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Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:416-424

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:416-424.)
© 1996 American Heart Association, Inc.


Articles

Contrary Effects of Lightly and Strongly Oxidized LDL With Potent Promotion of Growth Versus Apoptosis on Arterial Smooth Muscle Cells, Macrophages, and Fibroblasts

Barbro Björkerud; Sören Björkerud

From the Department of Pathology, Institute of Laboratory Medicine, Göteborg University, Sahlgrenska University Hospital, Göteborg, Sweden.


*    Abstract
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*Abstract
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Abstract The inhibition of experimental atherosclerosis by antioxidants and the presence of oxidized LDL (oxLDL) in atherosclerotic lesions indicate that oxLDL may play what is perhaps a primary role in atherogenesis. LDL promotes the growth of arterial smooth muscle cells (SMCs), and oxLDL has cytotoxic effects. Since excessive intimal growth alternating with necrosis is typical of atherosclerotic lesions, we wondered whether these extreme changes in the lesions could be related to the extreme effects of LDL and oxLDL on cells. We therefore examined the effects of increasing LDL oxidation on its capacity to induce cell growth or cell death and whether the latter could be due to apoptosis. Cells of the types present in the atherosclerotic artery were used, ie, SMCs (human arterial), macrophages (human macrophage-like cell line THP-1), and human fibroblasts. Growth was evaluated by measuring the synthesis of DNA and culture size (MTT method) and apoptosis by using the in situ labeling of internucleosomally degraded DNA and, in the case of SMCs, the appearance of chromatin condensation. The oxidation of LDL was mediated by UV or Fe ions. Shortly oxidized LDL had a markedly increased growth-promoting effect on all cell types. With prolonged exposure to UV, but not to Fe, LDL became increasingly cytotoxic, and this toxicity was paralleled by the appearance of apoptosis in all cell types. After prolonged UV treatment, low-molecular-weight material from the partially degraded LDL was responsible for the induction of apoptosis. The dual effect of oxLDL, ie, its strong growth-promoting effect or the induction of cell death by apoptosis, depending on the degree of change by oxidation, is compatible with the notion that oxLDL plays a role not only in atherogenesis but also more extensively in the development of the structure typical of the atherosclerotic lesion, with focal excessive growth alternating with necrosis.


Key Words: arterial smooth muscle cell • macrophage • cell growth • apoptosis • oxidized LDL


*    Introduction
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up arrowAbstract
*Introduction
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The constituents of the body are continuously exposed to the risk of accidental oxidative damage. In addition, many components are very susceptible to oxidation, and this is especially true in the case of the unsaturated lipids of the membrane systems of the cell (for a review see Reference 1). More stable constituents, such as proteins and even nucleic acids, may also be damaged by oxidation. There are several lines of defense against oxidative damage in the form of multiple antioxidants in the tissues,2 and in normal circumstances, the effects of oxidative injury are not apparent. In certain pathological changes, such as atherosclerotic lesions (for a review see Reference 3), oxidatively changed material originating from LDLs that are especially sensitive to oxidation4 has been found. There is also good reason to suppose that oxLDL could in fact play a primary atherogenic role, as the supply of antioxidants appears to have an inhibitory effect on the development of experimental atherosclerosis (for a review see Reference 3).

It is well established that LDL promotes the growth of arterial SMCs and that oxidatively changed LDL has cytotoxic properties (for review see Reference 5). Excessive intimal growth alternating with focal massive cell death are characteristics typical of the atherosclerotic lesion,6 and the aim of this study was to investigate whether these extreme changes in the lesions could be related to the extreme effects of LDL and oxLDL on cells. The type of cell damage induced by oxLDL is not known, and an additional purpose of the study was to determine whether the cytotoxic reaction of oxLDL reflects nonspecific cell death or the possible induction of programmed cell death, apoptosis, in cells of the types present in the atherosclerotic artery.

The most important results of this study are that shortly oxidized LDL had a very powerful mitogenic effect that was many times greater than that of the parent unoxidized LDL. LDL oxidized for a longer period induced apoptosis in human arterial SMCs, in a macrophage cell line, and in human fibroblasts, ie, in cells of the types present in the atherosclerotic artery.


