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Divisions of
* Experimental Pathology,
Sensor Science and Molecular Physics,
Occupational and Environmental Medicine and
Pharmacology, Faculty of Health Sciences, Linköping University, Linköping, Sweden; and
|| James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky, USA
1Correspondence: Division of Experimental Pathology, Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden. E-mail: yuan.ximing{at}inr.liu.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: atherosclerosis • apoferritin • apoptosis • carbonyls • ceroid • iron • macrophages
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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that the interior of such lesions may constitute a "death zone" containing toxic materials that may cause dysfunction and demise of invading phagocytes. This would lead to a progressive accumulation of dead or dying phagocytic cells incapable of resolving the lesion but still quite capable of releasing chemokines and cytokines, thereby promoting further phagocyte ingress and growth of the plaque (2
, 3)
.
In partial support of this idea, we earlier reported that 7-oxysterols, known to be present within atherosclerotic plaques (4)
, are potent cytotoxins capable of causing foam cell formation and apoptotic death of macrophages via labile iron-driven oxidative cellular damage (1
, 5)
. Indeed, apoptosis of macrophages has been observed within human atherosclerotic lesions (6)
, suggesting that a similar process might be operative in vivo. However, these earlier studies left open the question of the nature of the toxic elements within atherosclerotic lesions.
In the present investigation we have assessed the cytotoxic effects of nonfractionated gruel and insoluble (ceroid-like) material derived from advanced human atherosclerotic lesions, with particular emphasis on the effects of these preparations on a macrophage-like cell line and human macrophages. After extensive proteolytic digestion and aqueous and organic extraction of plaque material, an autofluorescent, calcium- and iron-rich residue was obtained. Small amounts of this insoluble material, as well as preparations of nonfractionated gruel, were added to cultured macrophages. We observed that although both were cytotoxic, the nonfractionated material was especially toxic, even more so (on a weight to weight basis) than pure 7-beta hydroxycholesterol (7ß-OH), a previously identified cytotoxic component of plaque. These observations lend further support to the concept that the interior of atheromatous lesions is indeed a death zone that contains powerful cytocidal substances capable of blocking plaque resolution by invading macrophages.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In situ visualization of ceroid in atheroma lesions
Segments of normal human mammary arteries (n=4) and advanced atherosclerotic lesions of human abdominal aortas (n=5) or carotid arteries (n=15) were collected from autopsy cases and carotid endarterectomy. The fatty streak lesions of aortas from Watanabe heritable hyperlipidemic (WHHL) rabbits were prepared as described elsewhere (7)
. Frozen (10 µm) and paraffin (5 µm) sections were serially cut according to standard procedures. To visualize ceroid in atheroma lesions, sections from both human and rabbit lesions were stained, or not, with Hoechst 3334 (10 µg/ml, 15 min), then examined by fluorescence microscopy (AH3-RFC, Olympus).
The studies were approved by institutional review boards at Linköping University (Ethical approval number Dnr 29–97 for WHHL rabbits and Dnr 03–499 for human atheroma lesions).
Preparation of nonfractionated atheroma gruel- and ceroid-depleted fractions of gruel
Atherosclerotic gruel (1.5 g per preparation) from human atheroma lesions was removed by scraping advanced lesions. The material was homogenized using a glass-on-glass homogenizer and suspended in ethanol (10 mg/ml as a stock solution). In some experiments, atheroma gruel was preincubated with freshly prepared sodium borohydride (1 mmol/L stock solution in 0.1 mmol/L NaOH) for 4 h at 22°C. The ceroid-depleted fractions of gruel were prepared by collecting the supernatants of atheroma homogenates after centrifugation at 2000 g for 10 min.
Preparation of insoluble ceroid from atheromatous human arteries
Atheroma gruel derived from human aortas or carotid arteries was scraped and weighed as described above and a similar weight of tissue from normal mammary arteries was used for controls. The homogenized material from lesions was centrifuged at 10,000 g and washed three times in sterile PBS, pH 7.4. The homogenized material was then exposed to proteinase K (1 mg/ml) overnight at 55°C. Proteinase K was removed by centrifugation at 10,000 g and the insoluble material was again washed in PBS. Residual lipids in the preparation were extracted by vigorous mixing with a mixture of chloroform and methanol (1:2) followed by centrifugation at 10,000 g. This step was repeated three times and followed by a final wash in PBS. The resultant aqueous- and organic-insoluble, protease-indigestible sediment was collected. Before use, the dried material was suspended in PBS.
