© 1999 American Heart Association, Inc.
Vascular Biology |
Characterization and Comparison of the Mode of Cell Death, Apoptosis Versus Necrosis, Induced by 7ß-Hydroxycholesterol and 7-Ketocholesterol in the Cells of the Vascular Wall
From the Laboratoire de Biochimie des Lipoprotéines, INSERM U 498, Faculté de Médecine (G.L., S.M., L.G., V.D., S.G., L.L., P.G.), and Unité de Nutrition Lipidique, INRA (C.C.), Dijon, France.
Correspondence to Gérard Lizard, Laboratoire de Biochimie Médicale, INSERM U 498, CHU/Hôpital du Bocage, BP 1542, 21034 Dijon Cedex, France. E-mail Gerard.Lizard{at}u-bourgogne.fr
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Abstract |
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Abstract—Oxidized low density lipoproteins (LDLs) play a central role in atherosclerosis, and their toxicity is due, at least in part, to the formation of oxysterols that have been shown to induce apoptosis in various cell types. As 7ß-hydroxycholesterol and 7-ketocholesterol are the major oxysterols found in oxidized LDLs, we have investigated and compared the mode of cell death, apoptosis versus necrosis, that they induce in the cells of the vascular wall, ie, endothelial cells, smooth muscle cells, and fibroblasts. To this end, human vascular endothelial cells from umbilical cord veins (HUVECs), human artery smooth muscle cells, A7R5 rat smooth muscle cells, MRC5 human fibroblasts, and human fibroblasts isolated from umbilical cord veins were taken at confluence and incubated for 48 hours with 7ß-hydroxycholesterol or 7-ketocholesterol (concentration range, 5 to 80 µg/mL). In all cells, both 7ß-hydroxycholesterol and 7-ketocholesterol exhibited toxic effects characterized by a loss of cell adhesion and an increased permeability to propidium iodide. In oxysterol-treated endothelial and smooth muscle cells, typical features of apoptosis were revealed: condensed and/or fragmented nuclei were detected by fluorescence microscopy after staining with Hoechst 33342, oligonucleosomal DNA fragments were visualized in situ in the cell nuclei by the TdT-mediated dUTP-biotin nick-end labeling (TUNEL) method, and internucleosomal DNA fragmentation was found on agarose gel. In contrast, in oxysterol-treated fibroblasts, fragmented and/or condensed nuclei were never revealed, and no DNA fragmentation was observed either by the TUNEL method or by DNA analysis on agarose gel, indicating that these oxysterols induced necrosis in these cells but not apoptosis. In addition, acetylated Asp-Glu-Val-L-aspartic acid aldehyde (an inhibitor of Asp-Glu-Val-L-aspartic acid–sensitive caspases) prevented 7ß-hydroxycholesterol– and 7-ketocholesterol–induced cell death in HUVECs and smooth muscle cells but not in fibroblasts. Thus, 7ß-hydroxycholesterol and 7-ketocholesterol have dual cytotoxic effects on the cells of the vascular wall by their ability to induce apoptosis in endothelial and smooth muscle cells and necrosis in fibroblasts.
Key Words: cell death • endothelial cells • smooth muscle cells • fibroblasts • oxysterols
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Introduction |
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Oxidized LDLs play a critical role in the development of atherosclerotic lesions,1 2 and in vitro experiments have revealed that they are toxic to different cell types, including endothelial cells,3 smooth muscle cells,4 5 and fibroblasts.6 The cytotoxicity of oxidized LDLs has been alternatively linked to the formation of various aldehydes,7 to the conversion of phospholipids to lysophospholipids,8 as well as to the oxidation of cholesterol to oxysterols.9 More specifically, a number of in vivo and in vitro studies have suggested a determinant role of oxysterols in inducing cytotoxicity in the vascular wall.10 11 12 13 14 In addition, increased levels of oxysterols were reported in hypercholesterolemic human plasma,15 16 17 in the arterial wall from hypercholesterolemic rabbits,18 and in atheromatous plaques from hypercholesterolemic patients.19
The mode of cell death, ie, apoptosis versus necrosis, mediated by oxysterols in the cells of the vascular wall was only recently addressed. Apoptotic cells were observed in vivo in atherosclerotic lesions,20 21 22 and in our previous works, we have shown that sterols oxidized at position 7, ie, 7ß-hydroxycholesterol and 7-ketocholesterol, were highly cytotoxic to cultured bovine aortic endothelial cells and induced a mode of cell death by apoptosis characterized by the occurrence of cells with condensed and/or fragmented nuclei and internucleosomal DNA fragmentation23 ; in human vascular endothelial cells isolated from umbilical cord veins (HUVECs), we described additional similar features of apoptosis under treatment with 7-ketocholesterol.24 However, it is noteworthy that variations in the cytotoxicity of oxysterols were reported from one cell type to another,25 suggesting that the characteristics of apoptosis and of the mode of cell death might differ among cell types of the vascular wall. In addition, cell death by necrosis and apoptosis can lead to the activation of serine proteases and of the cysteine proteases named caspases, respectively,26 27 28 and in vivo necrosis is associated with an inflammatory response, but apoptosis is not.29 According to these considerations, characterization of cell death induced by oxysterols has some pathophysiological and therapeutic ramifications for the understanding of the atherosclerosis process as well as for treatment of the lesions.
Therefore, the aim of the present work was to characterize the cytotoxicity of 7ß-hydroxycholesterol and 7-ketocholesterol in a comparative study involving the different cell types of the vascular wall, ie, endothelial cells, smooth muscle cells, and fibroblasts. To this end, cultured HUVECs, human artery smooth muscle cells, A7R5 rat smooth muscle cells, MRC5 human fibroblasts, and human fibroblasts from umbilical cord veins were taken at confluence and incubated in the presence of increasing concentrations of either 7ß-hydroxycholesterol or 7-ketocholesterol (concentration range, 5 to 80 µg/mL). Subsequently, cellular viability was assessed by counting adherent and nonadherent cells as well as by measuring the cellular permeability to propidium iodide (PI). The mode of cell death, necrosis versus apoptosis,30 was characterized by the morphology of the cell nuclei after staining the specimens with Hoechst 33342, by in situ detection of oligonucleosomal DNA fragments with the TdT-mediated dUTP-biotin nick-end labeling (TUNEL) method,31 and by the DNA fragmentation pattern by electrophoresis on 1.8% agarose gel. Moreover, as 7-ketocholesterol and 25-hydroxycholesterol induce apoptosis in smooth muscle cells from rabbit aortas through the activation of caspase-3 (CPP32),32 which cleaves and inactivates the enzyme poly(ADP-ribose) polymerase implicated in DNA repair and genome maintenance,33 we investigated the effect of the tetrapeptide acetylated Asp-Glu-Val-L-aspartic acid aldehyde (Ac-DEVD-CHO), an inhibitor of DEVD-sensitive caspases such as caspase-3,34 during 7ß-hydroxycholesterol– and 7-ketocholesterol–induced cell death on endothelial cells, smooth muscle cells, and fibroblasts. Under those conditions, the characteristics of cell death induced by 7ß-hydroxycholesterol or 7-ketocholesterol were found to vary according to the cell type of the vascular wall.