*    Methods
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*Methods
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Cell Culture
Human arterial SMCs were obtained from aortic segments from young adult kidney donors (traffic accident victims). Small samples of the inner third of the media were obtained by microdissection under a stereomicroscope, and cultures were obtained by primary explantation as described in detail previously.7 8 9 10 In short, the primary explants were seeded onto fibronectin-coated flasks (15 µg/cm2) with 5% WBS in Williams' medium E (GIBCO) and Ham's medium F-12 (1:1), with 20 mmol of HEPES buffer, pH 7.3, 2.6 mmol of L-glutamine, 50 mg of neomycin, and 10 mg of ascorbic acid per liter. Serum fibronectin was isolated from plasma as described by Vuento and Vaheri.11 The cells were passaged by trypsinization, and further cultivation was performed in nutrition medium MCDB 104 (GIBCO), with 10% WBS and with L-glutamine and ascorbic acid, as above, and with 105 U of penicillin and 100 mg of streptomycin per liter. The SMCs were used up to the 7th passage.

Human fetal lung fibroblasts were obtained from Statens Bakteriologiska Laboratorium and used up to the 20th passage. They were cultivated in Ham's medium F-12 (GIBCO) with 5% donor calf serum and with penicillin, streptomycin, and ascorbic acid, as above.

The macrophage-like cell line THP-1 (acute monocytic leukemia, human) was obtained from American Type Culture Collection and was cultivated in RPMI 1640 (GIBCO), with 7.5% fetal calf serum and with L-glutamine, penicillin, and streptomycin, as above, with the addition of 2 mmol of sodium pyruvate (GIBCO) and 50 µmol of mercaptoethanol (E. Merck) per liter.

For experiments in which apoptosis was to be visualized by the in situ detection of internucleosomal DNA degradation or by DNA staining with gallocyanin-chrome alum stain (see below), the cells were plated onto chamber slides (Lab-Tek Chamber-slides, catalog No. 177445, Nunc Inc) that had been coated with plasma fibronectin11 (15 µg/cm2) and under the conditions described below for plating in multiwells.

Assays of Growth and Cytotoxic Effects
For growth measurements, fibroblasts or SMCs detached by trypsinization and THP-1 cells were suspended in Iscove's modified Dulbecco's medium (GIBCO), which does not contain copper and iron components, with 2.6 mmol of L-glutamate per liter, 100 µg of streptomycin, and 100 U of penicillin per milliliter. The plating medium also contained 5% donor calf serum (GIBCO) for fibroblasts and 3% human platelet-poor plasma serum12 for SMCs and THP-1 cells. The cells were plated into multiwells (96-well plates for tissue culture; Falcon) that were coated with 15 µg/cm2 of fibronectin isolated from human plasma.11 The number of cells plated was low enough (the precise number is given in the description for each separate experiment) to keep the cultures well under confluence during the experiments. After 24 hours, the platelet-poor plasma serum–containing medium was changed to serum- and plasma-free medium, and the cells were incubated for at least 72 hours to obtain quiescence. For this purpose, Iscove's medium was used with 2.6 mmol of L-glutamate, 100 mg of streptomycin, 105 U of penicillin, 10 mg of transferrin (Collaboratory Research), and 2 g of ovalbumin per liter (Grade V, Sigma). For the subsequent period of exposure to samples, this medium was used, with the addition of 5 µg of cholesterol per milliliter (purified by chromatography on aluminum oxide5 ).

For studies of DNA replication, the cultures were exposed to the samples and to 5 µCi of methyl[3H]thymidine (NET-027, NEN, DuPont) per well for 24 hours, after which the cells were detached by trypsinization and loaded onto a glass filter by using a cell harvester. The activity was measured by liquid scintillation counting.

The culture size was determined by measuring mitochondrial activity using the MTT method, as described,5 13 by the reduction of MTT to blue formazan product. In short, a solution of MTT in PBS (5 mg/mL) was added to each well culture, and the cultures were incubated for 5 hours. When MTT is reduced, it is converted to blue formazan crystals during incubation. After incubation, these crystals were dissolved by adding SDS during an additional incubation period of 16 hours. The absorbency was read at 590 nm in an enzyme immunoassay multiwell photometer. Growth, as measured by the MTT method, was recorded as the net increase in absorbency after the subtraction of the background absorbency and expressed as "MTT units." Control experiments, described in a previous report,5 had shown that there was a direct and linear relationship between the number of cells and the MTT values.