Fourier transform infrared spectroscopy (FTIR) of plaque ceroid
Atheroma ceroid samples from different types of lesions, including fatty streaks (type 1), intact fibrotic lesions (type 2), and ruptured plaques (type 3), were prepared for FTIR analyses by grinding 1 mg of ceroid-like material with 300 mg KBr in an agate mortar. This preparation was subsequently pressed into a pellet (surface area=1.3 cm2) using a Perkin-Elmer pressure device. The pellet was analyzed with a Bruker IFS 48 FTIR spectrometer operating at a resolution of 4 cm–1 by averaging 100 scans.
Raman spectroscopy of plaque ceroid
Ceroid samples from different types of lesions, as described above, were also prepared for Raman spectroscopy. Approximately 1 mg of the ceroid-like material was placed in a cavity on an aluminum disk. The disk was then placed in a Bruker FRA 106 Raman spectrometer (laser 
=1.064 nm) and the spectra from 200 scans over 6 min were recorded at a power setting of 50 mW and a resolution of 4 cm–1.
Atomic absorption spectroscopy
Atheroma ceroid was dried and further digested in concentrated nitric acid at 50°C for 12 h (8)
. The samples were then diluted with double-distilled water for determination of iron and calcium. Pellets of cells preincubated with ceroid were collected and the concentrations of iron and calcium in the samples were determined by atomic absorption spectrophotometry (Z-8270 Polarized Zeeman, Hitachi).
ELISA for carbonyl content in atherma gruel and ceroid
Protein concentrations were adjusted to 200 µg/ml by dilution with PBS, and protein carbonyl contents were determined by ELISA as described previously (9)
. Briefly, samples were reacted with 2,4-dinitrophenylhydrazine (DNPH; Sigma) and derivatized proteins were adsorbed onto an ELISA plate (0.5 µg protein per well). The primary antibody (Ab) was a rabbit anti-DNPH IgG (Molecular Probes, Eugene, OR, USA) and the secondary Ab was an anti-rabbit IgE conjugated to horseradish peroxidase (DakoCytomation A/S, Glostrup, Denmark). Carbonyl content (nmol/mg protein) in samples was calculated using a standard curve that was generated using oxidized BSA.
Cell culture and experimental conditions
The murine macrophage-like cell line, J-774, was cultured in Ham’s F10 complete medium. The cells were subcultivated once a week. Confluent cells were seeded in 35 mm culture dishes at a density of 5 x 105/dish and grown to near confluence before being exposed to isolated ceroid (stock suspended in PBS at 1 mg/ml). Typically, cells were exposed to 50–100 µg/ml ceroid in culture medium for 24 to 48 h. Human monocyte-derived macrophages were prepared and cultured as described previously (5)
.
In other experiments, similarly prepared cells were exposed to nonfractionated atherosclerotic plaque gruel at a final concentration of 0.5–20 µg/ml (expressed as original weight of the material). Additional samples of cells were exposed to atheroma ceroid or 7ß-OH with various additions. Some cultures were preincubated with apoferritin (1 mg/ml) for 16 h and subsequently exposed to 7ß-OH, plaque gruel, and atheroma ceroid.
Assessment of viability and apoptosis
Loss of plasma membrane integrity (reflecting necrosis or postapoptotic necrosis) was determined by Trypan blue dye exclusion. Following the different procedures, detached and adherent cells were stained with 0.2% Trypan blue for 5 min, then viewed and counted by light microscopy.
For morphological assessment of apoptosis, cells were stained with Wright-Giemsa (15:1 dilution of Giemsa, 2 min) or Hoechst 3334 (10 µg/ml, 15 min). Using these stains, apoptotic cells can be distinguished from viable cells based on their cellular and nuclear morphology. Shrunken cells with condensed or fragmented nuclei were considered apoptotic. In each dish, 200 cells were randomly counted under light microscopy, and the percentage of apoptotic cells were calculated for each condition. Digitized images of the cells were obtained using a Nikon Microphot-SA microscope (Nikon, Tokyo, Japan) equipped with a Hamamatsu C4742–95 digital camera (Bridgewater, NJ, USA).