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Methods |
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Cell Culture
HUVECs were obtained from umbilical cord veins as previously described.35 36 They were isolated by enzymatic digestion from 20-cm-long umbilical cord vein segments filled with 0.5% collagenase (Boehringer Mannheim). After a 15-minute incubation at 37°C, umbilical cord vein segments were perfused with 40 mL PBS for collecting the cells. After centrifugation for 5 minutes at 900g, the cell pellet was resuspended in Ham's F10 medium (Gibco) supplemented with antibiotics (100 U/mL penicillin, 100 mg/mL streptomycin [Gibco], and 0.25 mg/mL amphotericin B [Gibco]) and 10% heat-inactivated FCS (Boehringer Mannheim), and HUVECs were seeded in tissue-culture flasks at 4x104 cells/cm2. At confluence, HUVECs were briefly treated with a solution of 0.05% trypsin–0.02% EDTA (Gibco). At the first passage, they were seeded at 3x104 cells/cm2 in the previously described culture medium containing 30 µg/mL endothelial cell growth supplement (Sigma) and 90 µg/mL heparin (Sigma). HUVECs were characterized by the intracellular detection of factor VIII–related antigen (von Willebrand factor) as revealed by immunofluorescence with a rabbit polyclonal antibody (Sigma) as previously described.37 As observed by flow cytometry, the proportion of cells expressing factor VIII–related antigen at the first and second passages was 98%.38 For all experiments, HUVECs were used at confluence after 3 days of culture at the first or second passage, and they were cultured in plastic flasks coated with human collagen type IV (Sigma). Collagen type IV–coated tissue-culture plastic flasks were made by incubating 8 µg of collagen type IV in 100 µL PBS per cm2 of surface area of tissue-culture flask for 1 hour at room temperature.
Smooth Muscle Cells
Human artery smooth muscle cells isolated from umbilical
arteries (BioWhittaker/Clonetics, Walkersville, Md) were cultured in
smooth muscle growth medium 2 (BioWhittaker) containing 10 µg/mL
bovine insulin, 2 µg/mL human recombinant fibroblast growth factor,
0.2 µg/mL human recombinant epidermal growth factor, antibiotics
(gentamicin, 100 µg/mL; amphotericin B, 10 µg/mL), and 5% FBS (all
from BioWhittaker). The cells were seeded at
3x104/cm2 in the
previously described culture medium. For all experiments, cells were
used at confluence after 10 days of culture, and they were used at
either the first or second passage.
Rat smooth muscle cells (A7R5) were isolated from the thoracic aortas of 14- to 17-day-old embryonic BDIX rats.39 The cells were obtained from the American Type Culture Collection (Manassas, Va). A7R5 cells were cultured in Dulbecco's modified Eagle's medium (Gibco) containing 2.5 mmol/L glutamine, 40 U/mL penicillin, 40 mg/mL streptomycin (Gibco), and 10% heat-inactivated FCS (Boehringer Mannheim). They were seeded at 3x104/cm2 in the previously described culture medium. Cultures were passaged once a week by trypsinization with a solution of 0.05% trypsin–0.02% EDTA (Gibco). For all experiments, cells were used at confluence after 3 days of culture, and they were used between passages 20 and 30.
Fibroblasts
Human fibroblasts were isolated from umbilical cord veins as
follows. After endothelial cells were removed from
umbilical cord veins as previously described, the umbilical segments
were successively incubated twice with 0.5% collagenase
(Boehringer Mannheim) for 15 minutes at 37°C and washed with
PBS. At the end of the second incubation with collagenase,
cells were collected in PBS by centrifugation for 5
minutes at 900g. The resulting cell pellet was resuspended
in modified Eagle's medium with Earle's salts (Gibco) containing
2 mmol/L glutamine (Gibco) and 10% heat-inactivated
FCS (Boehringer Mannheim), and the cells were seeded in
tissue-culture flasks at
4x104/cm2.
At confluence, cells isolated under these conditions
consistently had a spindle shape and exhibited a monolayer
pattern of growth without any hill-and-valley features. In addition,
immunostaining reactions were negative with both the
mouse monoclonal antibody directed against human muscle 
-actin
(clone HHF35, Dako) and the mouse monoclonal antibody directed against
human desmin (clone D33, Dako), but they were positive with the
monoclonal antibody directed against vimentin (clone V9, Dako). These
morphological and antigenic characteristics, which are specific for
fibroblast-like cells,40 41 42 led us to consider that the
cells isolated and amplified under our conditions were indeed
fibroblasts. At the first passage and later, fibroblasts isolated from
umbilical cord veins as well as MRC5 human fibroblasts (Eurobio, Les
Ulis, France) were cultured at
3x104/cm2 in modified
Eagle's medium with Earle's salts (Gibco) containing 2 mmol/L
glutamine (Gibco) and 10% heat-inactivated FCS
(Boehringer Mannheim). For all experiments, the cultures were
passaged once a week by trypsinization with a solution of 0.05%
trypsin–0.02% EDTA (Gibco); human fibroblasts from umbilical cord
veins and MRC5 human fibroblasts were used at confluence after 3 days
of culture and were taken between passages 1 and 5 or between passages
26 and 30, respectively. All the cells used were cultured at 37°C in
a humidified air atmosphere containing 5% CO2.