Isolation and Fractionation of Serum Lipoproteins
The serum lipoproteins were isolated and fractionated with density gradient ultracentrifugation in sodium bromide density solutions, as previously described in detail.5 14 The lipoproteins were protected from proteolytic degradation and from oxidation by a number of proteolytic inhibitors, bacteriocide, EDTA, and an antioxidant (BHT) during the entire isolation and fractionation procedure, which was carried out at 10°C.

Total lipoproteins were isolated by density-cushion ultracentrifugation at 302 000g max for 26 hours in a Beckman L8M ultracentrifuge with an angle rotor (50.2 Ti), essentially as described by Rudel et al,15 with minor modifications.5 The total lipoproteins were further fractionated by density-gradient ultracentrifugation at 288 000g max for 22 hours in a swinging-out rotor (SW 41 Ti, Beckman). The density gradient was shaped by layering density cushions to cover a density range of 1.06 to 1.21, as described by Pitas and Mahley.16 If not used within 2 days, the lipoprotein fractions were stored at -135°C under nitrogen after cryopreservation, as described by Rumsey et al.17 18 LDL was measured by its content of protein, with a modification19 of the method described by Lowry et al.20

Oxidation of LDL and Fractionation of OxLDL
LDL was oxidized by exposure to UV light at 257 nm with an intensity of 1 mW/cm2 (Osram HNF 200 W and a 16-cm distance between sample cuvette and lamp). Before exposure to UV light, the LDL was extensively dialyzed in a Pierce microdialysis apparatus at 4°C against 5 mmol of Tris buffer, pH 7.4, per liter in saline followed by saline or against PBS during the whole dialysis for experiments with unfractionated oxLDL. For experiments in which oxLDL was fractionated into a low-molecular-weight and high-molecular-weight fraction, LDL was dialyzed against a volatile buffer, viz 0.15 mol of ammonium bicarbonate buffer, pH 7.8 per liter, to remove EDTA and nonvolatile buffer material. The buffer solutions had been deoxygenated with oxygen-free nitrogen. Before exposure to UV, the dialyzed LDL was diluted with respective buffer to a protein concentration of {approx}0.25 mg/mL and transferred to a quartz cuvette.

The oxidation of LDL mediated by Fe ions was carried out as follows. LDL was extensively dialyzed in a Pierce microdialysis apparatus at 4°C against buffered saline followed by saline, as described above. The concentration of LDL was adjusted to 0.25 mg of protein per milliliter and FeCl3 added, to 0.2 mmol/L concentration, and the sample was incubated at +37°C for the periods indicated for each experiment.

The oxidation was monitored by the TBARS method, as described by Wallin et al21 and by the increased motility of oxLDL in agarose electrophoresis.22 The latter was performed with a kit for agarose gel electrophoresis for the diagnostic determination of lipoproteins (Hydragel LIPO+Lp(a); Sebia) and used under the conditions recommended by the manufacturer.

Changes induced by exposure to UV light were also examined by using PAGE with minigels (PhastGel gradients 4 to 15 or 8 to 25), using the Phast-System (Pharmacia LKB Biotechnology) as recommended by the manufacturer and with inclusion of appropriate molecular-weight standards (high and low range, Bio-Rad; polypeptides, Pharmacia Biotech). The gels were silver stained using procedures slightly modified from that described by Heukeshoven and Dernick23 and as recommended by the manufacturer of the gels.

High- and low-molecular-weight derivatives of LDL on oxidation were separated by ultrafiltration with an Amicon ultrafiltration device with filter cutoff of 30 000 D (YM30, Amicon Division, W.R. Grace & Co). The low-molecular fraction was concentrated in a vacuum in a Speed-Vac (Hetovac, VR-1, Heto-Holten), by which excessively volatile buffer material was also eliminated.

