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Mildly oxidized LDL particle subspecies are distinct in their capacity to induce apoptosis in endothelial cells: role of lipid hydroperoxides¹

ANATOL KONTUSH², LAURENT CHANCHARME², ISABELLE ESCARGUEIL-BLANC^*, PATRICE THEROND, ROBERT SALVAYRE^*, ANNE NÈGRE-SALVAYRE^* and M. JOHN CHAPMAN³

INSERM U.551, Pavillon Benjamin Delessert, Hôpital de la Pitié, 75651 Paris Cedex 13;
^* INSERM U.466, CHU Rangueil, 34054 Toulouse Cedex; and
INSERM U.347, CHU Kremlin Bicetre, 94276 Le Kremlin-Bicetre, France

³Correspondence: INSERM U.551, Hôpital de la Pitié, 75651 Paris Cedex 13, France. E-mail: chapman{at}infobiogen.fr

SPECIFIC AIMS

Atherosclerosis risk is intimately related to the heterogeneity of low density lipoprotein (LDL) particles, but the potential relationship between oxidative modification of distinct LDL particle subspecies and induction of apoptosis in arterial wall cells is indeterminate. Our objective was to determine whether differences in molecular species of oxidized lipids in mildly oxidized large, intermediate LDL (oxLDL3) vs. small, dense LDL (oxLDL5) could affect 1) the induction of apoptosis and necrosis in endothelial cells as a function of the concentration and number of LDL particles and of the degree of oxidation, and 2) whether the nature and extent of oxLDL-induced cell death (apoptosis and necrosis) was related to the physicochemical properties of oxidatively modified LDL subfractions and, more specifically, to the lipid hydroperoxide (LOOH) content of LDL.

PRINCIPAL FINDINGS

1. OxLDL3 is uniquely distinct from oxLDL5 in its elevated content of LOOH
The time course of the oxidation of LDL subfractions by Cu²⁺ at 37°C was monitored continuously at 234 nm; lag time (T_lag), which corresponds to the end of the lag phase, and propagation half-time (T_1/2), which corresponds to the midpoint of the propagation phase, were analyzed. OxLDL-induced cytotoxicity was determined as a function of oxLDL concentration in the medium or LDL oxidation state. In the first case, oxidation was terminated at the respective T_1/2 for each subfraction; the duration of oxidation was different for the two subspecies, but particles were at an equivalent stage of oxidation, i.e., at the middle of the propagation phase. In the second case, samples were taken at the same time point from the reaction mixture of each subfraction; in this case, corresponding samples of LDL3 and LDL5 were at distinct stages of oxidation at each time point.

OxLDL3 and oxLDL5 at the respective T_1/2 revealed no significant difference in oxycholesterol and TBARS content. TBARS content of oxidized LDL was within the range previously reported for minimally (mildly) modified LDL. At T_1/2, oxLDL3 and oxLDL5 displayed increased electrophoretic mobility (up to twofold) compared with the respective native particles, but again no difference was observed between oxLDL3 and oxLDL5, suggesting that their net negative charge was similar. Native LDL subfractions did not reveal significant difference in their copper binding capacity.

Cholesteryl ester-derived hydroperoxides (CEOOH) were more abundant than phosphatidylcholine-derived hydroperoxide (PCOOH) at all stages of oxidation; moreover, only a minor increase in PCOOH levels (from 0 to a maximum of 4.7 mol/mol LDL in LDL3) was observed. By contrast, CEOOH increased from 0 to 31 and to 15 mol/mol LDL in LDL3 and in LDL5, respectively. When oxLDL3 and oxLDL5 were at the same stage of oxidation (i.e., at their respective T_1/2) or after an equal duration of oxidation, we detected consistently higher (P<0.001) levels of CEOOH in oxLDL3, compared with oxLDL5.

2. OxLDL3 exerts potent cytotoxicity in endothelial cells, whereas oxLDL5 lacks cytotoxic action
Aliquots of oxLDL3 and oxLDL5 at their respective T_1/2 oxidized under condition A [160 µg/mL of total LDL cholesterol (CT), 3.2 µM Cu²⁺, i.e., similar amounts of lipid] were incubated for 24 h with two different models of endothelial cells: human endothelial-like cells ECV304 and human microvascular endothelial cells HMEC-1. The MTT test revealed that the cytotoxic response to oxLDL3 was dose dependent, with < 25% cell viability remaining at a concentration of 48 µg CT/mL. Under the same oxidation conditions, oxLDL5 lacked cytotoxic action; indeed, when incubated with 48 µg CT/mL of oxLDL5, 72% of cells were viable vs. 82% in control assays. No significant alteration in toxicity (72–83%) was observed as the amount of oxLDL5 increased. These data were confirmed by the determination of LDH leakage into the culture medium, which attained a ninefold increase in the presence of oxLDL3 compared with controls, thereby suggesting the occurrence of a necrotic or postapoptotic necrotic process; in contrast, LDH release remained negligible in the presence of oxLDL5. The cytotoxic effect of copper-oxidized LDL3 and LDL5 as a function of time was found to depend on the oxidative state of LDL particles, but remained consistently higher in LDL3.