Cell Treatments
For all experiments, 7ß-hydroxycholesterol and
7-ketocholesterol stock solutions were freshly prepared at
concentrations of 50, 100, 200, 400, and 800 µg/mL as previously
described.23 24 To establish the initial solutions,
oxysterols were dissolved in 1 volume of absolute ethanol, and 25
volumes of culture medium were added. One volume of these initial
solutions was further introduced into 10 volumes of culture medium to
obtain 7ß-hydroxycholesterol and
7-ketocholesterol at final concentrations of 5, 10, 20, 40,
or 80 µg/mL. Under these experimental conditions, the ethanol
concentration in the culture medium was 0.4%. The purity of
7ß-hydroxycholesterol and 7-ketocholesterol
(both from Steraloids Inc) as determined by gaseous phase
chromatography–mass spectrometry was 100%.
A stock solution of Ac-DEVD-CHO (BACHEM Biochimie) was prepared in distilled water at 10 mmol/L. Ac-DEVD-CHO was used at a final concentration of 100 µmol/L and was introduced into the culture medium 1 hour before the addition of oxysterols.
Cell Counting
HUVECs, human artery smooth muscle cells, A7R5 rat smooth muscle
cells, human fibroblasts isolated from umbilical cord veins, and MRC5
human fibroblasts were seeded at 3x105 cells per
well of 6-well plates (Nunc) containing 3 mL of culture medium. Cell
counting of adherent and nonadherent cells was performed by using a
hemacytometer. Adherent cells were collected by trypsinization with a
solution of 0.05% trypsin–0.02% EDTA (Gibco). Under these
conditions, cell detachment, which constitutes an index of
cytotoxicity, could be quantified.4
IC50 corresponding to the concentration required
to reduce by 50% the number of adherent cells was calculated after
fitting each curve according to a sigmoidal equation of the form
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Determination of Cell Viability
Cell viability was determined with PI
(
Ex max, 540 nm; Em
max, 620 nm), which stains dead cells only.44 A stock
solution of PI was prepared in PBS at a concentration of 100 µg/mL
and kept in the dark at room temperature. PI was used at a final
concentration of 4 µg/mL on the cell suspension (adjusted to
106 cells/mL), and the fluorescence was
immediately measured by flow cytometry with an FACScan flow cytometer
(Becton Dickinson) equipped with a 15-mW argon laser operating at a
wavelength of 488 nm. The red fluorescence of PI was collected
through a 585/42-nm band-pass filter, and the fluorescence
signals were measured on a logarithmic scale comprising four orders of
magnitude. For each sample, 10 000 cells were acquired, and the data
were analyzed with LYSYS I software (Becton
Dickinson).
Analysis of Cell Proliferation by Flow Cytometry
Analysis of cell proliferation was determined by flow
cytometry by estimating the proportion of cells in the different phases
of the cell cycle. To this end, the cells were trypsinized with a
solution of 0.05% trypsin–0.02% EDTA (Gibco), washed twice in PBS,
fixed in 70% ethanol, and stained with PI according to the method
described by Vindelov et al.45 Cell suspensions were then
analyzed with an FACScan flow cytometer (Becton Dickinson). The
red fluorescence of PI was collected through a 585/42-nm
band-pass filter, and the fluorescence signals were measured on
a linear scale of 256 channels. For each sample, counts for 10 000
cells were acquired, and the data were analyzed with
LYSYS I software (Becton Dickinson).
Light and Fluorescence Microscopy
The cellular and nuclear morphology of treated and untreated
cells was studied by phase-contrast microscopy and by
fluorescence microscopy with an inverted IX70 microscope
(Olympus). Hoechst 33342 excited by UV light was used to observe
nuclear morphology by fluorescence microscopy:
apoptotic cells were characterized by fragmented and/or
condensed nuclei and necrotic cells by diffuse and irregular
nuclei.46 Hoechst 33342 was prepared in distilled water at
1 mg/mL and added to the culture medium at a final concentration of 10
µg/mL for 1 hour at 37°C. Nonadherent cells in the culture medium
were collected by centrifugation and adherent cells by
trypsinization with a solution of 0.05% trypsin–0.02% EDTA (Gibco).
Cells were washed twice in PBS and resuspended at a concentration of
106 cells/mL in PBS containing 1% (wt/vol)
paraformaldehyde. Deposits of 
40 000 cells were
applied to glass slides by cytocentrifugation for 5
minutes at 1000 rpm with a cytospin 2 (Shandon), mounted in buffered
glycerin (Bio-Mérieux), and coverslipped. For each sample, 300
cells were examined to determine the percentage of apoptotic
and necrotic cells on the basis of the morphological aspect of cell
nuclei.
In Situ Detection of DNA Fragmentation
In situ visualization of DNA fragmentation at the single-cell
level was performed by the TdT-mediated dUTP-biotin nick-end labeling
(TUNEL) method developed by Gavrieli et al31 with the use
of the MEBSTAIN apoptosis kit (Immunotech) and according to the
manufacturer's procedure. In brief, cells were trypsinized with a
solution of 0.05% trypsin–0.02% EDTA (Gibco), washed twice in PBS,
resuspended at a concentration of 106 cells/mL in
PBS, and applied to glass slides (40 000 cells per slide) by
centrifugation for 5 minutes at 1000 rpm with a
cytospin 2 (Shandon). After fixation at 4°C for 15 minutes with a 4%
paraformaldehyde solution, the cells were
permeabilized at room temperature for 15 minutes with a
0.5% Tween 20–0.2% BSA solution, washed 3 times with deionized
water, and incubated with the TdT solution for 1 hour at 37°C in a
humidified atmosphere. The signal of TUNEL was then detected by
incubation for 30 minutes at room temperature with
peroxidase-conjugated streptavidin (Dako) diluted 1/300 in PBS–0.2%
BSA, and revelation of peroxidase activity was performed with
3,3'-diaminobenzidine with the Dako liquid DAB substrate chromogen
system (Dako). Cells were counterstained with methylene blue
(RAL/Rhône-Poulenc), and the slides were mounted in buffered
glycerol, coverslipped, and stored at 4°C. Observations were made
with an inverted Laborlux IX70 microscope (Olympus).
DNA Fragmentation
DNA fragmentation was assessed by electrophoresis on 1.8%
agarose gel. To this end, cellular DNA was extracted as previously
described by using a DNA extraction kit
(Stratagene).23 24 47 After electrophoresis, gels were
examined under UV light and photographed or stored digitally with an
image analysis system (Biocom).