Apoptotic cells were also evaluated using the pan-caspase inhibitor caspACETM*FITC-VAD-FMK in situ marker (10 µmol/L in complete medium for 20 min at 20°C) and annexin V/propidium iodide (PI) staining. Apoptotic cells showed only annexin V green fluorescence whereas both annexin V- and PI-positive cells were considered necrotic (5)
. Cells containing activated caspases turned intensely green on blue light excitation and were documented using the above photo microscope-based equipment or analyzed by flow cytometry.
Apoptosis was also detected by the terminal dUTP nick end-labeling (TUNEL) technique using an ApopTag in situ apoptosis detection kit according to the manufacturer’s instructions (Oncor Inc., Gaithersburg, MD, USA).
Statistics
Data are expressed as means ± SE. Statistical comparisons were made using ANOVA. Differences were considered statistically significant at a P value < 0.05. All experiments were repeated at least three times unless otherwise indicated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A, B). Prominent yellow-brown granular pigments, particularly in the necrotic core regions, showed ceroid/lipofuscin-type autofluorescence in sections from advanced lesions (Fig. 1B, C
). This ceroid-like material was particularly abundant in areas rich in apoptotic foam cells (Fig. 1D
). The autofluorescence was present in human paraffin sections (Fig. 1B-D
) and rabbit frozen sections (Fig. 1E
). This, together with the granular morphology of ceroid under light microscopy, excludes the possibility that the observed autofluorescence may be due to tissue fixation or paraffin processing. The frozen sections of aortas from WHHL rabbits also showed formation of atheroma ceroid even in early fatty streak lesions. Such granular ceroid was predominantly intracellular although some was also extracellular (Fig. 1D
), likely released from dead foam cells.
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After extensive homogenization and aqueous/organic extraction of the atheromatous material, a yellowish fluorescent substance that resembled the ceroid seen in atheroma lesions was obtained (Fig. 1F, G
). This substance represented 
50% by weight of the starting material from advanced lesions.
Figure 2
A shows Raman spectra of the yellowish solid substance obtained from lesions of progressive severity (lesion types 1, 2, 3). These Raman spectra were recorded with a near-infrared laser excitation source operating at wavelength of 1.064 nm making the spectra essentially free of interfering background intensity from fluorescent trace molecules. The peaks at 1080, 960, 590, and 430 cm–1 all increased in intensity with progression of the atheromatous lesions and resembled the most prominent peaks found in the Raman spectra of calcium hydroxyapatite (hemagglutin, Ca10(PO4)6(OH)2) and dentin. The 1080 cm–1 peak, for example, is attributed to the symmetric CO32–/asymmetric PO42– vibration and the 960, 590, and 430 cm–1 peaks are due to vibrations of PO43– species (9)
(Table 1
for assignments; ref. 10
). Thus, as expected from much earlier observations (11)
, this insoluble ceroid-like material contains abundant calcium phosphate and calcium carbonate. The abundant calcium content of this substance was further confirmed with FTIR analyses (Fig. 2B
). The peaks in the 1300 to 1800 cm–1 region, which are also seen in dentin but not in hemagglutinin (HA), are attributed to CH2 and amide vibrations (amide I at 1650 cm–1 representing C=O groups and amide II at 1550 cm–1 representing C-N-H groups) characteristic of proteinase K-resistant polymerized protein residues (most likely collagen), while the peaks in the 550 to 650 cm–1 and 1000 to 1200 cm–1 ranges belong to PO43– species (10
, 12
, 13)
. Moreover, in accordance with the Raman data, the relative intensity of the phosphate to protein peaks, and thereby the mineral content, increased with progressive severity of the lesions. Apart from small changes in relative intensity and frequency, the spectrum of material isolated from type 3 lesions is virtually indistinguishable from that of dentin. Thus, the yellowish substance extracted from atheroma lesions is composed predominantly of calcium phosphate, calcium carbonate, and polymerized lipofuscin-like residues of collagen and lipid.