3. OxLDL3 is cytotoxic in endothelial cells through apoptotic mechanisms
The type of cell death—necrosis or apoptosis—triggered by oxidized LDL was determined. ECV304 cells treated with oxLDL3 (T_1/2, 40 µg TC/mL), died mainly through an apoptotic process, as suggested by the number of cells exhibiting nuclear morphological changes characteristic of apoptosis such as chromatin condensation and nuclear fragmentation visualized by SYTO13/IP labeling (Fig. 1 a, b). oxLDL3 induced the appearance of numerous cells exhibiting fragmented nuclei, which were permeable to propidium iodide, a vital DNA marker excluded from intact nuclei. These data are indicative of a late stage of apoptosis (post-apoptotic necrotic patterns), consistent with the cellular release of LDH. Transmission electron microscopy (TEM) studies (Fig. 1c ) showed characteristic morphological features of apoptosis with cytoplasmic condensation and cell or nuclear fragmentation. Quantitative DAPI experiments and electrophoresis of cellular DNA revealed the progressive formation of DNA fragments characterized by the apoptotic ladder (Fig. 1d, e ) in oxLDL3-treated ECV304 cells. In contrast, both the number of apoptotic cells and the level of chromatin fragmentation remained markedly lower (<10%) in cells treated by oxLDL5 under the same conditions. A similar cytotoxic effect of oxLDL3 (but not of oxLDL5) was observed on HMEC-1 cells. The mechanism of apoptosis triggered by oxLDL3 was studied on ECV304 and HMEC-1 cells and involved cytoplasmic release of cytochrome c, which correlated with a concomitant increase in caspase-3-like protease (DEVDase) activity. The same experimental studies (MTT analysis, LDH activity, fluorescence microscopy, and DAPI) were performed on cells incubated with LDL subfractions oxidized under condition B. In this case, equivalent amounts of LDL protein (100 µg/mL) were added to the culture medium. Findings under condition B were indistinguishable from those obtained under condition A. Ox-LDL subspecies activity on endothelial cells was therefore equivalent when particles were oxidized on the basis of either cholesterol substrate concentration or LDL particle number, indicating that oxLDL-induced cytotoxicity was primarily a function of the physicochemical properties of the two particle subfractions of LDL.

View larger version (86K): [in this window] [in a new window]   Figure 1. Cytotoxic effect of oxLDL3 and oxLDL5 on ECV304 cells. Cells were incubated for 24 h with oxLDL3 or oxLDL5 (T1/2 of oxidation, 40 µg CT/mL). a) Percentage of apoptotic vs. necrotic cells determined by morphological counting as a function of oxLDL3 or oxLDL5 concentration. b) Double fluorescent staining of cell nuclei by Syto 13 and propidium iodide to discriminate normal HMEC cells and cells undergoing apoptosis, necrosis, or postapoptotic necrosis in the presence of oxLDL3 and oxLDL5. c) TEM of ECV304 cells. Cells were treated with oxLDL3, trypsinized, fixed, and stained for TEM. Upper panel: control cells; lower panel: apoptotic cells showing chromatin condensation and cell and nuclear fragmentation. d) Quantitative determination of DNA fragmentation by fluorometric DAPI procedure in ECV304 cells incubated 24 h with oxLDL3 or oxLDL5. e) Gel electrophoresis of DNA fragments showing typical apoptotic ladder. M, molecular weight markers; Co, control; L3, oxLDL3; L5, oxLDL5; T, taxol used as positive apoptotic inducer (1 µg/mL, 24 h incubation). *P < 0.05 vs. control; open star = P < 0.01.

4. NaBH₄ treatment prevents oxLDL cytotoxicity
Marked differences in LOOH (mainly CEOOH) level and stability during copper-mediated oxidation distinguished small, dense LDL5 from intermediate LDL3. To determine whether CEOOH could be implicated in the cytotoxicity of oxidized LDL particles, oxLDL were treated with sodium borohydride (NaBH₄; final concentration 10 µM) in order to degrade lipid (and protein) hydroperoxides. The cytotoxicity of oxLDL5 and oxLDL3 subfractions, determined by the MTT test and by residual [³H]thymidine incorporation, was markedly decreased in NaBH₄-pretreated oxLDL3 (95% residual cell viability vs. 57% in untreated oxLDL3). However, NaBH₄ treatment of oxLDL5 exerted a minor effect on cell viability (75–85% viability; MTT test). Hydroperoxide removal therefore leads to significant loss of oxLDL cytotoxicity, thereby indicating that hydroperoxides might play a pivotal role in oxLDL-induced cytotoxicity.