Statistical Methods
A one-way ANOVA followed by Dunnett's t test was
used to evaluate the effects of 7ß-hydroxycholesterol and
7-ketocholesterol. Statistical analyses were
performed with SYSTAT software. The cutoff value
of significance was P<0.05.
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Results |
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Effect of Ethanol on Cell Growth
In the present study, ethanol was used to dissolved 7ß-hydroxycholesterol and 7-ketocholesterol. To ensure that the solvent vehicle, ie, 0.4% ethanol, did not affect cell growth, HUVECs, human artery smooth muscle cells, A7R5 rat smooth muscle cells, MRC5 human fibroblasts, and human fibroblasts from umbilical cord veins were cultured for 48 hours in 6-well plates containing either the appropriate culture medium alone or culture medium supplemented with 0.4% ethanol. Under these conditions, the number of cells per well, the proportion of cell death, as well as the percentage of cells in the different phases of the cell cycle were similar in the absence or presence of 0.4% ethanol. Thus, in the absence or presence of 0.4% ethanol, the number of cells per well did not significantly differ: for HUVECs, the control value was 7.73±0.83x105; ethanol treated 8.36±0.39x105; for A7R5 rat smooth muscle cells, control 4.60±0.03x105; ethanol treated 4.54±0.03x105; for human artery smooth muscle cells, control 5.51±0.10x105; ethanol treated 5.60±0.38x 105; for human fibroblasts from umbilical cord veins, control 3.90±0.32x105; ethanol treated 4.11±0.03x105; and for MRC5 human fibroblasts, control 6.47±0.54x105; ethanol treated 6.28±0.22x105. Moreover, after the cells were stained with PI, nonsignificant differences were found in the percentages of cell death: for HUVECs, the control value was 7.58±0.53; ethanol treated 8.06±0.30; for A7R5 rat smooth muscle cells, control 2.15±0.39; ethanol treated 1.88±0.54; for human artery smooth muscle cells, control 8.81±1.00; ethanol treated 9.08±1.11; for human fibroblasts from umbilical cord veins, control 8.37±2.34; ethanol treated 7.84±1.06; and for MRC5 human fibroblasts, control 8.81±1.00; ethanol treated 9.08±1.11. In addition, in the absence or presence of 0.4% ethanol, similar proportions of cells in the different phases of the cell cycle were observed: for HUVECs, the control G0/G1, S, and G2+M values were 82.33±0.81, 8.91±0.71, and 8.75±0.28, respectively; ethanol treated G0/G1, S, and G2+M 82.33±0.81, 9.01±0.51, and 8.65±0.40; for A7R5 rat smooth muscle cells, control G0/G1, S, and G2+M 84.02±0.54, 7.36±0.30, and 8.06±0.42; ethanol treated G0/G1, S, and G2+M 83.46±0.87, 7.60±0.96, and 8.56±0.41; for human artery smooth muscle cells, control G0/G1, S, and G2+M 92.00±6.00, 5.00±2.00, and 3.00±1.00; ethanol treated G0/G1, S, and G2+M 93.00±5.00, 5.00±2.00, and 2.00±1.00; for human fibroblasts from umbilical cord veins, control G0/G1, S, and G2+M 81.66±0.57, 10.00±1.00, and 8.33±057; ethanol treated G0/G1, S, and G2+M 81.66±0.57, 10.00±1.00, and 8.33±057; and for MRC5 human fibroblasts, control G0/G1, S, and G2+M 85.00±0.00, 6.75±0.50, and 8.25±0.50; ethanol treated G0/G1, S, and G2+M 85.50±0.57, 6.25±0.50, and 8.25±0.50. Thus, over the 48-hour experimental period, cell growth of endothelial cells, smooth muscle cells, and fibroblasts was not affected by the presence of 0.4% ethanol, and analysis of the cell cycle indicated that treatments with oxysterols were performed mainly on nonproliferating cells, as shown by the high percentages of cells in the G0/G1 phase of the cell cycle, varying from 81.66% to 93%.
Effects of 7ß-Hydroxycholesterol and
7-Ketocholesterol on the Number of Adherent and
Nonadherent Cells
When 7ß-hydroxycholesterol and
7-ketocholesterol were added to confluent
endothelial cells, smooth muscle cells, or fibroblasts,
a concentration-dependent decrease in the number of adherent cells was
observed in all cases (Figure 1
).
In the meantime, the number of nonadherent cells progressively
increased in the culture medium (Figure 1). As determined from
concentration-dependent curves, the mean concentrations required to
reduce by 50% the number of adherent endothelial cells
(IC50) were 15.2 and 19.6 µg/mL for
7ß-hydroxycholesterol and 7-ketocholesterol,
respectively. The corresponding IC50 values
obtained with smooth muscle cells from umbilical artery as well as with
rat smooth muscle cells (A7R5) were 21.2 and 28.2 µg/mL for
7ß-hydroxycholesterol and 7-ketocholesterol,
respectively. In fibroblasts, the decrease in adherent cells clearly
appeared at lower concentrations with
7ß-hydroxycholesterol than with
7-ketocholesterol, and with each oxysterol, the number of
adherent fibroblasts strongly decreased while the number of nonadherent
cells slightly increased, probably because rapid cell lysis occurred
when fibroblasts were floating in the culture medium. Therefore, in
contrast to endothelial and smooth muscle cells, only a
few nonadherent fibroblasts were present in the culture medium
after treatment with oxysterols, and their number slightly but
significantly increased (P<0.01) only with the highest
concentration of 7ß-hydroxycholesterol or
7-ketocholesterol (80 µg/mL; Figure 1). The
corresponding IC50 values with human fibroblasts
from umbilical cord veins were 14.8 and 29.5 µg/mL for
7ß-hydroxycholesterol and 7-ketocholesterol,
respectively, and those obtained with MRC5 human fibroblasts were 20.8
µg/mL for 7ß-hydroxycholesterol and 29.5 µg/mL for
7-ketocholesterol.
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Effects of 7ß-Hydroxycholesterol and
7-Ketocholesterol on Cell Viability
Cell viability was assessed by flow cytometry after staining with
PI of both adherent and nonadherent cells that were incubated for 48
hours with either 7ß-hydroxycholesterol or
7-ketocholesterol (Figure 2). All nonadherent cells were
shown to be permeable to PI, with at least 90% of total nonadherent
cells being stained with PI, regardless of cell type and oxysterol
concentration (Figure 2). With endothelial cells
(Figure 2A and 2B), the proportion of PI-stained adherent cells
gradually increased along the oxysterol concentration scale, and
consistent observations were made with either
7ß-hydroxycholesterol or 7-ketocholesterol.
