oxidized LDLs exhibit a cytotoxic effect on cultured vascular cells. Toxic doses of mildly oxidized LDLs elicited massive apoptosis in both primary and immortalized cultures of endothelial cells as shown by characteristic morphological and biochemical changes. Cytoplasmic and nucleic modifications (eg, chromatin condensation and nucleus fragmentation) were visualized by using electron and fluorescence microscopy of intact cells labeled by the fluorescent DNA probe SYTO-11. DNA fragmentation was quantified by ultracentrifugation of chromatin fragments, evaluated in situ by using the TUNEL (Terminal transferase-mediated dUTP-biotin nick end labeling) procedure, and visualized by electrophoresis of radiolabeled DNA fragments showing the characteristic apoptotic ladder. Apoptotic cells became rapidly detached and underwent postapoptotic necrosis that led to cell disintegration. Apoptosis was subsequent to a sustained and delayed peak of cytosolic calcium. Both the calcium peak and apoptosis were blocked by chelating the extracellular calcium with EGTA or by inhibiting the calcium influx by the calcium-channel blockers nifedipine and nisoldipine, thus suggesting that the apoptotic process induced by oxidized LDLs is clearly calcium dependent. Aurintricarboxylic acid, an inhibitor of endonucleases, also blocked the apoptotic process without blocking the calcium peak. These results suggest that toxic doses of mildly oxidized LDLs induce massive apoptosis of endothelial cells through a calcium-dependent mechanism and that this apoptotic process can be prevented by inhibiting the rise of cytosolic calcium or by inhibiting cellular endonucleases by aurintricarboxylic acid.
Key Words: apoptosis • necrosis • endonuclease • oxidized LDL • calcium
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Introduction |
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In addition to playing a physiological role in delivering cholesterol and other lipids to peripheral cells, LDLs play an important role in atherogenesis after undergoing oxidative modifications.1 2 3 4 5 6 LDL oxidation is mediated in vitro by various types of cultured vascular cells and is thought to occur in vivo in the subendothelial area, as suggested by the presence of ox-LDLs in atherosclerotic areas (for review, see References 3 through 6). Ox-LDLs exhibit a wide variety of biological properties, such as formation of foam cells and fatty streaks, induction of gene expression, alterations of coagulation pathways, and arterial vasomotor properties (for review, see Reference 6). Ox-LDLs exhibit a dramatic cytotoxic effect on cultured cells.7 8 9 10 The morphological changes of cultured ECs treated by ox-LDLs11 12 exhibit some similarities (cell retraction and defects) with those of the endothelial cover of atherosclerotic areas.13 14 Such endothelial injury may explain the changes that occur during the progression of fatty streaks to more advanced lesions.13
Toxic cell injury induces a complex sequence of events that leads to cell death.15 Two types of cell death, necrosis and apoptosis, have been discriminated on the basis of morphological studies.16 Apoptosis is characterized by DNA fragmentation, alterations of nucleus morphology, such as chromatin condensation and nucleus fragmentation, organelle relocalization, and cell fragmentation without increased permeability of the plasma membrane.16 17 18 In contrast, necrosis is characterized by cellular swelling, organelle alterations, rupture of the plasma membrane, and finally cell lysis and leakage of the cellular components.16
During toxic cell injury, disruption of Ca2+ homeostasis seems to play a critical role by triggering activation of calcium-dependent degradative enzymes, which results in irreversible damage to cellular components and leads to cell death.19 20 21 Calcium-dependent cytoplasmic proteases are involved in cytoskeletal alterations and blebbing of the plasma membrane19 and necrosis,22 whereas calcium-dependent nuclear endonucleases induce chromatin cleavages and apoptosis.17 18 19 20 21 22 23
The toxic effect of ox-LDLs is mediated by an intense and sustained rise of cytosolic calcium11 24 that in turn activates calcium-dependent enzymes involved in the cellular events leading to necrosis or apoptosis in lymphoid and macrophagic cells.22 25 The cytotoxic effect of ox-LDLs on ECs has been clearly demonstrated,7 8 11 but the type of cell death elicited by ox-LDLs in ECs remains to be elucidated.