CONCLUSIONS

Distinct LDL particles of the light, intermediate, and dense subclasses differ significantly in their atherogenic activity at the arterial wall, but the relative potency of distinct LDL subfractions to induce cytotoxicity on oxidative modification remains indeterminate.

Our data reveal that for a defined oxidative state or for equal concentrations of LDL cholesterol or protein, intermediate oxLDL3 displayed significantly higher cytotoxicity than dense oxLDL5. The cytotoxic effect of oxLDL3 was dose dependent and related to the degree of oxidation of LDL particles; in contrast, endothelial cells displayed minor levels of cytotoxicity (<10%) in the presence of oxLDL5. OxLDL3 induced typical apoptosis as evidenced by morphological changes such as nuclear fragmentation, cytoplasmic condensation, DNA fragmentation, dysfunction of the mitochondrial membrane, cytochrome c release into the cytosol, and activation of DEVDase. Such activity was significantly greater than that of oxLDL5 at comparable stages of oxidation and for equivalent cholesterol concentrations and particle numbers.

Oxidized LDL is known to contain a variety of bioactive components, mainly LOOH, oxycholesterols, and short-chain aldehydes. OxLDL3 and oxLDL5 did not differ in oxycholesterol and TBARS content, the latter representing an overall measure of short-chain aldehydes. Clearly, then, oxycholesterols and TBARS content are a function of the degree of oxidation of LDL but do not reflect their biological properties, which appear to be primarily related to their content of other oxidation products. Distinct copper binding behavior by LDL subfractions cannot account for the observed differences in their cytotoxicity, as LDL3 and LDL5 bind similar amounts of copper.

In contrast, oxLDL3 contained markedly higher levels (up to 2.8-fold) of LOOH than oxLDL5 at all stages of copper-mediated oxidation. This is of critical importance, as previous studies have revealed a direct relation between LOOH levels in oxLDL and their propensity to induce apoptosis. When LDL hydroperoxides were removed by treatment with NaBH₄, oxLDL-induced cytotoxicity was significantly decreased, especially for oxLDL3. These results indicate that LOOH might play a pivotal role in oxLDL-induced apoptosis and necrosis.

It is established that dense LDL5 display elevated atherogenicity compared with intermediate LDL3. Whereas LDL5 predominate in the LDL density profile in mixed hyperlipidemia, hypertriglyceridemia, and type II diabetes, LDL3 preponderates in patients presenting heterozygous or homozygous forms of familial hypercholesterolemia. Intermediate LDL3 particles possess not only the potential to enhance cholesterol accumulation in atherosclerotic plaques, but also to elicit endothelial cell apoptosis, especially when present at elevated circulating levels as in hypercholesterolemia. Apoptotic cells present in atherosclerotic plaques may contribute significantly to lesion progression, vascular remodeling, and plaque rupture. Our results reveal that the intermediate LDL subclass is able to induce apoptosis of endothelial cells on oxidative modification and may therefore be implicated in fragilization of the fibrous cap. Such prothrombogenic action could be enhanced by the ability of endothelial cells to accumulate LOOH, which can trigger cellular responses ranging from induction of antioxidant enzyme production to apoptotic cell death.

In conclusion, these data provide the earliest evidence that LDL of the intermediate density subclass exert potent proapoptotic activity on endothelial cells on mild oxidative modification and are equally relevant to the premature coronary artery disease of subjects presenting familial hypercholesterolemia, in which elevated levels of intermediate LDL3 predominate.

View larger version (27K): [in this window] [in a new window]   Figure 2. Schematic diagram of the capacity of oxLDL3 vs. oxLDL5 to induce cytotoxicity in endothelial cells. OxLDL3 induces cytotoxicity through apoptotic mechanisms involving release of cytochrome c from mitochondria, increase in caspase-3-like protease activity and accumulation of DNA fragments in the nucleus. oxLDL5 is unable to induce apoptosis. Differential effects of oxLDL3 and oxLDL5 in endothelial cells are related to the elevated content of LOOH in oxLDL3 compared with oxLDL5. Reductants able to destroy LOOH block apoptotic effects of oxLDL3.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0293fje; to cite this article, use FASEB J. (November 15, 2002) 10.1096/fj.02-0293fje

² These authors contributed equally to this publication.

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