In both cases, the highest proportion of adherent PI-stained cells was
obtained with the highest oxysterol concentration studied (80 µg/mL),
and the proportion reached 70% with both
7ß-hydroxycholesterol and 7-ketocholesterol
(Figure 2A and 2B). With A7R5 rat smooth muscle cells as well as
with human artery smooth muscle cells (Figure 2C and 2D), the
proportion of PI-stained adherent cells rose significantly
(P<0.01) at a lower concentration of
7ß-hydroxycholesterol (5 µg/mL) than of
7-ketocholesterol (40 µg/mL) (Figure 2C and 2D).
The maximal proportions of PI-permeable adherent A7R5 cells, 35%,
was reached with 10 µg/mL 7ß-hydroxycholesterol, and
this plateau value was maintained with the highest concentrations
studied (Figure 2C); with human artery smooth muscle cells, the
percentage of PI-permeable adherent cells rose progressively, and the
highest proportion of PI-permeable adherent cells reached 50% with
80 µg/mL 7ß-hydroxycholesterol (Figure 2C). With
7-ketocholesterol, the proportion of PI-stained adherent
human and rat smooth muscle cells rose also progressively, and a
maximum of 35% was reached with the 80 µg/mL concentration
(Figure 2D). With human fibroblasts isolated from umbilical cord
veins as well as with MRC5 human fibroblasts, PI-stained adherent cells
were also detected earlier with 7ß-hydroxycholesterol (10
µg/mL) than with 7-ketocholesterol (40 µg/mL). In
contrast to endothelial and smooth muscle cells,
virtually all adherent fibroblasts were stained with PI in the presence
of the highest oxysterol concentrations, and >95% of cells died after
treatment with 40 or 80 µg/mL 7ß-hydroxycholesterol
(Figure 2E) or 7-ketocholesterol, respectively
(Figure 2F).
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Characterization of 7ß-Hydroxycholesterol– and
7-Ketocholesterol–Induced Cell Death by Phase-Contrast
Microscopy, Fluorescence Microscopy After Nuclear Staining With
Hoechst 33342, and the TUNEL Method
Endothelial cells, smooth muscle cells, and
fibroblasts were incubated for 48 hours in the presence of either
7ß-hydroxycholesterol or 7-ketocholesterol,
and they were subsequently observed and characterized by phase-contrast
microscopy, fluorescence microscopy after nuclear staining with
Hoechst 33342, and the TUNEL method to identify in situ DNA
fragmentation.
As shown by phase-contrast microscopy, incubation of
endothelial cells with
7ß-hydroxycholesterol caused some morphological changes
(Figure 3A and 3B). Compared with
control cultures (Figure 3A), loss of the typical cobblestone
aspect of confluent endothelial cells was observed in
the presence of 7ß-hydroxycholesterol, and the number of
round cells floating in the culture medium increased (Figure 3B). The cellular morphology of oxysterol-treated human artery
smooth muscle cells and rat smooth muscle cells (A7R5) was similarly
and markedly altered, and a 48-hour exposure to 40 µg/mL
7-ketocholesterol induced cell shrinkage, cell detachment,
and a loss of cell connections (Figure 3E and 3F). In human
fibroblasts isolated from umbilical cord veins or in MRC5 human
fibroblasts, 7ß-hydroxycholesterol and
7-ketocholesterol also induced morphological changes. Thus,
compared with untreated fibroblasts (Figure 3I), some
morphological modifications were detected among counterparts treated
with 40 µg/mL 7-ketocholesterol, with a loss of cell
connections and the appearance of nonbirefringent fibroblasts attached
to the plastic culture dish (Figure 3J); in addition, some cells
and cellular debris floating in the culture medium were observed.
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Characterization of cell death, apoptosis versus necrosis, was
also performed by examining the morphological aspect of cell nuclei as
assessed by fluorescence microscopy after staining with Hoechst
33342 and by the TUNEL method, which allows detection of in situ DNA
fragmentation. Under these conditions, as shown in Figure 3D
, cells with fragmented and/or condensed nuclei typical of
apoptotic cells appeared among nonadherent
endothelial cells treated with 40 µg/mL
7ß-hydroxycholesterol, whereas cells with regular nuclei
were observed in control cultures (Figure 3C). In
7ß-hydroxycholesterol– and
7-ketocholesterol–treated rat smooth muscle cells (A7R5),
as well as in human artery smooth muscle cells, typical features of
apoptosis were also found by the TUNEL method. This latter
method revealed the presence of fragmented DNA in some condensed and/or
fragmented nuclei among nonadherent, oxysterol-treated smooth muscle
cells (Figure 3H), whereas no DNA fragmentation was found in
untreated cells (Figure 3G). In contrast, in untreated as well
as oxysterol-treated fibroblasts, no DNA fragmentation was detected by
the TUNEL method (Figure 3K and 3L); in addition,
oxysterol-treated fibroblasts had no fragmented or condensed nuclei,
but they did exhibit irregular cell contours and diffuse nuclei, as
visualized by counterstaining with methylene blue (Figure 3L),
resembling necrotic cells previously characterized by staining with
Hoechst 33342.46
Quantitation of Apoptotic or Necrotic Cells After Treatment
With 7ß-Hydroxycholesterol and 7-Ketocholesterol
Apoptotic and necrotic cells were counted either after
nuclei staining with Hoechst 33342 or by the TUNEL method. Under these
conditions, in untreated cells the proportions of apoptotic or
necrotic cells was <5% by simultaneously taking into
account both adherent and nonadherent cells (Figures 4 and 5). Apoptotic
endothelial cells were mainly present among
nonadherent cells after treatment with
7ß-hydroxycholesterol or 7-ketocholesterol
(Figure 4A and 4B), and the increase in nonadherent
apoptotic cells became significant with 10 µg/mL
7ß-hydroxycholesterol (P<0.01) or with 20
µg/mL 7-ketocholesterol (P<0.05). The maximal
proportion of apoptosis among nonadherent
endothelial cells was obtained with an oxysterol
concentration of 40 µg/mL, and this value was 50% with
7ß-hydroxycholesterol and 40% with
7-ketocholesterol. Among adherent
endothelial cells, the proportion of apoptotic
cells was significantly increased with
7ß-hydroxycholesterol (P<0.01) and with
7-ketocholesterol (P<0.05) at 40 and 80
µg/mL, respectively. With smooth muscle cells, both
7ß-hydroxycholesterol and 7-ketocholesterol
induced apoptosis mainly among nonadherent cells (Figure 4C and 4D). The increase in nonadherent, apoptotic
smooth muscle cells became significant with 10 µg/mL
7ß-hydroxycholesterol (P<0.01) and with 40
µg/mL 7-ketocholesterol (P<0.01). The maximal
proportion of apoptotic, nonadherent smooth muscle cells tended
to be higher after treatment with 7ß-hydroxycholesterol
(40%) than with 7-ketocholesterol (20%; Figure 4). In contrast to endothelial and smooth muscle
cells, necrosis was identified among adherent and nonadherent
fibroblasts after treatment with 7ß-hydroxycholesterol or
7-ketocholesterol (Figure 5). The increase in
necrotic cells became significant with 10 µg/mL
7ß-hydroxycholesterol or 40 µg/mL
7-ketocholesterol among nonadherent fibroblasts and with 20
µg/mL or 40 µg/mL 7ß-hydroxycholesterol among
adherent fibroblasts. Virtually all of the fibroblasts became necrotic
with the highest concentrations of oxysterols (40 and 80 µg/mL,
Figure 5).