Extraction and Fractionation of LDL Lipids
The lipid component of LDL was extracted as described by Bligh and Dyer,24 as modified by Parthasarathy et al,25 with the addition of BHT to the solvents26 to avoid oxidation. LDL corresponding to 2 milligrams of protein was exhaustively dialyzed in a Pierce microdialysis device against PBS and diluted to 2.2 mL with PBS. Ice-cooled methanol (2.0 mL) with 0.1% BHT was rapidly added to 1.8 mL of the chilled LDL material in a glass centrifuge tube, the mixture was vortexed, 2.0 mL of ice-cooled chloroform was added, and the contents were once again mixed by vortexing. The mixture was centrifuged at 4°C, 1800g for 10 minutes, and the subnatant layer, which contained the lipids, was collected. The solvent was evaporated under oxygen-free nitrogen, and the lipids were dissolved in toluene and stored under nitrogen, at 4°C in the dark. For experiments on the distribution of apoptosis-inducing material in the methanol-water phase and the subnatant, lipid phase, the phases were collected and washed with chloroform and methanol-water, respectively. The methanol-water phase was concentrated in a vacuum in a Speed-Vac and the lipid phase under oxygen-free nitrogen, as described above.

The total LDL lipids were further fractionated into neutral lipids and phospholipids with chromatography on silicic acid.27 In short, the total lipids were dried under nitrogen and redissolved in 2.0 mL methyl-tert-butyl ether/hexane (1:9) and applied to a silica Sep-Pak cartridge (Sep-Pak Classic Silica, catalog No. 51900, Millipore Intertech) preequilibrated with hexane. The neutral lipids were eluted with 20 mL of methyl-tert-butyl ether/hexane (1:1) and the phospholipids with 20 mL methanol. For incubation with cells, the total lipids and the lipid fractions were dried under nitrogen and suspended in PBS with 2% lipid-free bovine serum albumin (A8806, Sigma) by vigorous vortexing under nitrogen and with slight warming. Before use, the albumin solution in PBS had been neutralized.

Detection of Apoptosis
Detection of Internucleosomal DNA Fragmentation With Methods for the In Situ Labeling of Degraded DNA
In the first part of this study, in situ nonisotopic labeling techniques for apoptosis involving avidin-biotin binding28 29 were used, but the reproducibility was not satisfactory with our material and in our hands. We therefore applied a technique involving the template-independent addition by terminal deoxynucleotidal transferase (TdT) of digoxigenin-dUTP to 3'-OH DNA ends generated by the internucleosomal degradation of DNA in apoptosis (TdT-mediated dUTP nick end labeling [TUNEL]) and the visualization of the digoxigenin-labeled DNA by direct immunoperoxidase detection (ApopTag, Oncor).

Detection of Light-Microscopic Nuclear Changes in Apoptosis
The standard May-Grünwald-Giemsa method has been used for the detection of apoptosis in cell cultures, especially for cells that do not exhibit endonuclease activity in conjunction with apoptosis, such as nontransformed vascular SMCs.30 31 The human arterial SMCs flatten out very strongly in the cultures, and the staining of the very attenuated cells with May-Grünwald-Giemsa was too weak and indistinct for our purpose to permit the detection of apoptosis.

Different specific DNA-staining techniques were therefore tried. Staining of DNA with Einarson's gallocyanin-chrome alum method32 was found to be satisfactory. It was performed essentially as previously described33 with cultures on chamber slides fixed in cacodylate-buffered formalin solution prepared from paraformaldehyde.34 The staining solution was prepared with 5 g of chrome alum and 0.15 g of gallocyanin in 100 mL of distilled water that was boiled for 5 minutes, cooled, and filtered (final pH 1.64). The volume was adjusted to 100 mL and staining was performed for 96 hours at room temperature.

Apoptotic cells were counted as follows. The counting was guided by a grid mounted in one of the oculars and covering {approx}80% of the microscope field. Apoptotic cells contained within the grid were counted, and the grid was then moved to a new field. Approximately 500 cells were counted for each chamber culture, and care was taken to cover the culture as evenly as possible.