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Additional FTIR analyses were performed on atheroma gruel samples in order to investigate the effect of NaBH4 –exposure and possible differences between the gruel and ceroid samples. In Fig. 2C
, spectra of three gruel samples are presented: one untreated control sample (Fig. 2C, a
) and two samples exposed to 3 mmol/L (Fig. 2C
, 2b)
and 6 mmol/L NaBH4 (Fig. 2C, c
), respectively (see Materials and Methods for details). The effects of NaBH4 treatment are illustrated by two spectra resulting from subtracting the control spectrum from the spectra of exposed samples (indicated as Fig. 2C
, b–a, c–a, respectively). A clear reduction of the C=O intensity (1650–1900 cm–1) is demonstrated as negative peaks, indicating decreased amounts of C=O groups (e.g., in esters or aldehydes). This effect is more pronounced in the sample treated with 6 mmol/L NaBH4 than in the sample exposed to 3 mmol/L (more negative peaks). For both samples, the O-H stretching region increases in intensity, possibly due to a reduction of carbonyls to alcohols. When the FTIR spectra of ceroid and gruel samples are compared, the main difference (Fig. 2D
) is an alteration of the relative ratio of HA-related vibrations (1000–1200 cm–1) to the protein-related peaks (1450–1700 cm–1). This undoubtedly shows that the ceroid samples are richer than gruel samples in HA-like species. Other less pronounced differences are seen in the 1400–1700 cm–1 range, which includes carbonyls, protein contents, and related structures in the samples.
Atomic absorption assays showed that compared with normal arterial tissues (iron 0.57±0.38 nmol/mg tissue, calcium 0.18±0.02 µmol/mg tissue) substantial increases in iron and calcium were found in human atheroma ceroid (iron 38.3±30 nmol/mg tissue, calcium 72.2±80 µmol/mg tissue).
The indigestible plaque ceroid is taken up by macrophages and engenders apoptosis
Samples of the insoluble ceroid-like material were added to cultured J774 macrophages at concentrations of 50–100 µg/ml of culture medium. It was engulfed over a period of 24 h, and easily recognized intracellularly due to its granularity and autofluorescence (using phase contrast and fluorescence microscopy, see Fig. 3
). More than 600 cells were counted from four different areas of each culture dish after treatment with atheroma ceroid. Ceroid-containing cells were 20.7 ± 2% of total cells after 24 h and remained at the same levels after 48 h (21.3±3%), indicating that uptake of ceroid by cells mainly took place during the first 24 h. After an incubation period of >48 h, we observed progressive apoptotic death characterized by distinctly condensed and fragmented nuclei, which appeared mainly in cells that had phagocytosed the ceroid material (Fig. 3C, D
). Phagocytosed ceroid in the cells showed similar ceroid-type autofluorescence as described above for atheroma tissues (Fig. 3D-F
).
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Plasma membrane integrity of J774 cells was assayed after exposure to atheroma ceroid and gruel by Trypan blue dye exclusion tests. After exposure to ceroid (100 µg/ml, 48 h), plasma membranes largely remained intact (>90%) while 0.5 µg/ml atheroma gruel caused a significant loss of cell viability (necrosis 28.01±6.27%; apoptosis 61%±8.67%, n=3). In human macrophages, compared with controls, 20 µg/ml gruel also induced a significant increase in apoptosis (31.6±6.8% vs. 5.42±2.3%, n=3). Apoptotic cells were further determined by assay of phosphatidylserine exposure after annexin V/PI staining, nuclear morphological alterations after Hoechst 3334 staining (Fig. 3D
), pan-caspase inhibitor caspACETM*FITC-VAD-FMK in situ marker, and TUNEL assays. As shown in Fig. 4
, compared with controls (Fig. 4A, B
), 100 µg/ml ceroid induced distinctive phosphatidylserine exposure (Fig. 4C, D
) and significant increases in caspase activation after 24 h (Fig. 4E
, black line), further enhanced by 7ß-OH (Fig. 4E
, dotted line). Ceroid-induced nuclear fragmentation was also confirmed by TUNEL assays. Significant alterations in TUNEL-positive cells were seen after treatment with 50–100 µg/ml ceroid for 48 h (Fig. 4F
).
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Crude gruel from atherosclerotic plaques is more cytotoxic than atheroma ceroid and 7ß-OH
Although the above results clearly indicate that the ceroid-like plaque material exerts significant toxicity and induces macrophage apoptosis, we wanted to compare this with the toxic effects of the starting lipid-rich material. As shown in Fig. 5
A, atherosclerotic gruel was more toxic than the atheroma ceroid after 24 h incubation. Compared with control cells, at a concentration of 0.5 µg/ml gruel, a majority of exposed J774 cells were killed whereas 100 µg/ml ceroid or 10.3 µg/ml (28 µmol/L) 7ß-OH were substantially less toxic. These results suggest that the extractable, perhaps oxidized, lipidaceous materials within the gruel are the most cytotoxic components in human atheroma lesions.