In this article we report that ox-LDLs elicit a massive apoptosis but no appreciable necrosis in cultured ECs, that apoptotic cells detach rapidly and undergo a rapid postapoptotic necrosis, and that this apoptosis is mediated by the sustained rise of cytosolic calcium evoked by ox-LDLs.
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Chemicals and Reagents
SYTO-11 was purchased from Molecular Probes, [
-32P]dCTP (800 Ci/mmol) from Isotopchim, quin-2/AM, fura-2/AM, Trypan blue dye, bovine serum albumin, ACA, DAPI, MTT, 2,4,6-trinitrobenzene sulfonic acid, agarose, nifedipine, extra-avidin peroxidase, and diaminobenzidine from Sigma, RPMI 1640, phenol red–free RPMI 1640, fetal calf serum, L-glutamine, penicillin, and streptomycin from GIBCO, Hydragel from Sebia, PGEM DNA ladder and Klenow polymerase from Promega, G-Nome kit (Bio 101) and acrylamide/bisacrylamide solution from Bioprobe, and other chemicals from Merck, Sigma, or Prolabo. Nisoldipine was a generous gift from Bayer AG.
Cell Culture
The human umbilical vein EC line CRL-1998 was obtained from the American Type Culture Collection; the BAEC line GM7372A was from the National Institute of Genetics Human Mutant Cell Repository. Nonimmortalized BAECs were obtained by following the procedure of Gospodarowicz et al26 as previously used.27 The human EBV-transformed lymphoblastoid cell lines P2 and NPA were from our laboratory. Cells were grown in RPMI 1640 (CRL-1998 and lymphoblastoid cells) and Dulbecco's modified Eagle's medium (GM7372A cells and BAECs were used between the 5th and 10th passages). All passages were made by using a splitting ratio of 1:4. Under the standard conditions, ECs (0.4x106 cells/mL) were seeded in six multiwell plates or in falcons (Nunc) when required and grown in their respective media with Glutamax supplemented with 10% heat-inactivated fetal calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. The culture medium of BAECs was supplemented with 1 ng/mL basic fibroblast growth factor. Cells were incubated in a humidified incubator (Heraeus) (5% CO2 at 37°C). Twenty-four hours before LDL incorporation, this medium was replaced by serum-free medium.
LDL Isolation and Oxidation
LDLs from human pooled sera were isolated by using ultracentrifugation according to the method of Havel et al,28 dialyzed against 150 mmol/L NaCl with 0.1 mmol/L EDTA, sterilized on a 0.2-µm Millipore membrane, and stored at 4°C under nitrogen for up to 3 weeks. LDL purity was tested by electrophoresis on Hydragel, and apoB was determined by immunonephelometry.
Mildly oxidized LDLs were obtained by UV- and copper/EDTA-mediated oxidation under mild conditions. LDL solution (2 mg apoB/mL containing 2 µmol/L CuSO4) was irradiated for 2 hours as a thin film (5 mm) in an open becher placed 10 cm under the UV-C source (HNS 30W OFR Osram UV-C tube, 
max 254 nm, 0.5 mW/cm2 determined by using a Scientech thermopile, model 360001) under standard conditions.22 After the irradiation, aliquots were taken for analyses, and ox-LDLs (200 µg apoB/mL under standard conditions or at the indicated concentrations) were immediately incorporated into the culture medium.
Copper-oxidized LDLs were obtained by oxidizing LDL (2 mg apoB/mL) for 2 hours in the presence of 5 µmol/L CuSO4 after extensive dialysis against 150 mmol/L NaCl, ie, in the absence of EDTA during oxidation. EDTA (0.3 mmol/L) was added after the oxidation.
Cell-mediated oxidation was performed by incubating LDLs (0.2 mg apoB/mL) for 16 hours with lymphoblastoid cells in RPMI medium.29 After the oxidation, ECs were transferred to the cell-culture medium containing cell-oxidized LDLs. An LDL-free culture medium of lymphoblastoid cells was used as a control.