*    Results
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up arrowIntroduction
up arrowMethods
*Results
down arrowDiscussion
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Low-density lipoprotein is sensitive to oxidation,3 and there is evidence of the presence of oxidized LDL in vivo both in plasma and in atherosclerotic lesions.4 The oxidation of LDL is accelerated in the presence of ions of transition metals such as Fe and Cu and by exposure to UV light (for a review see Reference 3). A certain selectivity for the oxidation of the protein and the lipid component with treatment with Fe and UV, respectively, has been reported.35 We wondered whether this behavior might also be reflected in selectivity in terms of the biological effects of material formed from LDL treated with Fe or UV. LDL was treated with Fe or UV for different periods of time, and the effect of the oxLDL was tested on fibroblast cultures (Fig 1Down). With a relatively short period of exposure to Fe or UV, oxLDL exhibited a far more pronounced growth-promoting effect than native LDL (Fig 1Down). With prolonged exposure to UV, this effect decreased and changed to a cytotoxic effect. With prolonged exposure to Fe, LDL essentially maintained its strong growth-promoting capacity, and cytotoxic effects were not observed.



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Figure 1. Graph showing the effect on the size of fibroblast cultures of oxidized LDL prepared by exposure to UV or Fe3+ for different periods of time. To facilitate a comparison with the control group that did not receive LDL or oxLDL, the values of the other groups were normalized to the value of this group set to zero on the y axis. The size of the cultures was measured using the MTT method (see "Methods"). Native unoxidized LDL (time of treatment=0 on x axis) was added, to a final protein concentration of 450 µg/mL, and oxLDL was added, to a concentration that corresponded to that of the parent LDL. Each culture group contained four well cultures to which 9500 cells per well had been plated. The cultures were incubated for 24 hours. For the series with Fe3+-treated LDL, the cultures receiving zero-time parent control LDL and Fe3+-treated LDL were exposed to Fe3+ at a final concentration of 20 µmol/L. No reaction to this concentration of Fe3+ had been observed for fibroblast cultures in a preceding control experiment.

The development of TBARS is shown in Fig 2ADown. With Fe, TBARS developed more rapidly and reached a higher concentration than with UV treatment. Consequently, there was no relationship between the development of TBARS and the degree of cytotoxicity. As evaluated with SDS-PAGE of UV-treated LDL (Fig 2BDown), very-high-molecular-weight, intact apoB decreased as the duration of treatment increased, with the formation of degradation products of varying molecular sizes. With prolonged exposure (25 hours), almost all the medium-sized molecules also disappeared, and fairly small amounts of very-high-molecular-weight material (apoB) remained (Fig 2BDown).



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Figure 2. Graph (A) showing the development of TBARS during the UV- and Fe3+-mediated oxidation of LDL and SDS-PAGE (B) of LDL exposed to UV for different periods of time. A, TBARS were measured on triplicate samples. B, SDS-PAGE: lane 1, native untreated LDL; lanes 2, 3, and 4, LDL treated with UV for 2, 14, and 25 hours, respectively; lane 5, standard mixture of peptides.

The cytotoxic effects of oxLDL on fibroblasts (Fig 1Up) were very pronounced in the case of LDL exposed to UV, in contrast to that of LDL treated with Fe. The exposure of LDL to UV in the specified conditions (see "Methods") for 25 hours was therefore chosen for the studies of the induction of apoptosis. Fig 3Down illustrates the effect of LDL exposed to UV for different time intervals on the replication of DNA in fibroblasts and the macrophage-like cell line THP-1. Replication was stimulated for both cell types by LDL exposed to UV for short and moderately long time intervals. After prolonged UV treatment, the synthesis of DNA was reduced, thus indicating cytotoxicity. In the same way as for the fibroblasts (Fig 1Up), analogous results were obtained with the macrophage-like cell line for the effects on culture size (Fig 4Down). The macrophages were more sensitive to the cytotoxic effects of oxLDL than the fibroblasts, as demonstrated by the reduction in culture size and the replication of the former, even with LDL that had been exposed to UV during a somewhat short time interval (Figs 1Up, 3Down, and 4Down).



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Figure 3. Graph depicting the effect of LDL exposed to UV for different periods of time on the synthesis of DNA in fibroblasts and in the THP-1 macrophage line. As in Fig 1Up, the value for the control group that did not receive LDL or oxLDL was set to zero on the y axis, and the values of the other groups were normalized accordingly. In the case of fibroblast cultures, 9750 cells per well and in the case of THP-1, 9000 cells per well were plated. Native unoxidized LDL (time of treatment=0) was added, to a final concentration of protein of 430 µg/mL, and oxidized LDL was added, to a concentration that corresponded to that of the parent LDL. The cultures were incubated with the LDL samples for 24 hours, and each group contained six cultures.