|
|
Analysis of DNA Fragmentation by Electrophoresis on
Agarose Gel
Because the highest proportions of apoptotic
endothelial and smooth muscle cells were present
among nonadherent cells, DNA was extracted from nonadherent
7ß-hydroxycholesterol– or
7-ketocholesterol–treated cells. To this end,
endothelial cells, smooth muscle cells, and fibroblasts
were treated or not with 20 µg/mL 7ß-hydroxycholesterol
or 40 µg/mL 7-ketocholesterol for 48 hours, because these
concentrations gave simultaneously high proportions of
apoptotic and necrotic cells, and the DNA was analyzed
by electrophoresis on 1.8% agarose gel. As shown in Figure 6, a typical internucleosomal DNA
fragmentation into 180 to 200 bp and multiples thereof was observed in
nonadherent, treated endothelial and smooth muscle
cells. In contrast, no DNA fragmentation was found among nonadherent,
treated human fibroblasts isolated from umbilical cord veins or in MRC5
human fibroblasts exposed to either 7ß-hydroxycholesterol
or 7-ketocholesterol (Figure 6).
|
Effect of Ac-DEVD-CHO, an Inhibitor of DEVD-Sensitive
Caspases, on 7ß-Hydroxycholesterol– and
7-Ketocholesterol–Induced Cell Death
The effect of the synthetic inhibitor Ac-DEVD-CHO used
at 100 µmol/L on DEVD-sensitive caspases such as caspase-3 was
investigated in 7ß-hydroxycholesterol– or
7-ketocholesterol–treated HUVECs, smooth muscle cells, and
fibroblasts by quantifying the proportion of apoptotic or
necrotic cells by the TUNEL method. 7ß-Hydroxycholesterol
and 7-ketocholesterol were used at 20 and 40 µg/mL,
respectively, because these concentrations yielded similar high
proportions of apoptosis in HUVECs and smooth muscle cells and
high proportions of necrotic cells in fibroblasts. Interestingly,
Ac-DEVD-CHO provided an effective protection against
7ß-hydroxycholesterol– and
7-ketocholesterol–induced apoptosis, as shown by
the significantly decreased proportions of apoptotic HUVECs and
smooth muscle cells; however, no reduction in the proportions of
necrotic cells was observed (the
Table).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The cores of advanced atherosclerotic lesions have been shown to contain lipid deposits, apoptotic and/or necrotic cells, and cell debris.48 In addition, fibrosis also occurs as atherosclerosis progresses, often yielding a lesion containing a dense, extracellular matrix with a relatively low cell density.48 49 Although in the latter process cell death provides a means for conversion of a hypercellular lesion to the more fibrotic atheroma, little is known about the agents inducing cell death in the vascular wall and about its mechanisms, but in this complex process, LDLs probably play a critical role.2 Indeed, LDLs accumulate at the subendothelial level,2 and their exposure to not only endothelial cells50 but also macrophages localized in the vessel wall51 (which produces various radical oxygen species) leads to the formation of oxidized LDLs containing various toxic lipid oxidation products such as oxysterols.4 6 The present in vitro study on the different cells of the vascular wall, ie, endothelial cells, smooth muscle cells, and fibroblasts, brings new insight to the role of oxysterols in promoting cytotoxicity, and the current results indicate that the mode of cell death induced by oxysterols oxidized on carbon 7, ie, 7ß-hydroxycholesterol and 7-ketocholesterol, can vary according to the cell type in the vascular wall being considered.
In agreement with our previous studies,23 24 treatment of HUVECs with 7ß-hydroxycholesterol and 7-ketocholesterol enhanced cell detachment, reduced cellular viability, and was accompanied by the appearance of condensed and/or fragmented nuclei characteristic of apoptotic cells (mainly among nonadherent cells), as well as by internucleosomal DNA fragmentation. Overall, these latter observations indicated that 7ß-hydroxycholesterol and 7-ketocholesterol constitute 2 potent inducers of apoptosis in HUVECs. In contrast to previous studies in which the toxicity of oxysterols was investigated in endothelial cells that were cultured directly on plastic culture plates,23 24 we chose to culture HUVECs on collagen IV. Indeed, collagen IV was reported to protect cultured sheep pulmonary artery endothelial cells from apoptosis induced by lipopolysaccharides,52 indicating that experimental conditions, in particular, the culture substrate used, might influence the cytotoxicity of exogenous compounds. In fact, we did not observe any protective effect of collagen IV on the apoptotic potency of 7ß-hydroxycholesterol and 7-ketocholesterol, and very similar observations were made when HUVECs were cultured either on plastic23 24 or on a collagen IV substrate. However, the possibility of protective effects of some other components of the extracellular matrix cannot be excluded. For instance, laminin but not fibronectin was reported to protect HR9 mouse endodermal carcinoma cells from apoptosis when cultured in the absence of serum.53 In fact, only some components of the extracellular matrix might be able to regulate the behavior of specific cell types,54 55 56 possibly by acting on apoptosis-related genes encoding interleukin-1ß–converting enzyme and Bcl-2,57 as well as P21WAF1/CIP1 and P27KIP1.58 Because apoptosis of endothelial cells can be associated with thrombin activation,59 IL-1ß secretion, and paracrine induction of the adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1,60 61 we can envision a link between apoptosis at the endothelial cell level, procoagulant activity, and monocytic infiltration of the vessel wall. Taken together, these data reinforce the hypothesis that apoptosis of endothelial cells certainly plays a critical role at different stages of development of atherosclerotic plaques.