The level of LDL oxidation was evaluated by monitoring the formation of thiobarbituric acid–reactive substances according to the method of Yagi,30 the relative electrophoretic mobility was assessed by using Hydragel (Sebia), and the level of trinitrobenzene sulfonic acid–reactive amino groups was determined by using the method of Steinbrecher.31
Determination of [Ca2+]i
[Ca2+]i was determined by using the permeant calcium probes quin-2/AM or fura-2/AM, which are hydrolyzed by intracellular carboxylesterases to liberate quin-2 or fura-2. Briefly, cells were incubated for 15 minutes at 37°C in RPMI medium buffered with 20 mmol/L HEPES that contained 0.5% bovine serum albumin and quin-2/AM (20 µmol/L) or fura-2/AM (2 µmol/L). After dilution and incubation in RPMI for 45 minutes, the cells were washed twice in PBS, and their fluorescence F was recorded. For quin-2 (excitation 340 nm, emission 395 nm), the calibration was done in very low and high calcium to calculate the absolute value of [Ca2+]i, as described by Arslan et al.32 [Ca2+]i determination for fura-2 was performed at the dual excitation wavelengths of 340 and 380 nm (emission 510 nm). [Ca2+]i was calculated by the ratio method according to the protocol of Thomas and Delaville.33 Because both fluorescent calcium probes gave quite similar results, we report only results obtained with one probe.
For [Ca2+]i microphotography, cells were grown on microscope glass cover slides and incubated with the permeant fluorescent calcium probe fluo-3/AM (used at a final concentration of 2 µmol/L and solubilized in 0.5% dimethyl sulfoxide and 0.02% Pluronic F-127 as indicated by the manufacturer) for 30 minutes before examination.34 Cells were examined by using an epifluorescence microscope (Leica model Diaplan with fluorescein filters).
Determination of Cytotoxicity and Indices of Necrosis and Apoptosis
The whole cytotoxic effect was evaluated by using the MTT test.35
The Trypan blue exclusion test was performed22 to test for the loss of plasma membrane integrity (ie, necrosis). Cellular debris was not counted, thus probably excluding the late steps of the apoptotic and necrotic processes.
The percentage of apoptotic cells was determined microscopically after staining the nuclei of the living cells with 5 µmol/L SYTO-11 (a fluorescent permeant DNA probe dissolved in dimethyl sulfoxide) and immediately examining them by using fluorescence microscopy (Leica model Diaplan with fluorescein filters). Alternatively, apoptotic nuclei were visualized after the cells were fixed in 3% paraformaldehyde for 15 minutes, washed in 150 mmol/L PBS, pH 7.4, stained by the DNA intercalating fluorescent probe DAPI (0.1 µg/mL in [pH 7.0] Tris/EDTA/NaCl [10:10:100] mmol/L), and mounted in Fluoprep for fluorescence microscopy (Leica model Diaplan).22
DNA fragmentation was also visualized in situ on fixed cells by using the TUNEL procedure of Gavrielli et al36 with the terminal transferase kit of Boehringer Mannheim. Briefly, cells grown on glass cover slides were fixed in 3% buffered paraformaldehyde, and endogenous peroxidases were inactivated by applying 2% H2O2. After the slides were rinsed, the cells were treated with 150 µL terminal transferase (0.3 U/µL), biotinylated dUTP in terminal transferase buffer (150 mmol/L potassium cacodylate, 25 mmol/L Tris-HCl, pH 6.6, and 0.25 mg/mL bovine serum albumin), and 2 mmol/L CoCl2 for 1 hour at 37°C. After the reaction was stopped the slides were rinsed four times.36 They were then covered by 150 µL extra-avidin peroxidase diluted 1:15 in water, incubated for 30 minutes at 37°C, washed twice, and finally stained with 1 mg/mL diaminobenzidine for 5 minutes at 37°C. The positive control was treated by DNase I (1 µg/mL; Sigma) for 10 minutes36 before being subjected to the TUNEL procedure.