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Figure 4. Graph showing the effect on the size of THP-1 macrophage line cultures of LDL exposed to UV for different periods of time. The construction of the figure and the conditions were the same as in Fig 3Up, apart from the fact that each group contained four well cultures.

Apoptosis could be readily visualized in fibroblasts and in the macrophage-like cell line THP-1 by the in situ staining of the internucleosomal degradation of DNA. The combination with phase-contrast microscopy also permitted the detection of other signs of apoptosis such as blebbing, retraction, and fragmentation. Fig 5Down shows micrographs in the phase contrast (Fig 5ADown) and light field (Fig 5BDown) of chamber-slide fibroblast cultures that had been exposed to UV-oxLDL at a concentration corresponding to 400 µg of parent LDL per milliliter and fixed and stained for internucleosomal DNA degradation. The numerical values for the appearance of apoptosis at different concentrations of oxLDL in this experiment are given in Table 1Down.



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Figure 5. Photomicrographs in phase contrast (A) and light field (B) of chamber-slide fibroblast culture exposed to oxLDL for 19 hours and fixed and stained in situ for the internucleosomal degradation of DNA for the detection of apoptosis. LDL treated with UV for 25 hours, to a final concentration corresponding to 400 µg/mL of parent unoxidized LDL, had been added to the culture. Magnification x365.


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Table 1. Effect of OxLDL on Incidence of Apoptosis in Fibroblasts Visualized With In Situ Staining of Internucleosomal Degradation of DNA

The internucleosomal degradation of DNA could not be detected in arterial SMCs by agarose electrophoresis of the extracted DNA (not shown) or by the in situ staining of internucleosomally degraded DNA, in agreement with other reports.30 31 Apoptosis could be readily detected, however, from chromatin condensation visualized by staining DNA using the gallocyanin-chrome alum method (Fig 6Down). Even the degree of chromatin condensation could be roughly estimated. In an experiment with graded exposure to oxLDL, the degree of chromatin condensation was roughly proportional to the concentration of oxLDL in the culture medium (Fig 6ADown through 6D). Additional information on changes indicating apoptosis could be obtained by combination with phase-contrast microscopy (see above).



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Figure 6. Photomicrographs of human arterial SMCs in culture in control cultures without oxLDL (A) and in cultures exposed to increasing concentrations of the low-molecular-weight fraction of UV-oxidized LDL (B through D). The cultures were fixed and stained with gallocyanin-chrome alum for DNA for the detection of apoptosis by the condensation of chromatin. The cells were cultivated in medium with 10% WBS. LDL was exposed to UV for 25 hours. The low-molecular fraction was separated as described in "Methods" and added to cultures shown in B, C, and D, to final concentrations corresponding to 75, 150, and 300 µg/mL of protein, respectively, of the parent unoxidized LDL. The incubation time with oxLDL was 20 hours. The photomicrographs were taken on cultures from an experiment for which the numerical results are given in Table 3Up. Magnification x1900.

The effect on fibroblasts of exposure to increasing concentrations of low- and high-molecular derivatives of oxLDL is shown in Table 1Up. Apoptosis was visualized by the in situ staining of the internucleosomal degradation of DNA. The incidence of apoptotic cells increased gradually as the concentration of low-molecular derivatives of oxLDL increased and reached a level of {approx}10% at the highest concentration of low-molecular oxLDL derivatives. Exposure to the high-molecular fraction of oxLDL was not followed by the appearance of apoptotic cells.

The incidence of apoptosis in SMCs in ordinary cultures without oxLDL and in the presence of 10% WBS was slightly higher than 1%, as evaluated from the appearance of chromatin condensation with gallocyanin-chrome alum stain (Table 2Down). Lipids from native unoxidized LDL apparently did not promote apoptosis in SMCs even at concentrations corresponding to high levels of unoxidized LDL (Table 2Down).