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The cytocidal effects of atheroma gruel were also tested on human macrophages. We found that crude gruel (20 µg/ml) caused a significant increase in apoptotic cell death. The toxicity of such gruel was substantially reduced after removal of the insoluble ceroid-like material (via centrifugation at 2000 g for 10 min). The apoptosis frequency of human macrophages treated with 20 µg/ml crude gruel was 31.6 ± 6.8%, but was only 8 ± 0.03% in cells treated with ceroid-depleted gruel (P<0.05, n=3). This result indicates that the full toxic effect of gruel is dependent on the simultaneous presence of both the soluble and insoluble fractions.
The high iron and calcium content of the ceroid-like material suggested that redox-active iron and calcium might be important in the toxicity of crude gruel and ceroid. Atomic absorption analyses of total metals showed that after uptake of ceroid, the iron and calcium content of J-774 cells was markedly increased compared with untreated cells (iron: 0.62±0.08 nmol/mg protein in untreated cells vs. 14.8±2.7 nmol/mg in ceroid-treated cells; calcium: 1.73±1.36 µmol/mg in untreated cells vs. 33.3±8.4 µmol/mg in ceroid-treated cells). The importance of elevated intracellular iron in gruel-induced toxicity was tested by preincubating J-774 cells with apo-ferritin (1 mg/ml) for 16 h before exposure to atheroma gruel, atheroma ceroid, or 7ß-OH. When cells are exposed to apo-ferritin, it is taken up by fluid phase endocytosis and finally delivered to the lysosomal compartment as described previously (14)
. Examination of Giemsa-stained cells showed that exposure to apo-ferritin alone caused vacuolization compared with control cells. Such preexposure to apo-ferritin before the plaque gruel efficiently protected against the apoptotic/necrotic cell death otherwise induced by gruel. Preexposure to apo-ferritin also resulted in a distinct protection against ceroid and 7ß-OH-induced apoptosis (Fig. 5A
). To further assess possible roles of iron and calcium in this model, cells were exposed to ceroid together with the iron or calcium chelators SIH and BAPTA/acetoxymethyl ester. Ceroid-induced apoptosis was enhanced by 7ß-OH and significantly reduced by the iron chelator SIH and the calcium chelator BAPTA (Fig. 5B
). However, we should note that the protective effects of BAPTA do not necessarily indict calcium in this effect inasmuch as this chelator has been shown to bind both ferrous and ferric iron (15)
. Enhanced apoptosis was also observed in cells treated with a combination of 7ß-OH, iron, and hydroxyapatite (Fig. 5B
). Similar trends were seen in assays of activated caspase. 7ß-OH significantly enhanced ceroid-induced caspase activation (Fig. 4E
, Fig. 5C
). These results strengthen the idea that the presence of cellular iron or calcium and oxidized lipids such as oxysterols may contribute to the macrophage apoptosis and death zone formation in atherogenesis (1
, 5)
.
Oxidation products such as aldehydes, carbonyls, and lipid hydroperoxides may be major toxic components in atheroma gruel
Given the abundant thiobarbituric acid-reactive aldehydic substances within atheroma plaque (16
, 17)
, we reasoned that aldehydes might be at least one toxic component of crude atheroma gruel. Therefore, some samples were preincubated with sodium borohydride, which will reduce aldehydes, carbonyls, and lipid peroxides to alcohols. As shown in Fig. 6
, pretreatment of atheroma gruel with sodium borohydride (after 4 h incubation at room temperature) led to a significant reduction of toxicity. However such treatment had less effect on ceroid-induced cell death because of the relatively less abundance of carbonyls in ceroid compared with gruel (Fig. 2D
). Furthermore, the content of carbonyl compounds in atheroma gruel (21.1 nmol/mg proteins) was dramatically reduced by incubation with sodium borohydride (5.4 or 4.2 nmol/mg proteins after treatment with 3 mmol and 6 mmol of sodium borohydride, respectively) at 37°C for 4 h. This result was also confirmed by Fourier transform infrared spectroscopy as shown in Fig. 2C
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
5
6)
, supporting the idea that the interior of these lesions might represent a death zone for macrophages and other cells ordinarily responsible for the resolution of wounds and lesions. If so, a better knowledge of the nature of these toxic materials might lead to the rational development of anti-atherogenic agents.