Determination of Chromatin Fragments
DNA fragmentation assays were essentially derived from the procedure of McConkey et al23 under the previously used conditions.22 Cells were allowed to lyse for 15 minutes in 1 mL lysis buffer (5 g/L Triton X-100, 20 mmol/L EDTA, and 5 mmol/L Tris, pH 8.0) and were then ultracentrifuged for 20 minutes at 27 000g to separate the chromatin pellet from cleavage products. The pellet (resuspended in 1 mL of 10 mmol/L Tris-HCl buffer, pH 8.0, containing 1 mmol/L EDTA) and the supernatant were assayed for DNA determination by the fluorometric DAPI procedure according to the method of Kapuscinski and Skooczylas.37
Labeling and Electrophoresis of DNA Fragments
Alternatively, DNA fragmentation of radiolabeled cellular DNA was visualized by gel electrophoresis on a 1.8% agarose gel for 2 hours at 50 V. DNA was extracted by using the G-Nome kit and labeled according to the procedure of Rosl38 : 0.5 to 1 µg DNA was treated with 5 U Klenow polymerase by using 0.5 µCi [32P]dCTP in a 10 mmol/L Tris-HCl buffer, pH 7.5, containing 5 mmol/L MgCl2. After labeling, DNA was precipitated three times by 2.5 mol/L ammonium acetate–isopropanol, resuspended in 10 mmol/L Tris-HCl, pH 7.5, containing 1 mmol/L EDTA, and subjected to electrophoresis and autoradiography.
Electron Microscopy
ECs were treated for 16 hours with 200 µg apoB/mL of native LDLs or ox-LDLs and used for electron microscopy studies. After the cells were carefully trypsinized (trypsin-EDTA) and neutralized with fetal calf serum, they were pelleted by centrifugation for 5 minutes at 600g, fixed in 2.5% glutaraldehyde and 0.1 mol/L cacodylate-HCl buffer, pH 7.2, at room temperature, and treated for TEM.10 After postfixation in 2% osmium tetroxide in cacodylate, pH 7.2, the pellets were embedded in Epon-812, stained by uranyl acetate and lead citrate, and examined by using a Hitachi H-300.
Western Blot Analysis of Bcl-2
Bcl-2 was detected in the human EC line used here (and in EBV-transformed lymphoblastoid cell lines for comparison) by Western blotting performed according to standard techniques.39 Crude extracts from ECs and lymphoblastoid cells were prepared by overnight lysis in hypotonic buffer (20 mmol/L Tris-HCl, pH 7.4, containing 50 mmol/L NaCl, 50 mmol/L NaF, 5 mmol/L EDTA, 20 mmol/L sodium pyrophosphate, 1 mmol/L sodium orthovanadate, 100 µg/mL phenylmethylsulfonyl fluoride, 1 µg/mL leupeptin, 10 mg/mL Triton-X100, and 1 mg/mL sodium dodecyl sulfate). Samples were sonicated (MSE Soniprep-150) for 5 seconds and centrifuged at 13 000g for 10 minutes at 4°C. An aliquot (5 µg as protein) of the supernatant was used for separation by sodium dodecyl sulfate–polyacrylamide gel (12%) electrophoresis at 100 V for 90 minutes. After electrotransfer (200 V for 60 minutes) to a nitrocellulose membrane (Pharmacia), proteins were visualized with Ponceau S (Sigma) to confirm equal loading of protein. The membrane was blocked overnight with 2% nonfat milk in 0.15 mol/L PBS, pH 7.4, containing 0.05% Tween 20 (PBS–Tween 20) at 4°C, after which it was incubated for 90 minutes at room temperature with anti–bcl-2 monoclonal antibody (Dako; dilution, 1:200). After being washed in PBS–Tween 20, the membrane was incubated with a secondary antibody (anti-mouse sheep IgG conjugated with horseradish peroxidase) for 1 hour at room temperature and developed by using a chemiluminescence reagent (Amersham) according to the instructions of the manufacturer; the enhanced chemiluminescence film (Amersham) was exposed for 30 seconds.
Protein concentrations were determined by using the procedure of Smith et al.40
Statistical significance was estimated by using Student's t test.