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Table 2. Effect of Total Lipids and Lipid Fractions From Unoxidized LDL on Incidence of Apoptosis in Arterial Smooth Muscle Cells Compared With Serum Controls

As for the fibroblasts (Table 1Up), oxLDL induced apoptosis in SMCs (Table 3Down), and this capacity was confined to the low-molecular derivatives of oxLDL. The effect on SMCs was very marked, and at the highest concentration of low-molecular oxLDL derivatives, all cells were apoptotic (Table 3Down).


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Table 3. Effect of Low- and High-Molecular-Weight Fractions of OxLDL on Incidence of Apoptosis in Arterial Smooth Muscle Cells1

Exposure of SMCs to the low-molecular fraction of oxLDL under serum-free conditions had a stronger apoptosis-inducing effect than in the presence of 10% WBS (Table 4Down).


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Table 4. Effect of Low-Molecular-Weight Fraction of OxLDL on Incidence of Apoptosis in Arterial Smooth Muscle Cells Under Serum-Free Conditions and With Serum1

OxLDL was separated into a water-methanol and a chloroform (lipid-soluble) fraction by phase separation during the extraction of lipids from oxLDL, but the apoptosis-inducing effect of the fractions was not clearly different (not shown).


*    Discussion
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up arrowAbstract
up arrowIntroduction
up arrowMethods
up arrowResults
*Discussion
down arrowReferences
 
There is a distinct relationship between oxidative stress and the induction of programmed cell death, apoptosis. Many oxidants initiate apoptosis and several antioxidants inhibit it.36 There are several lines of defense against unwanted oxidation in tissues and cells, and overt manifestations of oxidation in tissue components are rare under normal conditions. In some pathological states, however, such as atherosclerosis, the effects of uncontrolled oxidation are more apparent, and it has even been suggested that they play a pathophysiological role in the development of the disease. Irregular metabolism of LDL is strongly related to the development of atherosclerosis, and recent evidence suggests that this phenomenon may be mediated by effects from the uncontrolled oxidation of LDL (for a review see Reference 4). There is, in fact, direct evidence of the presence of oxLDL in plasma37 and in atherosclerotic lesions.37 38 The focal excessive death of arterial tissue is very prominent in atherosclerotic lesions, and one of the aims of the present work was to elucidate whether this process could be due to the effects of oxLDL, possibly by the induction of apoptosis.

A wide variety of methods have been used for the experimental oxidation of LDL. Oxidation is commonly promoted by exposure to UV light35 39 40 or to transition metal ion–mediated oxidation with Fe or Cu ions (for a review see Reference 4). Exposure to UV light was found to be far more effective when it came to the production of cytotoxic and apoptosis-inducing material from LDL than exposure to Fe ions, and the former method was therefore used for LDL oxidation in the present work.

The detection of apoptosis by using extracted DNA material was not suitable for the present purpose, but the in situ staining was very useful, except for SMCs in which the internucleosomal degradation of DNA did not accompany the structural changes of apoptosis.30 41 For this cell type, we applied the gallocyanin-chrome alum stain for DNA to visualize the condensation of the chromatin as an indication of apoptosis. This method proved to be very sensitive and permitted a semiquantitative assessment of the degree of chromatin condensation.

Relatively short exposure of LDL to UV or Fe ions increased the mitogenic effect of oxLDL to several times that of the native LDL. This effect was preserved even after the prolonged treatment of LDL with Fe. The fact that oxidation strongly increased the mitogenicity of LDL for SMCs and for macrophages may be a very important observation pathophysiologically. The increased growth of SMCs is regarded as a key mechanism for the development of intimal thickening and for the development of the thickened intima to a fibroatheroma or fibrous plaque. Macrophages tend to clear away accumulations of lipoproteins to become foam cells, and many of them may end up as cell debris and extracellular lipids to add instability to the atherosclerotic plaque. These changes are important components of the atherogenic process (for a review see References 6 and 42), and the above-mentioned results of the present work may therefore contribute to a pathophysiological explanation for one of the potential roles of oxLDL in atherogenesis. There could be several mechanisms underlying the strongly increased mitogenicity of shortly oxidized LDL. It has recently been shown that the mitogenic and growth-promoting effects of native, unoxidized LDL require the presence of LDL receptors.14 It is known that lightly oxidized LDL, in contrast to native LDL, may be internalized by multiple receptor-mediated pathways (for a review see Reference 3), which could increase the efficiency of internalization and thereby the effects on the cells. In addition, there is evidence that low concentrations of oxLDL may increase the production of PDGF-AA and the expression of PDGF receptors in SMCs and could thereby perhaps increase growth by autocrine stimulation.43