As with endothelial cells, smooth muscle cells can also
die by apoptosis when exposed to either
7ß-hydroxycholesterol or 7-ketocholesterol.
Thus, 7ß-hydroxycholesterol and
7-ketocholesterol induced the appearance of fragmented and
condensed nuclei typical of apoptosis, both among treated human
artery smooth muscle cells taken at the first passage and among A7R5
rat smooth muscle cells, whereas these latter cells could display
different characteristics than smooth muscle cells taken at earlier
passages. Like oxysterol-treated endothelial cells,
oxysterol-treated smooth muscle cells exhibited loss of cell adhesion
and increased permeability to PI, revealing the occurrence of some
membrane damage similar to that previously reported by studies
involving increased permeability to albumin of porcine
endothelial cells12 or modification of gap
junctions in human smooth muscle cells after oxysterol
treatments.14 It is, however, noteworthy that only a
fraction of adherent smooth muscle cells, 40% to 50% of the total,
became permeable to PI while the remaining cells were not stained, even
with the highest oxysterol concentrations used. These observations
would suggest either that adherent smooth muscle cells constitute a
heterogeneous population of cells with different
sensitivities to oxysterols or, more probably, that an atherogenic
stimulus recruits only a subset of smooth muscle cells.62
In addition, in the present study, internucleosomal DNA
fragmentation into 180 to 200 bp and multiples thereof was detected
among nonadherent human and rat smooth muscle cells, as has also been
observed in smooth muscle cells from thoracic aortas of Japanese white
rabbits treated with 7-ketocholesterol or
25-hydroxycholesterol.32 By contrast, rat
smooth muscle cells exposed to calphostin C,63
interferon-
, heparin, and cyclic nucleotide
analogues64 did not show any signs of internucleosomal
fragmentation, despite the detection of morphologically
apoptotic cells. Therefore, the DNA fragmentation pattern could
vary not only from one cell type to another65 but
also, with smooth muscle cells, on the inducer of cell death used. As
it has been previously reported that in atherosclerotic plaques of
cholesterol-fed rabbits the foam cells of
macrophage origin express cell proliferation markers while
adjacent smooth muscle cells show apoptosis,66
foam cells could be relatively more resistant to oxysterols
when compared with smooth muscle cells. In addition, as the smooth
muscle cells in atherosclerotic plaques show increased expression of
the proapoptotic protein Bax67 and as
apoptosis triggered by 7-ketocholesterol is
associated in vitro with a decrease of Bcl-2,32 we
hypothesize that dysregulation of the Bax to Bcl-2 ratio could be
involved in oxysterol-induced cell death.
The present study also demonstrated that 7ß-hydroxycholesterol and 7-ketocholesterol can exert a potent cytotoxic effect on the third cell type investigated, ie, fibroblasts (fibroblasts from human umbilical cord veins and MRC5 human fibroblasts). However, in contrast to endothelial and smooth muscle cells, no evidence for apoptosis was observed among oxysterol-treated fibroblasts. In fact, both 7ß-hydroxycholesterol and 7-ketocholesterol treatment induced the emergence of necrotic fibroblasts, which exhibited irregular and diffuse nuclei after being stained with Hoechst 33342. In further support of the absence of apoptotic cells among oxysterol-treated fibroblasts, no DNA fragmentation was observed in situ with the TUNEL method and by agarose gel electrophoresis. It is noteworthy that the results obtained in oxysterol-treated fibroblasts are rather puzzling, but in a previous work it had been shown that exposure of human fetal lung fibroblasts to oxidized LDLs, known to contain high levels of 7ß-hydroxycholesterol and 7-ketocholesterol,3 6 induced only 10% apoptotic cells68 ; therefore, we cannot exclude the possibility that the remaining 90% dead cells might correspond to necrotic ones, as suggested by aspects of the cells presented by phase-contrast and bright-field microscopy. Interestingly, the membrane damage revealed in our study by PI staining constitutes a common feature of both the apoptotic and necrotic process induced by 7ß-hydroxycholesterol and 7-ketocholesterol. However, major differences in the shape of the PI permeability curves were observed with increasing concentrations of oxysterols between endothelial cells, smooth muscle cells, and fibroblasts. In particular, whereas all adherent fibroblasts became permeable to PI after a threshold cytotoxic concentration of oxysterol was reached, the proportion of permeable endothelial and smooth muscle cells increased progressively along the oxysterol concentration range, and significant proportions of both adherent endothelial and adherent smooth muscle cells remained unstained with PI, even with the highest oxysterol concentrations studied. These observations strongly suggest that the metabolic pathways leading to increased permeability to PI might differ between apoptotic and necrotic cells. Therefore, we attempted to define the molecular mechanisms implicated in oxysterol-induced cell death.
As caspase-3 is highly expressed within apoptotic cells of human atherosclerotic plaques69 and 7-ketocholesterol– and 25-hydroxycholesterol–induced apoptosis is inhibited by Ac-DEVD-CHO,32 a tetrapeptide inhibiting DEVD-sensitive caspases such as caspase-3,34 we investigated the effect of this molecule on 7ß-hydroxycholesterol– and 7-ketocholesterol–induced cell death. Interestingly, 7ß-hydroxycholesterol– and 7-ketocholesterol–induced apoptosis in HUVECs and smooth muscle cells was significantly inhibited by Ac-DEVD-CHO, whereas no effect was observed on necrosis in oxysterol-treated fibroblasts. Thus, these data suggest the involvement of DEVD-sensitive caspases34 70 in the apoptotic process triggered by these oxysterols in HUVECs and smooth muscle cells but not in the necrosis observed in fibroblasts. It is noteworthy that these observations are in agreement with previous investigations reporting that necrosis, unlike apoptosis, was not accompanied by caspase-mediated proteolysis.26
In conclusion, the cytotoxic concentrations of 7ß-hydroxycholesterol and 7-ketocholesterol were in the range of those measured in plasma from hypercholesterolemic patients17 as well as of those found in atherosclerotic plaques.19 Therefore, our data not only reinforce the hypothesis that 7ß-hydroxycholesterol and 7-ketocholesterol probably play critical roles at different stages of the atherosclerotic plaques71 72 but also demonstrate that these oxysterols can induce both apoptosis and necrosis depending on the cell type of the arterial wall being considered.
|
Acknowledgments |
|---|
This work was supported by the Université de Bourgogne, the Conseil Régional de Bourgogne, the Institut National de la Santé et de la Recherche Médicale (INSERM), and the Comité Français de Coordination des Recherches sur l'Athérosclérose et le Cholestérol (ARCOL).