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Ox-LDLs Induce Apoptosis of Cultured ECs
Cultured ECs incubated in the presence of ox-LDLs (200 µg apoB/mL UV+copper-oxidized LDLs containing 4.5±0.9 nmol thiobarbituric acid–reactive substances/mg apoB) exhibited a progressive cytotoxic effect (beginning after 12 hours' incubation) as shown by the MTT test (Fig 1A
). In contrast, native LDLs did not alter cell viability. During the same time, we observed a progressively increasing number of cells with DNA fragmentation as shown by the TUNEL procedure (Fig 1B) and of cells with morphologically apoptotic nuclei as shown by SYTO-11 labeling (Fig 1B) as well as an increasing level of chromatin fragmentation (Fig 1C). Electrophoresis of radiolabeled DNA fragments showed the progressive formation of the characteristic apoptotic ladder (Fig 1, right). The cytotoxic effects induced by ox-LDLs began at 150 µg apoB/mL and were dose dependent (Fig 2). Under the standard conditions (cells incubated for 24 hours with 200 µg apoB/mL), the index of apoptosis (54±7% of TUNEL-stained cells) was three times higher than the index of necrosis (17±4% of Trypan blue–stained cells).
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As the DNA fragmentation was concomitant with the toxic effect, it may be that ox-LDLs induce an apoptotic process in cultured ECs. This hypothesis was supported by morphological studies (Figs 3 and 4). Nuclear morphological changes characteristic of apoptosis, such as chromatin condensation and nucleus fragmentation, were clearly visualized by staining of intact cells with the permeant fluorescent DNA probe SYTO-11 (Fig 3A and 3B). DNA fragmentation of apoptotic cells was also clearly demonstrated in situ by the TUNEL staining procedure (Fig 3C). TEM preparations (Fig 4) showed the characteristic morphological features of apoptosis, ie, chromatin condensation, nucleus fragmentation, and cytoplasm condensation with intact organelles.
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Time-course studies of the apoptotic process showed that under the conditions used in Fig 1
the apoptotic process of the EC population began 12 hours after the beginning of the pulse with toxic doses of ox-LDLs. At that time, the apoptotic ECs were not stained by Trypan blue dye (data not shown), suggesting that the integrity of the plasma membrane was not altered (as confirmed by the lack of lactate dehydrogenase leakage; data not shown). The apoptotic cells became relatively rapidly detached (in 4 to 6 hours) and were progressively stained by Trypan blue (
3 hours after detachment). This suggests the loss of integrity of the plasma membrane of apoptotic cells after cell detachment, ie, postapoptotic necrosis (data not shown). Finally, the detached apoptotic cells progressively (in 5 to 10 hours) disintegrated into small fragments, which was consistent with the smear observed at 24 hours on DNA electrophoresis (Fig 1).
In order to investigate whether the toxic and apoptotic effects were dependent on the mode of LDL oxidation, we compared the toxic effect of ox-LDLs prepared by three different procedures: physical and/or chemical oxidation (UV+copper/EDTA or copper) and cell-mediated oxidation. Oxidation by UV+copper/EDTA and cell-mediated oxidation led to the formation of mildly oxidized LDLs, whereas copper-promoted LDL oxidation led to the formation of extensively oxidized LDLs. All three preparations of ox-LDLs were cytotoxic to ECs, and in agreement with our previous observations,41 the toxic effect was clearly related to the oxidation levels (Table 1). UV+copper/EDTA-promoted oxidation was preferentially used because this method allows superior control of the oxidation level and the preparation of mildly oxidized LDLs more easily than copper-promoted oxidation; it also allows potentially interfering compounds (such as cytokines or growth factors secreted by cells and potentially present in cell-mediated ox-LDL preparations) to be excluded.
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To examine whether immortalized and nonimmortalized ECs differ in their regulation of cell death induced by ox-LDLs, we compared the toxic and apoptotic effects of increasing doses of ox-LDLs on three types of ECs: the immortalized human EC line CRL-1998, the immortalized BAEC line GM7372A, and primary cultures of BAECs. All three types of ECs were susceptible to the cytotoxic and apoptotic effects of ox-LDLs (Table 2); nonimmortalized BAECs were significantly more susceptible to ox-LDLs than the immortalized cell lines CRL-1998 and GM7372A (P<.01 at 100 and 150 µg apoB/mL), but the levels of apoptosis (ie, DNA fragmentation) were similar in both immortalized and nonimmortalized cells.