The cytotoxic effects of experimentally oxidized LDL and even of LDL that had not been intentionally oxidized have been described in many reports,5 40 43 44 45 46 47 48 but the nature of the cell death has not been clarified. Death by apoptosis induced by various means in vitro has been reported for macrophages49 50 51 52 53 and fibroblasts (for a review see Reference 54) of different origins and for arterial SMCs.30 31 55 Little information is available on the apoptosis-inducing effects of oxLDL. OxLDL was found to induce apoptosis in a macrophage-like murine cell line,49 in mouse peritoneal macrophages,50 and in lymphoblastoid cells.56 57 To our knowledge, no data on the apoptosis-inducing effects of oxLDL on arterial SMCs and fibroblasts have previously been presented. Sparse information is available on the occurrence of apoptosis in conditions more like those in the arterial wall in vivo. The occurrence of apoptosis was reported for reconstituted (spheroidal) human arterial smooth muscle tissue55 and for SMCs in human saphenous vein grafts.58 Evidence was recently presented for the occurrence of apoptosis in atherosclerotic lesions.59 60 Apoptosis has been described for myofibroblasts in granulation tissue,61 which have many properties in common with the modulated, synthetic phenotype of the arterial SMCs.

The generation of cytotoxic and apoptosis-inducing material on prolonged UV treatment was related to the degradation of apoB to low-molecular-weight material, in agreement with several reports on the oxidative degradation of apoB after various types of oxidative treatment.25 35 62 63 The results of the present study clearly demonstrate that the prolonged oxidation of LDL promoted by UV generates derivatives with apoptosis-inducing effects in SMCs, in a macrophage line and in fibroblasts. The apoptosis-inducing effect was confined to the low-molecular-weight material after prolonged exposure to UV.

Apoptosis was induced in vitro at a concentration of oxLDL material corresponding to slightly above the concentration of LDL in the interstitial fluid (100 to 150 µg of apoB per milliliter64 ). This is clearly within the pathophysiological range for the arterial wall tissue, especially in certain atherogenic situations such as endothelial injury or hyperlipidemic states.

Serum had a protective effect on apoptosis (Table 4Up), in agreement with other studies in which serum65 or growth factors in serum, such as insulin-like growth factor-I66 and PDGF,67 inhibited the appearance of apoptosis. Since oxLDL contained both lipid- and water-soluble components with the capacity to induce apoptosis, it was not possible to conclude from the present results whether the apoptosis-inducing material was derived from the lipid68 or the apoB component of LDL.

Proliferative tissue changes alternating with necrosis are apparent in atherosclerotic lesions. Such changes are the basis of several of the most severe complications of atherosclerosis, such as arterial stenosis or arterial rupture with hemorrhage or plaque rupture or fissuring with mural hemorrhage, atheroembolism, and/or thrombosis with subsequent occlusion. OxLDL is currently believed to play an important atherogenic role. The dual effect of oxLDL, ie, its strong growth-promoting effect or the induction of cell death by apoptosis, depending on the degree of change by oxidation, as demonstrated in this report is compatible with the conclusion that oxLDL plays a role not only in atherogenesis but also more extensively in the development of the structure typical of the atherosclerotic lesion and thereby contributes to the important clinical effects of the lesion.


*    Selected Abbreviations and Acronyms
 
oxLDL = oxidized LDL
PAGE = polyacrylamide gel electrophoresis
PDGF = platelet-derived growth factor
SMC(s) = smooth muscle cell(s)
TBARS = thiobarbituric acid–reactive substances
WBS = human whole-blood serum


*    Acknowledgments
 
This study was supported by the Swedish Medical Research Council (project No. 2589) and by the LUA fund of the Medical Faculty of the Göteborg University. The authors thank Eva Carlsson for excellent technical assistance.


*    Footnotes
 
Reprint requests to Sören Björkerud, MD, PhD, Department of Pathology, Sahlgrenska University Hospital, S-413 45, Göteborg, Sweden.

Received May 8, 1995; accepted November 7, 1995.


*    References
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*References
 
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