Consequently, we have further investigated the nature of the toxic materials within atherosclerotic lesions and find that both soluble and insoluble plaque components exhibit pronounced cytotoxicity toward a macrophage-like cell line and primary cultures of human monocyte/macrophages. Human atherosclerotic lesions are characterized by the presence of "ceroid" in macrophage-derived foam cells (18)
, although no previous effort has been made to isolate this material from human atheroma lesions in order to characterize its biological properties. This substance is composed of polymerized oxidation products of lipids and proteins (18
, 19)
and is resistant to digestion. There is a considerable body of evidence indicating that oxidative stress is a causal factor in ceroid formation, and ceroid can be used as a marker of oxidative stress seen in aging-related diseases, including atherosclerosis, as proposed earlier (20)
. It is generally believed that inside macrophages and foam cells, ceroid forms at least in part as a result of LDL oxidation in reactions, which are likely transitional-metal catalyzed (21
, 22)
. In the presence of redox-active iron, lipid peroxides form and decompose yielding aldhydes. These reaction products are toxic and, as we have argued elsewhere, induce lysosomal destabilization with release of lysosomal material and ensuing apoptosis (1
, 5
, 23
, 24)
.
In the present instance we have isolated plaque "ceroid" as the insoluble material remaining after protease digestion of crude atheromatous gruel, followed by extensive aqueous and organic extraction. An analysis of this material revealed that, despite its fluorescence, it is comprised predominantly of calcium phosphate and calcium carbonate with residua of protein, lipid, and, more important, iron. Enriched hydroxyapatite in isolated ceroid may associate with elastin residues, as described in a much earlier study of aortas from young and older individuals using X-ray diffraction (25)
. The implications of such an association in plaque stability require further investigation. The origin of the relatively abundant iron within atheroma ceroid remains incompletely understood but could arise from heme and heme degradation products (23
, 26)
. Earlier suggestions regarding the importance of lesion-associated iron in atherogenesis (23
24)
have been supported by a recent clinical pathological study (27)
.
When atheroma ceroid is engulfed by macrophages or macrophage-like cell lines, apoptosis ensues. Cell death is likely a result of at least two toxic principles. First, as has been appreciated for many years, many types of insoluble microparticulates are cytotoxic by virtue of disrupting the barrier function of biological membranes. Second, a significant fraction of cytotoxicity appears to derive from ceroid-associated iron. This is supported by our observations that the cytotoxic effects of this isolated ceroid are substantially reduced by either preincubation with apo-ferritin (a "natural" iron chelator) or addition of the iron chelator, SIH. Precisely how chelatable iron is involved in ceroid-mediated cytotoxicity is by no means clear. Protection by iron chelation could arise from either the neutralization of ceroid-associated iron or the chelation of intralysosomal iron, which we and others have shown prevents apoptotic cell death caused by a number of agents (5
, 28
29
30)
.
An even more potent toxic effect was exerted by homogenates of crude atheromatous gruel. This material has pronounced cytotoxic effects, with <20 µg/ml of crude gruel killing most macrophages after 24 h exposure under otherwise normal cell culture conditions. Given this extreme toxicity, it is almost a certainty that macrophages could not survive within the core of an advanced atherosclerotic lesion. Most of the toxic effect of this crude material evidently arises from aldehydes, carbonyls, or hydroperoxides because preincubation of this material with borohydride simultaneously diminishes cytotoxicity and reduces most C=O groups. Finally, through mechanisms yet to be explained, it appears that the soluble and insoluble components of crude gruel exert an additive toxicity wherein removal of the insoluble (ceroid-containing) fraction considerably diminishes the cytotoxicity of ethanolic gruel preparations. This may well suggest that toxicity of atheroma materials depends on coordinate effects of metals such as iron that are abundant in ceroid and carbonyls present in crude gruel. Possible interactions between redox-active iron (e.g., heme derivatives) and carbonyls may contribute to the formation of oxidative stress and cytotoxicity in the atheroma death zone (31)
.
In conclusion, our results lend further support to the concept that the interior of atherosclerotic lesions constitutes a death zone for incoming inflammatory cells. Our present results indicate that the toxic elements within this zone include both an insoluble ceroid-like substance and a soluble aldehyde-rich fraction. One observation of potential importance is that reduction of these carbonyls or hydroperoxides with borohydride blocks almost all cytotoxicity of the lipid-rich gruel. A more detailed investigation of these toxic plaque components may help explain the persistence and progression of atherosclerotic lesions.
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ACKNOWLEDGMENTS |
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Received for publication April 3, 2006. Accepted for publication June 19, 2006.
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