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Ox-LDL–Induced Apoptosis of ECs Is Calcium Dependent
As shown by the fluorometric determination of [Ca2+]i levels of the whole cell population, which were monitored every 30 minutes during the first 15 hours of the pulse with ox-LDLs by using the Ca2+ probes fura-2/AM and quin-2/AM (Fig 5A) and as visualized by fluorescence microscopy of cells loaded with fluo-3/AM (Fig 5A, inset), toxic doses of ox-LDLs induced a sustained and intense [Ca2+]i peak (maximum 2000±400 nmol/L at 9±1 hours). The relative time course of the [Ca2+]i peak and the subsequent cytotoxicity showed a progressively increasing number of apoptotic cells after the [Ca2+]i rise.
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) or native (
) LDLs (200 µg apoB/mL) were added to CRL-1998 ECs at time 0, and [Ca2+]i levels were fluorometrically determined on the whole cell population; inset, visualization by fluorescence microscopy using fluo-3/AM of [Ca2+]i in cells incubated for 10 hours with 200 µg apoB/mL mildly oxidized (top) or native (bottom) LDLs. B, Bar graph shows effect of 0.6 mmol/L EGTA (E) or 10 µmol/L of the calcium-channel blockers nifedipine (Nf) or nisoldipine (Ns) on the [Ca2+]i levels in cells incubated 10 hours with 200 µg apoB/mL mildly oxidized (ox) or native (n) LDLs. C, Line graph shows effect of 0.6 mmol/L EGTA (
) or 10 µmol/L of the calcium-channel blockers nifedipine (
indicates oxidized LDL only). Values are mean±SEM of three experiments. Right, Effect of 0.6 mmol/L EGTA or 10 µmol/L of the calcium-channel blocker nifedipine on DNA fragmentation as assessed by gel electrophoresis (under conditions described in Fig 1
When the free calcium of the culture medium (0.4 mmol/L) was chelated by adding 0.6 mmol/L EGTA at 8 hours (ie, just before the [Ca2+]i rise elicited by the ox-LDLs), the [Ca2+]i peak was blocked, and the DNA fragmentation was significantly inhibited (P<.01) (Fig 5). Similarly, when the calcium influx was inhibited by calcium-channel blockers (10 µmol/L of either nifedipine or nisoldipine), both the [Ca2+]i rise and the DNA fragmentation elicited by ox-LDLs were inhibited (P<.01) (Fig 5B and 5C).
All these data strongly suggest that the [Ca2+]i rise is involved in the activation of the apoptotic process induced by ox-LDLs.
Prevention of Apoptosis by ACA
As shown by morphological and biochemical studies (Fig 6), ACA, an inhibitor of endonucleases,42 prevented the cytotoxic effect (apoptosis and cell detachment) of toxic doses of ox-LDLs on ECs. The inhibition of the apoptotic process was not counterbalanced by any significant increase in the necrotic index, thus demonstrating that the inhibition of the final steps of the apoptotic process allows the cytotoxic effect of ox-LDLs and cell death to be blocked. Under the present experimental conditions, the [Ca2+]i rise elicited by ox-LDLs was not blocked by ACA (Fig 6A).
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) 1 mmol/L ACA. Line graphs show (A) calcium levels and (B) levels of chromatin fragmentation in the presence or absence of 1 mmol/L ACA. Values are mean±SEM of four experiments. Right, Effect of varying concentrations (1, 0.5, and 0 mmol/L) of ACA on DNA fragmentation as assessed by gel electrophoresis (under the conditions described in the legend to Fig 1
Level of Bcl-2 in ECs
As we have shown22 that toxic doses of ox-LDLs induce mainly necrosis in lymphoblastoid cells immortalized by EBV transformation and as bcl-2 is overexpressed in lymphoblastoid cells and protects them against apoptosis,43 44 45 46 we hypothesized that bcl-2 levels may be involved in the mechanism of cell death induced by ox-LDLs. Comparison of the levels of bcl-2 protein in lymphoblastoid cells and ECs revealed a much higher bcl-2 level in lymphoblastoid cells (Fig 7).