Received June 15, 1998; accepted October 7, 1998.
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 K. Yaraei, L. A. Campbell, X. Zhu, W. C. Liles, C.-c. Kuo, and M. E. Rosenfeld Chlamydia pneumoniae Augments the Oxidized Low-Density Lipoprotein-Induced Death of Mouse Macrophages by a Caspase-Independent Pathway Infect. Immun., July 1, 2005; 73(7): 4315 - 4322. [Abstract] [Full Text] [PDF]
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A. J. O'Sullivan, Y. C. O'Callaghan, and N. M. O'Brien Differential Effects of Mixtures of Cholesterol Oxidation Products on Bovine Aortic Endothelial Cells and Human Monocytic U937 Cells International Journal of Toxicology, May 1, 2005; 24(3): 173 - 179. [Abstract] [Full Text] [PDF]
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 E. Pedruzzi, C. Guichard, V. Ollivier, F. Driss, M. Fay, C. Prunet, J.-C. Marie, C. Pouzet, M. Samadi, C. Elbim, et al. NAD(P)H Oxidase Nox-4 Mediates 7-Ketocholesterol-Induced Endoplasmic Reticulum Stress and Apoptosis in Human Aortic Smooth Muscle Cells Mol. Cell. Biol., December 15, 2004; 24(24): 10703 - 10717. [Abstract] [Full Text] [PDF]
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 W. Martinet, M. De Bie, D. M. Schrijvers, G. R.Y. De Meyer, A. G. Herman, and M. M. Kockx 7-Ketocholesterol Induces Protein Ubiquitination, Myelin Figure Formation, and Light Chain 3 Processing in Vascular Smooth Muscle Cells Arterioscler Thromb Vasc Biol, December 1, 2004; 24(12): 2296 - 2301. [Abstract] [Full Text] [PDF]
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 C. I. Seye, M. W.M. Knaapen, D. Daret, C. Desgranges, A. G. Herman, M. M. Kockx, and H. Bult 7-Ketocholesterol induces reversible cytochrome c release in smooth muscle cells in absence of mitochondrial swelling Cardiovasc Res, October 1, 2004; 64(1): 144 - 153. [Abstract] [Full Text] [PDF]
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 R. Ringseis and K. Eder Insufficient Dietary Vitamin E Increases the Concentration of 7{beta}-Hydroxycholesterol in Tissues of Rats Fed Salmon Oil J. Nutr., December 1, 2002; 132(12): 3732 - 3735. [Abstract] [Full Text] [PDF]
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A. Matsuda, Y. Suzuki, K. Kondo, Y. Ikeda, and K. Umemura Hypercholesterolemia induces regression in neointimal thickening due to apoptosis of vascular smooth muscle cells in the hamster endothelial injury model Cardiovasc Res, February 1, 2002; 53(2): 512 - 523. [Abstract] [Full Text] [PDF]
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M. E. Higgins and Y. A. Ioannou Apoptosis-induced release of mature sterol regulatory element-binding proteins activates sterol-responsive genes J. Lipid Res., December 1, 2001; 42(12): 1939 - 1946. [Abstract] [Full Text] [PDF]
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P. M. Yao and I. Tabas Free Cholesterol Loading of Macrophages Is Associated with Widespread Mitochondrial Dysfunction and Activation of the Mitochondrial Apoptosis Pathway J. Biol. Chem., November 2, 2001; 276(45): 42468 - 42476. [Abstract] [Full Text] [PDF]
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S. R. Panini, L. Yang, A. E. Rusinol, M. S. Sinensky, J. V. Bonventre, and C. C. Leslie Arachidonate metabolism and the signaling pathway of induction of apoptosis by oxidized LDL/oxysterol J. Lipid Res., October 1, 2001; 42(10): 1678 - 1686. [Abstract] [Full Text] [PDF]
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 T.-S. Lee and L.-Y. Chau Fas/Fas ligand-mediated death pathway is involved in oxLDL-induced apoptosis in vascular smooth muscle cells Am J Physiol Cell Physiol, March 1, 2001; 280(3): C709 - C718. [Abstract] [Full Text] [PDF]
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C.-C. Hsieh, M.-H. Yen, C.-H. Yen, and Y.-T. Lau Oxidized low density lipoprotein induces apoptosis via generation of reactive oxygen species in vascular smooth muscle cells Cardiovasc Res, January 1, 2001; 49(1): 135 - 145. [Abstract] [Full Text] [PDF]
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A. E. Rusinol, L. Yang, D. Thewke, S. R. Panini, M. F. Kramer, and M. S. Sinensky Isolation of a Somatic Cell Mutant Resistant to the Induction of Apoptosis by Oxidized Low Density Lipoprotein J. Biol. Chem., March 15, 2000; 275(10): 7296 - 7303. [Abstract] [Full Text] [PDF]
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 M. Overhaus, J. Heckenkamp, S. Kossodo, D. Leszczynski, and G. M. LaMuraglia Photodynamic Therapy Generates a Matrix Barrier to Invasive Vascular Cell Migration Circ. Res., February 18, 2000; 86(3): 334 - 340. [Abstract] [Full Text] [PDF]
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W. L van Heerde, S. Robert-Offerman, E. Dumont, L. Hofstra, P. A Doevendans, J. F.M Smits, M. J.A.P Daemen, and C. P.M Reutelingsperger Markers of apoptosis in cardiovascular tissues: focus on Annexin V Cardiovasc Res, February 1, 2000; 45(3): 549 - 559. [Abstract] [Full Text] [PDF]
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