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Discussion |
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The cytotoxic effect of ox-LDLs on cultured ECs was reported almost 20 years ago,7 8 but to our knowledge, few studies have investigated the type and the mechanism of cell death induced by ox-LDLs.
The data reported here show that toxic doses of ox-LDLs induce a massive apoptosis of cultured ECs. Typical apoptosis and necrosis are discriminated on the basis of morphological features.16 Necrotic cells are characterized by loss of plasma membrane integrity and staining by nonpermeant dyes (Trypan blue) and swelling and rounding of the cell and cellular organelles without any nucleus condensation. Apoptotic cells exhibit morphological alterations of the nucleus (chromatin margination and condensation and nucleus fragmentation) that are easily visualized by fluorescent DNA staining without any increase of plasma membrane permeability (thus without Trypan blue staining). ECs treated by toxic doses of ox-LDLs exhibit the biochemical (DNA ladder) and morphological features of apoptosis. These apoptotic cells lose their adherence to the culture flask, undergo relatively rapid plasma membrane alterations (characteristic of postapoptotic necrosis), and progressively disintegrate into small fragments. This characteristic sequence of alterations is consistent with the classic description of apoptosis and postapoptotic alterations (for reviews, see References 16, 47, and 48).
Comparison of the cytotoxic and apoptotic effects of ox-LDLs on various immortalized EC lines and nonimmortalized ECs showed that ox-LDLs were more toxic to nonimmortalized ECs, induced apoptosis of nonimmortalized BAECs as well as immortalized cell lines, and were similarly toxic to human and bovine ECs. These data suggest that apoptosis induced by toxic doses of ox-LDLs may be studied on immortalized ECs.
Because nonimmortalized BAECs are relatively fragile and are dependent on supplementation with exogenous basic fibroblast growth factor, there may be a possible artifact in the study of cell death, as growth factor withdrawal often induces apoptosis of growth factor–dependent cells.47 48 In contrast, the established EC lines CRL-1998 and GM7372A are not dependent on exogenous growth factor supplementation, are not subjected to senescence, and are phenotypically stable with time and passages. These factors led us to use immortalized cell lines in the experiments reported here because they are more robust and are less susceptible to death from artifactual events than nonimmortalized BAECs.
The time course of the [Ca2+]i peak, which occurs before the cytotoxic effect, supports the claim that the [Ca2+]i rise is not a consequence but rather a cause of the cytotoxicity. The mechanism of the [Ca2+]i peak elicited by ox-LDLs very probably involves a calcium influx, as suggested by the inhibition of the [Ca2+]i peak by EGTA and by the dihydropyridine calcium-channel blockers nifedipine or nisoldipine, in agreement with our previous reports.22 49 In contrast to the [Ca2+]i rise observed in chemical or anoxic cell injury,5 6 7 the [Ca2+]i peak induced by ox-LDLs did not result from an intracellular oxidative stress, since up to the end of the [Ca2+]i peak we observed no depletion of glutathione and ATP, no defect of the calcium pumps,50 and no significant increase of the permeability of the cellular membrane to ions.11
The sustained [Ca2+]i peak elicited by ox-LDLs seems to be a common trigger of cytotoxic events leading to cell death, in agreement with reports on the role of calcium in lethal cell injury.19 21 The role of the sustained [Ca2+]i peak in the apoptotic process induced by ox-LDLs is strongly supported by the fact that the [Ca2+]i peak occurs before the cytotoxicity and by the protective effect of agents (ie, EGTA and dihydropyridine calcium-channel blockers) that concomitantly block the [Ca2+]i peak and the apoptotic process.
It is noteworthy that the sustained [Ca2+]i peak elicited by ox-LDLs triggered mainly necrosis in lymphoblastoid cell lines22 and apoptosis in ECs (present study). Necrosis results (at least in part) from the activation of calcium-dependent proteases, whereas calcium-dependent endonucleases are involved in the apoptotic process,22 but the mechanisms regulating the balance between the two pathways remain largely unknown. In ECs, ox-LDLs induced the activation of calcium-dependent endonucleases involved in the apoptotic process (as suggested by the internucleosomal DNA cleavage and its inhibition by ACA), in agreement with the data in lymphoid cells.22
As bcl-2 protein is overexpressed in lymphoblastoid cells and protects them against apoptosis,43 we compared bcl-2 levels in lymphoblastoid cells and ECs. Since bcl-2 is a potent antiapoptotic protein,43 44 45 46 we hypothesize that the relatively low expression of bcl-2 in ECs may be related to their high susceptibility to apoptosis (present study) in contrast to lymphoblastoid cells.22
From a pathophysiological point of view, the cytotoxic effect of ox-LDLs on ECs may be involved in the initiation and progression of arteriosclerosis and thrombosis.13 14 These toxic effects could explain, at least in part, the morphological changes of ECs and the focal defects in the integrity of the EC lining that occur in atherosclerotic arteries.14 51 52 53 The endothelial defects are associated with platelet adhesion, fibrin deposition, and microthrombi formation14 52 53 and are potentially involved in more extensive thrombotic events.
The fact that apoptosis is the prevailing type of EC death induced by ox-LDLs may have an additional pathophysiological significance, since apoptosis is probably less dangerous than necrosis. Generally, apoptotic cells are rapidly cleared by phagocytic cells in vivo and do not induce any explosive inflammatory events.16 48
One of the most striking results of the present study is the efficacy of ACA in protecting ECs against ox-LDL–induced apoptosis. ACA, an inhibitor of endonucleases and topoisomerase II,42 54 55 inhibited not only the apoptotic ladder formation but also the morphological changes that occur during ox-LDL–induced apoptosis, in agreement with data on apoptosis induced by various agents.22 42 55 These data strongly suggest (if ACA inhibits only endonucleases) that endonuclease activation and DNA fragmentation precede the morphological changes of apoptosis. However, we cannot definitively exclude the alternative hypothesis that ox-LDLs may first induce a shape change in ECs that in turn induces apoptosis. Indeed, the sustained [Ca2+]i peak elicited by ox-LDLs triggers morphological changes (rounding) that suggest a decreased adherence to the substrate and changes in actin microfilaments (A.N.-S. and M.-T.P., unpublished data, 1996); such shape changes of ECs with disorganized actin microfilaments are reported to trigger apoptosis.56 As the effective concentration of ACA was relatively high (1 mmol/L), it may inhibit molecular targets other than endonucleases, and therefore the observed apoptosis may be subsequent to a loosened contact of cells with the substrate.56 57 58
When apoptosis was blocked by ACA, the necrosis (as assessed by Trypan blue uptake) usually observed a few hours after cell detachment did not occur (data not shown), thus supporting the assumption that in the present case necrosis is a postapoptotic process. The inhibition by ACA of ox-LDL–induced apoptosis was associated with an inhibition of the whole cytotoxic effect of ox-LDLs and was not counterbalanced by an increase in the level of necrosis, thus demonstrating that in the present study ACA exhibited a true cytoprotective effect on ECs. This protective effect of ACA against endothelial injury may protect the integrity of the EC lining and therefore may explain, at least in part, the demonstrated antithrombotic effect of ACA in vivo.59
In conclusion, we report that toxic doses of ox-LDLs induce a sustained [Ca2+]i peak that in turn triggers a massive apoptosis, cell detachment, and postapoptotic necrosis. This calcium-dependent apoptotic process and the subsequent events can be prevented by blocking the sustained [Ca2+]i rise and by inhibiting endonucleases.
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Acknowledgments |
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This work was supported by grants from INSERM (CJF-9206), Universite Paul Sabatier-Toulouse III (MENESR JE-174), Fondation pour la Recherche Medicale, Region Midi-Pyrenees (9308181), and the European Communities (PL 931790). Dr Escargueil-Blanc received a fellowship from the "Ligue contre le Cancer." The authors wish to thank J.C. Thiers for microscopy iconography, C. Mora for lipoprotein preparations, and department SCMEAB for electron microscopy.
Received December 5, 1995;
revision received May 31, 1996;
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